Proteoglycan-induced Arthritis Research Articles

Tissue specific CD4+ T cell priming determines the requirement for interleukin-23 in experimental arthritis.

IntroductionRheumatoid arthritis (RA) is a chronic inflammatory disease with striking heterogeneity in (i) clinical presentation, (ii) autoantibody profiles and (iii) responses to treatment suggesting that distinct molecular mechanisms may underlie the disease process. Proteoglycan-induced arthritis (PGIA) is induced by two pathways either by intraperitoneal (i.p.) or subcutaneous (s.c.) exposure to PG. CD4+ T cells primed by the i.p. route are T helper (Th)1 cells expressing interferon gamma (IFN-γ) whereas CD4+ T cells primed by the s.c. route are Th17 cells expressing interleukin (IL)-17. IL-23 is necessary for maintaining the phenotype of Th17 cells; however, IL-23 is inflammatory independent of IL-17. The aim of this study was to determine if PGIA induced by different routes of immunization is dependent on IL-23.MethodsBALB/c wild type (WT), IL-12p40−/− and IL-23p19−/− littermate mice were immunized with recombinant G1 (rG1) domain of human PG in adjuvant either i.p. or s.c. and development of arthritis monitored. Joint histology was assessed. CD4+ T cell cytokines in spleen, lymph node (LN), and joint were assessed by intracellular staining and cytokine enzyme-linked immunosorbent assay. RNA transcripts for cytokines and transcription factors were examined.ResultsPGIA was suppressed in the p40−/− and p19−/− mice immunized by the s.c. route but only inhibited in p40−/− mice by the i.p. route. The joints of s.c. but not i.p. sensitized mice contained a population of CD4+ T cells expressing single positive IFN-γ and IL-17 and double positive IFN-γ/IL-17 which were dependent on IL-23 expression. The IFN-γ and IL-17 response in spleen and inguinal LN was inhibited in p19−/− mice and p40−/− mice after s.c. immunization, whereas in i.p. immunized p19−/− mice, IL-17 but not IFN-γ was reduced. Inguinal LN CD11c+ dendritic cells (DC) from s.c. immunized, but not spleen DC from i.p. immunized mice, produced IL-23, IL-1β, and IL-6 and activated T cells to produce IL-17.ConclusionIL-23 is necessary for the activity of Th17 after s.c. immunization and does not play a role independent of IL-17 after i.p. immunization. These data demonstrate that the molecular pathways IL-23/17 and IL-12/IFN-γ may represent subtypes of arthritis determined by the mode of induction.Electronic supplementary materialThe online version of this article (doi:10.1186/s13075-014-0440-1) contains supplementary material, which is available to authorized users.

Assessment of B cell depletion in combination with IL-2/anti-IL-2 mAb complexes in experimental arthritis (THER5P.824)

Abstract Autoimmunity is often associated with a deficiency in functional CD4+CD25+Foxp3+ T regulatory cells (Tregs). In our mouse model of rheumatoid arthritis (RA), proteoglycan induced arthritis (PGIA); we have shown that B cell depletion using a monoclonal antibody against CD20 suppresses disease along with an increase in functional Tregs. In an effort to enhance these Tregs we combined anti-mCD20 with administration of IL-2/anti-IL-2 mAb complexes and investigated the effects of the combination on PGIA. We show in G1 primed mice that IL-2/anti-IL-2 mAb complexes increase the percentage and numbers of Tregs higher than anti-mCD20 or control ab. To determine the effects of the complex on arthritis, we induced PGIA in BALB/c mice. Mice treated with the complex displayed an accelerated severe disease as compared to B cell depleted or control ab treated mice. This was due to an early increase in antigen specific CD4+T cells. Mice that received anti-mCD20 together with IL-2/anti-IL-2 mAb however displayed a less severe disease as compared to the group that only received the complex. Anti-mCD20 treated mice displayed the least arthritic severity. To determine the suppressive capacity of Tregs from the different treatment groups we setup Treg suppression assays. Tregs isolated from B cell depleted mice displayed the highest suppressive capacity. Our findings demonstrate that B cell depletion generates better Tregs and ameliorates PGIA better than IL-2/anti-IL-2 mAb complexes.

Brief Report: Autologous Stem Cell Transplantation Restores Immune Tolerance in Experimental Arthritis by Renewal and Modulation of the Teff Cell Compartment

Autologous stem cell transplantation (ASCT) induces long-term drug-free disease remission in patients with juvenile idiopathic arthritis. This study was undertaken to further unravel the immunologic mechanisms underlying ASCT by using a mouse model of proteoglycan-induced arthritis (PGIA). For initiation of PGIA, BALB/c mice received 2 intraperitoneal injections of human PG in a synthetic adjuvant on days 0 and 21. Five weeks after the first immunization, the mice were exposed to total body irradiation (7.5 Gy) and received (un)manipulated bone marrow (BM) grafts from mice with PGIA. Clinical scores, T cell reconstitution, (antigen-specific) T cell cytokine production, and intracellular cytokine expression were determined following autologous BM transplantation (ABMT). ABMT resulted in amelioration and stabilization of arthritis scores. BM grafts containing T cells and T cell-depleted grafts provided the same clinical benefit, with similar reductions in PG-induced T cell proliferation and the number of PG-specific autoantibodies. In vivo reexposure to PG did not exacerbate disease. Following ABMT, basal levels of disease-associated proinflammatory cytokines (interferon-γ [IFNγ], interleukin-17 [IL-17], and tumor necrosis factor α [TNFα]) were reduced. In addition, restimulation of T cells with PG induced a strong reduction in disease-associated proinflammatory cytokine production. Finally, although the remaining host T cells displayed a proinflammatory phenotype following ABMT, IFNγ, IL-17, and TNFα production by the newly reconstituted donor-derived T cells was significantly lower. Taken together, our data suggest that ABMT restores immune tolerance by renewal and modulation of the Teff cell compartment, leading to a strong reduction in proinflammatory (self antigen-specific) T cell cytokine production.

Mesenchymal stem cell therapy in proteoglycan induced arthritis

ObjectivesTo explore the immunosuppressive effect and mechanism of action of intraperitoneal (ip) and intra-articular (ia) mesenchymal stem cell (MSC) injection in proteoglycan induced arthritis (PGIA).MethodsMSC were administered ip or ia...

Commercial bovine proteoglycan is highly arthritogenic and can be used as an alternative antigen source for PGIA model.

Rheumatoid arthritis (RA) is the most common systemic autoimmune disease. It affects mainly the joints, causing synovitis, cartilage destruction, and bone erosion. Many experimental models are used to study the mechanisms involved in immunopathogenesis and new therapies for this disease. Proteoglycan-induced arthritis (PGIA) is a widely used model based on the cross-reactivity of injected foreign (usually human) PG and mice self-PG. Considering the complexity of the extraction and purification of human PG, in this study we evaluated the arthritogenicity of bovine PG that is commercially available. Bovine PG was highly arthritogenic, triggering 100% incidence of arthritis in female BALB/c retired breeder mice. Animals immunized with bovine PG presented clinical symptoms and histopathological features similar to human RA and other experimental models. Moreover, bovine PG immunization determined higher levels of proinflammatory and anti-inflammatory cytokines in arthritic mice compared to healthy ones. As expected, only the arthritic group produced IgG1 and IgG2a antibodies against PG. Thus, commercial bovine PG can be used as an alternative antigenic source to PGIA for the study of many RA aspects, including the immunopathogenesis of the disease and also the development of new therapies.

Proteoglycan Aggrecan Conducting T Cell Activation and Apoptosis in a Murine Model of Rheumatoid Arthritis

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.

B Cell–Specific Expression of Inducible Costimulator Ligand Is Necessary for the Induction of Arthritis in Mice

Inducible costimulator (ICOS)-ICOSL interactions are necessary for activation of Teff cells and follicular helper T (Tfh) cells. ICOSL is expressed on B cells, macrophages, and dendritic cells and can be induced on nonhematopoietic cells. The aim of this study was to determine whether expression of ICOSL on B cells is necessary for the development of proteoglycan (PG)-induced arthritis (PGIA). PGIA was initiated by immunizing wild-type and ICOSL-deficient (ICOSL(-/-) ) or B cell-specific ICOSL(-/-) chimeric BALB/c mice with human PG in adjuvant. The onset and severity of arthritis were monitored over time. CD4+ T cell proliferation and CD4+ T cell cytokine production were measured in vitro after the cells were restimulated with PG. Germinal center (GC) B cells, plasma cells, Tfh cells, and Treg cells were identified by staining with specific antibodies. Arthritis progression was completely inhibited in both ICOSL(-/-) mice and B cell-specific ICOSL(-/-) chimeric mice. Production of the Teff cell-produced cytokines interferon-γ and interleukin-17 (IL-17) and the antiinflammatory cytokine IL-4 was suppressed. The reduced percentages of GCs and Tfh cells and the decreased production of IL-21 correlated with a decrease in the anti-mouse PG antibody response. However, the percentage of plasma cells was not reduced despite a reduction in IgG responses. These data indicate that the signals provided by ICOSL-expressing B cells to Teff cells and Tfh cells are necessary for the development of arthritis. Thus, therapeutic blockade of ICOSL-ICOS interactions may be an effective strategy for the treatment of rheumatoid arthritis.

T helper cells plasticity in inflammation

CD4+ T cells can be subdivided from a functional point of view into two main subsets: effector cells, which provide protection against exogenous offending agents, and regulatory T (Treg) cells whose function is to avoid autoimmune reactions and to stop the effector response against exogenous antigens, when the response itself becomes dangerous for the host. Human effector CD4+ T lymphocytes can be additionally classified into lineages based mainly on their immunological functions that are supported by distinct profile of cytokine, transcription factor, and homing receptors expression. In the last years, beyond the well known populations of human T helper (Th) lymphocytes, Th1 and Th2 cells, other populations have been discovered and phenotypically characterized. These include the Th17 subset, which is certainly the most intensively studied, but also Th22, Th9, and T follicular helper (Tfh) lymphocytes. In addition to their protective functions, these T helper populations are also involved in the pathogenesis of several inflammatory immune-mediated disorders. Th1 and Th17 cells are involved in the pathogenesis of organ-specific autoimmune diseases and other chronic inflammatory disorders, whereas allergen-specific Th2 lymphocytes play a crucial role in allergy. Although classically viewed as distinct lineages, recent evidence indicate that CD4+ T cells, particularly the Th17 subset, are more plastic than previously thought. It is not fully understood how often such plasticity occurs in the course of physiologic responses to pathogens and what its importance is in protective immunity, but in inflammatory conditions Th17 lymphocytes that have shifted towards a Th1 or Th2 phenotype, acquiring the ability to produce IFN-γ or IL-4, and seem to be particularly aggressive and more pathogenic than the unshifted cells. In this context, the possibility to interfere with this modulation of phenotype can be considered a possible target for developing novel therapeutic strategies in the above mentioned diseases.

Location of CD4+ T Cell Priming Regulates the Differentiation of Th1 and Th17 Cells and Their Contribution to Arthritis

Th cytokines IFN-γ and IL-17 are linked to the development of autoimmune disease. In models of rheumatoid arthritis, that is, proteoglycan (PG)-induced arthritis, IFN-γ is required, whereas in collagen-induced arthritis, IL-17 is necessary for development of arthritis. In this study we show that the route of immunization determines the requirement for either IFN-γ or IL-17 in arthritis. Intraperitoneal immunization with PG induces a CD4(+) T cell IFN-γ response with little IL-17 in the spleen and peripheral lymph nodes. However, s.c. immunization induces both an IFN-γ and an IL-17 CD4(+) T cell response in spleen and lymph nodes. The failure to induce a CD4(+) T cell IL-17 response after i.p. immunization is associated with T cell priming, as naive T cells activated in vitro were fully capable of producing IL-17. Moreover, PG-induced arthritis is converted from an IFN-γ to an IL-17-mediated disease by altering the route of immunization from i.p. to s.c. The histological appearance of joint inflammation (cellular inflammation and bone erosion) is similar in the i.p. versus s.c. immunized mice despite the presence of CD4(+) T cells producing IL-17 in joint tissues only after s.c. immunization. These data indicate a critical role for the site of initial T cell priming and the Th cytokines required for susceptibility to arthritis. Our findings suggest that T cell activation at different anatomical sites in rheumatoid arthritis patients may skew the T cells toward production of either IFN-γ or IL-17.

LEAPS peptide vaccination alters T cell phenotype and suppresses joint inflammation in a murine model of rheumatoid arthitis (P5223)

Abstract Introduction. A major therapeutic goal in autoimmune inflammatory diseases such as rheumatoid arthritis (RA) is to suppress responses against (auto)antigens present in the target organs. Antigen-specific therapies such as the Ligand Epitope Antigen Presentation System (LEAPS) technology offer such a possibility. Materials and Methods. We evaluated LEAPS therapy in the cartilage proteoglycan (PG) induced arthritis (PGIA) model of RA. After the onset of PGIA, mice were divided in 3 groups, each with a similar mean arthritis index (AI). One group was vaccinated with adjuvant only, and the other groups with one of two LEAPS conjugates of a PG epitope 70 (J-PG70 or derG-PG70) in adjuvant. The same vaccination was repeated two weeks later. AI was measured by visual scoring every other day for 5 weeks. Upon euthanization, the proportions of Th1, Th2, Th17, and regulatory T cells (Tregs) in the spleen were determined by flow cytometry, and PG-specific lymphocyte proliferation by [3H]thymidine uptake. Results. Disease severity (AI) was dramatically suppressed in mice vaccinated with the LEAPS conjugate derG-PG70 as compared with controls or animals receiving J-PG70 vaccine. Protection was associated with reduced PG-specific lymphocyte proliferation, decreased proportions of Th1/Th17 cells, and increases in Th2 cells and Tregs. Conclusion. The derG-PG70 vaccine is therapeutically effective in PGIA (a Th1 dominated disease) by inducing immune deviation in favor of Th2 and Treg cells.

The role of citrullination of an immunodominant proteoglycan (PG) aggrecan T cell epitope in BALB/c mice with PG-induced arthritis

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 present in 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.

Genetics of Rheumatoid Arthritis — A Comprehensive Review

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.

Irreversible Heavy Chain Transfer to Hyaluronan Oligosaccharides by Tumor Necrosis Factor-stimulated Gene-6

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 an HC acceptor and an HC donor. In the present study, we show that transfer of HCs to low molecular weight 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 monoarticular mouse models of rheumatoid arthritis. 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 rheumatoid arthritis patients (in vitro).

Mycobacterial and mouse HSP70 have immuno-modulatory effects on dendritic cells

Previously, it has been shown that heat shock protein 70 (HSP70) can prevent inflammatory damage in experimental autoimmune disease models. Various possible underlying working mechanisms have been proposed. One possibility is that HSP70 induces a tolerogenic phenotype in dendritic cells (DCs) as a result of the direct interaction of the antigen with the DC. Tolerogenic DCs can induce antigen-specific regulatory T cells and dampen pathogenic T cell responses. We show that treatment of murine DCs with either mycobacterial (Mt) or mouse HSP70 and pulsed with the disease-inducing antigen induced suppression of proteoglycan-induced arthritis (PGIA), although mouse HSP70-treated DCs could ameliorate PGIA to a greater extent. In addition, while murine DCs treated with Mt- or mouse HSP70 had no significantly altered phenotype as compared to untreated DCs, HSP70-treated DCs pulsed with pOVA (ovalbumin peptide 323-339) induced a significantly increased production of IL-10 in pOVA-specific T cells. IL-10-producing T cells were earlier shown to be involved in Mt HSP70-induced suppression of PGIA. In conclusion, this study indicates that Mt- and mouse HSP70-treated BMDC can suppress PGIA via an IL-10-producing T cell-dependent manner.

The choice of adjuvant determines the cytokine profile of T cells in proteoglycan‐induced arthritis but does not influence disease severity

Rheumatoid arthritis (RA) is a debilitating autoimmune disease characterized by chronic inflammation of the synovial joints. Collagen-induced arthritis (CIA) and proteoglycan-induced arthritis (PGIA) are mouse models of inflammatory arthritis; CIA is a T helper type 17 (Th17) -dependent disease that is induced with antigen in complete Freund's adjuvant, whereas PGIA is Th1-mediated and is induced using antigen in dimethyldioctadecyl-ammonium bromide (DDA) as an adjuvant. To investigate whether the type of adjuvant determines the cytokine profile of the pathogenic T cells, we have compared the effect of CFA and DDA on T-cell responses in a single arthritis model. No differences in incidence or disease severity between aggrecan-T-cell receptor transgenic mice immunized with aggrecan in either CFA or DDA were observed. Immunization with CFA resulted in a higher proportion of Th17 cells, whereas DDA induced more Th1 cells. However, the levels of interleukin-17 (IL-17) produced by T cells isolated from CFA-immunized mice after antigen-specific stimulation were not significantly different from those found in DDA-immunized mice, indicating that the increased proportion of Th17 cells did not result in significantly higher ex vivo IL-17 levels. Hence, the choice of adjuvant can affect the overall proportions of Th1 and Th17 cells, without necessarily affecting the level of cytokine production or disease incidence and severity.

Suppression of dendritic cell maturation and T cell proliferation by synovial fluid myeloid cells from mice with autoimmune arthritis

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.

Regulatory T cells that recognize a ubiquitous stress-inducible self-antigen are long-lived suppressors of autoimmune arthritis

Reestablishing self-tolerance in autoimmunity is thought to depend on self-reactive regulatory T cells (Tregs). Exploiting these antigen-specific regulators is hampered by the obscure nature of disease-relevant autoantigens. We have uncovered potent disease-suppressive Tregs recognizing Heat Shock Protein (Hsp) 70 self-antigens, enabling selective activity in inflamed tissues. Hsp70 is a major contributor to the MHC class II ligandome. Here we show that a conserved Hsp70 epitope (B29) is present in murine MHC class II and that upon transfer, B29-induced CD4(+)CD25(+)Foxp3(+) T cells suppress established proteoglycan-induced arthritis in mice. These self-antigen-specific Tregs were activated in vivo, and when using Lymphocyte Activation Gene-3 as a selection marker, as few as 4,000 cells sufficed. Furthermore, depletion of transferred Tregs abrogated disease suppression. Transferred cells exhibited a stable phenotype and were found in joints and draining lymph nodes up to 2 mo after transfer. Given that (i) B29 administration by itself suppressed disease, (ii) our findings were made with wild-type (T-cell receptor nontransgenic) Tregs, and (iii) the B29 human homolog is presented by HLA class II, we are nearing translation of antigen-specific Treg activation as a promising intervention for chronic inflammatory diseases.

Wnt signaling genes of murine chromosome 15 are involved in sex‐affected pathways of inflammatory arthritis

Sex disparities in rheumatoid arthritis (RA) are well documented despite the lack of any known major RA susceptibility genes mapped to sex chromosomes. Murine chromosome 15 carries the sex-affected Pgia8 locus that mediates proteoglycan-induced arthritis, and homologous human loci are associated with RA. This study was undertaken to identify genes/mechanisms implicated in sex disparities in arthritis. Gene expression analysis was performed using RNA isolated from the paws of male and female Pgia8-congenic mice with collagen antibody-induced arthritis. Results were corroborated by reverse transcription-polymerase chain reaction, and mice were also studied prior to disease onset. Ingenuity Pathways Analysis of the expression patterns and gene functions was used to discover locus-specific and sex-affected signature transcripts. We found that the Pgia8 locus regulates antibody-mediated inflammatory arthritis differently in males and females. In Pgia8-congenic males, arthritis severity was 30% less (P < 0.005) than in wild-type males, but the antiinflammatory effect was similar in wild-type and congenic females. Transcriptome analysis indicated that 12 genes within the locus were significantly dysregulated in arthritic joints of congenic mice; expression of these genes was also sex specific. The genes that correlated most highly with arthritis severity included those for collagen triple-helix repeat-containing 1 (Cthrc1), metalloproteinase (Adamts12), R-spondin (Rspo2), and syndecan (Sdc2) (r = 0.87-0.91). The level of Cthrc1 message also correlated with that of the genes for the proinflammatory cytokines interleukin-1β and interleukin-6. These results indicate that sex-specific disparities in RA are linked to transcriptional regulation of genes involved in cartilage degradation (Adamts12) and canonical and noncanonical Wnt signaling (Cthrc1, Rspo2, Sdc2).

Disease-promoting and -protective genomic loci on mouse chromosomes 3 and 19 control the incidence and severity of autoimmune arthritis

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.

Presentation of the candidate rheumatoid arthritis autoantigen aggrecan by antigen‐specific B cells induces enhanced CD4+ T helper type 1 subset differentiation

Effective immune responses require antigen uptake by antigen-presenting cells (APC), followed by controlled endocytic proteolysis resulting in the generation of antigen-derived peptide fragments that associate with intracellular MHC class II molecules. The resultant peptide-MHC class II complexes then move to the APC surface where they activate CD4(+) T cells. Dendritic cells (DC), macrophages and B cells act as efficient APC. In many settings, including the T helper type 1 (Th1) -dependent, proteoglycan-induced arthritis model of rheumatoid arthritis, accumulating evidence demonstrates that antigen presentation by B cells is required for optimal CD4(+) T cell activation. The reasons behind this however, remain unclear. In this study we have compared the activation of CD4(+) T cells specific for the proteoglycan aggrecan following antigen presentation by DC, macrophages and B cells. We show that aggrecan-specific B cells are equally efficient APC as DC and macrophages and use similar intracellular antigen-processing pathways. Importantly, we also show that antigen presentation by aggrecan-specific B cells to TCR transgenic CD4(+) T cells results in enhanced CD4(+) T cell interferon-γ production and Th1 effector sub-set differentiation compared with that seen with DC. We conclude that preferential CD4(+) Th1 differentiation may define the requirement for B cell APC function in both proteoglycan-induced arthritis and rheumatoid arthritis.