[Show abstract][Hide abstract] ABSTRACT: The potential role of fungal organisms and their co-aggregation with either periodontopathogens or opportunistic pathogens at peri-implantitis sites is unknown. The aim of the present study was to qualitatively/quantitatively analyze and correlate fungal organisms and bacterial species at peri-implantitis sites.
In a total of 29 patients, submucosal/subgingival plaque samples were collected at peri-implantitis and healthy implant sites as well as teeth with a history of periodontitis (controls). A real-time PCR assay was established for the qualification of fungal organisms and a TaqMan assay for the quantification of Porphyromonas gingivalis, Parvimonas micra, Tannerella forsythia, Mycoplasma salivarium, Veillonella parvula, and Staphylococcus aureus.
Fungal organisms were more frequently identified at peri-implantitis (31.6%) (i.e., Candida albicans, Candida boidinii, Penicillium spp., Rhodotorula laryngis, Paelicomyces spp., Saccharomycetes, Cladosporium cladosporioides) and healthy implant sites (40% - Candida dubliniensis, C. cladosporioides) than at selected teeth (20% - C. albicans, Fusarium solani). At implant sites, fungal organisms were significantly correlated with P. micra and T. forsythia.
Candida spp. and other fungal organisms were frequently identified at peri-implantitis as well as healthy implant sites and co-colonized with P. micra and T. forsythia.
[Show abstract][Hide abstract] ABSTRACT: Background:
The viral regulatory protein Tat is essential for establishing a productive transcription from the 5′-LTR promoter during the early phase of viral gene expression. Formation of the Tat-encoding mRNAs requires splicing at the viral 3′ss A3, which has previously been shown to be both negatively and positively regulated by the downstream splicing regulatory elements (SREs) ESS2p and ESE2/ESS2. However, using the novel RESCUE-type computational HEXplorer algorithm, we were recently able to identify another splicing enhancer (ESE5807-5838, henceforth referred to as ESEtat) located between ESS2p and ESE2/ESS2. Here we show that ESEtat has a great impact on viral tat-mRNA splicing and that it is fundamental for regulated 3′ss A3 usage.
Mutational inactivation or locked nucleic acid (LNA)-directed masking of the ESEtat sequence in the context of a replication-competent virus was associated with a failure (i) to activate viral 3′ss A3 and (ii) to accumulate Tat-encoding mRNA species. Consequently, due to insufficient amounts of Tat protein efficient viral replication was drastically impaired. RNA in vitro binding assays revealed SRSF2 and SRSF6 as candidate splicing factors acting through ESEtat and ESE2 for 3′ss A3 activation. This notion was supported by coexpression experiments, in which wild-type, but not ESEtat-negative provirus responded to higher levels of SRSF2 and SRSF6 proteins with higher levels of tat-mRNA splicing. Remarkably, we could also find that SRSF6 overexpression established an antiviral state within provirus-transfected cells, efficiently blocking virus particle production. For the anti-HIV-1 activity the arginine-serine (RS)-rich domain of the splicing factor was dispensable.
Based on our results, we propose that splicing at 3′ss A3 is dependent on binding of the enhancing SR proteins SRSF2 and SRSF6 to the ESEtat and ESE2 sequence. Mutational inactivation or interference specifically with ESEtat activity by LNA-directed masking seem to account for an early stage defect in viral gene expression, probably by cutting off the supply line of Tat that HIV needs to efficiently transcribe its genome.
[Show abstract][Hide abstract] ABSTRACT: The AIM2 inflammasome detects double-stranded DNA in the cytosol and induces caspase-1-dependent pyroptosis as well as release of the inflammatory cytokines interleukin 1β (IL-1β) and IL-18. AIM2 is critical for host defense against DNA viruses and bacteria that replicate in the cytosol, such as Francisella tularensis subspecies novicida (F. novicida). The activation of AIM2 by F. novicida requires bacteriolysis, yet whether this process is accidental or is a host-driven immunological mechanism has remained unclear. By screening nearly 500 interferon-stimulated genes (ISGs) through the use of small interfering RNA (siRNA), we identified guanylate-binding proteins GBP2 and GBP5 as key activators of AIM2 during infection with F. novicida. We confirmed their prominent role in vitro and in a mouse model of tularemia. Mechanistically, these two GBPs targeted cytosolic F. novicida and promoted bacteriolysis. Thus, in addition to their role in host defense against vacuolar pathogens, GBPs also facilitate the presentation of ligands by directly attacking cytosolic bacteria.
[Show abstract][Hide abstract] ABSTRACT: Rationale:
Lymphotoxin β receptor (LTbR) regulates immune cell trafficking and communication in inflammatory diseases. However, the role of LTbR in atherosclerosis is still unclear.
The aim of this study was to elucidate the role of LTbR in atherosclerosis.
Methods and results:
After 15 weeks of feeding a Western-type diet, mice double-deficient in apolipoprotein E and LTbR (apoE(-/-)/LTbR(-/-)) exhibited lower aortic plaque burden than did apoE(-/-) littermates. Macrophage content at the aortic root and in the aorta was reduced, as determined by immunohistochemistry and flow cytometry. In line with a decrease in plaque inflammation, chemokine (C-C motif) ligand 5 (Ccl5) and other chemokines were transcriptionally downregulated in aortic tissue from apoE(-/-)/LTbR(-/-) mice. Moreover, bone marrow chimeras demonstrated that LTbR deficiency in hematopoietic cells mediated the atheroprotection. Furthermore, during atheroprogression, apoE(-/-) mice exhibited increased concentrations of cytokines, for example, Ccl5, whereas apoE(-/-)/LTbR(-/-) mice did not. Despite this decreased plaque macrophage content, flow cytometric analysis showed that the numbers of circulating lymphocyte antigen 6C (Ly6C)(low) monocytes were markedly elevated in apoE(-/-)/LTbR(-/-) mice. The influx of these cells into atherosclerotic lesions was significantly reduced, whereas apoptosis and macrophage proliferation in atherosclerotic lesions were unaffected. Gene array analysis pointed to chemokine (C-C motif) receptor 5 as the most regulated pathway in isolated CD115(+) cells in apoE(-/-)/LTbR(-/-) mice. Furthermore, stimulating monocytes from apoE(-/-) mice with agonistic anti-LTbR antibody or the natural ligand lymphotoxin-α1β2, increased Ccl5 mRNA expression.
These findings suggest that LTbR plays a role in macrophage-driven inflammation in atherosclerotic lesions, probably by augmenting the Ccl5-mediated recruitment of monocytes.
Circulation Research 03/2015; 116(8). DOI:10.1161/CIRCRESAHA.116.305723 · 11.02 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Rationale: Lymphotoxin beta receptor (LTbR) regulates immune cell trafficking and communication in inflammatory diseases. However, the role of LTbR in atherosclerosis is still unclear. Objective: Aim of the present study was to elucidate the role of LTbR in atherosclerosis. Methods and Results: After 15 weeks of feeding a Western-type diet, mice double-deficient in apolipoprotein E and LTbR (apoE(-/-)/LTbR(-/-)) exhibited lower aortic plaque burden than did apoE(-/-) littermates. Macrophage content at the aortic root and in the aorta was reduced, as determined by immunohistochemistry and flow cytometry. In line with a decrease in plaque inflammation, chemokine (C-C motif) ligand 5 (Ccl5) and other chemokines were transcriptionally down-regulated in aortic tissue from apoE(-/-)/LTbR(-/-) mice. Moreover, bone marrow chimeras demonstrated that LTbR deficiency in hematopoietic cells mediated the atheroprotection. Furthermore, whereas during atheroprogression apoE(-/-) mice exhibited increased concentration
[Show abstract][Hide abstract] ABSTRACT: Unlabelled:
Skin keratinocytes represent a primary entry site for herpes simplex virus 1 (HSV-1) in vivo. The cellular proteins nectin-1 and herpesvirus entry mediator (HVEM) act as efficient receptors for both serotypes of HSV and are sufficient for disease development mediated by HSV-2 in mice. How HSV-1 enters skin and whether both nectin-1 and HVEM are involved are not known. We addressed the impact of nectin-1 during entry of HSV-1 into murine epidermis and investigated the putative contribution of HVEM. Using ex vivo infection of murine epidermis, we showed that HSV-1 entered the basal keratinocytes of the epidermis very efficiently. In nectin-1-deficient epidermis, entry was strongly reduced. Almost no entry was observed, however, in nectin-1-deficient keratinocytes grown in culture. This observation correlated with the presence of HVEM on the keratinocyte surface in epidermis and with the lack of HVEM expression in nectin-1-deficient primary keratinocytes. Our results suggest that nectin-1 is the primary receptor in epidermis, while HVEM has a more limited role. For primary murine keratinocytes, on which nectin-1 acts as a single receptor, electron microscopy suggested that HSV-1 can enter both by direct fusion with the plasma membrane and via endocytic vesicles. Thus, we concluded that nectin-1 directs internalization into keratinocytes via alternative pathways. In summary, HSV-1 entry into epidermis was shown to strongly depend on the presence of nectin-1, but the restricted presence of HVEM can potentially replace nectin-1 as a receptor, illustrating the flexibility employed by HSV-1 to efficiently invade tissue in vivo.
Herpes simplex virus (HSV) can cause a range of diseases in humans, from uncomplicated mucocutaneous lesions to life-threatening infections. The skin is one target tissue of HSV, and the question of how the virus overcomes the protective skin barrier and penetrates into the tissue to reach its receptors is still open. Previous studies analyzing entry into cells grown in vitro revealed nectin-1 and HVEM as HSV receptors. To explore the contributions of nectin-1 and HVEM to entry into a natural target tissue, we established an ex vivo infection model. Using nectin-1- or HVEM-deficient mice, we demonstrated the distinct involvement of nectin-1 and HVEM for HSV-1 entry into epidermis and characterized the internalization pathways. Such advances in understanding the involvement of receptors in tissue are essential preconditions for unraveling HSV invasion of skin, which in turn will allow the development of antiviral reagents.
Journal of Virology 10/2014; 89(1). DOI:10.1128/JVI.02917-14 · 4.44 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Background:
Tumor necrosis factor/tumor necrosis factor receptor superfamily members conform a group of molecular interaction pathways of essential relevance during the process of T-cell activation and differentiation toward effector cells and particularly for the maintenance phase of the immune response. Specific blockade of these interacting pathways, such as CD40-CD40L, contributes to modulate the deleterious outcome of allogeneic immune responses. We postulated that antagonizing the interaction of LIGHT expression on activated T cells with its receptors, herpesvirus entry mediator and lymphotoxin β receptor, may decrease T cell-mediated allogeneic responses.
A flow cytometry competition assay was designed to identify anti-LIGHT monoclonal antibodies capable to prevent the interaction of mouse LIGHT with its receptors expressed on transfected cells. An antibody with the desired specificity was evaluated in a short-term in vivo allogeneic cytotoxic assay and tested for its ability to detect endogenous mouse LIGHT.
We provide evidence for the first time that in mice, as previously described in humans, LIGHT protein is rapidly and transiently expressed after T-cell activation, and this expression was stronger on CD8 T cells than on CD4 T cells. Two anti-LIGHT antibodies prevented interactions of mouse LIGHT with its two known receptors, herpesvirus entry mediator and lymphotoxin β receptor. In vivo administration of anti-LIGHT antibody (clone 10F12) ameliorated host antidonor short-term cytotoxic response in wild type B6 mice, although to a lesser extent than that observed in LIGHT-deficient mice.
The therapeutic targeting of LIGHT may contribute to achieve a better control of cytotoxic responses refractory to current immunosuppressive drugs in transplantation.
[Show abstract][Hide abstract] ABSTRACT: IFN receptor signaling induces cell-autonomous immunity to infections with intracellular bacterial pathogens. Here, we demonstrate that IFN-inducible guanylate binding protein (Gbp) proteins stimulate caspase-11-dependent, cell-autonomous immunity in response to cytoplasmic LPS. Caspase-11-dependent pyroptosis is triggered in IFN-activated macrophages infected with the Gram-negative bacterial pathogen Legionella pneumophila. The rapid induction of pyroptosis in IFN-activated macrophages required a cluster of IFN-inducible Gbp proteins encoded on mouse chromosome 3 (Gbp(chr3)). Induction of pyroptosis in naive macrophages by infections with the cytosol-invading ΔsdhA L. pneumophila mutant was similarly dependent on Gbp(chr3), suggesting that these Gbp proteins play a role in the detection of bacteria accessing the cytosol. Cytoplasmic LPS derived from Salmonella ssp. or Escherichia coli has recently been shown to trigger caspase-11 activation and pyroptosis, but the cytoplasmic sensor for LPS and components of the caspase-11 inflammasome are not yet defined. We found that the induction of caspase-11-dependent pyroptosis by cytoplasmic L. pneumophila-derived LPS required Gbp(chr3) proteins. Similarly, pyroptosis induced by cytoplasmic LPS isolated from Salmonella was diminished in Gbp(chr3)-deficient macrophages. These data suggest a role for Gbp(chr3) proteins in the detection of cytoplasmic LPS and the activation of the noncanonical inflammasome.
Proceedings of the National Academy of Sciences 04/2014; 111(16). DOI:10.1073/pnas.1321700111 · 9.67 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The Janus kinase / signal transducer and activator of transcription (Jak/STAT) pathway can be activated by many different cytokines, among them all members of the Interleukin (IL-)6 family. Dysregulation of this pathway, resulting in its constitutive activation, is associated with chronic inflammation and cancer development. In the present study, we show that activity of protein kinase II (CK2), a ubiquitously expressed serine/threonine kinase, is needed for induced activation of STAT1 and STAT3 by IL-6 classic and trans-signaling, IL-11, IL-27, oncostatin M (OSM), leukemia inhibitory factor (LIF) and cardiotrophin-1 (CT-1). Inhibition of CK2 efficiently prevented STAT phosphorylation and inhibited cytokine-dependent cell proliferation in a Jak1-dependent manner. Conversely, forced activation of CK2 alone was not sufficient to induce activation of the Jak/STAT signaling pathway. Inhibition of CK2 in turn inhibited Jak1-dependent STAT activation by oncogenic gp130 mutations. Furthermore, CK2 inhibition diminished the Jak1- and Src kinase-dependent phosphorylation of a constitutively active STAT3 mutant recently described in human large granular lymphocytic leukemia. In conclusion, we characterize CK2 as an essential component of the Jak/STAT pathway. Pharmacologic inhibition of this kinase is therefore a promising strategy to treat human inflammatory diseases and malignancies associated with constitutive activation of the Jak/STAT pathway.
[Show abstract][Hide abstract] ABSTRACT: CD8(+) T-cell functions are critical for preventing chronic viral infections by eliminating infected cells. For healthy immune responses, beneficial destruction of infected cells must be balanced against immunopathology resulting from collateral damage to tissues. These processes are regulated by factors controlling CD8(+) T-cell function, which are still incompletely understood. Here, we show that the interferon regulatory factor 4 (IRF4) and its cooperating binding partner B-cell-activating transcription factor (BATF) are necessary for sustained CD8(+) T-cell effector function. Although Irf4(-/-) CD8(+) T cells were initially capable of proliferation, IRF4 deficiency resulted in limited CD8(+) T-cell responses after infection with the lymphocytic choriomeningitis virus. Consequently, Irf4(-/-) mice established chronic infections, but were protected from fatal immunopathology. Absence of BATF also resulted in reduced CD8(+) T-cell function, limited immunopathology, and promotion of viral persistence. These data identify the transcription factors IRF4 and BATF as major regulators of antiviral cytotoxic T-cell immunity.Cell Death and Differentiation advance online publication, 14 February 2014; doi:10.1038/cdd.2014.19.
Cell death and differentiation 02/2014; 21(7). DOI:10.1038/cdd.2014.19 · 8.18 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The DC-derived chemokine CCL17, a ligand of CCR4, has been shown to promote various inflammatory diseases such as atopic dermatitis, atherosclerosis, and inflammatory bowel disease. Under steady state conditions, and even after systemic stimulation with LPS, CCL17 is not expressed in resident splenic DCs as opposed to CD8α(-) CD11b(+) LN DCs, which produce large amounts of CCL17 in particular after maturation. Upon systemic NKT cell activation through α-galactosylceramide stimulation however, CCL17 can be upregulated in both CD8α(-) and CD8α(+) splenic DC subsets and enhances cross-presentation of exogenous antigens. Based on genome-wide expression profiling, we now show that splenic CD11b(+) DCs are susceptible to IFN-γ-mediated suppression of CCL17, whereas LN CD11b(+) CCL17(+) DCs downregulate the IFN-γR and are much less responsive to IFN-γ. Under inflammatory conditions, particularly in the absence of IFN-γ signaling in IFN-γRKO mice, CCL17 expression is strongly induced in a major proportion of splenic DCs by the action of GM-CSF in concert with IL-4. Our findings demonstrate that the local cytokine milieu and differential cytokine responsiveness of DC subsets regulate lymphoid organ specific immune responses at the level of chemokine expression. This article is protected by copyright. All rights reserved.
European Journal of Immunology 02/2014; 44(2). DOI:10.1002/eji.201343820 · 4.03 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We have previously characterized mouse CMV (MCMV)-encoded immune-evasive IFN signaling inhibition and identified the viral protein pM27 as inducer of proteasomal degradation of STAT2. Extending our analysis to STAT1 and STAT3, we found that MCMV infection neither destabilizes STAT1 protein nor prevents STAT1 tyrosine Y701 phosphorylation, nuclear translocation, or the capability to bind γ-activated sequence DNA-enhancer elements. Unexpectedly, the analysis of STAT3 revealed an induction of STAT3 Y705 phosphorylation by MCMV. In parallel, we found decreasing STAT3 protein amounts upon MCMV infection, although STAT3 expression normally is positive autoregulative. STAT3 phosphorylation depended on the duration of MCMV infection, the infectious dose, and MCMV gene expression but was independent of IFNAR1, IL-10, IL-6, and JAK2. Although STAT3 phosphorylation did not require MCMV immediate early 1, pM27, and late gene expression, it was restricted to MCMV-infected cells and not transmitted to bystander cells. Despite intact STAT1 Y701 phosphorylation, IFN-γ-induced target gene transcription (e.g., IRF1 and suppressor of cytokine signaling [SOCS] 1) was strongly impaired. Likewise, the induction of STAT3 target genes (e.g., SOCS3) by IL-6 was also abolished, indicating that MCMV antagonizes STAT1 and STAT3 despite the occurrence of tyrosine phosphorylation. Consistent with the lack of SOCS1 induction, STAT1 phosphorylation was prolonged upon IFN-γ treatment. We conclude that the inhibition of canonical STAT1 and STAT3 target gene expression abrogates their intrinsic negative feedback loops, leading to accumulation of phospho-tyrosine-STAT3 and prolonged STAT1 phosphorylation. These findings challenge the generalization of tyrosine-phosphorylated STATs necessarily being transcriptional active and document antagonistic effects of MCMV on STAT1/3-dependent target gene expression.
The Journal of Immunology 12/2013; 192(1). DOI:10.4049/jimmunol.1203516 · 4.92 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Intestinal microbes provide multicellular hosts with nutrients and confer resistance to infection. The delicate balance between pro- and anti-inflammatory mechanisms, essential for gut immune homeostasis, is affected by the composition of the commensal microbial community. Regulatory T cells (Treg cells) expressing transcription factor Foxp3 have a key role in limiting inflammatory responses in the intestine. Although specific members of the commensal microbial community have been found to potentiate the generation of anti-inflammatory Treg or pro-inflammatory T helper 17 (TH17) cells, the molecular cues driving this process remain elusive. Considering the vital metabolic function afforded by commensal microorganisms, we reasoned that their metabolic by-products are sensed by cells of the immune system and affect the balance between pro- and anti-inflammatory cells. We tested this hypothesis by exploring the effect of microbial metabolites on the generation of anti-inflammatory Treg cells. We found that in mice a short-chain fatty acid (SCFA), butyrate, produced by commensal microorganisms during starch fermentation, facilitated extrathymic generation of Treg cells. A boost in Treg-cell numbers after provision of butyrate was due to potentiation of extrathymic differentiation of Treg cells, as the observed phenomenon was dependent on intronic enhancer CNS1 (conserved non-coding sequence 1), essential for extrathymic but dispensable for thymic Treg-cell differentiation. In addition to butyrate, de novo Treg-cell generation in the periphery was potentiated by propionate, another SCFA of microbial origin capable of histone deacetylase (HDAC) inhibition, but not acetate, which lacks this HDAC-inhibitory activity. Our results suggest that bacterial metabolites mediate communication between the commensal microbiota and the immune system, affecting the balance between pro- and anti-inflammatory mechanisms.
[Show abstract][Hide abstract] ABSTRACT: Toxoplasma gondii is an obligate intracellular protozoan parasite responsible for a common infection of the central nervous system. Interferon (IFN) γ is the key cytokine of host defence against T. gondii. However, T. gondii strains differ in virulence and T. gondii factors determining virulence are still poorly understood. In astrocytes IFN γ primarily induces immunity-related GTPases (IRGs), providing a cell-autonomous resistance system. Here, we demonstrate that astrocytes prestimulated with IFN γ inhibit the proliferation of various avirulent, but not virulent, T. gondii strains. The two analyzed immunity-related GTPases Irga6 and Irgb6 accumulate at the PV only of avirulent T. gondii strains, whereas in virulent strains this accumulation is only detectable at very low levels. Both IRG proteins could temporarily be found at the same PV, but did only partially colocalize. Coinfection of avirulent and virulent parasites confirmed that the accumulation of the two analyzed IRGs was a characteristic of the individual PV and not determined by the presence of other strains of T. gondii in the same host cell. Thus, in astrocytes the accumulation of Irga6 and Irgb6 significantly differs between avirulent and virulent T. gondii strains correlating with the toxoplasmacidal properties suggesting a role for this process in parasite virulence.
The Scientific World Journal 11/2013; 2013:480231. DOI:10.1155/2013/480231 · 1.73 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Janus kinases (Jak) play essential roles in cytokine and growth factor signaling. Conventional gene targeting of Jak2, creating a null allele, leads to a block in definitive erythropoiesis as a result of failing signal transduction at the homomeric Erythropoietin receptor (EpoR) and at the heteromeric Interferon γ receptor (IFNGR). To investigate the in vivo relevance of the activation loop of Jak2, a Jak2-YY1007/1008FF knock-in mutation was introduced into the germline of mice. The phenotype of the Jak2(FF/FF) mouse line reveals that tyrosine residues 1007/1008 are absolutely essential for kinase function and signal transduction at the homomeric EpoR. Detailed studies using the Jak2 activation loop mutant uncover an essential scaffolding function of Jak2 within the IFNGR receptor complex and reveal that Jak1 can mediate a semi-redundant function for IFNGR signal transduction. These studies are highly important for the molecular understanding of cytokine and growth factor signaling and provide new insights for future strategies in the design of pharmacological blockers of Jak2.