Cell Responses In Rheumatoid Arthritis Research Articles

Trichinella spiralis Paramyosin Alleviates Collagen-Induced Arthritis in Mice by Modulating CD4+ T Cell Differentiation.

Rheumatoid arthritis (RA) is an autoimmune disease that significantly impacts quality of life by disrupting CD4+ T cell immune homeostasis. The identification of a low-side-effect drug for RA treatment is urgently needed. Our previous study suggests that Trichinella spiralis paramyosin (Ts-Pmy) has immunomodulatory effects, but its potential effect on CD4+ T cell response in RA remains unclear. In this study, we used a murine model to investigate the role of rTs-Pmy in regulating CD4+ T cell differentiation in collagen-induced arthritis (CIA). Additionally, we assessed the impact of rTs-Pmy on CD4+ T cell differentiation towards the Th1 and Th17 phenotypes, which are associated with inflammatory responses in arthritis, using in vitro assays. The results demonstrated that rTs-Pmy administration reduced arthritis severity by inhibiting Th1 and Th17 response while enhancing Treg response. Prophylactic administration of Ts-Pmy showed superior efficacy on CIA compared to therapeutic administration. Furthermore, in vitro assays demonstrated that rTs-Pmy could inhibit the differentiation of CD4+ T cells into Th1 and Th17 while inducing the production of Tregs, suggesting a potential mechanism underlying its therapeutic effects. This study suggests that Ts-Pmy may ameliorate CIA by restoring the immune balance of CD4+ T cells and provides new insights into the mechanism through which helminth-derived proteins exert their effects on autoimmune diseases.

The role of posttranslational modifications in generating neo-epitopes that bind to rheumatoid arthritis-associated HLA-DR alleles and promote autoimmune T cell responses

While antibodies to citrullinated proteins have become a diagnostic hallmark in rheumatoid arthritis (RA), we still do not understand how the autoimmune T cell response is influenced by these citrullinated proteins. To investigate the role of citrullinated antigens in HLA-DR1- and DR4-restricted T cell responses, we utilized mouse models that express these MHC-II alleles to determine the relationship between citrullinated peptide affinity for these DR molecules and the ability of these peptides to induce a T cell response. Using a set of peptides from proteins thought to be targeted by the autoimmune T cell responses in RA, aggrecan, vimentin, fibrinogen, and type II collagen, we found that while citrullination can enhance the binding affinity for these DR alleles, it does not always do so, even when in the critical P4 position. Moreover, if peptide citrullination does enhance HLA-DR binding affinity, it does not necessarily predict the generation of a T cell response. Conversely, citrullinated peptides can stimulate T cells without changing the peptide binding affinity for HLA-DR1 or DR4. Furthermore, citrullination of an autoantigen, type II collagen, which enhances binding affinity to HLA-DR1 did not enhance the severity of autoimmune arthritis in HLA-DR1 transgenic mice. Additional analysis of clonal T cell populations stimulated by these peptides indicated cross recognition of citrullinated and wild type peptides can occur in some instances, while in others cases the citrullination generates a novel T cell epitope. Finally, cytokine profiles of the wild type and citrullinated peptide stimulated T cells unveiled a significant disconnect between proliferation and cytokine production. Altogether, these data demonstrate the lack of support for a simplified model with universal correlation between affinity for HLA-DR alleles, immunogenicity and arthritogenicity of citrullinated peptides. Additionally they highlight the complexity of both T cell receptor recognition of citrulline as well as its potential conformational effects on the peptide:HLA-DR complex as recognized by a self-reactive cell receptor.

T Cell-Dependent Affinity Maturation and Innate Immune Pathways Differentially Drive Autoreactive B Cell Responses in Rheumatoid Arthritis.

Rheumatoid arthritis (RA) is characterized by the activation of B cells that produce anti-citrullinated protein antibodies (ACPAs) and rheumatoid factors (RFs), but the mechanisms by which tolerance is broken in these B cells remain incompletely understood. We undertook this study to investigate whether ACPA+ and RF+ B cells break tolerance through distinct molecular mechanisms. We developed antigen-tetramers to isolate ACPA+ and RF+ B cells and performed single-cell RNA sequencing on 2,349 B cells from 6 RA patients and 1 healthy donor to analyze their immunoglobulin repertoires and transcriptional programs. Prominent immunoglobulins were expressed as monoclonal antibodies and tested for autoantigen reactivity. ACPA+ and RF+ B cells were enriched in the peripheral blood of RA patients relative to healthy controls. Characterization of patient-derived monoclonal antibodies confirmed ACPA and RF targeting of tetramer-specific B cells at both antigen-inexperienced and affinity-matured B cell stages. ACPA+ B cells used more class-switched isotypes and exhibited more somatic hypermutations relative to RF+ B cells, and these differences were accompanied by down-regulation of CD72 and up-regulation of genes that promote class-switching and T cell-dependent responses. In contrast, RF+ B cells expressed transcriptional programs that stimulate rapid memory reactivation through multiple innate immune pathways. Coexpression analysis revealed that ACPA+ and RF+ B cell-enriched genes belong to distinct transcriptional regulatory networks. Our findings suggest that ACPA+ and RF+ B cells are imprinted with distinct transcriptional programs, which suggests that these autoantibodies associated with increased inflammation in RA arise from 2 different molecular mechanisms.

Increased Binding of Specificity Protein 1 to the IL21R Promoter in B Cells Results in Enhanced B Cell Responses in Rheumatoid Arthritis.

B cells are implicated in rheumatoid arthritis (RA) based on the presence of autoantibodies and the therapeutic response to B cell depletion. IL-21 has a significant role in B cell development and function. Here we assess B cell responses to IL-21 and the mechanisms responsible for altered IL-21R expression in RA. Flow cytometry of PBMC and cultured B cells was used to quantify protein and mRNA levels of IL-21R, IL-21 signaling through pSTAT3, specificity protein 1 (SP1) and to determine cytokine production (IL-6) and maturation status of B cells in RA and healthy control subjects. SP1 binding to the IL21R promoter region in B cells was assessed with ChIP-qPCR. We demonstrate an increase in IL-21R expression in total and memory B cells from RA subjects, which correlated with responsiveness to IL-21 stimulation. Stimulation of naïve RA B cells with IL-21 and CD40L resulted in an increase in differentiation into plasmablasts and an increase in IL-6 production in comparison to healthy controls, which was dose dependent on IL-21 stimulation. IL-21R expression on memory B cells in RA synovial fluid was comparable to peripheral blood making our study pertinent to understanding B cell responses in the joint and site of inflammation. We identified an increase in SP1 protein and mRNA in RA B cells and demonstrate an increase in binding of SP1 to the IL21R promoter region, which suggests a mechanism by which IL-21R expression is enhanced on B cells in RA. Taken together, our results indicate a mechanism by which IL-21 enhances B cell development and function in RA through an SP1 mediated increase in IL-21R expression on B cells.

The Transcriptional Coactivator Bob1 Is Associated With Pathologic B Cell Responses in Autoimmune Tissue Inflammation.

The molecular mechanisms steering abnormal B cell responses in autoimmune diseases remain poorly understood. We undertook this study to identify molecular switches controlling pathologic B cell responses in rheumatoid arthritis (RA). Candidate molecules were identified by gene expression profiling of RA synovitis and validated by quantitative polymerase chain reaction and immunohistochemistry. B cell-specific expression was confirmed by immunofluorescence, immunoblotting, and flow cytometry. The role of Bob1 in pathologic B cell responses was assessed in collagen-induced arthritis (CIA). Transcriptional profiling of RA synovitis revealed a prominent B cell signature, with the transcriptional coactivator Bob1 and its putative target BCMA being among the most up-regulated genes. Further analysis confirmed the microarray data and demonstrated elevated levels of Bob1 in B cells in RA synovium. A functional study showed that Bob1-deficient mice failed to produce pathogenic anti-type II collagen (anti-CII) antibodies and were resistant to CIA. Adoptive transfer of cells from Bob1-deficient and Bob1-sufficient mice to recombination-activating gene 1-null mice demonstrated that Bob1 deficiency exclusively in B cells abrogated germinal center (GC) B cell formation, anti-CII antibody production, and CIA development. Consistent with data from animal studies, immunophenotyping of human B cell subsets revealed increased expression of Bob1, predominantly in centrocytes and centroblasts. Correspondingly, Bob1 expression in RA synovitis was strongly correlated with CD21L, a molecular marker of GCs. In addition, similar Bob1 overexpression and correlation with CD21L expression was evidenced in parotid salivary gland tissue from patients with primary Sjögren's syndrome. These expression and functional data identify the transcriptional coactivator Bob1 as a candidate molecular switch of pathogenic B cell responses in autoimmune diseases in humans.

Citrulline‐Specific Th1 Cells Are Increased in Rheumatoid Arthritis and Their Frequency Is Influenced by Disease Duration and Therapy

Rheumatoid arthritis (RA) is thought to be a T cell-mediated disease, based on its strong association with HLA class II alleles, clinical responsiveness to T cell-directed therapies, and the presence of CD4+ T cells in rheumatoid joints. The presence of anti-citrullinated protein antibodies (ACPAs) in RA serum and the association of these antibodies with HLA-DR4 alleles implicate citrulline-specific autoreactive T cells in the development and progression of RA. The goal of this study was to determine the characteristics and specificity of autoreactive T cell responses in RA. We developed a panel of HLA-DRB1*04:01 tetramers, selecting citrullinated peptides from synovial antigens and verifying their immunogenicity in DRB1*04:01-transgenic mice. Seven tetramers were used to examine the ex vivo frequency and surface phenotype of citrulline-specific (Cit-specific) T cells in patients with RA and healthy subjects with DRB1*04:01 haplotypes, using a magnetic enrichment procedure. Cit-specific T cells were detectable in peripheral blood samples from both healthy subjects and RA patients. In comparison to healthy subjects, RA patients had significantly higher frequencies of Cit-specific T cells, and a greater proportion of these cells displayed a Th1 memory phenotype. Among RA patients, the frequency of Cit-specific T cells was highest within the first 5 years after diagnosis of RA and was decreased in patients taking biologic agents, irrespective of disease duration. These findings link the presence of ACPAs in RA with Th1 cells specific for citrullinated epitopes and provide tools for disease-specific immunomonitoring of autoreactive T cells.

Toll-like receptors expressed by synovial fibroblasts perpetuate Th1 and th17 cell responses in rheumatoid arthritis.

Rheumatoid arthritis (RA) is a chronic inflammatory disease characterized by synovial fibroblast hyperplasia and bone and cartilage erosion. Synovial fibroblast- and T cell-mediated inflammation plays crucial roles in the pathogenesis of RA. However how this inflammation is initiated, propagated, and maintained remains controversial. Here, we systemically examined the contribution of toll-like receptors (TLRs) to the inflammatory mediator production as well as Th1 and Th17 cell hyperactivity in RA. Our results show that rheumatoid arthritis synovial fibroblasts (RASF) express a series of TLRs, including TLR2, TLR3, TLR4, and TLR9, with the predominant expression of TLR3. Moreover, the expression levels of these TLRs were higher than those in osteoarthritis synovial fibroblasts (OASF). Ligation of TLR3, as well as TLR2 and TLR4, resulted in vigorous production of inflammatory cytokines, matrix metalloproteinases (MMPs), and vascular endothelial growth factor (VEGF) in RASF, with activation of the NF-κB, MAPK, and IRF3 pathways. More important, activation of these TLRs expressed by RASF exacerbated inflammatory Th1 and Th17 cell expansion both in cell-cell contact-dependent and inflammatory cytokine-dependent manners, which induced more IFN-γ and IL-17 accumulation. Targeting TLRs may modulate the inflammation in RA and provide new therapeutic strategies for overcoming this persistent disease.

B cell resistance to Fas-mediated apoptosis contributes to their ineffective control by regulatory T cells in rheumatoid arthritis

ObjectiveTo investigate whether regulatory T cells (Treg) can control B cell function in rheumatoid arthritis (RA) and if not to explore the basis for this defect.MethodsSuppression of B cell responses...

Participation of T Lymphocytes in the Development and Perpetuation of Rheumatoid Arthritis

The emphasis on T lymphocytes (TL) as an engine or perpetuating factor in rheumatoid arthritis (RA) has changed over time. The TL constitute the most plastic specific or adaptive immune system cells, both in their ability to recognize different antigens as in their various forms of response. Thus TL are responsible for orchestrating the most powerful and specific defensive responses, but also overall autoimmune responses, them being antigen-specific (autoantigens). However, insufficient identification of autoantigens responsible for autoimmune T-cell responses, particularly in RA, has been a major constraint to understand these responses. While rheumatoid factor and more recently the anti-CCP antibodies (antiCCP) are well known B responses as well as easy to detect in patients, this does not happen with any T cell response in RA. Some animal models illustrate, however, that it is possible to induce Tdependent arthritis in the absence of antigen-specific T cells by simply expanding or increasing the activation in a poly-specific manner. This occurs, for example, in response to genetic manipulation of factors that amplify global intracellular activation of the TCR receptor (ZAP70) or cytokine receptors (gp130).1,2 Another source of skepticism about T cells dependence in RA was the development of T immunosuppressive drugs and strategies that have proven very effective in preventing transplant rejection, but had little impact on RA therapy. The advent of effective antiTNF, paradoxically a TL activator, moved the attention to innate immune response cells. Despite these questions, two facts strongly support the importance of TL participation in both susceptibility and the development of the disease. The first is related to advances of genetic studies, particularly whole genome studies. Many other associations, whose functional significance point unequivocally to a TL response, have joined the classic association of RA and the shared epitope by different alleles of HLA class II genes. While the shared epitope is the platform for interaction between HLAantigen for presentation to T-cell receptors (TCR), other genes are known to encode elements such as receptors, or intracellular soluble factors that regulate multiple functions specific to TL.

PAN–DR‐Binding Hsp60 self epitopes induce an interleukin‐10–mediated immune response in rheumatoid arthritis

Human Hsp60 is expressed in the joints of patients with rheumatoid arthritis (RA) and can elicit a regulatory T cell response in the peripheral blood and synovial fluid. However, Hsp60 can also trigger strong proinflammatory pathways. Thus, to understand the nature of these Hsp60-directed responses in RA, it is necessary to study such responses at the molecular, epitope-specific level. This study was undertaken to characterize the disease specificity and function of pan-DR-binding Hsp60-derived epitopes as possible modulators of autoimmune inflammation in RA. Lymphocyte proliferation assays (using (3)H-thymidine incorporation and carboxyfluorescein diacetate succinimidyl ester [CFSE] staining) and measurement of cytokine production (using multiplex immunoassay and intracellular staining) were performed after in vitro activation of peripheral blood mononuclear cells from patients with RA, compared with healthy controls. A disease (RA)-specific immune recognition, characterized by T cell proliferation as well as increased production of tumor necrosis factor alpha (TNFalpha), interleukin-1beta (IL-1beta), and IL-10, was found for 3 of the 8 selected peptides in patients with RA as compared with healthy controls (P < 0.05). Intracellular cytokine staining and CFSE labeling showed that CD4+ T cells were the subset primarily responsible for both the T cell proliferation and the cytokine production in RA. Interestingly, the human peptides had a remarkably different phenotype, with a 5-10-fold higher IL-10:TNFalpha ratio, compared with that of the microbial peptides. These results suggest a disease-specific immune-modulatory role of epitope-specific T cells in the inflammatory processes of RA. Therefore, these pan-DR-binding epitopes could be used as a tool to study the autoreactive T cell response in RA and might be suitable candidates for use in immunotherapy.

The study of inhibitory peptides on T cell activation in rheumatoid arthritis

To study the inhibitory role of altered HA308 - 317 peptides in T cell responses in patients with rheumatoid arthritis (RA). Peripheral blood mononuclear cells (PBMC) were obtained from 27 HLA-DRB1 positive RA patients. T cell responses to altered HA308-317 peptides were examined by using (3)H incorporation assay. The level of IL-2 and IFNgamma in the supernatants was identified by an enzyme-linked immunosorbent assay. The CD69 expression on T cell surface was studied by using flow cytometry. Compared to wild type CII263 - 272 or HA308 - 317, altered HA308 - 317 peptides failed to stimulate T cell proliferation (P < 0.05) and production of IL-2 as well as IFNgamma and resulted in lower expression of CD69 in RA patients (P < 0.05). SI values for T cell coincubated with altered HA308 - 317 peptides and CII263 - 272 or wild type HA308 - 317 were significantly lower than coincubated with CII263 - 272 and wild type HA308 - 317 alone (P < 0.05). Substitution of TCR-binding residue of HA308 - 317 yielded weak or non-T-cell stimulating peptides, which might be potentially effective in blocking abnormal T cell responses in RA.

TCRB Clonotypes Are Present in CD4 T Cell Populations Prepared Directly from Rheumatoid Synovium

The identification of clonal T cells at sites of inflammation is hampered by the large number of polyclonal T cells that nonspecifically accumulate. In this report, we combine the use of T cell sorting with spectratyping of the third complementarity determining region (CDR3) and direct sequence analysis to rapidly screen for and identify clonal expansions of T cells from synovial tissue specimens from patients with rheumatoid arthritis (RA). Initially, we used a polymerase chain reaction specific for the variable region gene of the T cell receptor β chain (TCRBV) to compare the TCRBV repertoire expressed by CD4+ T cells from the peripheral blood and synovium of five patients with long-standing RA. Each patient had several TCRBV genes that were amplified to a greater degree from synovium. Extensive sequence analysis ( n > 170) showed that each patient contained junctional sequences that occurred more than once, implying the presence of T cell clones within the starting CD4+ T cell population. To assess a more straightforward approach to identifying clones, six additional patients were recruited and CD4+, TCRBV2+ synovial T cells were positively selected and analyzed by CDR3 spectratyping. Bands deviating from a normal distribution were excised from the gel and sequenced directly. Clones were detected in half of the patients. These data are consistent with the possibility of an antigen-driven T cell response in RA that remains present in the setting of advanced disease.

The role of T-lymphocytes in rheumatoid arthritis.

The role of T lymphocytes in the pathology of rheumatoid arthritis (RA) is controversial. To some extent this has resulted from contradictory data, but even where specific points of fact are not in dispute, their interpretation often is. Nevertheless, the basic idea of a pathological T cell response in RA receives significant support from the work of several groups who have removed them by thoracic duct drainage, lymphapheresis or total lymphoid irradiation (TLI). Each of these procedures ameliorates RA; in the case of TLI which induces a profound and sustained decrease in CD4+ cells, the effect on synovitis was prolonged. These observations are in line with the general assumption that the strong association of RA with HLA-DR molecules containing a particular conserved region of amino acids implies an important role for T cells in RA, since the physiological role of HLA-DR molecules is to present antigens to T cells. In addition the association with Dw4 and related alleles is strongest for the most severe and persistent forms of disease, arguing that T cells may be important not just in initiation of RA but also in its perpetuation. However, the infiltrating T cells seem to be remarkably inactive and as a population to lack specificity for any particular antigen, leading some to conclude that their role is either passive or irrelevant, while others contend that it is precisely this inactivity which is responsible for the persistence of RA.

Antibodies in rheumatoid synovial fluids bind to a restricted series of protein antigens in rheumatoid synovial tissue.

By searching the synovial fluid of patients with rheumatoid arthritis (RA) for antibodies that react to protein antigens in synovial tissue, we sought to identify putative antigens present in RA synovial tissue that might drive the pathologic immune response believed to be responsible for the joint inflammation. Synovial tissue was homogenized in sodium dodecyl sulfate polyacrylamide gel buffer, electrophoresed, and analyzed by immunoblotting. Antibodies from synovial fluids of patients with RA bound to several proteins in rheumatoid synovial tissues, including a series of low (27.5-, 29-, and 30-kd), middle (43- and 53-kd), and high (140-, 164-, and 182-kd) molecular weight proteins. Most of these antigens were also detected in normal synovial tissue, and the high molecular weight proteins were also present in normal dermal, muscle, and liver tissues. The low and middle molecular weight proteins were detected in some, but not all, of the other normal tissues and in Jurkat cell lysates. Antibodies to the low and high series of proteins were present in all rheumatoid synovial fluids tested, but were generally absent from synovial fluids from patients with other arthritic diseases. These results show that antibodies in synovial fluids consistently react to several proteins in RA and normal synovial tissues. These antigens are possibly the same antigens provoking the T cell response in RA; therefore, understanding the mechanism of the immune response against these proteins will likely lead to important insight into the etiology of RA.