Research Items (222)
Background: Fibrosis may be considered the hallmark of systemic sclerosis (SSc), the end stage triggered by different pathological events. Transforming growth factor-β (TGF-β) and platelet-derived growth factor BB (PDGF-BB) are profibrotic molecules modulating myofibroblast differentiation and proliferation, respectively. There is evidence linking CD248 with these two molecules, both highly expressed in patients with SSc, and suggesting that CD248 may be a therapeutic target for several diseases. The aim of this work was to evaluate the expression of CD248 in SSc skin and its ability to modulate SSc fibrotic process. Methods: After ethical approval was obtained, skin biopsies were collected from 20 patients with SSc and 10 healthy control subjects (HC). CD248 expression was investigated in the skin, as well as in bone marrow mesenchymal stem cells (MSCs) treated with TGF-β or PDGF-BB, by immunofluorescence, qRT-PCR, and Western blotting. Finally, in SSc-MSCs, the CD248 gene was silenced by siRNA. Results: Increased expression of CD248 was found in endothelial cells and perivascular stromal cells of SSc skin. In SSc-MSCs, the levels of CD248 and α-smooth muscle actin expression were significantly higher than in HC-MSCs. In both SSc- and HC-MSCs, PDGF-BB induced increased expression of Ki-67 when compared with untreated cells but was unable to modulate CD248 levels. After CD248 silencing, both TGF-β and PDGF-BB signaling were inhibited in SSc-MSCs. Conclusions: CD248 overexpression may play an important role in the fibrotic process by modulating the molecular target, leading to perivascular cells differentiation toward myofibroblasts and interfering with its expression, and thus might open a new therapeutic strategy to inhibit myofibroblast generation during SSc.
Background We aimed to evaluate the phenotype, function, and microRNA (miRNA)17–92 cluster expression in Vγ9Vδ2 T-cell subsets and the correlation with immune response in rheumatoid arthritis (RA) patients. Methods Peripheral blood from 10 early RA untreated patients and 10 healthy donors (HD) was obtained. Polyclonal Vγ9Vδ2 T-cell lines were generated and analysed by flow cytometry. Analysis of miRNA17–92 cluster expression was performed by real-time polymerase chain reaction (RT-PCR), and expression of mRNA target genes was also studied. Results A remarkable change in the distribution of Vγ9Vδ2 T-cell functional subsets was observed in the peripheral blood of RA patients compared with HD, with an expansion of effector subsets and reduction of naive cells which was accompanied by modifications in proinflammatory cytokine expression. Vγ9Vδ2 T cells with a TEM (effector memory) phenotype and producing proinflammatory cytokines were correlated with disease activity score (DAS28). The comparison of miRNA expression among Vγ9Vδ2 T-cell subsets from RA patients and HD showed a lower level of miR-106a-5p and miR-20a-5p, and a higher level of miR-21a-5p, among Vγ9Vδ2 TEM cells, and a lower level of miR-19b-3p among Vγ9Vδ2 TCM (central memory) cells was also found. These differentially expressed miRNAs correlated with higher levels of expression of interleukin (IL)-8, IL-6, and PDCD4 genes. Conclusions Our results provide evidence for a role of miR-106a, miR-19-3p, miR-20a, and miR-21a in the regulation of Vγ9Vδ2 T-cell function in RA patients and suggest the possibility that the miRNA17–92 family and Vγ9Vδ2 T cells contribute to the pathogenesis of RA.
- Oct 2018
Objectives: To review the available evidence concerning the possibility of discontinuing and/or tapering the dosage of TNF inhibitors (TNFi) in RA patients experiencing clinical remission or low disease activity. Methods: A systematic review of the literature concerning the low dosage and discontinuation of TNFi in disease-controlled RA patients was performed by evaluation of reports published in indexed international journals (Medline via PubMed, EMBASE), in the time frame from 8 April 2013 to 15 January 2016. Results: We analysed the literature evaluating the efficacy and the safety of two different strategies using TNFi, decreasing dosage or discontinuation, in patients experiencing clinical remission or low disease activity. After the analysis of online databases, 25 references were considered potentially relevant and 16 references were selected. The majority of data concerned etanercept and adalimumab. Results suggested the induction of stable clinical remission or low disease activity by using TNFi followed by a dosage tapering and/or discontinuation of such drugs may be associated with the maintenance of a good clinical response in a subset of patients affected by early disease. Conclusion: RA patients treated early with TNFi and achieving their therapeutic clinical targets seem to maintain their clinical response after tapering or discontinuing TNFi. These data may allow physicians a more dynamic and tailored management of RA patients.
- Apr 2018
Objective: Macrophage activation syndrome (MAS) is a reactive form of hemophagocytic lymphohistiocytosis, which can complicate adult-onset Still disease (AOSD). We investigated AOSD clinical features at the time of diagnosis, to assess predictors of MAS occurrence. Further, we analyzed the outcomes of patients with AOSD who experience MAS. Methods: Patients with AOSD admitted to any Gruppo Italiano di Ricerca in Reumatologia Clinica e Sperimentale center were retrospectively analyzed for features typical of AOSD, MAS occurrence, and their survival rate. Results: Of 119 patients with AOSD, 17 experienced MAS (12 at admission and 5 during followup). Twelve patients with MAS at first admission differed from the remaining 107 in prevalence of lymphadenopathy and liver involvement at the time of diagnosis. In addition, serum ferritin levels and systemic score values were significantly higher in the patients presenting with MAS. At the time of diagnosis, the 5 patients who developed MAS differed from the remaining 102 in the prevalence of abdominal pain, and they showed increased systemic score values. In the multivariate analysis, lymphadenopathy (OR 7.22, 95% CI 1.49-34.97, p = 0.014) and abdominal pain (OR 4.36, 95% CI 1.24-15.39, p = 0.022) were predictive of MAS occurrence. Finally, MAS occurrence significantly reduced the survival rate of patients with AOSD (p < 0.0001). Conclusion: MAS occurrence significantly reduced the survival rate in patients with AOSD. Patients with MAS at baseline presented an increased prevalence of lymphadenopathy and liver involvement, as well as high serum ferritin levels and systemic score values. The presence of lymphadenopathy and abdominal pain was associated with MAS occurrence.
Objective To investigate the role of IL‐25/IL‐17RB axis in experimental Sjögren's syndrome (ESS) and in patients with primary Sjögren Syndrome (pSS) and pSS‐associated lymphoma. Methods Expression of IL‐25, IL‐17RB, IL‐17B and TRAF6 was analyzed on minor salivary glands (MSG) from pSS patients and on parotid glands from pSS‐associated B cell non‐Hodgkin lymphomas (NHL). IL‐17RB expression and the frequencies of natural type 2 innate lymphoid cells (nILC2), inflammatory ILC2 (iILC2) and M2‐polarized macrophages were assessed by flow cytometry in salivary gland mononuclear cells (SGMC) and peripheral blood mononuclear cells (PBMC). Tissue distribution of ILC2 cells was studied by confocal microscopy. The role of recombinant IL‐25 and of Rituximab in modulating IL‐25 expression was investigated in in vitro studies. IL‐25/IL‐17RB and TRAF6 expression and the role of IL‐25 inhibition were also studied in the ESS murine model. Results activation of IL‐25/IL‐17RB/TRAF6 axis correlated with the focus score, was observed in pSS patients and in pSS‐associated NHL. A significant increase in the frequency of iILC2 was observed in both SGMC and PBMC. IL‐25 stimulation of isolated SGMC and PBMC from patients and controls resulted in both iILC2 expansion and increased autoantibodies production. Rituximab modulated iILC2 and IL‐25 expression in pSS. Salivary gland protein‐immunised mice developed overt SS symptoms with increased IL‐25 expression and increased frequency of CD4⁺IL‐17RB⁺TRAF6⁺ cells. IL‐25 neutralization attenuated disease progression and tissue pathology in ESS mice. Conclusions IL‐25 may promote the inflammatory state in pSS and may be a potential target for novel disease‐modyfing therapeutic strategies in pSS patients. This article is protected by copyright. All rights reserved.
Background: A close relationship between rheumatic diseases and cardiovascular disease (CVD) has been reported, accounting for the higher mortality and morbidity observed in these patients. In the last years, it has been clearly reported that patients affected by primary Sjögren's syndrome (pSS) experienced an increased risk of CVD. Objective: This review aimed at investigating CVD, traditional cardiovascular (CV) risk factors and possible targeted therapeutic strategies in pSS patients. Method: Available literature concerning CV risk factors in pSS patients has been selected and discussed. Conclusions: Disease-related characteristics and traditional CV risk factors contribute to observed atherosclerotic damage in pSS patients. The pro-inflammatory mediators together with hypertension and type 2 diabetes, which are more prevalent in pSS patients, determine the increased CV burden. Future studies are needed to fully elucidate the role of possible targeted therapies.
Background: Although in the past, prevention of the joint destruction and disability were strongly emphasised in rheumatoid arthritis (RA), at present, a growing body of evidence is focused at identifying the best management of associated comorbidities, such as type 2 diabetes (T2D). Recently, the hypothesis that blocking pro-inflammatory activity may be helpful in the treatment of some comorbidities has been proposed in RA patients. Objective: We reviewed the role of IL-1β during RA and T2D, the efficacy of IL-1 blocking agents in controlling both diseases and, possible, decreasing the concomitant enhanced atherosclerotic process. Method: After literature search, the available evidence has been selected and commented in the text. Results: During RA, it is well known that different inflammatory cytokines, such as interleukin-1β (IL-1β), are pivotal pathogenic mediators and their role has been largely confirmed in clinical settings. Similarly, it has been shown that the excess of nutrients, secondary to over-nutrition, may activate the immune system, leading to an increased production of inflammatory cytokines, including IL-1β, suggesting new possible therapeutic targets. Conclusions: Although further studies are needed to fully investigate the pathogenic interplay between inflammation and metabolic disorders, IL-1β has been implicated in both RA and T2D pathogenic mechanisms. Intriguingly, the potential role of anti-IL-1 drugs has been proposed in RA patients affected by T2D.
- Feb 2018
Objective: The early diagnosis of inflammatory bowel disease (IBD)-associated spondyloarthritis (SpA/IBD) in patients affected by IBD represents a major topic in clinical practice; in particular, to date there are no available serum biomarkers revealing the presence of joint inflammation in these patients. Sclerostin (SOST), an antagonist of the Wnt/β-catenin pathway, and antisclerostin-immunoglobulin G (anti-SOST-IgG) have been recently studied in patients with ankylosing spondylitis (AS) as a putative marker of disease activity. Methods: SOST and anti-SOST-IgG serum levels were assayed in 125 patients with IBD, 85 with axial or peripheral SpA, and in control groups (patients with AS and rheumatoid arthritis, and healthy individuals). The diagnostic performance in discriminating the presence of SpA/IBD was assessed for both candidate biomarkers. Results: Patients affected by SpA/IBD with axial involvement displayed significantly lower levels of SOST and higher levels of anti-SOST-IgG compared to patients with only peripheral arthritis, IBD, and controls. Moreover, SOST and anti-SOST-IgG serum levels were inversely correlated and were associated with the duration of articular symptoms. Both biomarkers showed good accuracy in predicting the presence of axial SpA in patients with IBD. Conclusion: We demonstrated that in patients with IBD, SOST and anti-SOST-IgG might represent novel biomarkers to assess the presence of axial joint involvement. Moreover, the development of anti-SOST-IgG and the subsequent decrease of SOST serum levels could play a role in the pathogenesis of SpA/IBD.
Background Primary Sjögren’s syndrome (pSS) is a common chronic autoimmune disease characterized by lymphocytic infiltration of exocrine glands and peripheral lymphocyte perturbation. In the current study, we aimed to investigate the possible pathogenic implication of autophagy in T lymphocytes in patients with pSS. Methods Thirty consecutive pSS patients were recruited together with 20 patients affected by sicca syndrome and/or chronic sialoadenitis and 30 healthy controls. Disease activity and damage were evaluated according to SS disease activity index, EULAR SS disease activity index, and SS disease damage index. T lymphocytes were analyzed for the expression of autophagy-specific markers by biochemical, molecular, and histological assays in peripheral blood and labial gland biopsies. Serum interleukin (IL)-23 and IL-21 levels were quantified by enzyme-linked immunosorbent assay. ResultsOur study provides evidence for the first time that autophagy is upregulated in CD4+ T lymphocyte salivary glands from pSS patients. Furthermore, a statistically significant correlation was detected between lymphocyte autophagy levels, disease activity, and damage indexes. We also found a positive correlation between autophagy enhancement and the increased salivary gland expression of IL-21 and IL-23, providing a further link between innate and adaptive immune responses in pSS. Conclusions These findings suggest that CD4+ T lymphocyte autophagy could play a key role in pSS pathogenesis. Additionally, our data highlight the potential exploitation of T cell autophagy as a biomarker of disease activity and provide new ground to verify the therapeutic implications of autophagy as an innovative drug target in pSS.
To report adalimumab (Ada) efficacy on articulargastrointestinal disease and health-related quality of life (HRQoL) in patients with enteropathic spondyloarthritis(ES). METHODS A cohort of 52 patients with ES was evaluated in the departments of gastroenterology and internal medicine. At baseline, all patients underwent assessment by an integrated gastro-rheumatologic evaluation of articular and gastrointestinal activity, as well patient reported outcomes (PROs) of the HRQoL questionnaires. After this integrated evaluation and following a specific working flowchart, the Ada anti-tumor necrosis factor (TNF)-inhibitor was assigned to a cohort of 30 patients and its clinical efficacy was evaluated at baseline and after 6-mo and 12-mo treatment by the following tests: (1) Ankylosing Spondylitis Disease Activity Score- C-Reactive Protein (ASDAS-CRP); Bath Ankylosing Spondylitis Disease Activity Index (BASDAI), Bath Ankylosing Spondylitis Functional Index (BASFI) and Bath Ankylosing Spondylitis Metrology Index (BASMI) for articular activity; (2) Inflammatory Bowel Disease Questionnaire (IBDQ), Crohn’s Disease Activity Index (CDAI) and partial Mayo (pMayo) score for gastrointestinal symptoms and activity; and (3) Health Assessment Questionnaire (HAQ), Patient Global Assessment (PGA) and Short Form-36 health survey (SF-36) questionnaires for PROs of the HRQoL. RESULTS Integrated evaluation and management of the patients affected by ES, carried out simultaneously by a gastroenterologist and a rheumatologist, allowed clinicians to choose the optimal therapeutic strategy. In a cohort of 30 ES patients affected by active articular and gastrointestinal disease, or axial active articular inflammation, Ada led to fast and sustained improvement of both articular and gastrointestinal disease activities. In fact, all the clinimetric evaluation tests exploring articular or gastrointestinal activity, as well as all the HRQoL scores, showed a significant improvement having been achieved at the earliest (6-mo) assessment. This important clinical improvement was maintained at the 12-mo follow-up. Importantly, global and gastrointestinal quality of life significantly correlated with articular disease activity, providing evidence to support that the integrated evaluation is the best option to manage patients with ES. CONCLUSION Ada treatment, upon multidisciplinary (gastrorheumatologic) evaluation, significantly improves both articular and gastrointestinal inflammation, thereby improving the HRQoL in patients affected by ES.
Several studies have pointed out a significant association between rheumatoid arthritis (RA) and accelerated atherosclerosis. At the best of our knowledge, no such study has been carried out in a large Italian series and, in this study, we aimed to investigate the prevalence of both subclinical atherosclerosis and history of cardiovascular events (CVEs), in patients consecutively admitted from January 1, 2015 to December 31, 2015 to Rheumatology Units throughout the whole Italy. Centers members of GIRRCS (Gruppo Italiano di Ricerca in Reumatologia Clinica e Sperimentale) were invited to enrol patients consecutively admitted from January 1, 2015 to December 31, 2015 and satisfying American College of Rheumatology/ European League Against Rheumatism criteria for RA and to investigate each of them for: traditional cardiovascular risk factors: sex, age, smoking habit, total cholesterol, triglycerides, glycaemia, high blood pressure, metabolic syndrome (MS), type 2 diabetes (T2D); RA features: disease duration as assessed from the first symptom, disease activity as evaluated by DAS28, radiographic damage as assessed by hands and feet x-ray, and previous joint surgery; prevalence of both subclinical atherosclerosis and history of CVEs. Eight centers participated to the study. From January 1, 2015 to December 31, 2015, the 1176 patients, who had been investigated for all the items, were enrolled in the study. They were mostly women (80.52%), with a median age of 60 years (range, 18–91 years), a median disease duration of 12 years (range, 0.8–25 years), seropositive in 69.21%. Nineteen percent were in remission; 17.51% presented low disease activity; 39.45% moderate disease activity; 22.61% high disease activity. Eighty-two patients (6.9%) had a history for CVEs (58 myocardial infarction, 38 heart failure, 10 ischemic transitory attack, and 7 stroke). This figure appears to be lower than that reported worldwide (8.5%). After excluding the 82 patients with a history of CV events, subclinical atherosclerosis was detected in 16% of our patients, (176 patients), a figure lower than that reported worldwide (32.7%) and in previous Italian studies. This is the first Italian multicenter study on subclinical and clinical atherosclerosis in patients with RA. We pointed out a low prevalence of both subclinical atherosclerosis and history of CV events.
- Sep 2017
Macrophage activation syndrome (MAS) is hyper-inflammatory life-threatening syndrome, typically associated with high levels of serum ferritin. This is an iron storage protein including heavy (H) subunits and light (L) subunits, categorised on their molecular weight. The H-/L-subunits ratio may be different in tissues, depending on the specific tissue and pathophysiologic status. In this work, we analysed the bone marrow (BM) biopsies of adult MAS patients to assess the presence of: i. H-ferritin and L-ferritin; ii. CD68+/H-ferritin+ and CD68+/L-ferritin+; iii. IL-1β, TNF, IFN-γ. We also explored possible correlations of these results with clinical data. H-ferritin, IL-1β, TNF and IFN-γ resulted to be significantly increased in MAS. Furthermore, an increased number of CD68+/H-ferritin+ cells and an infiltrate of cells co-expressing H-ferritin and IL-12, suggesting an infiltrate of M1 macrophages, were observed. H-ferritin levels and CD68+/H-ferritin+ cells resulted to be correlated with haematological involvement of the disease, serum ferritin and C-reactive protein. L-ferritin and CD68+/L-ferritin+ cells did not correlate with these parameters. In conclusion, during MAS, H-ferritin, CD68+/H-ferritin+ cells and pro-inflammatory cytokines resulted to be significantly increased in the BM inflammatory infiltrate, pointing out a possible vicious pathogenic loop. To date, H-ferritin and CD68+/H-ferritin+ were significantly associated with haematological involvement of the disease, suggesting bio-markers assessing severity of clinical picture. This article is protected by copyright. All rights reserved.
- Aug 2017
MAIT cells are expanded in salivary glands of patients with Sjogren's syndrome and are IL-17 polarized. IL-7 and IL-23 induce IL-17 production activating two different pathways: IL-7 stimulation induces in fact a significant STAT3 and HIF1alpha upregulation, conversely, IL-23 stimulation significantly induces RORc overexpression in MAIT cells of patients with Sjogren's syndrome.
Although the better management of rheumatoid arthritis (RA) has significantly improved the long-term outcome of affected patients, a significant proportion of these may develop associated comorbidities including cardiometabolic complications. However, it must be pointed out that a comprehensive cardiometabolic evaluation is still poorly integrated into the management of RA patients, due to a limited awareness of the problem, a lack of appropriate clinical studies, and optimal strategies for cardiovascular (CV) risk reduction in RA. In addition, although several studies investigated the possible association between traditional CV risk factors and RA, conflicting results are still available. On this basis, we planned this cross-sectional study, aimed at investigating the prevalence of type 2 diabetes (T2D) and impaired fasting glucose (IFG) in RA patients compared with age- and gender- matched control individuals. Furthermore, we analyzed the role of both traditional and RA-related CV risk factors in predicting T2D and IFG. We observed an increased prevalence of T2D in RA patients when compared with age- and gender-matched controls. Regression analyses demonstrated that the presence of high blood pressure (HBP), a longer disease duration, and exposure to corticosteroids (CCS) were significantly associated with an increased likelihood of being classified as T2D. In addition, we observed an increased prevalence of IFG in RA patients when compared with age- and gender-matched controls. Regression analyses demonstrated that a higher body mass index (BMI), the presence of metabolic syndrome (MetS), higher levels of total cholesterol, the presence of radiographic damage, and higher serum levels of C-reactive protein (CRP) were significantly associated with an increased likelihood of presenting IFG. In this cross-sectional study, we observed an increased prevalence of T2D and IFG in an Italian cohort of RA patients when compared with age- and gender-matched control individuals. Interestingly, both RA-specific features, such as disease duration, CCS exposure, and radiographic damage, and traditional CV risk factors, such as HBP and MetS, were significantly associated with glucose metabolism abnormalities.
Autoimmune diseases are a complex set of diseases characterized by immune system activation and, although many progresses have been done in the last 15 years, several unmet needs in the management of these patients may be still identified. Recently, a panel of international Experts, divided in different working groups according to their clinical and scientific expertise, were asked to identify, debate and formulate a list of key unmet needs within the field of rheumatology, serving as a roadmap for research as well as support for clinicians. After a systematic review of the literature, the results and the discussions from each working group were summarised in different statements. Due to the differences among the diseases and their heterogeneity, a large number of statements was produced and voted by the Experts to reach a consensus in a plenary session. At all the steps of this process, including the initial discussions by the steering committee, the identification of the unmet needs, the expansion of the working group and finally the development of statements, a large agreement was attained. This work confirmed that several unmet needs may be identified and despite the development of new therapeutic strategies as well as a better understanding of the effects of existing therapies, many open questions still remain in this field, suggesting a research agenda for the future and specific clinical suggestions which may allow physicians to better manage those clinical conditions still lacking of scientific clarity.
Objective: To evaluate occult cardiac involvement in asymptomatic systemic sclerosis (SSc) patients by pharmacological stress, rest perfusion and delayed enhancement cardiac magnetic resonance (CMR), for a very early identification of patients at higher risk of cardiac-related mortality. Methods: Sixteen consecutive patients with definite SSc, fulfilling the American College of Rheumatology/European League Against Rheumatism 2013 classification criteria in less than 1 year from the onset of Raynaud's phenomenon, underwent pharmacological stress, rest perfusion and delayed enhancement CMR. At enrollment, no patient showed signs and/or symptoms suggestive for cardiac involvement. No patient showed traditional cardiovascular risk factors. Both the 12-lead electrocardiogram examination and echocardiographic evaluation did not show any alterations in our cohort. Results: Stress perfusion defects of left ventricle were detected in six out of 16 (37.5%) patients and these defects did not match with the coronary flow distribution. The results showed the presence of two different patterns of stress perfusion defects: sub-endocardial and/or a midmyocardial. The presence of stress perfusion defects did not correlate with any clinical feature of enrolled patients. Conclusion: Myocardial stress perfusion defects may be detected early by pharmacological stress perfusion CMR, a reliable and sensitive technique for the noninvasive evaluation of SSc heart disease, in patients with SSc of recent onset. These defects seem to be independent from traditional risk factors and associated comorbidities, suggesting they are a specific hallmark of the disease.
Background Primary Sjogren Syndrome (pSS) is a chronic inflammatory disorder affecting exocrine glands. Both IL-23 and the downstream cytokines IL-17 and IL-22 are recognised as key players in the disease. Therefore, the identification of the cellular sources and inducers of IL-17 is crucial in the understanding of the drivers of inflammation in pSS. Mucosal-associated invariant T (MAIT) cells recognize riboflavin derivatives presented by the MHC class I-like molecule MR1. Objectives Recently, MAIT cells have been implicated in the pathogenesis of autoimmune disorders and found expanded in salivary glands of pSS patients. Their expression of IL7R and IL23R, makes them potential contributors to the pathogenesis of pSS. Methods Mononuclear cells from 16 patients with pSS and 14 subjects with non Sjogren Syndrome secca syndrome (nSS) were isolated from blood and salivary glands. We assessed the phenotype and cytokine expression of MAIT cells by surface and intracellular flow cytometry analysis. The function of MAIT cells was assessed upon in vitro stimulation with recombinant IL-7, IL-23 or their specific ligand lumazine (a Vitamin B2 precursor previously reported to be recognized by and specifically activate MAIT cells). Results MAIT cells were reduced in frequency in peripheral blood but not in minor salivary glands of patients with pSS, compared with nSS. In vitro stimulation of MAIT cells from pSS patients induced cytokine production which was dependent on priming with IL-7, IL-23 or specific antigen (lumazine) stimulation. Particularly, IL-7 and IL-23 guarantee IL-17 polarization of MAIT cells by two different pathways via the activation of STAT3 or RORc respectively. Conclusions This study confirms a potential role for MAIT cells in pSS and, for the first time, links IL-7 and IL-23 to the IL-17 polarization of MAIT cells in these patients. Disclosure of Interest None declared
Background During macrophage activation syndrome (MAS), an inflammatory life-threatening syndrome, extremely high levels of serum ferritin may be observed . Ferritin is an intracellular iron storage protein comprising 24 subunits that may be divided in heavy (H) subunits and light (L) subunits, based on their molecular weight . The H-/L-subunits ratio may change, depending on the specific tissue and the physiologic status of the cell. In the normal condition, ferritin enriched in L subunits (L-ferritin) has been found in the liver and in the spleen, whereas the ferritin enriched in H subunits (H-ferritin), may be mainly observed in the heart and kidneys . Objectives We investigated the tissue expression of both H-and L-ferritin as well as the macrophage subsets expressing these molecules, in the inflammatory BM infiltrate of MAS patients. In addition, the co-expression of IL-1β, TNF, IFN-γ and H- or L ferritin, within the inflammatory cells, was assessed. Finally, we explored if the imbalance between H-ferritin and L-ferritin as well as the number of ferritin positive cells may be considered helpful bio-markers to assess the severity of these patients. Methods We analysed the bone marrow (BM) biopsies, by immunofluorescence of 10 adult MAS patients affected by rheumatic disease to assess the presence of: i. both H- and L-ferritin; ii. the number of CD68+/H-ferritin+ and CD68+/L-ferritin+; iii. the tissue pro-inflammatory cytokines, IL-1β, TNF, IFN-γ; and we correlated these data with clinical and laboratory data. Furthermore, the presence of ferritins was assessed in the sera of the same patients by western blot analysis. Results We observed an increased tissue expression of H-ferritin and of pro-inflammatory cytokines (IL-1β, TNF, IFN-γ). Western blot analysis, in the sera, of H-ferritin mirrored data on the tissue. Furthermore, an increased number of CD68+/H-ferritin+ cells and an infiltrate of cells co-expressing H-ferritin and IL-12, suggesting an infiltrate of M1 macrophages, were observed. Tissue H-ferritin levels correlated with the decreased counts of WBC (p=0.01) and PLT (p=0.0001); with the increased values of serum ferritin (p=0.012) and C-reactive protein (CRP) (p=0.0058); and with the tissue expression of IL-1β (p=0.006). The number of the CD68+/H-ferritin+ cells correlated with the decreased counts of WBC (p=0.03) and PLT (p=0.0007), and with the increased serum ferritin levels (p=0.0088) and CRP (p=0.049). The analyses concerning tissues L-ferritin as well as the number of CD68+/L-ferritin+ cells and the same parameters failed to show any significant result. Conclusions We observed an increased tissue expression of H-ferritin associated with an increased expression of IL-1β. Interestingly, in the BM inflammatory infiltrate an increased number of CD68+/H-ferritin+ cells was shown. Of note, tissue expression of H-ferritin as well as the number of CD68+/H-ferritin+ significantly were associated with the hematological involvement of the disease, suggesting possible bio-markers to assess the severity of these patients. References • Ramos-Casals M, et al. Lancet. 2014;383:1503–16. • Rosário C, et al. BMC Med. 2013;11:185. References Disclosure of Interest None declared
Background miRNAs are non-coding RNAs which have significant roles in regulating gene expression. The miR17-92 cluster appears to be a key factor in the inflammatory pathways activated during RA. Objectives In this study we aimed to evaluate miR17–92 expression and functions in γδ T cell subsets in RA patients, γδ T cells, in fact produce proinflammatory cytokines such as IFN-g, IL-6 and IL-8 that may contribute to the inflammatory responses in RA. Methods Heparinized peripheral blood from 10 early RA untreated patients and 10 healthy donors was obtained for this study. Polyclonal Vγ9Vδ2 T cell lines were generated first by magnetic isolation followed by sorting (FACSAria) and further analysed by flow cytometry to define their phenotype and their pattern of cytokine production. Expression analysis of miRNA17–92 cluster was performerd by RT-PCR and after identification of relevant miRNA involved in RA, mRNA expression of miRNA target genes was studied. Results A remarkable change in the distribution of Vγ9Vδ2 T cell functional subsets with an expansion of effector subsets and a reduction of naïve cells, was observed in the peripheral blood of RA patients, as compared to healthy donors, which were accompanied by modifications in proinflammatory cytokine expression. Particularly, TEM (effector memory) and TEMRA (effector memory terminally differentiated) cells resulted the major souce of IL-6 and IL-8. A significant correlation between miRNA expression and levels of IL-6 and IL-8 was found. The comparative analysis of miRNA expression among Vγ9Vδ2 T cell subsets, between RA patients and healthy controls showed a downregulation of miR106a-5p and miR20a-5p and an upregulation of miR 21a-5p among Vγ9Vδ2 TEM cells; a downregulation of miR19–3p among Vγ9Vδ2 TEM and TcM (central memory) cells was also found. This miRNA expression regulated IL-8 and IL-6 gene transcription contributing to the survival of the pro-inflammatory pool reducing the expression of the PDCD4 gene. Conclusions Our results provide evidence of a role of miR106a, miR19–3p, 20a and 21a in the regulation of Vγ9Vδ2 T cells function in RA patients and suggest the possibility that the miR17–92 family and γδ T cells participate to the pathogenesis of RA. Disclosure of Interest None declared
Background Remission is the current aim of early RA treatment. In patients with early, aggressive RA, combination with adalimumab (ADA) plus methotrexate (MTX) was superior to either MTX or ADA alone in obtaining clinical remission. Moreover, it has been demonstrated that a short-term aggressive treatment with high-dose glucocorticoids (GC) plus conventional DMARDs lead to long-term (up to 5 years) benefits. Objectives To compare the proportion of patients who achieve remission at 12 months, between two groups of subjects treated with ADA + MTX + high dose GC (intensive) and ADA + MTX + placebo (standard), and to evaluate the proportion of those maintaining remission at 24 months, after discontinuation of GC at 6 and ADA at 12 months. Methods The main inclusion criteria were: active RA, disease duration ≤1 year, GC and MTX-naïvity, at least one predictor of aggressive disease. All subjects received ADA for 12 months and MTX (20 mg/w) to the end of month 24. Subjects were randomized to receive prednisone (orally, 50 mg/d, progressively tapered to 6.25 mg and stopped at month 6) or placebo. Response was evaluated using DAS28, CDAI, SDAI and ACR response criteria. The difference in rate and 95% CI will be computed with a binomial regression model and identity link, while clustering on center. An intention-to-treat analysis was performed. Results 118 patients were assigned to standard and 115 patients to intensive group. Remission (DAS28<2.6, SDAI≤3.3, CDAI≤2.8) at 1 year in standard vs intensive group were: 56.25% vs 45.28% (RD= -11%, 95% CI -23 to1, p=0.07), 30.36% vs 21.69% (RD= -9%, 95% CI -20 to 3, p=0.14), and 28.57% vs 22.64% (RD= -6%, 95% CI -18 to 6, p=0.34). DAS28 remission at 2 years was 36.84% in standard vs 30.93% in intensive (RD= -6%, 95% CI -17 to 5, p=0.28). No superiority of the intensive group was seen in ACR20–50–70 response rates at 4, 8, 12, 24 months. The overall frequency of adverse events (AE) in patients that completed the trial was comparable between groups. The percentage of patients who discontinued for AE was higher in the intensive group (9.32% in standard vs 16.52% in intensive, RD=7%, 95% CI 1 to 13, p=0.01). • Download figure • Open in new tab • Download powerpoint Conclusions Our results confirm that intensive treatment with biologics in early, aggressive RA might be considered to induce and maintain clinical remission. The addition of high-dose GC to a first line treatment with ADA and MTX did not prove to induce a further improvement in efficacy. Although these results should be tested with other biologic therapies, the high rate of drop out for AE in the intensive group should be carefully considered in the risk-benefit ratio. Disclosure of Interest None declared
Background Lupus nephritis (LN) is one of the most severe features of systemic lupus erythematosus (SLE). Several cytokines and chemokines are secreted locally in case of glomerular inflammation. Interleukin 32 (IL32) is a newly described cytokine that exhibits several properties typical of proinflammatory cytokines. Ex vivo and in vitro studies supported the role of Toll like receptors (TLRs) in LN pathogenesis and recent investigations demonstrated that Poly I:C, a ligand for (TLR) 3, strongly induced IL32 production from several cell populations. Objectives To investigate serum and urinary levels of IL32 in a cohort of LN patients compared to SLE patients without renal involvement and healthy controls (HC). In addition, we investigated kidney expression of IL32 and in vitro ability of LN patients' serum IgG to stimulate IL32 production by TLR3 activation in human embryonic kidney 293 cells line stably transfected with a TLR3 plasmid (Hek293/T3). Methods Serum and urinary IL32 concentrations were measured using ELISA; a polyclonal rabbit anti-human IL32 was used to evaluate the expression of IL32 in renal biopsies. To assess the production of IL32 induced by patients' IgG and the transduction pathway of TLR3 we performed Western Blot analysis for IL32, TBK1 and NFkB. Results We recruited 60 LN patients, 50 SLE patients without renal involvement and 30 HC; 40 LN patients had an active disease (a-LN) and the remaining 20 were in remission (r-LN). IL32 serum levels were significantly higher in patients with r-LN (median 1368, IQR 3910) and HC (median 721, IQR 2271) compared to SLE patients without renal involvement (median 203, IQR 662.8 pg/ml) (p=0.03 and p=0.018, respectively). There were no significant differences in urinary IL32 levels among LN patients, SLE patients without renal involvement and HC. In LN patients a direct association between IL32 serum levels and disease duration (p=0.02; r 0.2978) was observed. Immuno-histochemical analysis performed on renal biopsies of 20 a-LN and 8 HC showed that IL32 was strongly expressed in renal samples of LN patients, especially in patients with class IV, compared to controls. Western Blot analysis showed that antibodies isolated from LN patients induced in vitro production of IL32 in Hek293/T3 cells as well as the phosphorylation of NFkB and TBK1. Conclusions The results of the present study show increased IL32 serum levels in r-LN patients as well as increased expression of such cytokine in renal tissues of a-LN patients. IL32 renal expression and its production by Hek293/T3 cells after patients' IgG stimulation, probably mediated by TLR3, may suggest the production of IL32 directly at renal level in course of LN. Disclosure of Interest None declared
Background Several studies showed a close relationship between Rheumatoid Arthritis (RA) and accelerated atherosclerosis [1,2]. At the best of our knowledge, no such study has been carried out in a large Italian series. Objectives To investigate the prevalence of presence of subclinical atherosclerosis and history of cardio-cerebrovascular events (CVEs), in 1266 patients consecutively admitted to Rheumatology Units throughout the whole Italy. Methods From 01/01/2015 to 31/12/2015, 1266 consecutive patients admitted to GIRRCS centres, satisfying ACR/EULAR criteria for RA were investigated for: i. traditional cardiovascular risk factors: gender, age, smoking habit, cholesterol, triglycerides, glycemia, systemic arterial hypertension (SAH), metabolic syndrome (MS), type 2 diabetes (T2D); ii. RA aspects: disease duration as assessed from the first symptom, disease activity as evaluated by DAS28, radiographic damage by joint X-ray, and joint surgery; iii. subclinical atherosclerosis, as assessed by ultrasound technique and/or atherosclerotic peripheral lesions; iv. history of CVEs. Results We evaluated 1176 patients out of 1266, that were investigated for both CVEs and subclinical atherosclerosis. They were mostly females (80.52%), with a median age of 60 years (range 18–91 years), a median disease duration of 12 years (range 0.8–25 years), seropositive in 69.21%. Nineteen percent were in remission; 17.51% presented low disease activity; 39.45% moderate disease activity, 22.61% high disease activity. Out of 1176 patients, 217 (18%) showed evidence of subclinical atherosclerosis: a figure lower than that reported worldwide (32.7%) . Eighty-two patients (6.9%) had a history for CVEs (58 myocardial infarction, 38 heart failure, 10 ischemic transitory attack, 7 Stroke), too this figure is lower than that reported worldwide (8.5%) [3,4]. In multivariate analysis, older age (p=0.0001, OR:1.069, CI95%:1.05–1.09), MS (p=0.0001, OR:3.417, CI95%:2.16–5.40) and SAH (p=0.0001, OR:3.714, CI95%:2.23–6.17) and high disease activity (p=0.001, OR:2.117, CI95%:1.35–3.32) were significantly associated with the presence of subclinical atherosclerosis. Male gender (p=0.0001, OR:3.465, CI95%:1.94–6.185), MS (p=0.005, OR:2.542, CI95%:1.29–4.52), T2D (p=0.007, OR:2.324, CI95%:1.29–4.29) and SAH (p=0.001, OR:4.921, CI95%:2.14–11.45) and higher disease activity (p=0.003, OR:1.316, CI95%:1.15–1.68) were significantly associated with a history of CVEs. Conclusions This is the first Italian multicenter study on subclinical and clinical atherosclerosis in patients with RA. We pointed out a low prevalence of both subclinical atherosclerosis and history of CV events. Nonetheless, a high disease activity and presence of cardiovascular risk factors were found to play a role, similarly to other countries. References • Ruscitti P, et al. PLoS One. 2017;12:e0170108. • Ambrosino P, et al. Thromb Haemost. 2015;113:916–30. • Solomon D et al. Ann Rheum Dis. 2010;69:1920–5. • Avina-Zubieta JA, et al. Ann Rheum Dis. 2012;71:1524–9. References Disclosure of Interest None declared
Background Vascular adventitia is a major site of immune surveillance and inflammatory cell trafficking and is the most complex compartment of the vessel wall comprising fibroblasts, dendritic cells and macrophages, progenitor cells, vasa vasorum, pericytes and adrenergic nerves. It has been proposed that activation of adventitial nerves and release of sensory neuropeptides from their peripheral terminals may leads to neurogenic inflammation. Giant cell arteritis (GCA) is an immune-mediated disease of unknown etiology in which the inflammatory process seems to start from the adventitia of affected arteries. Objectives aim of the study was to evaluate the occurrence of adventitial nerves inflammation and to immunologically characterize the adventitial neuritis occurring in GCA patients. Methods Immunohistochemistry and RT-PCR were used to study the presence of neuritis in temporal artery samples obtained from 30 patients with temporal artery (TAB) positive GCA, 20 TAB negative GCA and 20 controls. Laser capture microdissection (LCM) was used to isolate adventitial nerves and nerve expression of pro-inflammatory cytokines involved in the pathogenesis of GCA such as IL-6, IL-17, IL-32, IL-9, IL-33 was assessed by RT-PCR. Expression of pro-inflammatory cytokines by adventitial nerves was further studied by immunohistochemistry and confocal microscopy. Autophagy, unfoled protein response (UPR) and inflammasomes pathways were also studied by RT-PCR and immunohistochemistry. Results Adventitial nerves showed infiltration of CD3⁺ T cells in all the TAB positive and in 12 out of 20 TAB negative arteries but were never observed in control arteries. RT-PCR expression analysis of different pro-inflammatory cytokines clearly demonstrated specific over-expression of IL-33 in LCM isolated inflamed nerves. Immunohistochemical and confocal microscopy analysis confirmed nerve IL-33 expression. RT-PCR and immunoistochemistry demonstrated that AIM2 and NLRP3, but not NLCR4, inflammasomes were activated in inflamed nerves of GCA patients. According to inflammasomes activation, increased IL-18 expression was observed. Autophagy-related genes such as ATG16L1 and LC3 and UPR-related genes such as XBP-1 and CHOP were also over-expressed in GCA adventitial nerves as demonstrated by RT-PCR and immunohistochemistry. Finally, increased expression of AIM2 and NLRP3 inflammasomes, autophagy and UPR was also observed outside the nerves, in the context of inflamed artery wall. Conclusions here we demonstrated that adventitial neuritis is present in both inflamed and non-inflamed arteries of GCA patients. GCA inflamed nerves specifically produce IL-33 a cytokine of the innate immunity that has been demonstrated to be involved in the pathogenesis of GCA. AIM2 and NLRP3 inflammasomes activation was also observed in GCA arteries and in particular in the inflamed adventitial nerves being accompanied by the increased expression of IL-18. Specific activation of autophagy and UPR pathways was also observed in the inflamed GCA nerves. Altogheter these findings seem to suggest a complex immune activation of adventitial nerves in GCA, suggesting a possible role of arterial neuritis in the initiation and perpetuation of GCA artery inflammation. Disclosure of Interest None declared
Background Sclerostin is an osteocyte-specific factor that binds to low-density lipoprotein receptor-related protein 5 (LRP5) inhibiting the Wnt signaling pathway and possibly contributing to the pathogenesis of Ankylosing spondylitis (AS). Subclinical gut inflammation observed in AS patients is characterized by the presence of dysbiosis and innate immune alterations. In the gut, LRP5 activation by unknown ligands inhibits serotonin production. Serotonin, by inducing glial derived neurotrophic factor (GDNF), controls ILC3 expansion, in the context of glial–ILC3–epithelial cell unit (GIECU). Sclerostin/serotonin axis has been never studied in AS. Objectives Aim of this study was to evaluate whether sclerostin is produced in the gut; to study the sclerostin/serotonin axis in AS and the effect of sclerostin on enterochromaffin cells (EC); to evaluate the presence of intestinal GIECU in AS and the role of serotonin in modulating the production of GDNF on isolated intestinal glial derived cells. We finally studied the effect of GDNF on ILC3. Methods Ileal, synovial and bone marrow (BM) expression of sclerostin, serotonin and GDNF were investigated by rt-PCR, immunohistochemistry and WB in 30 AS patients and 20 controls. Platelet and plasma unconjugated concentrations of serotonin were assessed by high-performance liquid chromatography (HPLC). Isolated bacteria from AS ileal biopsies were cultured with EC and serotonin expression evaluated by RT-PCR. Sclerostin and serotonin gut expression were evaluated in HLA-B27 TG rats before and after antibiotics treatment. EC were stimulated with sclerostin and the expression of THP1 assessed by RT-PCR. The presence of GIECU was studied by confocal microscopy analysis of GFAP/Tbet/Thy1. Isolated intestinal glial cells were stimulated with serotonin and the modulation of GDNF assessed by RT-PCR. The effect of GDNF on ILC3 was evaluated by flow cytometry. Results Sclerostin was produced in the gut and down-regulated in AS. Up-regulation of serotonin was observed in the gut, in the synovia and plasma, but not in BM of AS. Isolated intestinal bacteria from AS reduced EC serotonin production. Sclerostin down-regulation and serotonin over-expression were observed in the gut of HLA-B27 TG rats where Antibiotics increased intestinal sclerostin production and reduced serotonin expression. Treatment of isolated gut EC with sclerostin down-regulated the expression of THP1. GDNF was over-expressed in AS gut and confocal microscopy analysis demonstrated the existence of glial-ILC3-epithelial cells unit in AS patients. Finally, serotonin induces the release of GDNF by isolated intestinal glial cells and recombinant GDNF expanded RET⁺ILC3. Conclusions here we demonstrate for the first time that intestinal sclerostin is the ligand of LRP5 and modulates the release of serotonin. Sclerostin/serotin axis is dysregulated in AS patients and HLA-B27 TG rats. In HLA-B27 TG rats, antibiotics restored sclerostin production and serotonin expression indicating a role of dysbiosis in modulating sclerostin/serotonin axis. Serotonin seems to be an important regulator of ILC3 expansion by inducing the production of GDNF by enteric glial cells, in the context of glial-ILC3-epithelial cells unit. Disclosure of Interest None declared
Giant cell arteritis (GCA) is an inflammatory chronic disease occurring exclusively in elderly individuals. Until recently, the disease has been considered a unique disease resulting from the interaction in the walls of susceptible arteries, between an unknown infectious agents with local dendritic cells (DCs), activated CD4 T cells and effector macrophages. Recent evidence has shown that this view was too simplistic and has clarified many of the pathogenetic aspects of the disease. Many genetic studies recently published have identified different new genes, including cytokines, adhesion molecules and regulators of innate immunity, as crucial players in the development and progression of GCA. Recent evidence suggests that there is heterogeneity of histological lesions in GCA, that are correlated with different immunological Th9 and Th17 signature.The recent demonstration that Varicella-zoster virus (VZV) antigen is present in the 64% of GCA-negative TAs and in the 73% of GCA-positive TAs could represent an important point of arrival in the search for a causative agent in the pathogenesis of a metameric disease such as GCA. In this context, cytokines such as IL-32 and IL-33 that act as a danger signal following tissue damage and infection are over-expressed in GCA arteries. Artery tertiary lymphoid organs, present in up to 50% of GCA-positive arteries, could represent the sites were primary immune responses and T- and B-cell autoimmune responses against viral antigens are organized. The recently demonstrated disturbed distribution of B cells in GCA could be also relevant in the pathogenesis of the disease, possibly contributing to the enhanced IL-6 response. Altogether, these evidences may clarify many pathogenetic aspect of the disease, also suggesting complexity greater than first imagined.
Background Dysbiosis has been recently demonstrated in patients with ankylosing spondylitis (AS) but its implications in the modulation of intestinal immune responses have never been studied. The aim of this study was to investigate the role of ileal bacteria in modulating local and systemic immune responses in AS. Methods Ileal biopsies were obtained from 50 HLA-B27⁺ patients with AS and 20 normal subjects. Silver stain was used to visualise bacteria. Ileal expression of tight and adherens junction proteins was investigated by TaqMan real-time (RT)-PCR and immunohistochemistry. Serum levels of lipopolysaccharide (LPS), LPS-binding protein (LPS-BP), intestinal fatty acid-BP (iFABP) and zonulin were assayed by ELISA. Monocyte immunological functions were studied in in vitro experiments. In addition the effects of antibiotics on tight junctions in human leukocyte antigen (HLA)-B27 transgenic (TG) rats were assessed. Results Adherent and invasive bacteria were observed in the gut of patients with AS with the bacterial scores significantly correlated with gut inflammation. Impairment of the gut vascular barrier (GVB) was also present in AS, accompanied by significant upregulation of zonulin, and associated with high serum levels of LPS, LPS-BP, iFABP and zonulin. In in vitro studies zonulin altered endothelial tight junctions while its epithelial release was modulated by isolated AS ileal bacteria. AS circulating monocytes displayed an anergic phenotype partially restored by ex vivo stimulation with LPS+sCD14 and their stimulation with recombinant zonulin induced a clear M2 phenotype. Antibiotics restored tight junction function in HLA-B27 TG rats. Conclusions Bacterial ileitis, increased zonulin expression and damaged intestinal mucosal barrier and GVB, characterises the gut of patients with AS and are associated with increased blood levels of zonulin, and bacterial products. Bacterial products and zonulin influence monocyte behaviour.
- Jan 2017
Systemic sclerosis (SSc) has the highest fatality rate among connective tissue diseases and is characterized by vascular damage, inflammation and fibrosis of the skin and various internal organs. Interstitial lung disease (ILD) frequently complicates SSc and can be a debilitating disorder with a poor prognosis. ILD is the most frequent cause of death in SSc, and the management of SSc-ILD patients is a great challenge. Early detection of pulmonary involvement based on a recent decline of lung function tests and on the extent of lung involvement at high-resolution computed tomography is critical for the best management of these patients. This article summarizes classification, pathogenesis, diagnosis, prognosis, survival and finally current and future treatment options in SSc-ILD.
The aim was to establish how patients experience the impact of spondyloarthritis (SpA) on work disability and working life. The survey was performed in 17/20 regions in Italy (1 January to 31 March 2013). A multiple-choice questionnaire was published on the official website of the sponsor - the National Association of Rheumatic Patients (ANMAR) - and hard-copies were distributed at outpatient clinics for rheumatic patients. Respondents (n = 770) were of both sexes (56 % men), educated (62 % at high school or more), of working age (75 % aged ≤60 years), and affected by SpA. The most common types diagnosed were ankylosing spondylitis (AS) (39 %) and psoriatic arthritis (PsA) (36 %). Respondents were working full-time (45 %), part-time (8 %) or had retired (22 %); 15 % were unemployed (for reasons linked to the disease or for other reasons, students or housewives). Patients reported disability (39 %), were receiving disability benefits (34 %), were experiencing important limitations that were hindering their professional development/career (36 %) and some had to change/leave their job or lost it because of SpA (21 %). Employed respondents (n = 383) had worked on average 32.2 h in the last 7 days. More hours of work were lost over the last 7 days due to SpA (2.39 h vs 1.67 h). The indirect costs of the disease amounted to €106/week for patients reporting well-being/good physical conditions/improvement and €216/week for those reporting permanent impairment. Most patients were in the midst of their productive years and were experiencing considerable difficulties in carrying out their job because of the disease: half of them reported disability and one third were experiencing important limitations in their career perspective.
Background Adult-onset Still’s disease (AOSD) is rare inflammatory disease of unknown etiology that usually affects young adults. The more common clinical manifestations are spiking fevers, arthritis, evanescent rash, elevated liver enzymes, lymphadenopathy, hepatosplenomegaly, and serositis. The multi-visceral involvement of the disease and the different complications, such as macrophage activation syndrome, may strongly decrease the life expectancy of AOSD patients. Methods This study aimed to identify the positive and negative features correlated with the outcome of patients. A retrospective analysis of AOSD patients prospectively admitted to three rheumatologic centers was performed to identify the clinical features present at the time of diagnosis and to predict the possible outcome. Furthermore, we investigated the as yet to be validated prognostic value of the systemic score previously proposed. ResultsOne hundred consecutive AOSD patients were enrolled. The mean systemic score showed that the majority of patients had a multi-organ involvement. Sixteen patients showed different complications, mainly the macrophage activation syndrome. A strong increase of inflammatory markers was observed. All patients received steroids at different dosages, 55 patients in association with immunosuppressive drugs and 32 in association with biologic agents. Sixteen patients died during the follow-up. Regression analysis showed that the higher values of the systemic score and the presence of AOSD-related complications, assessed at the time of diagnosis, were significantly correlated with patient mortality. A prognostic impact of the systemic score of ≥ 7.0 was reported. Conclusions Our study showed that a higher systemic score and the presence of AOSD-related complications at the time of diagnosis were significantly associated with mortality. Of note, a cut-off at 7.0 of the systemic score showed a strong prognostic impact in identifying patients at risk of AOSD-related death.
- Dec 2016
Objectives: The basic mechanisms underlying ankylosing spondylitis (AS) pathogenesis remain unresolved. We previously reported an association of the single nucleotide polymorphisms (SNP) rs2549782 in Endoplasmic Reticulum Aminopeptidase 2 (ERAP2) with AS. It is known that patients homozygous for the G allele (GG) of another ERAP2 SNP rs2248374 have no ERAP2 expression (ERAP2-null). We utilized this information to study the impact of ERAP2 deficiency on HLA-B27 expression in AS patients. We studied the functional interaction of ERAP2 and HLA-B27 in peripheral blood mononuclear cells (PBMC) from AS patients and in vitro using cell lines. Methods: The expression of intact peptide-HLA-B27 (pB27) or the MHC-I free heavy chains (FHC) was assessed in PBMCs isolated from HLA-B27 positive AS patients. ERAP2 suppressed stable B27-expressing C1R (C1R-B27) cells were tested for pB27 and FHC as well as markers for unfolded protein response (UPR). Distribution of the single nucleotide polymorphisms (SNPs) in ERAP2, rs2549782 and rs2248374 in AS and Crohn's Disease patients was assessed. Results: PBMCs from AS patients lacking ERAP2 expressed higher FHC expression than those with ERAP2. This finding was replicated in C1R-B27 cells after ERAP2 suppression. In addition ERAP2 suppression led to increased UPR markers BiP, CHOP and XBP1-splicing compared to control-shRNA treated cells. There was strong linkage disequilibrium in the ERAP2 locus. All patients with the rs2549782 T allele (increased function) were homozygous for the G allele of rs2248374 (leads to the absence of ERAP2 protein). Conclusions: ERAP2 deficiency causes increased FHC expression and upregulation of the UPR pathway. This article is protected by copyright. All rights reserved.
- Nov 2016
Dysregulation of the intestinal epithelial barrier in genetically susceptible individuals may lead to both intestinal and extraintestinal autoimmune disorders. There is emerging literature on the role of microbiota changes in the pathogenesis of systemic rheumatic diseases such as rheumatoid arthritis, spondyloarthropathies, and connective tissue diseases. Although the role of the gastrointestinal tract in the pathogenesis of spondyloartropathies is well defined and many studies underline the importance of gastrointestinal inflammation in modulating local and systemic inflammation, the data are inconclusive regarding the effect of dysbiosis on rheumatoid arthritis and connective tissue diseases. This review aims to summarize current data on the role of the gastrointestinal involvement and intestinal microbiota in the pathogenesis of systemic rheumatic disease.
- Sep 2016
Macrophage activation syndrome (MAS) is a rare, life-threatening disease in which early diagnosis and aggressive therapeutic strategy may improve the outcome. Due to its rarity, epidemiologic data are still lacking. Hyperferritinemia is frequently associated with MAS and might modulate the cytokine storm, which is involved in the development of multiple organ failure. In this paper, we investigated clinical data, treatments, and outcome of a homogeneous cohort of 41 adult MAS patients, complicating autoimmune rheumatic diseases. MAS-related death occurred in 17 patients (42.5%) during the follow-up, and older age and increased serum ferritin levels, at the time of diagnosis, were significantly associated with mortality. In conclusion, adult MAS is associated with high mortality rate. Some clinical features at diagnosis may be predictive of MAS-associated death.
We describe the impact of α4-β1/7 blockade with natalizumab, a recombinant humanised immunoglobulin (Ig) G4κ monoclonal antibody (mAb) targeted to the α4 subunit of the α4β1 and α4β7 integrins, on the gut and spine inflammation in a patient with ankylosing spondylitis (AS) who developed multiple sclerosis after treatment with tumour necrosis factor (TNF)-blocking agents. A 45-year-old man with human leucocyte antigen (HLA)-B27-positive AS was admitted in January 2007. He had been diagnosed with AS 4 years earlier based on the presence of inflammatory back pain, peripheral arthritis, radiographic bilateral grade 2 sacroiliitis, HLA-B27 positivity. At that time, he had evidence of chronic intestinal inflammation upon histological evaluation of the gut. He was treated with adalimumab for 2 years, achieving a good clinical response. In March 2009, MRI of the brain was performed for the occurrence of …
Cytokines such as tumor necrosis factor-α (TNF-α), IL-12, interferon-γ (IFN-γ), IL-23 and more recently IL-9, have been implicated in the initiation/maintenance of inflammation in psoriasis and psoriatic arthritis (PsA). In the present study we aimed to characterize the role of γδ T cells in peripheral blood and synovial fluid of PsA patients and to investigate their response to in vitro stimulation with antigen or cytokines (IL-9 and IL-23). γδ T cells isolated from peripheral blood mononuclear cells and synovial fluid were analyzed by flow cytometry to evaluate the phenotype and cytokine production. IL-23R and IL-9R gene expression were also evaluated by RT-PCR. PBMC, sorted γδ T cells and γδ cell lines were also stimulated in vitro with IPP, recombinant IL-9 or recombinant IL-23. Our results show an expansion of γδ T cells with a predominant effector memory phenotype in peripheral blood and synovium of untreated PsA patients, which significantly reverses after treatment with anti-TNF-α or anti-IL-12/IL-23R monoclonal antibodies (mAbs). Moreover, in PsA patients γδ T cells activation is prevalently driven by IL-9/IL-9R interaction and not only by IL-23/IL-23R. Altogether these findings indicate γδ T cells and IL-9 as new players in the pathogenesis of PsA. This article is protected by copyright. All rights reserved.
Objective: Microvascular damage is pivotal in the pathogenesis of systemic sclerosis (SSc), preceding fibrosis, and whose trigger is not still fully understood. Perivascular progenitor cells, with profibrotic activity and function, are identified by the expression of the isoform 12 of ADAM (ADAM12) and this molecule may be upregulated by transforming growth factor-β (TGF-β). The goal of this work was to evaluate whether pericytes in the skin of patients with diffuse cutaneous SSc (dcSSc) expressed ADAM12, suggesting their potential contribution to the fibrotic process, and whether TGF-β might modulate this molecule. Methods: After ethical approval, mesenchymal stem cells (MSC) and fibroblasts (FB) were isolated from bone marrow and skin samples collected from 20 patients with dcSSc. ADAM12 expression was investigated in the skin and in isolated MSC and FB treated with TGF-β by immunofluorescence, quantitative real-time PCR, and western blot. Further, we silenced ADAM12 expression in both dcSSc-MSC and -FB to confirm the TGF-β modulation. Results: Pericytes and FB of dcSSc skin showed an increased expression of ADAM12 when compared with healthy control skin. TGF-β in vitro treatment induced a significant increase of ADAM12 in both SSc-MSC and -FB, with the higher levels observed in dcSSc cells. After ADAM12 silencing, the TGF-β ability to upregulate α-smooth muscle actin in both SSc-MSC and SSc-FB was inhibited. Conclusion: Our results suggest that in SSc, pericytes that transdifferentiate toward activated FB are present in the vascular tree, and TGF-β, while increasing ADAM12 expression, may modulate this transdifferentiation.
Adult onset Still's disease (AOSD) patients may show an evanescent salmon-pink erythema appearing during febrile attacks and reducing without fever. Some patients may experience this eruption for many weeks. During AOSD, exceptionally high serum levels of ferritin may be observed, it is an iron storage protein composed by 24 subunits, heavy (H) subunits and light (L) subunits. The ferritin enriched in L subunits (L-ferritin) and the ferritin enriched in H subunits (H-ferritin), may be observed in different tissues. In this work, we aimed to investigate the skin expression of both H-and L-ferritin and the number of macrophages expressing these molecules from AOSD patients with persistent cutaneous lesions. We observed an increased expression of H-ferritin in the skin, associated with an infiltrate in the biopsies obtained from persistent cutaneous lesions of AOSD patients. Furthermore, a positive correlation between H-ferritin skin levels as well as the number of CD68+/H-ferritin+ cells and the multi-visceral involvement of the disease was observed. Our data showed an increased expression of H-ferritin in the skin of AOSD patients, associated with a strong infiltrate of CD68+/H-ferritin+ cells. Furthermore, a correlation between the levels of H-ferritin as well as of the number of CD68+/H-ferritin+ cells and the multi-visceral involvement of the disease was observed. This article is protected by copyright. All rights reserved.
Background Adult onset Still's disease (AOSD) is rare inflammatory disease . Three different patterns of AOSD have been identified: i. monocyclic pattern, characterized by a systemic single episode; ii. polycyclic pattern, associated with multiple flares, separated by remissions; iii. chronic pattern, related to a persistently active disease with associated polyarthritis . Until now, the treatment of AOSD remains largely empirical, lacking controlled clinical trials . Objectives We aimed to investigate clinical data of AOSD patients and any possible correlation among these features and the outcome of patients. Methods Clinical data of 100 AOSD patients were recorded. All patients fulfilled the Yamaguchi AOSD diagnostic . Gender, age, clinical features, disease activity Pouchot's score , complications, serum ferritin, erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP), treatments, comorbidities, patterns and AOSD-associated death, were reported. Furthermore, we evaluated any possible correlation among these clinical features and the outcome of patients, by using linear regression analysis. Results One hundred consecutive AOSD patients (66 men, 34 women), whose age at diagnosis was 45.35±16.23 (mean±SD), were enrolled. All patients experienced fever, 86 patients showed joints involvement, 79 patients showed splenomegaly, skin rash was present in 78 patients, 62 patients showed hepatic involvement, 57 patients showed enlargement of lymph nodes. The Pouchot's score, 6.11±2.02 (mean±SD), confirmed the activity of the disease. Sixteen patients experienced different complications, mainly the macrophage activation syndrome. A strong increase of inflammatory markers (mean±SD) was observed: serum ferritin was 2560.07±3726.64 ng/mL, ESR was 67.28±26.65 mm/hr, CRP was 78.35±72.76. All the patients received steroids at different dosages, and 55 patients were treated with steroids and immunosuppressive drugs, mainly methotrexate and ciclosporin. Thirty-two patients were treated with biologic agents: 5 with anakinra, 8 with tocilizumab, and 19 with TNF inhibitors. Twenty-nine patients showed the monocyclic pattern, 22 patients showed the polycyclic pattern, 33 patients showed the chronic pattern. AOSD-associated death occurred in 16 patients. Regression analysis showed that Pouchot's score (p=0.011), the presence of any complication (p<0.0001) and associated comorbidities (p<0.0001) correlated with the unfavorable outcome. Conclusions This study provides information on the clinical, laboratory, therapeutic, and prognostic features in a large cohort of AOSD patients. The Pouchot's score, the presence of complications and comorbidities were significantly associated with the AOSD-associated death. • Gerfaud-Valentin M, et al. Adult-onset Still's disease, Autoimmun Rev 2014;13,708–722. • Cush JJ, et al. Adult-onset Still's disease. Clinical course and outcome. Arthritis Rheum 1987;30:186–9425. • Yamaguchi M, et al. Preliminary criteria for classification of adult Still's disease. J Rheumatol 1992;19:424–30. • Pouchot J, et al. Adult Still's disease: manifestations, disease course, and outcome in 62 patients. Medicine (Baltimore) 1991;70:118–36. Disclosure of Interest None declared
Background Type 2 innate lymphoid cells (ILC2) and M2 polarized macrophages are activated and produce cytokines in response to IL-25/IL-17RB, although the relevance of this axis to immune responses in primary Sjoren's syndrome (pSS) is unknown. Objectives We sought to investigate the role of the IL-25/IL-17RB axis and ILC2 and M2 macrophages in patients with pSS. Methods Expression analysis of IL-17B, IL-25, IL-33 and IL-17RB was performed by TaqMan real-time PCR and immunohistochemistry on salivary glands from 50 patients and 20 controls. Analysis of IL-17RB expression and the frequencies of natural type 2 innate lymphoid cells (nILC2), inflammatory ILC2 (iILC2), and M2-polarized macrophages were assessed by means of flow cytometry among salivary gland (SGMC) and peripheral blood (PBMC) mononuclear cells. Clusters of ILC2 cells and M2 macrophages were also studied by immunohistochemistry and confocal microscopy. The role of IL-25 on ILC2 was investigated in ex vivo studies. Results IL-25 and IL-17RB, but not IL-25, were significantly over-expressed in the MSG of pSS patients and correlated with the focus score and the degree of lymphoid organization. IL-17RB was mainly expressed by T lymphocytes, ILC2 cells and macrophages. No significant increased expression of IL-33 was observed in pSS. Significant increase in the frequency of iILC2, but not of nILC2, was observed in both PBMC and SGMC. Clusters of ILC2 cells, resembling fat associated lymphoid clusters (FLAC), and M2 polarization were also observed in pSS salivary glands and correlated with the focus score. IL-25 stimulation of isolated SGMC and PBMC resulted in the expansion of iILC2. Conclusions These findings suggest that IL-25 may promote, by interacting locally and sistemically with IL-17RB⁺ cells, an iILC2 and M2 inflammatory state in pSS. Blockade of the IL-25 axis could be clinically relevant in pSS. Disclosure of Interest None declared
Background Macrophage activation syndrome (MAS) is a rare, life-threatening disease in which early diagnosis and aggressive therapeutic strategy may improve the outcome . Due to its rarity, epidemiologic data are still lacking. Hyperferritinemia is frequently associated with MAS and might modulate the cytokines storm the latter contributing to the development of multi-organ failure . Objectives In the current study, we aimed to investigate clinical data, treatments, and outcome of adult MAS patients secondary to autoimmune rheumatic disease. Methods We retrospectively investigated clinical data, treatments, and outcome of 40 adult MAS patients secondary to autoimmune rheumatic disease. For all the MAS patients, the following clinical characteristics were recorded: age, gender, values of serum ferritin, erythsedimentation rate (ESR) and C-reactive protein (CRP), treatments, concomitant comorbidities, time of observation and outcome. Furthermore, we evaluated the correlations among these clinical features and MAS-associated death. Results Forty consecutive MAS patients, 15 men (37.5%) and 25 women (62.5%), whose mean age at diagnosis was 48.20±14.10 (mean±SD) were enrolled. Thirty out of 40 enrolled patients reported an history of adult onset Still's disease (AOSD) (75%), 8 patients an history of systemic lupus erythematosus (20%), 1 patient an history of systemic sclerosis (2.5%), and 1 an history of ankilosing spondylitis (2.5%). Twenty-four out of enrolled patients (60%) did not show any comorbidity, and MAS was diagnosed during an AOSD flare. A severe infections was associated with MAS in the other patients. The laboratory data (mean±SD) reported a strong increase of inflammatory markers: i. serum ferritin 6842.70±7569.70 ng/mL; ii. ESR 63.87±30.19 mm/hr; iii. CRP 51.59±46.92. All the patients were treated with pulsed high dosage of methylprednisolone (1000 mg/daily for a maximum of 6 cycles) and 16 patients (16, 40%) received a combination therapy with: cyclosporin (13, 32.5%); methotrexate (6, 15%); intravenous immunoglobulin therapy (3, 7.5%); anakinra (3, 7.5%); tocilizumab (1, 2.5%). MAS-associated death occurred in 17 patients (42.5%). By using Cox regression proportional hazard model of survival of enrolled MAS patients, we observed that: older age [p=0.017, EXP(B): 1.305, 95%CI: 1.006–1.365], increased serum ferritin levels [p=0.001, EXP(B): 1.776, 95%CI: 1.030–2.728], increased CRP levels [p=0.029, EXP(B): 1.511, 95CI%: 1.021–1.651], higher number of comorbidities [p=0.01, EXP(B): 1.751, 95%CI: 1.142–2.2686], and the use biologics [p=0.015, EXP(B): 2.915, 95%CI: 1.233–6892] were significantly associated with MAS-associated death. Conclusions Adult MAS is still associated with higher mortality rate. Some clinical features at diagnosis, such as older age, increased serum ferritin and CRP levels and higher number of comorbiditis, may be associated with MAS-associated death. • Ramos-Casals M et al, Adult haemophagocytic syndrome. Lancet 2014; 383: 1503–16. • Rosário C et al, The hyperferritinemic syndrome: macrophage activation syndrome, Still's disease, septic shock and catastrophic antiphospholipid syndrome, BMC Med 11, 2013,185. Disclosure of Interest None declared
Background Gut derived innate lymphoid cells of type 3 (ILC)3 are increased in number in the circulation and inflamed tissues of AS patients. Factors influencing the maintainace of ILC3 in an activated status are not clear. The atypical chemokine receptor D6 is a decoy and scavenger receptor for most inflammatory CC chemokines and acts preventing exacerbated inflammatory reactions. Mice lacking D6 expression in the non-hematopoietic compartment display a significant increase of pro-inflammatory monocytes in the peripheral blood and in secondary lymphoid tissues. The role of D6 in human inflammatory disorders has not been inverstigated. Objectives To evaluate whether modulation of D6 expression occurs in the gut and in BM of AS patients and accompanied with a selective increase of pro-inflammatory CXCR1highLy6highTL1A⁺CCR7⁺ cells in the circulation and inflamed tissues of AS and to study the effect of these cells in modulating ILC3 differentiation. Methods RT-PCR and immunohistochemistry were used to evaluate the expression of D6 in the gut and bone marrow of AS patients and controls. Different monocyte substes were analyzed by flow cytometry in the peripheral blood, gut, synovial fluids and bone marrow of AS patients and controls. Isolated peripheral CXCR1highLy6highTL1A⁺CCR7⁺ cells were co-coltured with isolated peripheral ILC3 and changes in ILC3 frequency were evaluated by flow cytometry. Results D6 was significantly down-regulated in the ileum and in the bone marrow of AS patients compared to controls. D6 deficiency was accompanied by a complex monocyte/macrophage signature. Tissue resident CXCR1highLy6low macrophages were expanded in the ileum of AS patients compared to controls. A significant increase in the frequency of pro-inflammatory CXCR1highLy6high monocytes producing high levels of TL1A and IL-23 was observed in the ileum of AS patients. In the peripheral blood a statistically significant increase in the frequencies of both bone marrow derived CXCR1lowLy6High and intestinal derived CXCR1highLy6highCCR7⁺TL1A⁺IL-23⁺ monocytes, the latest being also expanded in AS synovial fluids and bone marrow. Isolated pro-inflammatory CXCR1highLy6highTL1A⁺IL-23⁺CCR7⁺ monocytes from peripheral blood of AS induced the expansion and activation, evaluated through the production of IL-22 and IL-17, of ILC3. Conclusions In this study we provide the first demonstration that absence of D6 expression in the gut and in the inflamed tissues of AS patients selectively induced the intestinal accumulation and re-circulation of pro-inflammatory CXCR1highLy6highTL1A⁺IL-23⁺CCR7⁺ monocytes. Since the ability of these cells in promoting ILC3 expansion and activation, these cells may promote a sustained pro-inflammatory status in AS. Disclosure of Interest None declared
Background Arteries are immuno-privileged sites. In advanced atherosclerotic lesions, however, adventitial lymphoid infiltrates, sometimes aggregated in lymphoid follicles (the so called artery tertiary lymphoid organs, ATLO), occur together with marked neoangiogenesis and lymphangiogenesis, and with the extensive induction of high endothelial venules. Objectives To investigate if tertiary lymphoid organs (TLO) are present in GCA and their formation is associated with the ectopic expression of constitutive lymphoid tissue-homing chemokines. Methods RT-PCR, immunohistochemical and immunofluorescence analysis were used to determine the presence of ectopic TLO in GCA and the expression of chemokines/chemokine receptors and cytokines involved in lymphoneogenesis in the artery samples obtained from 50 GCA patients and 30 controls. The presence of lymphatic conduits, of follicular dendritic cells (FDC) precursors and lymphoid tissue inducer cells was also investigated. Finally, expression of CXCL13 and BAFF by isolated vascular smooth muscle cells was evaluated before and after stimulation with toll like receptors agonists and cytokines. Results Artery TLO (ATLOs) were observed in the media layer of 60% of GCA patients in close proximity to neo-formed PNAD+ high endothelial venules. Pesence of ATLOs was independent by the number of infiltrating mononuclear cells, the age of patients and the presence of atherosclerosis. ATLO formation was also accompanied by the expression of CXCL13, BAFF, APRIL, IL-7 and IL-17. CXLC13 and BAFF expression was manily observed in the context of ATLOs, in VSMC and endothelial cells of neo-formed high endothelial cells. IL-7 and IL-17 expression levels were significantly correlated with the levels of CXCL13 and BAFF. The presence of follicular dendritic cells (FDC) precursors and of lymphoid conduits was demonstrated by confocal microscopy analysis and immunohistochemistry, respectively. Stimulation of VSMC with TLR agonists (LPS and poly-IC) and pro-inflammatory cytokines (IL-1b, IL-6, IFN-g and IL-17) resulted in the up-regulation of BAFF and CXCL13. Conclusions ATLOs occur in the inflamed arteries of GCA patients possibly representing the immune sites where immune responses toward unknown arterial wall-derived antigens may be organized. Disclosure of Interest None declared
Background Systemic Sclerosis (SSc)-heart disease (SSc-HD), although often clinically silent, significantly reduces the life-expectancy in these patients . The cardiac magnetic resonance (CMR) is recognized useful tool for the diagnosis of SSc-HD and cardiac stress tests might be a helpful technique to highlight the occult myocardial involvement, which cannot be detected at rest due to compensatory mechanisms [2,3]. Objectives To evaluate the possible occult cardiac involvement in asymptomatic SSc patients by pharmacological stress, rest perfusion and delayed enhancement CMR, to identify as earlier as possible those patients at higher risk of cardiac related mortality and needing specific cardiovascular treatments. Methods Sixteen consecutive patients with definite SSc fulfilling the ACR/EULAR 2013 classificative criteria  in less than 1 year from the onset of Raynaud's phenomenon underwent pharmacological stress, rest perfusion and delayed enhancement CMR. No enrolled patient showed signs and/or symptoms suggestive for cardiac involvement, confirmed by both the 12-lead ECG examination and echocardiography. No patient showed traditional cardiovascular risk factors. Patients, in which CMR technique showed occult cardiac involvement, underwent to heart computed tomography (CT) scan to evaluate a possible coronary disease. Results Stress perfusion defects, in the left ventricle, were detected in 6 out of 16 (37.5%) patients. A sub-endocardial defects and/or a ventricular mid-myocardial layer stress perfusion defects were observed. Our results confirmed, in these 6 patients, a normal flow distribution of the explored coronary arteries by CT scan. The presence of CMR stress perfusion defects did not correlate with any clinical feature of patients. Conclusions Myocardial stress perfusion defects may be early detected, by pharmacological stress perfusion CMR, a reliable and sensitive technique for the non-invasive evaluation of SSc-HD, in patients with SSc of recent onset. These defects seems to be independent from traditional risk factors and associated comorbidities, suggesting that are a specific hallmark of the disease. An early detection of myocardial involvement might suggest starting of vasodilatative therapies in these patients. • Allanore Y, et al. Primary myocardial involvement in systemic sclerosis: evidence for a microvascular origin. Clin Exp Rheumatol 2010;28:S48–53. • Di Cesare E, et al. Early assessment of sub-clinical cardiac involvement in systemic sclerosis (SSc) using delayed enhancement cardiac magnetic resonance (CE-MRI). Eur J Radiol. 2013;82:e268–73. • Hachulla AL, et al. Cardiac magnetic resonance imaging in systemic sclerosis: a cross-sectional observational study of 52 patients. Ann Rheum Dis. 2009;68:1878–84. • van den Hoogen F, et al. 2013 Classification Criteria for Systemic Sclerosis An American College of Rheumatology/European League Against Rheumatism Collaborative Initiative. Arthritis Rheum. 2013;65:2737–47. Disclosure of Interest None declared
Background Adult onset Still's disease (AOSD) is an inflammatory disease, characterized by high spiking fevers, arthritis, salmon-pink erythema and multivisceral involvement . During AOSD, exceptionally high serum levels of ferritin may be observed and they might contribute to production of proinflammatory molecules . Ferritin is composed by 24 subunits, heavy (H) subunits and light (L) subunits. The ferritin enriched in L subunits (L-ferritin) and the ferritin enriched in H subunits (H-ferritin) may be recognized in different tissues . Objectives To investigate the skin tissue expression of both H-and L-ferritin and the number of macrophages expressing these molecules, in the inflammatory cutaneous infiltrate from 10 AOSD patients with persistent (more than 24–36 hours) cutaneous lesions. In addition, we performed statistical analysis to evaluate whether these results might correlate with the severity of the disease. Methods We obtained skin samples from 10 consecutive patients with active AOSD with persistent cutaneous lesions and 10 healthy controls (HC). An immunofluorescence analysis was performed evaluating the tissue expression of both H- and L-ferritin and the number of macrophages expressing these moleculkes. Serum levels of ferritin, ESR, CRP, Pouchot's score were also recorded. Results We observed an increased H-ferritin expression in the skin samples of AOSD patients, when compared with HC (p<0.001). On the contrary, no differences were observed for L-ferritin when compared to HC. Furthermore, a significant increase of H-ferritin was detected when compared with L-ferritin in AOSD patients (p<0.001) and when compared with both H-ferritin and L-ferritin in HC (p<0.001; p<0.001). Furthermore, a positive correlation between H-ferritin and the Pouchot's score was observed (p<0.01). We also reported a positive correlation among the tissue expression of H-ferritin and both serum ferritin (p<0.05) and CRP (p<0.05). The double staining between the H-ferritin and CD68 showed an increased presence of macrophages expressing H-ferritin in the inflammatory infiltrate when compared with HC (p<0.001). In the same samples, we did not observe any co-localization between CD68 and L-ferritin. However, our analysis failed to show any positive correlation among the number of CD68+/H-ferritin+ in the skin and disease activity, serum ferritin and inflammatory markers. Conclusions Our data show an imbalance between the production of L- and H-ferritin in the skin of AOSD patients, associated with a strong infiltrate of CD68+/H-ferritin+ cells in the same samples suggesting a possible pathogenic role. Furthermore, a correlation between the levels of H-ferritin in the skin of these patients and the disease activity was observed. • Gerfaud-Valentin M, et al. (2014) Adult-onset Still's disease. Autoimmun Rev 13:708–22. • Rosário C, et al (2013) The hyperferritinemic syndrome: macrophage activation syndrome, Still's disease, septic shock and catastrophic antiphospholipid syndrome. BMC Med 11:185. • Recalcati S, et al (2008) New functions for an iron storage protein: the role of ferritin in immunity and autoimmunity. J Autoimmun 30:84–9. Disclosure of Interest None declared
Adult-onset Still’s disease (AOSD) is a systemic inflammatory condition of unknown aetiology characterized by typical episodes of spiking fever, evanescent rash, arthralgia, leukocytosis and hyperferritinemia. Given the lack of data in Italian series, we promote a multicentric data collection to characterize the clinical phenotype of Italian patients with AOSD. Data from 245 subjects diagnosed with AOSD were collected by 15 centres between March and May 2013. The diagnosis was made following Yamaguchi’s criteria. Data regarding clinical manifestations, laboratory features, disease course and treatments were reported and compared with those presented in other published series of different ethnicity. The most frequent features were the following: arthritis (93 %), pyrexia (92.6 %), leukocytosis (89 %), negative ANA (90.4 %) and neutrophilia (82 %). As compared to other North American, North European, Middle Eastern and Far Eastern cohorts, Italian data show differences in clinical and laboratory findings. Regarding the treatments, in 21.9 % of cases, corticosteroids and traditional DMARDs have not been able to control the disease while biologics have been shown to be effective in 48 to 58 patients. This retrospective work summarizes the largest Italian multicentre series of AOSD patients and presents clinical and laboratory features that appear to be influenced by the ethnicity of the affected subjects.
IL-9 is a 28-30 kDa monomeric glycosylated polypeptide belonging to the IL-7/IL-9 family of proteins that bind to a composite receptor consisting of the private receptor IL-9R and the interleukin 2 receptor, gamma (IL2RG), a common gamma subunit shared by the receptors of many different cytokines. The IL-9R is widely expressed and IL-9 impacts a number of effector cells such as effector T cells, B cells, innate lymphoid cells, mast cells, polymorphonuclear cells, epithelial cells and smooth muscle cells playing an important role in regulating inflammatory immunity. The critical role of IL-9 in promoting cellular and humoral immune responses makes it an important focus of potential therapeutic interventions. Recently, a defined subset of T helper cells, Th9 cells has been identified by the potent production of IL-9. The involvement of Th9 cell subset has been described in many types of inflammatory diseases, namely atopic diseases, helminth infections, experimental autoimmune encephalomyelitis and ulcerative colitis. In this review, we summarize the IL-9 biological activities, highlighting roles for IL-9 and Th9 cells in rheumatoid and psoriatic arthritis, systemic vasculitis, systemic lupus erythematosus and systemic sclerosis. This article is protected by copyright. All rights reserved.
Endothelial Progenitor Cells (EPCs) are bone marrow derived cells able to differentiate in mature endothelial cells (EC) contributing to the generation of new vessels, connecting to fibronectin, and forming colonies and/or colony forming units. Since circulating EPCs can be actively considered part of endothelial damage in several cardiovascular diseases and autoimmune disorders the possibility to have a measure for endothelium damage should be considered of interest to predict the patient out-come. At the same time the EPCs proliferative and regenerative role could be considered for therapeutic applications. Studies have been performed to elucidate the role of EPCs in Systemic Sclerosis and many review and articles published on this topic. In the present paper we aimed to review the role of EPCs in other autoimmune disorders.
Purpose of review: Inflammatory innate and adaptive immune cell responses to commensal bacteria underlie the pathogenesis of human chronic inflammatory diseases. Intestinal dysbiosis has been described in patients with spondyloarthritis (SpA) and seems to be correlated with histologic and immunologic alterations. Purpose of this review is to discuss the relationship occurring between intestinal dysbiosis and innate immune responses in patients with axial SpA. Recent findings: Intestinal dysbiosis and differential activation of intestinal immune responses in patients with SpA have been demonstrated. Furthermore, innate cells that appear to be involved in the pathogenesis of SpA may control intestinal homeostasis through induction of apoptotic cell death and deletion of activated commensal bacteria-specific T cells. Summary: Although the evidence shows that dysbiosis occurs in SpA, it is not clear the role of dysbiosis in regulating innate immune responses in SpA. Relationships between cause and effect remain to be answered. VIDEO ABSTRACT: http://links.lww.com/COR/A34.
- Apr 2016
Objectives: To investigate whether artery tertiary lymphoid organs (ATLOs) are present in giant cell arteritis (GCA) and that their formation is associated with the ectopic expression of constitutive lymphoid tissue-homing chemokines. Methods: Reverse transcriptase PCR, immunohistochemical and immunofluorescence analysis were used to determine the presence of ectopic ATLOs in GCA and the expression of chemokines/chemokine receptors and cytokines involved in lymphoneogenesis in the temporal artery samples obtained from 50 patients with GCA and 30 controls. The presence of lymphatic conduits, of follicular dendritic cells (FDCs) precursors and lymphoid tissue inducer cells was also investigated. Finally, expression of CXCL13, B cell activating factor (BAFF), a proliferation-inducing ligand (APRIL) and CCL21 by isolated myofibroblasts was evaluated before and after stimulation with Toll-like receptors (TLRs) agonists and cytokines. Results: ATLOs were observed in the media layer of 60% of patients with GCA in close proximity to high endothelial venules and independently by the age of patients and the presence of atherosclerosis. ATLO formation was also accompanied by the expression of CXCL13, BAFF, a proliferation-inducing ligand (APRIL), lymphotoxin (LT)-β, interleukin (IL)-17 and IL-7, the presence of FDC precursors and of lymphoid conduits. Stimulation of myofibroblasts with TLR agonists and cytokines resulted in the upregulation of BAFF and CXCL13. Conclusions: ATLOs occur in the inflamed arteries of patients with GCA possibly representing the immune sites where immune responses towards unknown arterial wall-derived antigens may be organised.
- Mar 2016
Although in the past the prevention of joint destruction in rheumatoid arthritis (RA) was strongly emphasized, now a great interest is focused on associated comorbidities in these patients. Multiple data suggest that a large percentage of RA patients are affected by Type 2 Diabetes (T2D), whose incidence has reached epidemic levels in recent years, thus increasing the health care costs. A better knowledge about the pathogenesis of these diseases as well as the mechanisms of action of drugs may allow both policy designers and physicians to choose the most effective treatments, thus lowering the costs. This review will focus on the role of Interleukin (IL)-1β in the pathogenesis of both the diseases, the efficacy of IL-1 blocking molecules in controlling these diseases, and will provide information suggesting that targeting IL-1β, in patients affected by both RA and T2D, may be a promising therapeutic choice.
- Feb 2016
Objective: This study was undertaken to investigate the expression and tissue distribution of Th9 related cytokines in patients with psoriatic arthritis (PsA). Methods: Quantitative gene expression analysis of Th1, Th17, and Th9 cytokines was performed in intestinal biopsy samples obtained from patients with PsA, with HLA-27 positive-ankylosing spondylitis (AS), Crohn's disease (CD) and healthy controls. IL-23, IL-17, IL-22, IL-9 and IL-9R expression and tissue distribution were evaluated by immunohistochemistry and confocal microscopy. Flow cytometry was used to study the frequency of Th9 cells among peripheral blood (PBMC), lamina propria (LPMC) and synovial (SMC) mononuclear cells. The functional relevance of IL-9R expression on epithelial cells was assessed in functional in vitro studies. Th9 cells were also studied in synovial samples of PsA patients. Results: subclinical gut inflammation of PsA patients was characterized by a clear Th17 and Th22, but not Th1, polarized immune responses. Unlike AS and CD, a strong and significant up-regulation of IL-9 immunologically was observed in PsA gut, especially among infiltrating mononuclear cells, high endothelial venules and Paneth cells (PC). IL-9 mononuclear cells were demonstrated to be in large part Th9 cells. IL-9 over-expression was accompanied by significant PC hyperplasia. PC strongly over-expressed IL-9R and stimulation of epithelial cells, isolated from PsA patients, with IL-9 resulted in over-expression of α-defensin5 and IL-23p19. Peripheral and synovial expansion of α4β7(+) Th9 cells was also observed in patients with PsA. Increased expression of IL-9 and IL-9R was also found in synovial tissues. Conclusions: A strong IL-9/Th9 polarization seems to be the predominant immunological signature of patients with PsA. This article is protected by copyright. All rights reserved.
- Jan 2016
Purpose of reviewSubclinical gut inflammation has been described in a significant proportion of patients with ankylosing spondylitis (AS), up to 10% of them developing it during the time of clinically overt inflammatory bowel disease. Histologic, immunologic, and intestinal microbiota alterations characterize the AS gut.Recent findingsMicrobial dysbiosis as well as alterations of innate immune responses have been demonstrated in the gut of AS. Furthermore, a growing body of evidence suggests that the gut of AS patients may be actively involved in the pathogenesis of AS through the production of proinflammatory cytokines, such as IL-23p19, and the differentiation of potentially pathogenic innate lymphoid cells producing IL-22 and IL-17. Finally, a strong correlation between the presence of subclinical gut inflammation and the degree of spine inflammation have been also proved in AS.SummarySubclinical gut inflammation and innate immune responses in AS may be considered a possible consequence of microbial dysbiosis. Relationships between cause and effect remain, however, to be answered.
The aim of this study was to assess safety and efficacy of ultrasonography (US)-guided intra-articular injections using tumor necrosis factor (TNF) blockers compared to corticosteroids in rheumatoid arthritis (RA) or psoriatic arthritis (PsA) patients, experiencing refractory monoarthritis despite the current systemic therapy. Eighty-two patients were randomized to receive three intra-articular injections monthly of either corticosteroid or TNF blockers. Primary endpoints were the safety and an improvement greater than 20% for visual analogic scales of involved joint pain in patients injected with anti-TNFα. Further clinical, US, and magnetic resonance imaging (MRI) evaluations were considered secondary endpoints. Intra-articular TNF blockers are a safe strategy, determining a significant reduction of patient and physician reported clinical outcomes and US/MRI scores, in RA and PsA patients, when compared to intra-articular injections of corticosteroids. US guidance excluded the possibility to inject the drug in the wrong site, maximizing local effects, reducing systemic effects, and increasing the safety of the procedure. Patients with inflammatory monoarthritis could be successfully treated with US-guided intra-articular TNF blockers that are a safe and well tolerated procedure, to achieve a longstanding clinical and radiological good clinical response and/or disease remission.
Objectives: Invariant NKT (iNKT) cells play a role in regulating the function of autoreactive B cells before their entry into germinal centres. Absence and/or reduction of iNKT cells have been demonstrated in patients with systemic lupus erythematosus (SLE) together with an increase of autoreactive B cell activity. Primary Sjögren's syndrome (pSS) is a systemic autoimmune disease in which lymphocyte infiltration and organisation in lymphoid structures of inflamed salivary glands occurs. The aim of the study was to investigate the percentage and function of iNKT in the salivary glands and peripheral blood of patients with pSS. Methods: Minor salivary gland biopsies were obtained from patients with pSS and with non-specific chronic sialoadenitis (nSS). Flow cytometry analysis of CD1d/α-GalactosylCeramide (α-GalCer) tetramer positive cells, producing IFN-γ and IL-17, and quantitative gene expression analysis by TaqMan real-time PCR for Vα24 were performed on salivary glands biopsies and peripheral blood samples obtained from patients and controls. Flow cytometry and immunofluorescence analysis for autoreactive B lymphocytes and ELISA for anti-SSA antibodies (Ab) detection were also performed. Results: An increase of iNKT was detected ex vivo in peripheral blood of pSS patients; after α-GalCer stimulation this subset produce IL-17 and IFN-iNKT were undetectable in the salivary glands of pSS patients and anti-SSA specific B cells were found in target tissue. Invariant NKT cells were able to inhibit autoantibody production by B cells obtained from salivary glands of pSS. Conclusions: Impaired iNKT migration to inflamed sites might induce the activation of autoreactive B cells specific for SSA-antigen in salivary glands of pSS patients.
- Nov 2015
Objectives In this work, we aimed to evaluate the levels of ferritin enriched in H subunits (H-ferritin) and ferritin enriched in L subunits (L-ferritin) and the cells expressing these 2 molecules, in the lymph node (LN) biopsies obtained from adult onset Still's disease (AOSD) patients, and the possible correlation among these data and the severity of the disease. Methods Ten patients with AOSD underwent LN biopsy. All the samples were stained by immunofluorescence. A statistical analysis was performed to estimate the possible correlation among both H-ferritin and L-ferritin tissue expression and the clinical picture of the disease. Furthermore, the same analysis was performed to evaluate the possible correlation among the number of CD68+/H-ferritin+ or CD68+/L-ferritin+ cells and the clinical picture. Results Immunofluorescence analysis demonstrated an increased tissue H-ferritin expression, in the LNs of AOSD patients. This increased expression correlated with the severity of the disease. An increased number of CD68 macrophages expressing H-ferritin was observed in LN samples of our patients. Furthermore, we observed that the number of CD68+/H-ferritin+ cells significantly correlated with the severity of clinical picture. Conclusions Our data showed an imbalance between the levels of H- and L-ferritin in LNs of AOSD patients and the evidence of increased number of CD68+/H-ferritin+ cells in the same organs. Furthermore, a correlation among both the tissue H-ferritin levels and the CD68+/H-ferritin+ cells and the clinical picture was observed. This article is protected by copyright. All rights reserved.
Primary Sjögren’s syndrome (pSS) is an autoimmune disease, showing a broad heterogeneity concerning the symptoms, the involved organs, as well as the prognosis. The clinical presentation, in the majority of patients, is limited to the mucosal surfaces, although some patients may show a systemic involvement and/or immunological bio-markers strongly associated with a higher risk to develop lymphoma. The assessment of disease activity, by using validated scores, at the time of pSS diagnosis, has been shown to be helpful in identifying patients requiring a closer follow-up and more-intensive therapeutic management. At present, evidence-based therapy for pSS is limited to symptomatic drugs for dryness, and the choice of immunosuppressive or biologic agent treatment is mainly based on their efficacy in other systemic autoimmune diseases, as well as expert opinion and uncontrolled studies. In recent years, given the central role of B-lymphocytes in pSS pathogenesis, B-cell depleting therapy with anti-CD20 monoclonal antibody rituximab (RTX) has shown promising, results in clinical trials. Although data present in literature do not allow to draw definitive conclusion on the efficacy of RTX, B-cell depletion may be considered a therapeutic option for a subgroup of pSS patients. In particular, RTX treatment seems to be effective in early active disease, mainly on glandular swelling and in systemic manifestations.
IL-9 has been shown to be upregulated before the clinical onset of articular disease in RA. The exact role of IL-9 and Th9 cells in RA, however, has not yet been adequately studied. The aim of this study was to evaluate the expression of IL-9 and IL-9-expressing cells in RA patients. IL-9, IL-9R, PU.1, IL-9, thymic stromal lymphopoietin (TSLP), IL-4 and TGF-β expression was assessed by real-time-PCR in the synovial tissues of RA and OA patients. IL-9, IL-9R, IL-4, TSLP and TGF-β were also investigated by immunohistochemistry. Peripheral CD4(+) T cell subsets were studied by flow cytometry analysis before and after incubation with citrullinated peptides. IL-9 was overexpressed in RA synovial tissues and correlated with the degree of histological organization of B and T cells in ectopic lymphoid structures. The majority of IL-9-producing cells were identified as CD3(+) cells. Increased mRNA and protein expression of IL-9R, IL-4, TSLP and TGF-β was also observed in RA synovial tissue. Blood peripheral Th9 cells were expanded by citrullinated peptides. These results indicate that Th9 cells and IL-9 were frequently detected in peripheral blood mononuclear cells and synovia of RA patients. A possible pathogenic role for Th9 in RA is discussed. © The Author 2015. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For Permissions, please email: firstname.lastname@example.org.
A better understanding about the mechanisms involved in the pathogenesis of type 2 diabetes mellitus (T2D) showed that inflammatory cytokines such as tumor necrosis factor (TNF) and interleukin-1β (IL-1β) play a pivotal role, mirroring data largely reported in rheumatoid arthritis (RA). IL-1β is mainly produced by monocytes (MO) and hyperglycaemia may be able to modulate, in the cytoplasm of these cells, the assembly of a NLRP3-inflammosome, a cytosolic multiprotein platform, where, the inactive pro-IL-1β is cleaved into active form, via caspase-1 activity. In this paper, we evaluated the production of IL-1 β and TNF, in peripheral blood MO of patients affected by RA or T2D or both diseases, in order to understand if, an alteration of the glucose metabolism may influence their pro-inflammatory status. Our data showed, after 24 hours of incubation with different glucose concentrations, a significantly increased production of IL-1β and TNF in all evaluated groups when compared with healthy controls. However, a significant increase of IL-1β secretion by T2D/RA was observed when compared with other groups. The analysis of relative mRNA expression confirmed these data. Our results showed after 24 hours of incubation with different concentration of glucose a significant increase in NLRP3 expression. In this work, an increased production of IL-1β by MO obtained from patients affected by both RA and T2D via NLRP3-inflammasome activation may suggest a potential IL-1β targeted therapy in these patients. This article is protected by copyright. All rights reserved. © 2015 British Society for Immunology.
Background Intestinal dysbiosis has been recently demonstrated in the inflamed ileum of AS patients. Objectives To study the ileal localization of bacteria in AS patients and their relationship with local and systemic immune responses. Methods Consecutive gut biopsies obtained from 30 HLA-B27+ AS patients and 20 normal subjects were histologically classified in normal histology, acute inflammation and chronic inflammation. Giemsa and Silver stains were used to visualize bacteria and characterize their morphology. Intestinal bacteria were scored on the basis of the numbers of bacteria and their aggregation in clusters. The ileal expression and tissue distribution of claudin-2 and 4, Zonulin 1 and occludin were investigated by rt-PCR and immunohistochemistry. Serum levels of LPS and intestinal fatty acid binding protein (i-FABP) were also assayed by ELISA. The expression of HLA-DR, CD71 and CD14 on circulating monocytes and the monocyte immunologic behavior were studied by flow-cytometry. Results Cocci-like bacteria, but not segmented filamentous bacteria, were observed in the inflamed ileum of AS sometimes aggregated in clusters. Bacterial scores were significantly correlated with the percentages of infiltrating inflammatory cells (r2=0.56, p<0.0001). The presence of invadent bacteria in AS was invariably associated with histologic changes characterized by i) the detachment of epithelial cell layer from the basement membrane; ii) an edematous lamina propria with extravasated red blood cells and iii) the down-regulation of occludin, claudin 2 and 4 and Zonulin 1 (∼10, ∼12, ∼8 and ∼ 6 fold decrease, respectively; p<0.05) assessed by rt-PCR and confirmed by IHC. Higher serum levels of LPS and i-FABP significantly associated with the higher score of bacteria invasion and tissue inflammation were observed in AS patients. Circulating monocytes in AS displayed a reduced expression of CD14 (p>0.0001), HLA-DR (p<0.05) and CD71 (p<0.001). Ex vivo stimulation of monocytes with LPS increased the expression of IL-23 in CD14+ cells in both AS patients and controls that was, however significantly higher in AS patients. Production of IL-23 by CD14- monocytes in AS patients was however undetectable and not modified by LPS alone. Stimulation of monocytes with LPS+sCD14 increased IL-23 production in CD14+ cells only in controls, strongly inducing the production of IL-23+ in CD14- monocytes in AS. Neither LPS or LPS+CD14 modify the expression of UPR genes both in patients and controls. Conversely, LPS and more intensely, LPS+sCD14 stimulation significantly abrogated the expression of the autophagy genes ATG16L1 (∼5 fold decrease, p<0.05); IRGM (∼ 3 fold decrease, p<0.05) and MAP1LC3A (∼7 fold decrease, p<0.05) only in AS monocytes. Conclusions Bacterial ileitis, accompanied by damaged intestinal mucosal barrier, characterizes AS patients. i-FABP and LPS are increased in the peripheral blood of AS patients and associated with the down-regulation of LPS co-receptor CD14 and with an anergic monocyte phenotype. Ex vivo stimulation with sCD14 may confer LPS-responsiveness to the cells not expressing CD14. Dysregulated autophagy response is present in the peripheral blood of AS patients. Disclosure of Interest None declared
Background Autophagy is now considered as a major regulator in trafficking events that activates innate and adaptive immunity and consistent evidence supports its role in autoimmunity (1). Primary Sjogren's syndrome (pSS) is a systemic autoimmune disease characterized by infiltration of exocrine glands by T and B cells that, producing chemokines and cytokines, coordinate the chronic inflammatory process. No data on the role of autophagy in pSS are available in humans, although studies in mice demonstrated its involvement in the salivary and lacrimal gland homeostasis (2,3). Objectives We investigated the autophagy process in salivary gland tissue and in peripheral T lymphocytes from pSS patients to evaluate its possible implication in the pathogenesis of the disease. Methods 30 patients with pSS, 20 patients with sicca syndrome or non-specific-chronic-sialoadenitis and 30 healthy donors were studied. Peripheral T lymphocytes were isolated by standard procedures. Salivary gland biopsies were evaluated by i) H&E to assess histological pattern, the severity of inflammatory infiltrate and the presence of germinal centers, ii) RT-PCR for the expression of autophagy-related genes and IL-23p19 and IL-21 mRNA. Autophagy-related proteins (LC3, Atg5, p62/SQSTM1) were detected in peripheral T lymphocytes by western blot and in salivary gland by immunohistochemistry and immunofluorescence. IL-21 and IL-23p19 serum levels were measured by ELISA. Results Autophagy is up-regulated in T cells from the salivary glands, but not from the peripheral blood, of pSS patients and it is correlated with disease activity and damage indexes. Autophagy is also correlated with the local expression of the pro-inflammatory cytokines IL-21 and IL-23p19, but not with serum levels of these cytokines. Conclusions Our data show that, in pSS, T cells present high levels of autophagy, which may up-regulate the expression of pro-inflammatory cytokines, providing evidence for a role of this process in the pathogenesis of pSS and identifying a possible therapeutic target. References Disclosure of Interest None declared
Background IL-36a is a cytokine that predominantly acts on naive CD4+ T cells and gamma-delta T cells via the IL-36 receptor. IL-36a has been recently demonstrated to be involved in human disease, such as psoriasis, by modulating innate and adaptive immune responses. Objectives In this study we aimed to investigate the expression of IL-36 axis and to assess the role of γδ T cells in patients with primary Sjogren's syndrome (pSS). Methods Blood and minor labial salivary glands (MSG) biopsies were obtained from 35 pSS and 20 nSS patients. Serum IL-36a was assayed by ELISA. IL-36a, IL-36R, IL-36RA, IL-38, IL-22, IL-17, IL-23p19, expression in MSGs was assessed by rt-PCR and tissue IL-36a and IL-38 expression also investigated by immuno-histochemistry (IHC). ab and gδ T cells and CD68+ cells isolated from MSGs were also studied by flow cytometry and confocal microscopy analysis. Results IL-36a was significantly over-expressed in the serum and in the salivary glands of pSS. Salivary gland IL-36a mRNA expression was correlated with the expression levels of IL-17, IL-22 and IL-23p19 (r2=0.45, r2=0.66, r2=0.73, respectively; p<0.05). IL-38 mRNA that acts as inhibitor of IL-36a was also up-regulated in pSS. In pSS the expression of IL-36α was essentially observed in inflammatory infiltrates among lymphoid cells and cells displaying dendritic morphology. IL-38 was also up-regulated at protein level in the salivary glands of pSS being mainly expressed among acinar epithelial cells and infiltrating mononuclear cells. By confocal microscopy analysis and flow cytometry, ab+CD3+ T cells and CD68+ cells were confirmed to be the major source of IL-36a in minor salivary glands of pSS. gδ T cells were not significantly expanded in the salivary glands of pSS but produced more IL-17 being their percentage correlated with the focus score. Higher expression of IL-36a and IL-36R was also demonstrated in γδ T cells isolated from pSS compared to controls. Conclusions In this study we demonstrate that a significant increase in circulating and tissue levels of IL-36a occurs in pSS and provide also the first evidence of a putative role of IL-17+/IL-36+ γδ T lymphocytes. Disclosure of Interest None declared
Background We have shown an increase in the unfolded protein response (UPR) with decreased ERAP1 or ERAP2 function in an in vitro system. Similarly UPR has been demonstrated to correlate with onset of disease in the HLA-B27 rat model. UPR has been difficult to demonstrate in the gut of AS patients but autophagy is upregulated. ERAP2 is associated with both AS and inflammatory bowel disease (IBD). Objectives Here we explore the moderating effect of autophagy on UPR. Specifically we study the impact of suppressing autophagy on UPR. Methods Lamina Propria Mononuclear cells (LPMC) were isolated from terminal ileal biopsies of 10 AS patients. Autophagy was suppressed with 2 agents anisomycin and 3-MA. In parallel an in vitro system was established with C1R-B27 cells (B-lymphoblastoid cells with stable HLA-B27 expression) and the presence of autophagy in these cells were established with electron microscopy as well as by transfecting these cells with LC3-RFP followed by confocal microscopy. Autophagy was suppressed in C1R-B27 cells using 3-MA. In both LPMC and C1R B27 cells, suppression of autophagy was demonstrated by RT-PCR of appropriate markers. Changes in MHC-I free heavy chain (FHC) expression were tested by HC10 staining and flow cytometry. Changes in UPR following inhibition were tested by XBP1 splicing assay and RT-PCR for BiP, CHOP, PERK, GADD34 and PDIA6. Results Electron and confocal microscopy demonstrated autophagy in C1R-B27 cells. Autophagy was in a dynamic state in the C1R cells as demonstrated by changes with rapamycin a stimulator of autophagy. Significant suppression of autophagy was noted in both LPMCs and C1R-B27 cells. Following autophagy suppression there was a significant increase in FHC expression in both C1R cells and LPMCs. In parallel we demonstrated increase in UPR markers in both LPMCs and C1R cells. Conclusions The inability to demonstrate UPR in some in vivo studies could be due to compensation by autophagy. Autophagy and UPR regulate each other and perturbations of this fine balance can influence the pathogenesis of AS and IBD. Disclosure of Interest None declared
Background Lupus nephritis (LN) affects up to 70% of patients with systemic lupus erythematosus (SLE) and is a major cause of morbidity and mortality. There is compelling evidence demonstrating that resident renal cells can directly contribute to renal inflammation through their ability to secrete cytokines, chemokines, and glycosaminoglycans. Interleukin 32 (IL32) is a recently described cytokine with an important role in both innate and adaptive immune responses. IL32 exhibits several properties typical of proinflammatory cytokines by inducing the maturation and activation of dendritic cells, resulting in Th1 and Th17 polarization, and endothelial activation. In addition, overexpression of intracellular IL32 aggravated the secretion of interstitial collagenase, gelatinase, and collagenase 3 that are implicated in glomerular basement membrane damage. Objectives To evaluate serum concentration of IL32 in LN patients and its expression in renal biopsy samples. Methods Serum samples were obtained from 28 LN patients who underwent a renal biopsy as part of the routine clinical assessment, 30 SLE patients without renal involvement (non renal SLE), and 10 healthy controls (HC) matched for sex and age. Sera were collected and stored at -20°C until use. Serum IL32 concentrations were measured using ELISA (R&D system). IL32, expression was also evaluated in renal biopsy samples of patients with active LN (n=20) by using a polyclonal rabbit anti-human IL32. As control, normal perilesional kidney tissue from patients undergoing nephrectomy for kidney malignancies were used. Patients' demographic, clinical and serological data were recorded in an electronically-filled database. All the patients signed a written informed consent and local Ethical Committee approved the protocol. Data were expressed as mean ± standard deviation or median and interquartil range (IQR) when appropriate (non parametric distribution). A p value ≤0.05 was considered significant. Results The twenty-eight LN patients included in this study were all Caucasian (2 male and 26 women) with a mean age at renal biopsy of 36.8±11.5 years and a mean disease duration of 9.74±11.42 years. Non renal SLE patients (3 male and 27 female) showed a mean age of 46 years ±17 and a mean disease duration of 18±7 years. We found that IL32 serum levels were significantly higher in patients with LN (median 2210, IQR 2404) compared to non renal SLE patients (median 1254, IQR 1334 pg/ml) (p=0.019). No significant difference in IL32 serum levels between LN patients and HC was found. IL32 expression was strongly expressed in renal biopsy samples of LN patients, especially in patients with class III and IV LN, compared to controls (for detail view Figure.1). Analysis of tissue distribution demonstrated IL32 expression in epithelial cells of proximal and distal tubular segments and among mesangial cells. IL-32-expressing cells were also observed among infiltrating inflammatory mononuclear cells scattered in the peri-tubular interstitium. Conclusions Data from this study showed increased serum and tissue levels of IL32 in LN patients, suggesting a potential role for IL32 in the pathogenesis of LN. Disclosure of Interest None declared
The aim of the study was to better characterise the immunological origin and the behaviour of interleukin (IL)-23-responsive innate lymphoid cells (ILCs) in the gut, synovial fluid (SF) and bone marrow (BM) of patients with ankylosing spondylitis (AS). ILC1, ILC2 and ILC3 cells were determined and characterised by confocal microscopy and flow cytometry in ileal and BM biopsies, in peripheral blood (PB) and SF mononuclear cells obtained from patients with AS and controls. Mucosal vascular addressin cell adhesion molecule 1 (MADCAM-1), IL-7, IL-15 and aggregates of lymphoid tissue inducer cells (LTi) were evaluated by immunohistochemistry. The in vitro ability of epithelial cells in driving the differentiation of ILC3 and the effect of tumour necrosis factor inhibitors (TNFi) on the frequency of ILC3 and the expression of MADCAM1 were also assessed. ILC3 characterised as Lyn(-)RORc(-)Tbet(+) NKp44(+) cells were significantly expanded in the gut, SF and BM of patients with AS compared with controls, produced high levels of IL-17 and IL-22 and expressed α4β7. MADcAM1 was overexpressed in BM and ileal high endothelial venules. IL-7 was significantly increased in AS gut, especially in the context of Paneth cells, and accompanied by the presence of aggregates of c-kit/IL-7R(+) cells (LTi). In in vitro experiments, epithelial cells from patients with AS actively induced differentiation of ILC3 from LTi. TNFi efficacy was accompanied by a significant decrease in the percentage of intestinal and circulating ILC3 and in the expression of MADCAM1. Gut-derived IL-17(+) and IL-22(+)ILC3 are expanded in the peripheral blood, SF and inflamed BM of patients with AS, suggesting the presence of an active homing axis between the gut and the inflamed sacroiliac joints. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.
To investigate the expression of IL-36 axis in patients with primary Sjogren's syndrome (pSS). Blood and minor labial salivary glands (MSG) biopsies were obtained from 35 pSS and 20 nSS patients. Serum IL-36α was assayed by ELISA. IL-36α, IL-36R, IL-36RA, IL-38, IL-22, IL-17, IL-23p19, expression in MSGs was assessed by rt-PCR and tissue IL-36α and IL-38 expression also investigated by immuno-histochemistry (IHC). αβ and γδ T cells and CD68(+) cells isolated from MSGs were also studied by flow cytometry and confocal microscopy analysis. IL-36α was significantly over-expressed in the serum and in the salivary glands of pSS. Salivary gland IL-36α expression was correlated with the expression levels of IL-17, IL-22 and IL-23p19. IL-38 that acts as inhibitor of IL-36α was also up-regulated in pSS. αβ(+) CD3(+) T cells and CD68(+) cells were the major source of IL-36α in minor salivary glands of pSS. γδ T cells were not significantly expanded in the salivary glands of pSS but produced more IL-17 being their percentage correlated with the focus score. Higher expression of IL-36α and IL-36R was also demonstrated in γδ T cells isolated from pSS compared to controls. in this study we demonstrate that a significant increase in circulating and tissue levels of IL-36α occurs in pSS patients. This article is protected by copyright. All rights reserved. © 2015 British Society for Immunology.
The vasculitides are a highly heterogeneous group of disorders characterized by the presence of inflammatory leukocytes in the vessel walls and reactive inflammation. Giant cell arteritis (GCA) and Takayasu’s arteritis (TA) are the two primary large-vessel vasculitides. Two distinct cellular pathways have been identified in GCA: Th17 polarization and IL-17 secretion and generation of Th1 cells which secrete IFN-γ. These two pathways may play different roles in the pathogenesis of vasculitides. The antineutrophil cytoplasmic antibody (ANCA)-associated vasculitides (AAVs) are small vessel vasculitis associated with antibodies directly to myeloperoxidase (MPO-ANCA) such as eosinophilic granulomatosis with polyangiitis (EGPA) and microscopic polyangiitis (MPA) or with antibodies directly to proteinase 3 (PR3-ANCA) such as granulomatosis with polyangiitis (GPA). Both in vitro and in vivo experimental data have shown that MPO-ANCA can induce necrotizing smallvessel vasculitis; however the presence of granulomatous lesions suggests the involvement of cell-mediated immune responses. Behçet syndrome (BS) is a chronic relapsing vasculitis of arteries and veins with unclear etiology. Exogenous and endogenous antigens, innate immune cells such as dendritic, NK, neutrophils and adaptive-immune cells are involved.
Aim of this study was to better elucidate the role of interleukin (IL)-18 in modulating the IL-22 pathway in primary Sjogren's syndrome (pSS) patients and in pSS-associated lymphomas. Minor salivary glands (MSGs) from patients with pSS and non-specific chronic sialo-adenitis (nSCS), parotid glands biopsies from non-Hodgkin lymphomas (NHL) developed in pSS patients were evaluated for IL-18, IL-22, IL-22 Receptor 1 (IL-22R1), IL-22 binding protein (IL-22BP) and signal transducer and activator of transcription 3 (STAT3) expression. MSGs IL-22R1-expressing cells were characterized by confocal microscopy and flow cytometry in pSS, nSCS and healthy controls . The effect of recombinant IL-18 and IL-22 on PBMCs from pSS and nSCS was studied by flow cytometry and RT-PCR. MSGs of pSS and NHL were characterized by an imbalance between IL-22 and IL-22BP protein expression with IL-18 and IL-22BP being expressed in a mutually exclusive manner and IL-18 and IL-22R1 being directly correlated. Aberrant expression of IL-22R1, induced by IL-18, was observed only among tissue and circulating myeloid cells of pSS patients and macrophages of NHL tissues of pSS patients, but not nSCS. IL-22R1 expression on PBMC of pSS was functional since its stimulation with recombinant IL-22 significantly up-regulated the expression of STAT3, IL-17 and IL-22. an IL-18-dependent aberrant expression of IL-22R1 on cells of hematopoietic origin seems to be a specific immunological signature of patients with pSS and pSS associated lymphomas. This article is protected by copyright. All rights reserved. © 2015 British Society for Immunology.
GCA is a large- and medium-vessel arteritis characterized by a range of histological patterns of vascular wall injury. The aim of this study was to immunologically characterize the various histological patterns of GCA. Thirty-five consecutive patients with biopsy-proven GCA and 15 normal controls were studied. IL-8, IL-9, IL-9R, IL-17, IL-4, TGF-β and thymic stromal lymphopoietin expression was evaluated by RT-PCR and immunohistochemistry on artery biopsy specimens. Confocal microscopy was used to characterize the phenotypes of IL-9-producing and IL-9R-expressing cells. Five additional patients who had received prednisone when the temporal artery biopsy was performed were also enrolled to evaluate the effect of glucocorticoids on IL-9 and IL-17 expression. IL-17 overexpression was observed mainly in arteries with transmural inflammation and vasa vasorum vasculitis. IL-9 overexpression and Th9 polarization predominated in arteries with transmural inflammation and small-vessel vasculitis. The tissue expression of both IL-9 and IL-17 was correlated with the intensity of the systemic inflammatory response. IL-4, TGF-β and thymic stromal lymphopoietin, which are involved in the differentiation of Th9 cells, were overexpressed in arteries with transmural inflammation and small-vessel vasculitis. IL-9R was also overexpressed in GCA arteries with transmural inflammation and was accompanied by increased expression of IL-8. Herein we provide the first evidence that distinct populations of potentially autoreactive T cells, expressing different cytokines (Th17 vs Th9), characterize patients with particular histological subsets of GCA and may thus contribute to the heterogeneity of tissue lesions observed in these patients. © The Author 2015. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For Permissions, please email: email@example.com.
Patients with systemic sclerosis (SSc) often complain reduced capacity at submaximal exercise; conversely physical capacity in performing daily duties has never been measured in SSc. To evaluate this performance and its correlates, in patients with SSc compared to healthy controls, in a free-living setting. Twenty-seven outpatients with stable SSc and 11 controls were recruited. Physical activity was assessed by portable multiple sensor device (SenseWear Armband) worn for at least six days. Physical activity duration (PAD; in minutes) for non-sedentary activities and physical activity level (PAL= total daily energy/resting energy expenditure) per day were calculated. Nutritional status was estimated by bioelectrical impedance analysis, and pulmonary arterial hyperthension excluded by echocardiography. Daily physical activities (243±145 minutes per day versus 397±142 min, respectively; p=0.005) and PAL were significantly reduced in SSc compared to controls (1.5±0.4 vs 2±0.7, respectively; p=0.019). 74% of SSc patients showed PAL <1.70, whereas only 27% of controls were below this threshold for sedentary life style. Both PAD and PAL positively correlated with DLco. Patients and controls did not differ for spirometric parameters, BMI, phase angle at bioelectrical impedance analysis, fat mass or fat-free mass indexes. In SSc, exercise capacity during daily activity was reduced compared to controls, and was associated with early evidence of functional decay (decreasing DLco) but not with malnutrition (undernutrition). A reduction of daily physical activity is already present even in early stages of lung involvement in SSc, characterized by unaltered spirometry and well preserved nutritional status. This article is protected by copyright. All rights reserved.
- Feb 2015
In recent years, mesenchymal stromal cells (MSCs) have been largely investigated and tested as a new therapeutic tool for several clinical applications, including the treatment of different rheumatic diseases. MSCs are responsible for the normal turnover and maintenance of adult mesenchymal tissues as the result of their multipotent differentiation abilities and their secretion of a variety of cytokines and growth factors. Although initially derived from bone marrow, MSCs are present in many different tissues such as many peri-articular tissues. MSCs may exert immune-modulatory properties, modulating different immune cells in both in vitro and in vivo models, and they are considered immune-privileged cells. At present, these capacities are considered the most intriguing aspect of their biology, introducing the possibility that these cells may be used as effective therapy in autoimmune diseases. Therefore, stem cell therapies may represent an innovative approach for the treatment of rheumatic diseases, especially for the forms that are not responsive to standard treatments or alternatively still lacking a definite therapy. At present, although the data from scientific literature appear to suggest that such treatments might be more effective whether administered as soon as possible, the use of MSCs in clinical practice is likely to be restricted to patients with a long history of a severe refractory disease. Further results from larger clinical trials are needed to corroborate preclinical findings and human non-controlled studies, and advancement in the knowledge of MSCs might provide information about the therapeutic role of these cells in the treatment of many rheumatic diseases. Copyright © 2015 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.
- Jan 2015
In this paper, we aimed to evaluate the levels of ferritin enriched in H subunits (H-ferritin) and ferritin enriched in L subunits (L-ferritin) and the cells expressing these 2 molecules, in the bone marrow (BM) and liver biopsies obtained from adult onset Still's disease (AOSD) patients who developed macrophage activation syndrome (MAS), and correlating these data with the severity of the disease. Twenty-one patients with MAS-associated AOSD underwent BM biopsy and among them, 9 patients with hepatomegaly and elevated liver enzymes underwent liver biopsy. All the samples were stained by both immunohistochemistry and immunofluorescence. A statistical analysis was performed to estimate the possible correlation among both H-ferritin and L-ferritin tissue expression and the clinical picture of the disease. Furthermore, the same analysis was performed to evaluate the possible correlation among the number of CD68/H-ferritin or CD68/L-ferritin positive cells and the clinical picture. Both immunohistochemical and immunofluorescence analysis demonstrated an increased tissue H-ferritin expression, in the BM and liver samples of our patients. This increased expression correlated with the severity of the disease. An inflammatory infiltrate, enriched in CD68 macrophages, expressing H-ferritin was observed in both the BM and the liver samples of our patients. Furthermore, we observed, that this increased number of CD68/H-ferritin positive cells significantly correlated with the severity of clinical picture and this specific BM infiltrate correlated with the mortality rate, reported in our cohort. Our data showed an imbalance between the levels of H- and L-ferritin in different organs of patients with MAS-associated AOSD and the evidence of a strong infiltrate of CD68/H-ferritin positive cells in the same organs. Furthermore, a strong correlation among both the tissue H-ferritin and the CD68/H-ferritin positive cells and the clinical picture was observed.
- Jan 2015
Eicosanoids have an important role in the pathogenesis of rheumatic diseases and anumber of enzymes and receptors involved in eicosanoid biosynthesis or action constitutevaluable therapeutic targets. Among the pro-inflammatory eicosanoids, prostaglandin E2(PGE2) is considered a key mediator of various inflammations; this includes swelling,fever and inflammatory pain. Prostaglandin I2 and thromboxane A2 regulate plateletaggregation and leukotriene B4 is one of the most powerful chemotactic agents.Prostaglandins of the J2 series decreases pro-inflammatory cytokine release and areimplicated in the resolution phase of inflammation. In inflammatory rheumatic diseases,including rheumatoid arthritis, spondyloarthritis, osteoarthritis, gout and in autoimmunerheumatic diseases including systemic sclerosis, the arachidonic acid cascade is activatedby cytokine-dependent enzyme induction, and the activity and/or expression ofcomponents of several pathways. Several enzymes of the arachidonic acid cascade oreicosanoid receptors are well-recognized targets of anti-inflammatory drugs. Moresimply, they can reduce symptoms of inflammation in rheumatic diseases. A moreexciting idea is that PGD2 pathway might possess anti-inflammatory properties whichcould be utilized in future treatment strategies
- Dec 2014
Background iNKT cells represent a T cell subset at the bridge between innate and adaptive immunity, playing a role in regulating auto-antibody-producing B cells before their entry into germinal centers. Therefore the absence and/or reduction of iNKT cells seem to increase auto-reactive B cell activation. Primary Sjogren's syndrome (pSS) is a systemic autoimmune disease in which lymphocyte infiltration and organization in lymphoid structures of inflamed salivary glands occur. Objectives The aim of this study was to investigate the frequency of iNKT in the salivary glands and peripheral blood of patients with pSS and their function by using CD1d/aGalactosylceramide (aGalaCer) tetramers. Methods Flow cytometry analysis of ex vivo frequency of tetramer+ cells, producing IFN-g and IL-17, and quantitative gene expression analysis by real-time PCR for Va24 chain expression was performed on salivary glands and peripheral blood obtained from patients and controls. Flow cytometric analysis and immune fluorescence for autoreactive B lymphocytes and ELISA for anti-SSA detection were also performed. Results A significant expansion of IL-17+- and IFN-γ+-producing iNKT cells, expressing low levels of the chemokine receptors CXCR3, CCR6 and CCR5, was observed in the peripheral blood of pSS patients. In the inflamed salivary glands, iNKT were absent whereas a significant increase of SSA specific B cells occurs. Moreover, co-culture experiments in vitro demonstrate that activated iNKT inhibit autoantibody production by autoreactive B cells from pSS patients. Conclusions An impaired tissue migration of iNKT may lead to the expansion of autoreactive B cells specific for SSA -antigen in salivary glands of patients, suggesting an important role of iNKT in regulating B cells activation in pSS. References Disclosure of Interest None declared
The mechanism by which human leukocyte antigen B27 (HLA-B27) contributes to ankylosing spondylitis (AS) remains unclear. Genetic studies demonstrate that association with and interaction between polymorphisms of endoplasmic reticulum aminopeptidase 1 (ERAP1) and HLA-B27 influence the risk of AS. It has been hypothesised that ERAP1-mediated HLA-B27 misfolding increases endoplasmic reticulum (ER) stress, driving an interleukin (IL) 23-dependent, pro-inflammatory immune response. We tested the hypothesis that AS-risk ERAP1 variants increase ER-stress and concomitant pro-inflammatory cytokine production in HLA-B27(+) but not HLA-B27(-) AS patients or controls. Forty-nine AS cases and 22 healthy controls were grouped according to HLA-B27 status and AS-associated ERAP1 rs30187 genotypes: HLA-B27(+)ERAP1(risk), HLA-B27(+)ERAP1(protective), HLA-B27(-)ERAP1(risk) and HLA-B27(-)ERAP1(protective). Expression levels of ER-stress markers GRP78 (8 kDa glucose-regulated protein), CHOP (C/EBP-homologous protein) and inflammatory cytokines were determined in peripheral blood mononuclear cell and ileal biopsies. We found no differences in ER-stress gene expression between HLA-B27(+) and HLA-B27(-) cases or healthy controls, or between cases or controls stratified by carriage of ERAP1 risk or protective alleles in the presence or absence of HLA-B27. No differences were observed between expression of IL17A or TNF (tumour necrosis factor) in HLA-B27(+)ERAP1(risk), HLA-B27(+)ERAP1(protective) and HLA-B27(-)ERAP1(protective) cases. These data demonstrate that aberrant ERAP1 activity and HLA-B27 carriage does not alter ER-stress levels in AS, suggesting that ERAP1 and HLA-B27 may influence disease susceptibility through other mechanisms.Genes and Immunity advance online publication, 6 November 2014; doi:10.1038/gene.2014.62.
Objective Ankylosing spondylitis (AS) is a common, highly heritable immune-mediated arthropathy that occurs in genetically susceptible individuals exposed to an unknown but likely ubiquitous environmental trigger. There is a close relationship between the gut and spondyloarthritis, as exemplified in patients with reactive arthritis, in whom a typically self-limiting arthropathy follows either a gastrointestinal or urogenital infection. Microbial involvement in AS has been suggested; however, no definitive link has been established. The aim of this study was to determine whether the gut in patients with AS carries a distinct microbial signature compared with that in the gut of healthy control subjects.Methods Microbial profiles for terminal ileum biopsy specimens obtained from patients with recent-onset tumor necrosis factor antagonist-naive AS and from healthy control subjects were generated using culture-independent 16S ribosomal RNA gene sequencing and analysis techniques.ResultsOur results showed that the terminal ileum microbial communities in patients with AS differ significantly (P < 0.001) from those in healthy control subjects, driven by a higher abundance of 5 families of bacteria (Lachnospiraceae [P = 0.001], Ruminococcaceae [P = 0.012], Rikenellaceae [P = 0.004], Porphyromonadaceae [P = 0.001], and Bacteroidaceae [P = 0.001]) and a decrease in the abundance of 2 families of bacteria (Veillonellaceae [P = 0.01] and Prevotellaceae [P = 0.004]).Conclusion We show evidence for a discrete microbial signature in the terminal ileum of patients with AS compared with healthy control subjects. The microbial composition was demonstrated to correlate with disease status, and greater differences were observed between disease groups than within disease groups. These results are consistent with the hypothesis that genes associated with AS act, at least in part, through effects on the gut microbiome.
We read with interest the study by Neerinckx et al1 addressing the expression of interleukin (IL)-23p19 and of autophagy genes in the synovium and in the peripheral blood mononuclear cells of patients with ankylosing spondylitis (AS). Differently from our observation in the gut,2 the authors failed to demonstrate any significant increase by RT-PCR in the expression of synovium autophagy-related genes (ATG16L1, IRGM, MAP1LC3A, ATG5, HSPA8 and HSP90AA1) together with no significant overexpression of IL-23p19 compared with disease and healthy controls. We have previously demonstrated by immunohistochemistry that in the … [Full text of this article]
Primary Sjögren's syndrome (pSS) is an autoimmune disorder affecting exocrine glands and characterized in most cases by a rather mild clinical picture. However, a subgroup of pSS patients experience systemic extraglandular involvement leading to a worsening of disease prognosis. Current therapeutic options for the treatment of pSS are mainly empirical, often translated by other autoimmune diseases, and recent systematic reviews have highlighted the lack of evidence-based recommendations for most of the drugs commonly employed in the spectrum of extraglandular involvement. Because of the well-established role of B-lymphocytes in the pathogenesis of pSS, a B-cell targeting therapy may represent a new and intriguing therapeutic approach; in this context, growing evidence suggests that B-cell depletion by rituximab (RTX) is also effective in pSS. Of interest, besides clinical efficacy, RTX also showed biologic effects, consistently affecting the inflammation and the lymphoid organization that occur in target tissue. Moreover, the good results observed in the published trials after RTX treatment in pSS should represent the starting point to develop evidence-based guidelines for the use of biologic therapy in this disease.
- Jul 2014
Abstract INTRODUCTION: Rituximab (RTX) is a monoclonal anti-CD20 antibody approved for the treatment of rheumatoid arthritis (RA) in association with methotrexate (MTX). OBJECTIVES: To evaluate the efficacy and safety of RTX-MTX combination therapy compared with RTX alone in the treatment of RA. METHODS: We analyzed data from a prospective cohort study, the Italian biologic register GISEA, to investigate the efficacy and safety of rituximab. Moreover, the adverse events (AE) and the causes of discontinuation therapy were analyzed. RESULTS: We identified 338 RA patients, 162 treated with RTX and 176 with RTX-MTX. After 52 and 104 weeks of therapy the disease activity score in 28 joints and the Health Assessment Questionnaire Score were available in 168 patients (78 with RTX-MTX and 60 with RTX alone), showing significant reduction without differences among the two groups. AE were reported in 142 patients (42%), for a total of 368 recorded side effects. The majority (90.5%) of AE were mild to moderate in severity. Comparable percentages of severe AE were reported in the 2 groups (9.9% for RTX alone and 9.3% for RTX+MTX). A poor disease control was observed in 14.2% and 13.5% of patients treated with RTX+MTX and RTX, respectively; while 12 patients (4.5% in RTX+MTX, and 2.5% in RTX group) suspended therapy for AE. CONCLUSIONS: RTX showed a good efficacy and safety profile in the real-life management of RA patients regardless of the association with MTX.
Background Rheumatoid Arthritis (RA) is a chronic autoimmune disease characterized by joint erosion and damage. Several cytokines and recruitment of auto-reactive lymphocytes (characterized by a marked shift toward the Th1 and Th17 phenotype) to inflamed tissue is a defined feature of the disease. In addition to Th1/Th17 and Th2 cells, another subset of effector T cells, identified by the potent production of IL-9 and named Th9 cells, has been recently demonstrated. IL-9 was found in particular to be increased before the clinical onset of the articular disease in RA patients, and associated with the presence of RA-related autoantibodies and circulating biomarkers of inflammation. The exact role of Th9/IL-9 and related cytokines in RA however has not been yet adequately studied Objectives To evaluate the role of IL-9 and IL-9-producing cells in patients with Rheumatoid arthritis and in a murine model of collagen-induced arthritis (CIA) Methods IL-9, IL-9R and PU.1 expression was assessed by rt-PCR and IHC in the synovial tissue of RA and osteoarthritis (OA) patients. IL-9, IL-9R, IL-4, TSLP and TGF-b were investigated by immuno-histochemistry (IHC). Confocal microscopy was used to characterize the phenotype of IL-9-producing cells. The prevalence of Th-9 lymphocytes on peripheral blood mononuclear cells has been determined by flow cytometry analysis before and after incubation with citrullinated aggrecan (or not) peptide. Evaluation of CIA severity and IL-9 occurrence was also studied in normal and PU.1 deficient mice. Results IL-9 was over-expressed in the RA synovial tissues and correlated with the degree of histological organization of B and T cells in ectopic tertiary lymphoid structures. IL-9 producing cells were demonstrated to co-express PU.1 and were thus identified as Th9 cells. Increased expression of IL-9R was also observed in RA, with diffuse positive cells detected outside T-B cell aggregates and few cells detectable at the outside border of lymphoid aggregates. Strong IL-4, TSLP and TGF-b (cytokines involved in the induction of Th9 polarization) expression was demonstrated by IHC in RA. Blood peripheral Th9 cells were also increased in RA and specifically activated by a citrullinated peptide. In the CIA mouse model, the abrogation of the Th9 transcription factor PU.1 ameliorated arthritis and was associated with low levels of IL-9 expression. Conclusions Here we show for the first time that IL-9 and Th9 cells are over-expressed in the RA synovial tissue and correlated with the degree of histological organization of B and T cells in ectopic lymphoid structures. We also show that blood peripheral Th9 cells are increased in RA and in vitro expanded by citrullinated peptide incubation. Finally, in the CIA mouse model, the abrogation of the Th9 transcription factor PU.1 ameliorated arthritis. Altogether these results suggest that IL-9 and the Th9 cells may play an important role in the pathogenesis of RA. Disclosure of Interest None declared DOI 10.1136/annrheumdis-2014-eular.5190
Background Chronic gut inflammation occurring in AS patients has been linked to active axial inflammation and the gut has been proposed as the main site of IL-23 production in AS patients. IL-23 has been demonstrated to be essential in murine enthesitis by acting on a unique subset of entheseal resident T cells that share some immunological features with a subset of IL-23-responsive gut derived innate lymphoid cells (type III ILCs). Objectives Aim of the study was to better characterize the immunologic origin and the behavior of ILCs in the gut, synovial fluid and bone marrow of AS patients. Methods Consecutive ileal gut biopsies were obtained from 20 HLA-B27+ AS patients with axial active disease (BASDAI mean 6.7±2.2) and 15 healthy subjects. Paired samples of peripheral blood and synovial fluid (form knee joints) were obtained from 10 of the AS and 10 osteoarthritis patients. Bone marrow (BM) biopsies (N=5) from inflamed sacroiliac joints (SI) as demonstrated by MRI and from subjects undergoing BM biopsy for suspected monoclona gammopathy (N=5) were also obtained. IL-23R+CD3+CD4–CD8–CD56+T-bet+NKp44+ (type III ILCs) cells were determined and characterized for IL-17 and IL-22 production by flow cytometry and confocal microscopy on isolated lamina propria (LPMC), peripheral blood (PBMC), BM and synovial mononuclear cells (SMC). a4b7 integrin and its counter-receptor MADCAM-1 were evaluated in ileal and BM samples. Quantitative gene expression analysis by rt-PCR of IL-7 and IL-15 (involved in ILCs differentiation) and of IL-17, IL-22 and IL-23p19 was also performed. IL-17, IL-22, IL-23p19, IL-7 and IL-15 protein expression was also analyzed by immunohistochemistry (IHC). Tissue distribution of lymphoid tissue inducer cells (LTi) was also assessed by confocal microscopy. Results CD3–/CD3+CD4–CD8–RORc–Tbet+CD56+NKp44+ (type III ILCs) cells expressing the IL-23R were significantly expanded in the gut, synovial fluid, and bone marrow of AS patients compared to controls and produced high levels of IL-17 and IL-22. Type III ILCs isolated from the peripheral blood, synovial fluid and BM of AS displayed a significant over-expression of a4b7 suggesting an intestinal origin for these cells. MADcAM1 increased co-expression was demonstrated in both BM and ileal samples. Strong expression of both IL-17 and IL-22 was confirmed by IHC in AS BM. IL-7 was significantly increased in AS gut compared to control gut, especially in the context of Paneth cells (PC) and surrounded by aggregates of c-kit/IL-7R+ cells (LTi) that have been demonstrated to be precursors of gut type III ILC. Conclusions In this study we demonstrated that gut-derived IL-23R-expressing type III ILCs are expanded in the synovial fluid and inflamed BM of AS patients and produced IL-17 and IL-22, both cytokines suggested as key players in the pathogenesis of AS. The increased expression of IL-7 in AS gut and the presence of clusters of LTi cells close to Paneth cells suggest an important role of innate intestinal immunity in the differentiation of type III ILCs. The increased intestinal and BM expression of MADCAM-1 occurring in AS also suggests the presence of an active homing axis between the gut and the inflamed SI joints. Disclosure of Interest None declared DOI 10.1136/annrheumdis-2014-eular.5160
Background Subclinical gut inflammation has been demonstrated in patients with psoriatic arthritis (PsA) suggesting a role for the gut in the pathogenesis of inflammation in these patients. A key role for the IL-23, IL-17, IL-22 and IL-9 in the pathogenesis of psoriasis and psoriatic arthritis has been suggested, the immunologic abnormalities underlying subclinical gut inflammation in PsA are still undefined however. Objectives This study was undertaken to investigate the expression and tissue distribution of IL-23 and of Th17,Th22 and Th9 related molecules in the subclinical gut inflammation of patients with PsA. Methods Gut inflammation was assessed accordingly to De Vos et al (1). Quantitative gene expression analysis of Th1, IL-23/Th17, Th22 and Th9 responses was performed in intestinal biopsy samples obtained from 22 patients with PsA (12 with non-radiographic psoriatic axial SpA, 5 of them displaying HLA-27 positivity), 10 patients with HLA-27 positive-ankylosing spondylitis (AS), 10 patients with Crohn's disease (CD) and 20 healthy controls. IL-23, IL-17, IL-22 and IL-9 tissue distribution was also evaluated by immunohistochemistry. Results Chronic ileal inflammation was observed in 15 PsA patients with no difference regarding axial or peripheral disease. The terminal ileum of PsA patients was characterized by a complex immunological signature. IL-23p19 m-RNA was not significantly over-expressed compared to controls but a clear Th17 and Th22 polarized immune response was demonstrable, at levels comparable to those observed in CD. Differently from CD and similarly to AS, Th1 cytokines were not significantly over-expressed in PsA. Conversely, a strong IL-23p19 and IL-22 expression in the absence of Th17 polarization was confirmed in AS gut. Unlike AS and CD, a strong and significant up-regulation of IL-9 immunologically characterized the gut of PsA patients. In particular, IL-9 immune-reactivity was observed among infiltrating mononuclear cells, high endothelial venules and Paneth cells. IL-9 mononuclear cells were demonstrated to be in large part Th9 cells. IL-9 over-expression was accompanied by significant Paneth cells hyperplasia. Conclusions The strong IL-9/Th9 polarization seems to be the predominant immunological signature of subclinical gut inflammation in PsA. Our findings indicate however a complex immune-regulation occurring in the gut of PsA patients where Th17 and Th22 polarization seems also to occur. References Disclosure of Interest None declared DOI 10.1136/annrheumdis-2014-eular.5174
Background Adult onset Still's disease (AOSD) is a rare rheumatic disease with an estimated prevalence of less than 1 case per 100,000 population. The diagnosis is difficult and is often delayed due to the lack of specific diagnostic tests and the need to rule out other pathological entities. Objectives To describe the clinical characteristics of a multicenter Italian case series of patients with AOSD. Methods 14 Italian University Hospital centers participated in the study. A standardized medical record containing clinical data, laboratory investigations, disease patterns and the different therapies has been sent to all participating centers. Each center collected data retrospectively. Results In the first wave of data collection, from March to May 2013, 233 patients were included with AOSD according to Yamaguchi criteria (125 males, 53.6%, mean age at onset: 40.2±16.2 years, time between onset and diagnosis: 242±650 days). The most frequent manifestations at onset were: arthralgia (93.0%), fever (92.6%), leukocytosis (89.0%), typical rash (67.7%), sore throat (61.8%), lymphadenopathy/splenomegaly (60.4%), increased liver enzymes (53.5%). In 56.4% of patients ferritin was increased more than 5 times the normal threshold. Fever intensity was recorded up to 41 ° C (mean 39.1±0.7), with a single daily peak in 27.3% of cases and 2 peaks in 51.9% of cases. The average duration of febrile episodes was 10.3 days with variable interval between episodes (average 3.2 days). Based on clinical evolution the disease was classified polycyclic systemic in 40.8%, chronic articular polycyclicin 30.7%, systemic monocyclic in 23.9% and chronic articular monocyclic in 4.6%. In 21.9% of cases, corticosteroids and traditional DMARDs have not been able to control the disease while biological drugs have been shown to be effective. 56 cycles of biological drugs were used in 51 patients: anakinra in 22 cases, etanercept in 15 cases, infliximab in 8 cases, adalimumab in 7 cases, rituximab in 3 cases, tocilizumab in 2 cases, abatacept and golimumab in 1 case each (anti- TNFalpha 52.5%, 37.3% anakinra, other 10.2%). Conclusions This study presents the largest Italian multicentre series of AOSD patients. The main clinical and laboratory characteristics are in line with those reported in the literature. In more than 20% of patients biologics are the only drugs that allow to control the disease. Disclosure of Interest : None declared DOI 10.1136/annrheumdis-2014-eular.5300
Objectives: Several histological scoring systems, including the focus score, performed in minor salivary glands (MSGs) by hematoxylin-eosin (H&E) staining, have been employed in clinical practice to assess the inflammatory infiltrate and provide the diagnosis of primary Sjo¨gren׳s syndrome (pSS). Aims of this study were to integrate different scoring systems and identify potential differences in the molecular profile of lymphoid cytokines related to germinal center (GC) formation and clinical subsets in pSS. Methods: Overall, 104 pSS patients and 40 subjects with sicca non-pSS were retrospectively evaluated. MSG biopsies were evaluated by H&E and immunofluorescence to assess histological pattern, Chisholm and Mason grading system, Tarpley score, a grading for the severity of inflammatory infiltrate, T-/B-cell segregation, and the presence of GC. MSGs from 50 pSS patients and 30 sicca non-pSS patients were processed by real-time PCR to assess LTα, LTβ, BAFF, CXCR4, CXCL12, CXCR5, CXCL13, CCR7, CCL19, and CCL21. Results: GCs presence was associated with anti-Ro/SSA and anti-La/SSB antibodies, hypergammaglobulinemia, salivary gland swelling, higher Tarpley score and focus score, and extraglandular involvement but, at multivariate analysis, only extraglandular involvement was independently associated to GC. pSS patients displayed higher level of all cytokines compared to those with sicca symptoms. GC(+) pSS patients displayed higher level of all cytokines compared to those GC(-). Conclusions: Our study demonstrates that different histopathological patterns, including GC presence, reflect different cytokine expression and different clinical subsets. We believe that the combined immunofluorescence/molecular approach in MSGs would help to tailor diagnostic and therapeutic approach for different subsets of pSS patients.
The aim of our study was to evaluate methotrexate (MTX) and methylprednisolone (MP) effect on peripheral Th17 and Treg subsets in patients with rheumatoid arthritis (RA). We enrolled 15 patients (10 early RA and 5 long-standing disease) with active RA and 10 age-matched healthy donors as controls. Frequencies of Th17 and Treg were quantified using flow cytometry before and after in vitro addition of MTX, MP or both drugs. Our results showed a reduction in the overall Th17 population followed by an increase in Th17 IL-10(+) and Treg, after in vitro treatment of PBMCs with the drugs in patients with early RA. Long-standing disease patients showed a less evident increase in Treg cells and less enhancement of IL-10 Th17 cells. We suggest that the treatment with MTX and MP could ameliorate RA disease activity by normalizing the distribution/imbalance of Th17/Treg and indicate a new regulatory role of IL-17(+) cells in RA patients.
Objective: The aim of this study was to evaluate the role of rituximab (RTX) in modulating the expression of the IL-17/IL-23 pathway in the salivary glands (SGs) of patients with primary SS (pSS). Methods: Consecutive SG biopsies were obtained from 15 patients with pSS before and after 1 year of RTX therapy. The SG expression of IL-17, IL-23p19 and p-STAT3 was evaluated by immunohistochemistry at baseline and after RTX therapy. The role of mast cells in pSS patients in modulating the Th17 response and the immunologic effect of RTX on mast cells were also studied in in vitro experiments. Results: IL-17 was overexpressed in the SGs of patients with pSS mainly by infiltrating T cells and mast cells. After RTX therapy, the SG expression of IL-17, but not of IL-23p19 and p-STAT3, was significantly reduced and was accompanied by the depletion of tissue mast cells. In in vitro experiments with heterologous peripheral lymphocytes RTX significantly induced the apoptosis of isolated mast cells. Finally, mast cells isolated from peripheral blood mononuclear cells of pSS patients in vitro significantly increased Th17 lymphocytes. Conclusion: RTX acts on pSS patients by globally reducing the expression of IL-17 and specifically inducing a pronounced apoptotic depletion of mast cells.
Objective: The aim of this study was to assess the expression of IL-22, IL-22 receptor 1 (IL-22R1), IL-22 binding protein (IL-22BP) and p-STAT3 in muscle tissue from patients with PM and DM. Methods: Levels of IL-22, IL-22R1, IL-22BP and STAT3 mRNA were quantified by RT-PCR. The expression of IL-22, IL-22R1, IL-22BP and p-STAT3 was also analysed using immunohistochemistry. Results: Significant modulation of the IL-22 pathway was observed in inflammatory myopathic tissues. In particular, a significant overexpression of IL-22 at the protein but not the mRNA level was observed in PM/DM tissues and was correlated with myositis activity. IL-22R1 aberrant expression was also observed among infiltrating mononuclear cells and necrotic muscle cells. IL-22BP, which inhibits IL-22 signalling, was expressed only in some muscle fibres in PM/DM patients. Conclusion: Our findings indicate that the IL-22 pathway is activated in inflammatory myopathic tissues and may be involved in the induction of muscle inflammatory processes and muscle necrosis.
- Feb 2014
In primary Sjögren's syndrome (pSS) a complex of interconnections between epithelial barrier, innate and adaptive immunity occurs. IL-22 is a pleiotropic cytokine that in pSS may be placed at the intersection of the adaptive and innate branches of immunity. Some evidence suggests that, in pSS, IL-22 may play a prominent pro-inflammatory role driving the early phase of tissue and systemic inflammation and participating in the self-perpetuation of disease. Despite contradictory data in literature about the role of NK cells in pSS, recent data also suggest an important contribution of this subset of cells of the innate immune system in the development and perpetuation of inflammation. Here, we discuss the role of IL-22 in the pathogenesis of pSS and in epithelial barrier function.
Background Interleukin (IL)-22 is a potent mediator of cellular inflammatory responses that has been recently reported to play a role in the pathogenesis of primary Sjogren’s Syndrome (p-SS) (1, 2) and of T and B lymphomas. IL-22 biological activity is initiated by binding to a cell-surface complex composed of two subunits, IL-22R1 and IL-10R2 receptor chains, and further regulated by interactions with a soluble binding protein, IL-22BP. Unlike the IL-10R2, which is constitutively expressed in many human tissues, IL-22R1 is not detectable in immune cells. Objectives Aim of this study was to better characterize the role of IL-22 axis in the pathogenesis of p-SS and p-SS-associated lymphomas. Methods Minor salivary gland biopsies were obtained from 30 patients with p-SS and 20 with non-specific chronic sialo-adenitis (n-SS) and evaluated by RT-PCR for IL-22, IL-22R1 and IL-22BP expression. The protein expression of IL-22, IL-22R1 and p-STAT3 was evaluated by immunohistochemistry and IL-22R1-expressing cells were caharcterized by confocal microscopy. Flow cytometry analysis of IL-22R1 expression was also conducted on peripheral blood mononuclear cells (PBMCs) from p-SS (n=30), n-SS (n=20), SLE (n=20) and rheumatoid arthritis (n=20) patients and normal controls (n=30). PBMCs isolated from 10 p-SS and 10 controls were also cultured with (or without) recombinant IL-22 and the modulation of the transcripts for pro-inflammatory cytokines was assessed by RT-PCR. Paraffin embedded sections of non-Hodgkin lymphomas from 5 p-SS patients were finally evaluated for the expression of IL-22R1. Results IL-22, STAT3 and IL-22BP but not IL-22R1 transcript levels were significantly up-regulated in the inflamed salivary glands of p-SS but not in n-SS. Immunohistochemistry confirmed the increased salivary expression of IL-22 and p-STAT3 also demonstrating a significant increased protein levels of IL-22R1 in p-SS. Confocal microscopy analysis showed that IL-22R1 was aberrantly and strongly expressed in monocytes/macrophages infiltrating the p-SS salivary glands and these cells also co-expressed p-STAT3, suggesting the occurrence of autocrine activation of p-STAT3. CD68+ cells obtained from the peripheral blood also aberrantly expressed IL-22R1 in p-SS patients. IL-22R1 expression on cells of hematopoietic origin was never observed in control disease patients, n-SS and healthy controls. The stimulation with recombinant IL-22 of PBMCs from p-SS but not controls significantly up-regulated the expression of IL-17 and IL-22. Non-Hodgkin lymphoma tissues from p-SS patients were also characterized by the aberrant expression of IL-22R1 on macrophages and neoplastic B cells. Conclusions The aberrant expression of IL-22RA1 on cells of hematopoietic origin seems to be a specific immunological signature of patients with p-SS and p-SS associated lymphomas and suggests a fundamental role of IL-22 signaling in the pathogenesis of p-SS and its neoplastic evolution. Targeting of IL-22 pathway may represent a successful therapeutic strategy in p-SS patients. References References Disclosure of Interest None Declared
Background Subclinical gut inflammation occurs in patients with Ankylosing Spondylitis (AS) and long term evolution to overt Crohn’s disease (CD) has been described in these patients. Gut mucosal macrophages represent the largest pool of tissue macrophages in the body. Different pathways of macrophage activation have been described in humans. Objectives To study the macrophages polarization occurring in the inflamed gut of AS patients. Methods Twenty two consecutive HLA-B27+ Ankylosing Spondylitis (AS) patients, 15 Crohn’s Disease (CD) patients and 15 normal controls were included in this study. Four AS patients developed an overt CD during the follow-up and were included. Ileal macrophage subsets were characterized by immunohistochemistry and flow cytometry. Quantitative gene expression analysis, by Real Time-PCR of IFN-g, IRF5, IL-4, IL-5, IL-33 and STAT6 was also performed. Results CD68+ macrophages were expanded in both AS and CD patients but not in controls. Inducible nitric synthase (iNOS+) macrophages were increased in both AS and CD patients and comprised classical M1 macrophages (iNOS+IL-10-) and resolution phase macrophages (rMs) (iNOS+IL-10+). Adaptive immunity, valued through IFN-g expression, was the main M1-polarizing factor in CD whereas innate immune M1 response, valued through IRF-5 induction, predominates in AS. Increased numbers of CD163+ (M2) macrophages were observed in both AS and CD patients together with increased expression of Th2 cytokines and the Th2 transcription factor STAT6. Unlike in CD, CD14+ macrophages were virtually absent in the gut of AS patients and controls. On the other hand CD14+ macrophages were observed to be expanded in the four AS patients that developed CD. Conclusions M1, M2 macrophages and rMs expansion was observed in both AS and CD. IL-33 over-expression immunologically characterizes AS gut inflammation. CD14+ macrophages were observed in CD patients and appear to characterize evolution from subclinical towards clinically evident gut inflammation in AS. Disclosure of Interest None Declared
Background Rheumatoid arthritis (RA) is conventionally defined as moderate when the disease activity score assessed in 28 joints (DAS28) is ≥3.2, and high disease with DAS28 >5.1 Objectives Aim of this analysis was to evaluate the rate of remission and LDA (low disease activity), in RA patients starting etanercept therapy with moderate disease activity in the real world settings Methods The clinical records were retrospectively analysed from the Italian nationwide registry, called GISEA (Gruppo Italiano Studio Early Arthritis). RA patients with moderate (Mod-RA) and high (H-RA) disease activity (DAS28 >3.2) with incomplete response to conventional DMARDs and beginning a treatment with etanercept 50 mg/week subcutaneously (or 25 mg twice/week) over 10 years (2001-2010) were considered and compared. Clinical remission (DAS28 <2.6), LDA (DAS28 <3.2), HAQ, EULAR clinical response were assessed at 6 and 12 months of therapy, and etanercept survival rate at 1 and 4 years Results 963 patients with moderate to severe RA were selected. Out of 963, 320 had Mod-RA and 633 H-RA. At 1 year, the rate of patients achieving DAS28 remission were significantly higher in Mod-RA (47.9%) than in H-RA (24.5%, p<0.001), and patients achieving LDA was significantly higher in Mod-RA (65.9%) than in H-RA (36.7%, p<0.001). “Good” EULAR response was significantly higher in Mod-RA (58.9%) than in H-RA group (36.7%) (p<0.01). Surprisingly, the retention rate of etanercept was significantly higher in H-RA patients (84.5%) than in Mod-RA patients (75.4%), both at 1 year (p<0.01) and at 4 years, 58.4% and 48.8%, respectively (p<0.01). Also the HAQ response was intriguing. In H-RA patients, mean HAQ score was 1.6 at baseline and 0.83 at 12 months (mean difference 0.77), while in Mod-RA patients, mean HAQ score was 1.01 at baseline and fell down at 0.55 after 6 months (mean difference 0.46, p=0.0001).Apparently patients starting with high disease activity have more room for improvement in HAQ, but the patients starting with moderate RA reach better absolute levels.Univariate and multivariate logistic regression analysis did not detect any predictor of response differentiating patients with H-RA from those with Mod-RA Conclusions Consistently with previous reports, this preliminary data have provided evidence that in standard care setting Mod-RA patients had a high likelihood to achieve a DAS28 driven clinical remission as well as a condition of LDA when treated with etanercept Disclosure. This analysis was supported by Pfizer Inc. Disclosure of Interest F. Iannone Grant/Research support from: Pfizer Inc., G. Ferraccioli: None Declared, C. Ferri: None Declared, M. Galeazzi: None Declared, R. Gerli: None Declared, R. Giacomelli: None Declared, W. Grassi: None Declared, R. Gorla: None Declared, M. Govoni: None Declared, A. Marchesoni: None Declared, L. Punzi: None Declared, P. Sarzi-Puttini: None Declared, G. Triolo: None Declared, G. Lapadula: None Declared
To investigate the in vitro and ex-vivo effect of IL-6 inhibition on the balance between Th1, Th2, Th17 and Treg cells. Ten consecutive adult patients with active early rheumatoid arthritis (ERA) and ten healthy volunteers were included in the study. The percentages of Th1, Th2, Th17 and Treg cells were analysed by flow cytometry in the peripheral blood mononuclear cells obtained from controls and from RA patients at the time of first evaluation and just before the third TCZ infusion. The in vitro effect of TCZ on the different subsets of CD4+ T cells and the expression levels of Th1, Th2, Th17 and Treg-related cytokines was also assessed. Treatment with TCZ, both ex vivo and in vitro, resulted in a significant reduction of the percentage of Th1, Th17 and Treg cells with a concomitant significant increase of Th2 cell subsets. The reduction of the different subsets of T lymphocytes was associated with an intense staining with Annexin V, suggesting an apoptotic-related cell reduction. A significant decrease of Th1, Th17 and Treg cytokines and a concomitant increase of IL-4 was also observed after TCZ treatment in PBMC isolated from RA patients. TCZ could modify the immune imbalance in RA inducing apoptosis of Th1, Th17 and Treg cells and promoting the appearance of a Th2 response.