[Show abstract][Hide abstract] ABSTRACT: Myeloid-derived suppressor cells (MDSCs) are innate immune cells capable of suppressing T-cell responses. We previously reported the presence of MDSCs with a granulocytic phenotype in the synovial fluid (SF) of mice with proteoglycan (PG)-induced arthritis (PGIA), a T cell-dependent autoimmune model of rheumatoid arthritis (RA). However, the limited amount of SF-MDSCs precluded investigations into their therapeutic potential. The goals of this study were to develop an in vitro method for generating MDSCs similar to those found in SF and to reveal the therapeutic effect of such cells in PGIA.
PLoS ONE 11/2014; 9(11):e111815. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of innate immune cells with a granulocyte-like or monocyte-like phenotype and a unique ability to suppress T-cell responses. MDSCs have been shown to accumulate in cancer patients, but recent studies suggest that these cells are also present in humans and animals suffering from autoimmune diseases. We previously identified MDSCs in the synovial fluid (SF) of mice with experimental autoimmune arthritis. The goal of the present study was to identify MDSCs in the SF of patients with rheumatoid arthritis (RA).
[Show abstract][Hide abstract] ABSTRACT: The involvement of autoreactive T cells in the pathogenesis of rheumatoid arthritis (RA) as well as in autoimmune animal models of arthritis has been well established; however, unanswered questions, such as the role of joint-homing T cells, remain. Animal models of arthritis are superb experimental tools in demonstrating how T cells trigger joint inflammation, and thus can help to further our knowledge of disease mechanisms and potential therapies. In this Review, we discuss the similarities and differences in T-cell subsets and functions between RA and mouse arthritis models. For example, various T-cell subsets are involved in both human and mouse arthritis, but differences might exist in the cytokine regulation and plasticity of these cells. With regard to joint-homing T cells, an abundance of synovial T cells is present in humans compared with mice. On the other hand, local expansion of type 17 T-helper (TH17) cells is observed in some animal models, but not in RA. Finally, whereas T-cell depletion therapy essentially failed in RA, antibody targeting of T cells can work, at least preventatively, in most arthritis models. Clearly, additional human and animal studies are needed to fill the gap in our understanding of the specific contribution of T-cell subsets to arthritis in mice and men.
[Show abstract][Hide abstract] ABSTRACT: An increasing number of studies show that besides the inherited genetic architecture (that is, genomic DNA), various environmental factors significantly contribute to the etiology of rheumatoid arthritis. Epigenetic factors react to external stimuli and form bridges between the environment and the genetic information-harboring DNA. Epigenetic mechanisms are implicated in the final interpretation of the encoded genetic information by regulating gene expression, and alterations in their profile influence the activity of the immune system. Overall, epigenetic mechanisms further increase the well-known complexity of rheumatoid arthritis by providing additional subtle contributions to rheumatoid arthritis susceptibility. Although there are controversies regarding the involvement of epigenetic and genetic factors in rheumatoid arthritis etiology, it is becoming obvious that the two systems (genetic and epigenetic) interact with each other and are ultimately responsible for rheumatoid arthritis development. Here, epigenetic factors and mechanisms involved in rheumatoid arthritis are reviewed and new, potential therapeutic targets are discussed.
BMC Medicine 01/2014; 12(1):35. · 7.28 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Rheumatoid arthritis (RA) is a systemic autoimmune disease and its targeting of the joints indicates the presence of a candidate autoantigen(s) in synovial joints. Patients with RA show immune responses in their peripheral blood to proteoglycan (PG) aggrecan. One of the most relevant animal models of RA appears to be proteoglycan-induced arthritis (PGIA), and CD4(+) T cells seem to play a crucial role in the initiation of the disease. In this review, the role of various T cell epitopes of aggrecan in the induction of autoreactive T cell activation and arthritis is discussed. We pay special attention to two critically important arthritogenic epitopes, 5/4E8 and P135H, found in the G1 and G3 domains of PG aggrecan, respectively, in the induction of autoimmune arthritis. Finally, results obtained with the recently developed PG-specific TCR transgenic mice system showed that altered T cell apoptosis, the balance of activation, and apoptosis of autoreactive T cells are critical factors in the development of autoimmunity.
BioMed research international. 01/2014; 2014:942148.
[Show abstract][Hide abstract] ABSTRACT: OBJECTIVE: To identify epigenetic factors that are implicated in the pathogenesis of rheumatoid arthritis (RA), and to explore the therapeutic potential of the targeted inhibition of these factors.
METHODS: Polymerase chain reaction (PCR) arrays were used to investigate the expression profile of genes that encode key epigenetic regulator enzymes. Mononuclear cells from RA patients and mice were monitored for gene expression changes, in association with arthritis development in murine models of RA. Selected genes were further characterized by quantitative reverse transcription-PCR, Western blot, and flow cytometry methods. The targeted inhibition of the up-regulated enzymes was studied in arthritic mice.
RESULTS: A set of genes with arthritis-specific expression was identified by the PCR arrays. Aurora kinases A and B, both of which were highly expressed in arthritic mice and treatment-naive RA patients, were selected for detailed analysis. Elevated aurora kinase expression was accompanied by increased phosphorylation of histone H3, which promotes proliferation of T lymphocytes. Treatment with VX-680, a pan-aurora kinase inhibitor, promoted B cell apoptosis, provided significant protection against disease onset, and attenuated inflammatory reactions in arthritic mice.
CONCLUSION: Arthritis development is accompanied by changes in expression of a number of epigenome-modifying enzymes. Drug-induced down-regulation of the aurora kinases, among other targets, seems to be sufficient to treat experimental arthritis. Development of new therapeutics that target aurora kinases can potentially improve RA management.
[Show abstract][Hide abstract] ABSTRACT: The P70-84 peptide (also called 5/4E8 epitope) of the human cartilage proteoglycan (PG) aggrecan is the dominant/arthritogenic epitope in both humans and arthritis-prone BALB/c mice (PG-induced arthritis, PGIA). An elevated T cell reactivity was demonstrated to a citrullinated version of the P70-84 epitope in most of the patients with rheumatoid arthritis (RA). The goal of this study was to understand better how a T cell epitope, if citrullinated, may affect antigenicity/arthritogenicity in PGIA, a murine model of RA. T cell reactivity to differentially citrullinated versions of either the human PG aggrecan P70-84 peptide or the corresponding mouse sequence was assessed in peptide or aggrecan-immunized and arthritic BALB/c mice as well as in T cell receptor transgenic mice specific for peptide P70-84 sequence. Peripheral T cell responses were induced by priming BALB/c mice with either the human wild-type or its citrullinated versions. Unexpectedly, priming with the citrullinated self-peptide induced a higher T cell response compared to the wild-type sequence (p<0.001), and the citrullination of the human peptide abolished T cell reactivity in PGIA.Our data suggest that T cells reactive to the citrullinated P70-84 peptide escaped thymic selection and are presentin the peripheral T cell repertoire. Results of this study provide evidence that citrullination of an immunodominant T cell epitope may substantially alter, either increase or abolish, T cell recognition at the periphery in an experimental model of arthritis.
[Show abstract][Hide abstract] ABSTRACT: Pharmacogenetics and pharmacogenomics deal with possible associations of a single genetic polymorphism or those of multiple gene profiles with responses to drugs. In rheumatology, genes and gene signatures may be associated with altered efficacy and/or safety of anti-inflammatory drugs, disease-modifying antirheumatic drugs (DMARDs) and biologics. In brief, genes of cytochrome P450, other enzymes involved in drug metabolism, transporters and some cytokines have been associated with responses to and toxicity of non-steroidal anti-inflammatory drugs, corticosteroids and DMARDs. The efficacy of biologics may be related to alterations in cytokine, chemokine and FcγR genes. Numerous studies reported multiple genetic signatures in association with responses to biologics; however, data are inconclusive. More, focused studies carried out in larger patient cohorts, using pre-selected genes, may be needed in order to determine the future of pharmacogenetics and pharmacogenomics as tools for personalized medicine in rheumatology.
Immunologic Research 04/2013; · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The "Bermuda triangle" of genetics, environment and autoimmunity is involved in the pathogenesis of rheumatoid arthritis (RA). Various aspects of genetic contribution to the etiology, pathogenesis and outcome of RA are discussed in this review. The heritability of RA has been estimated to be about 60 %, while the contribution of HLA to heritability has been estimated to be 11-37 %. Apart from known shared epitope (SE) alleles, such as HLA-DRB1*01 and DRB1*04, other HLA alleles, such as HLA-DRB1*13 and DRB1*15 have been linked to RA susceptibility. A novel SE classification divides SE alleles into S1, S2, S3P and S3D groups, where primarily S2 and S3P groups have been associated with predisposition to seropositive RA. The most relevant non-HLA gene single nucleotide polymorphisms (SNPs) associated with RA include PTPN22, IL23R, TRAF1, CTLA4, IRF5, STAT4, CCR6, PADI4. Large genome-wide association studies (GWAS) have identified more than 30 loci involved in RA pathogenesis. HLA and some non-HLA genes may differentiate between anti-citrullinated protein antibody (ACPA) seropositive and seronegative RA. Genetic susceptibility has also been associated with environmental factors, primarily smoking. Some GWAS studies carried out in rodent models of arthritis have confirmed the role of human genes. For example, in the collagen-induced (CIA) and proteoglycan-induced arthritis (PgIA) models, two important loci - Pgia26/Cia5 and Pgia2/Cia2/Cia3, corresponding the human PTPN22/CD2 and TRAF1/C5 loci, respectively - have been identified. Finally, pharmacogenomics identified SNPs or multiple genetic signatures that may be associated with responses to traditional disease-modifying drugs and biologics.
[Show abstract][Hide abstract] ABSTRACT: The covalent transfer of heavy chains (HCs) from inter-α-inhibitor (IαI) to hyaluronan (HA) via the protein product of tumor-necrosis-factor-stimulated-gene-6 (TSG-6) forms the HC-HA complex, a pathological form of HA that promotes the adhesion of leukocytes to HA matrices. The transfer of HCs to high molecular weight (HMW) HA is a reversible event whereby TSG-6 can shuffle HCs from one HA molecule to another. Therefore, HMW HA can serve as both a HC acceptor and donor. In the present study, we show that transfer of HCs to low molecular weight (LMW) HA oligosaccharides is an irreversible event where subsequent shuffling does not occur, i.e. HA oligosaccharides from 8 to 21 monosaccharide units in length, can serve as HC acceptors, but are unable to function as HC donors. We show that the HC-HA complex is present in the synovial fluid of mice subjected to systemic and mono-articular mouse models of rheumatoid arthritis (RA). Furthermore, we demonstrate that HA oligosaccharides can be used, with TSG-6, to irreversibly shuffle HCs from pathological, HMW HC-HA to HA oligosaccharides, thereby restoring HC-HA matrices from the inflamed joint to their normal state, unmodified with HCs. This process was also effective for HC-HA in the synovial fluid of human RA patients (in vitro).
Journal of Biological Chemistry 11/2012; · 4.60 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Tendinopathy is a widespread and disabling condition characterized by collagen fiber disruption and accumulation of a glycosaminoglycan-rich chondroid matrix. Recent clinical reports have illustrated the potential of mechanical loading (exercise) therapies to successfully treat chronic tendinopathies. We have developed a new murine tendinopathy model which requires a single injection of TGF-β1 into the Achilles tendon midsubstance followed by normal cage activity for 2 weeks. At this time, tendon maximum stress showed a dramatic (66%) reduction relative to that of normal controls and this persisted at four weeks. Loss of material properties was accompanied by abundant chondroid cells within the tendon (closely resembling the changes observed in human samples obtained intra-operatively) and increased expression of Acan, Col1a1, Col2a1, Col3a1, Fn1 and Mmp3. Mice subjected to two weeks of daily treadmill exercise following TGF-β1 injection showed a similar reduction in tendon material properties as the caged group. However, in mice subjected to 4 weeks of treadmill exercise, tendon maximum stress values were similar to those of naive controls. Tendons from the mice exercised for 4 weeks showed essentially no chondroid cells and the expression of Acan, Col1a1, Col2a1, Col3a1, and Mmp3 was significantly reduced relative to the 4-week cage group. This technically simple murine tendinopathy model is highly amenable to detailed mechanistic and translational studies of the biomechanical and cell biological pathways, that could be targeted to enhance healing of tendinopathy.
Journal of biomechanics 11/2012; · 2.66 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Hyaluronan (HA) deposition is often correlated with mucosal inflammatory responses where HA mediates both protective as well as pathological responses. By modifying the HA matrix, tumor necrosis factor-alpha-induced protein-6 (Tnfip6; also known as tumor necrosis factor-stimulated gene-6 (TSG-6)), is thought to potentiate anti-inflammatory and anti-plasmin effects that are inhibitory to leukocyte extravasation. Our study examines the role of endogenous TSG-6 in the pathophysiological responses associated with acute allergic pulmonary inflammation. When compared to wild-type littermate controls, TSG-6-/- mice exhibited attenuated inflammation marked by a significant decrease of pulmonary HA concentrations measured in the bronchoalveolar lavage (BAL) and lung tissue. Interestingly, despite the equivalent induction of both humoral and cellular T helper type 2 (Th2) immunity, and the comparable levels of cytokines and chemokines typically associated with eosinophilic pulmonary inflammation, airway eosinophilia was significantly decreased in TSG-6-/- mice. Most importantly, contrary to their counterpart wild type littermates, TSG-6-/- mice were resistant to the induction of airway hyperresponsiveness (AHR) and manifested improved lung mechanics in response to methacholine challenge. Our study demonstrates that endogenous TSG-6 is dispensable for the induction of Th2 immunity, but is essential for the robust increase of pulmonary HA deposition, propagation of acute eosinophilic pulmonary inflammation, and the development of AHR. Thus, TSG-6 is implicated in the experimental murine model of allergic pulmonary inflammation and is likely to contribute to the pathogenesis of asthma.
Journal of Biological Chemistry 11/2012; · 4.60 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Recent imaging studies on intact lymph nodes (LNs) of naïve T cell receptor (TCR)-transgenic mice have reported that T cells reduce their motility upon contact with relevant antigen-presenting cells (APCs). Using in vivo two-photon imaging of T cells in joint-draining (JD) LNs, we examined whether similar changes in T cell motility are observed in wild type mice. Co-transfer of T cells from naïve mice and antigen-experienced T cells from mice with proteoglycan (PG)-induced arthritis into naïve or arthritic recipients resulted in prolonged interactions of antigen-experienced T cells with APCs upon intra-articular antigen (PG) injection, indicating that T cells from arthritic wild type mice recapitulate the motile behavior reported in naïve TCR-transgenic mice. However, naïve T cells also engaged in stable interactions with APCs in the JDLNs of arthritic recipients, suggesting an enhanced ability of APCs in the JDLNs of arthritic hosts to present antigen to either naïve or antigen-experienced T cells.
[Show abstract][Hide abstract] ABSTRACT: T cells orchestrate joint inflammation in rheumatoid arthritis (RA), but B cells/B cell-derived factors are also involved in disease pathogenesis. The goal of this study was to understand the role of antigen-specific T and B cells in the pathological events of arthritis, which is impossible to study in humans due to the small number of antigen-specific cells. To determine the significance of antigen-specific lymphocytes and antibodies in the development of an autoimmune mouse model of RA, we generated TCR transgenic (TCR-Tg) mice specific for the dominant arthritogenic epitope of cartilage proteoglycan (PG) and performed a series of combined transfers of T cells, B cells and autoantibodies into BALB/c.Scid mice. The adoptive transfer of highly purified T cells from naive TCR-Tg, arthritic TCR-Tg or arthritic wild-type mice induced arthritis in SCID recipients, but the onset and severity of the disease were dependent on the sequential events of the T cell-supported reconstitution of PG-specific B cells and autoantibodies. The presence of activated PG-specific T cells was critical for disease induction, establishing a unique milieu for the selective homeostasis of autoantibody-producing B cells. In this permissive environment, anti-PG autoantibodies bound to cartilage and induced activation of the complement cascade, leading to irreversible cartilage destruction in affected joints. These findings may lead to a better understanding of the complex molecular and cellular mechanisms of RA.
International Immunology 04/2012; 24(8):507-17. · 3.18 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Synovial inflammation, a feature of both osteoarthritis (OA) and meniscal injury, is hypothesized to be triggered in part via stimulation of Toll-like receptors (TLRs). We undertook this study to test whether a TLR-2- or TLR-4-stimulating factor in synovial fluid (SF) from patients with early knee OA with meniscal injury could lead to inflammatory activation of synoviocytes.
SF was obtained from patients with early OA cartilage damage undergoing arthroscopic meniscal procedures. SF was used to stimulate primary cultures of fibroblast-like synoviocytes (FLS) and cell lines transfected with TLR-2 or TLR-4. SF was used either alone or in combination with a TLR-2 stimulus (palmitoyl-3-cysteine-serine-lysine-4 [Pam3CSK4]) or a TLR-4 stimulus (lipopolysaccharide [LPS]). In blocking experiments, SF was preincubated with anti-CD14 antibody.
SF from these patients did not stimulate interleukin-8 (IL-8) release from TLR transfectants. Compared with SF on its own, SF (at concentrations of 0.09-25%) in combination with TLR-2 or TLR-4 ligands resulted in significant augmentation of IL-8 release from both transfectants and primary FLS. Soluble CD14 (sCD14), a coreceptor for TLRs, was measured in SF from patients with early OA at levels comparable to those in patients with advanced OA and patients with rheumatoid arthritis. Blockade with anti-CD14 antibody abolished the ability of SF to augment IL-8 production in response to LPS, and diminished Pam3CSK4 responses.
SF augments FLS responses to TLR-2 and TLR-4 ligands. This effect was largely due to sCD14. Our results demonstrate that sCD14 in the setting of OA and meniscal injury sensitizes FLS to respond to inflammatory stimuli such as TLR ligands.
[Show abstract][Hide abstract] ABSTRACT: To determine whether myeloid cells (such as granulocytes) present in the synovial fluid (SF) of arthritic joints have an impact on adaptive immunity. Specifically, we investigated the effects of SF cells harvested from the joints of mice with proteoglycan-induced arthritis (PGIA), on dendritic cell (DC) maturation and antigen-specific T cell proliferation.
We monitored DC maturation (MHCII and CD86 expression) by flow cytometry upon coculture of DCs with SF cells or spleen myeloid cells from mice with PGIA. The effects of these myeloid cells on T cell proliferation were studied using T cells purified from PG-specific T cell receptor (TCR)-transgenic (Tg) mice. Phenotype analysis of myeloid cells was performed by immunostaining, reverse transcription-polymerase chain reaction, Western blotting, and biochemical assays.
Inflammatory SF cells significantly suppressed the maturation of DCs upon coculture. PG-TCR-Tg mouse T cells cultured with antigen-loaded DCs showed dramatic decreases in proliferation in the presence of SF cells. Spleen myeloid cells from arthritic mice did not have suppressive effects. SF cells were unable to suppress CD3/CD28-stimulated proliferation of the same T cells, suggesting a DC-dependent mechanism. SF cells exhibited all of the characteristics of myeloid-derived suppressor cells (MDSCs) and exerted suppression primarily through the production of nitric oxide and reactive oxygen species by granulocyte-like cells.
SF in the joints of mice with PGIA contains a population of granulocytic MDSCs that potently suppress DC maturation and T cell proliferation. These MDSCs have the potential to limit the expansion of autoreactive T cells, thus breaking the vicious cycle of autoimmunity and inflammation.
[Show abstract][Hide abstract] ABSTRACT: Existing literature demonstrates that fibroblast growth factor-2 (FGF-2) exerts opposing, contradictory biological effects on cartilage homeostasis in different species. In human articular cartilage, FGF-2 plays a catabolic and anti-anabolic role in cartilage homeostasis, driving homeostasis toward degeneration and osteoarthritis (OA). In murine joints, however, FGF-2 has been identified as an anabolic mediator as ablation of the FGF-2 gene demonstrated increased susceptibility to OA. There have been no previous studies specifically addressing species-specific differences in FGF-2-mediated biological effects. In this study, we provide a mechanistic understanding by which FGF-2 exerts contradictory biological effects in human versus murine tissues. Using human articular cartilage (ex vivo) and a medial meniscal destabilization (DMM) animal model (in vivo), species-specific expression patterns of FGFR receptors (FGFRs) are elucidated between human and murine articular cartilage. In the murine OA model followed by intra-articular injection of FGF-2, we further correlate FGFR profiles to changes in behavioral pain perception, proteoglycan content in articular cartilage, and production of inflammatory (CD11b) and angiogenic (VEGF) mediators in synovium lining cells. Our results suggest that the fundamental differences in cellular responses between human and murine tissues may be secondary to distinctive expression patterns of FGFRs that eventually determine biological outcomes in the presence of FGF-2. The complex interplay of FGFRs and the downstream signaling cascades induced by FGF-2 in human cartilage should add caution to the use of this particular growth factor for biological therapy in the future.
Journal of Cellular Biochemistry 03/2012; 113(7):2532-42. · 3.37 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Proteoglycan (PG)-induced arthritis (PGIA) is a murine model of rheumatoid arthritis. Arthritis-prone BALB/c mice are 100% susceptible, whereas the major histocompatibility complex-matched DBA/2 strain is completely resistant to PGIA. To reduce the size of the disease-suppressive loci for sequencing and to find causative genes of arthritis, we created a set of BALB/c.DBA/2-congenic/subcongenic strains carrying DBA/2 genomic intervals overlapping the entire Pgia26 locus on chromosome 3 (chr3) and Pgia23/Pgia12 loci on chr19 in the arthritis-susceptible BALB/c background. Upon immunization of these subcongenic strains and their wild-type (BALB/c) littermates, we identified a major Pgia26a sublocus on chr3 that suppressed disease onset, incidence and severity via controlling the complex trait of T-cell responses. The region was reduced to 3 Mbp (11.8 Mbp with flanking regions) in size and contained gene(s) influencing the production of a number of proinflammatory cytokines. Additionally, two independent loci (Pgia26b and Pgia26c) suppressed the clinical scores of arthritis. The Pgia23 locus (∼3 Mbp in size) on chr19 reduced arthritis susceptibility and onset, and the Pgia12 locus (6 Mbp) associated with low arthritis severity. Thus, we have reached the critical sizes of arthritis-associated genomic loci on mouse chr3 and chr19, which are ready for high-throughput sequencing of genomic DNA.
Genes and immunity 03/2012; 13(4):336-45. · 4.22 Impact Factor