Role of Citrullinated Collagen in Autoimmune Arthritis.

Citrullination of proteins plays an important role in protein function and it has recently become clear that citrullinated proteins play a role in immune responses. In this study we examined how citrullinated collagen, an extracellular matrix protein, affects T-cell function during the development of autoimmune arthritis. Using an HLA-DR1 transgenic mouse model of rheumatoid arthritis, mice were treated intraperitoneally with either native type I collagen (CI), citrullinated CI (cit-CI), or phosphate buffered saline (PBS) prior to induction of autoimmune arthritis. While the mice given native CI had significantly less severe arthritis than controls administered PBS, mice receiving cit-CI had no decrease in the severity of autoimmune arthritis. Using Jurkat cells expressing the inhibitory receptor leukocyte-associated immunoglobulin-like receptor-1 (LAIR-1), Western blot analysis indicated that while CI and cit-CI bound to LAIR-1 with similar affinity, only CI induced phosphorylation of the LAIR ITIM tyrosines; cit-CI was ineffective. These data suggest that cit-CI acts as an antagonist of LAIR-1 signaling, and that the severity of autoimmune arthritis can effectively be altered by targeting T cells with citrullinated collagen.

Leukocyte-associated immunoglobulin-like receptor 1 inhibits T-cell signaling by decreasing protein phosphorylation in the T-cell signaling pathway.

Multiple observations implicate T-cell dysregulation as a central event in the pathogenesis of rheumatoid arthritis. Here, we investigated mechanisms for suppressing T-cell activation via the inhibitory receptor leukocyte-associated immunoglobulin-like receptor 1 (LAIR-1). To determine how LAIR-1 affects T-cell receptor (TCR) signaling, we compared 1) T cells from LAIR-1-sufficient and -deficient mice, 2) Jurkat cells expressing either LAIR-1 mutants or C-terminal Src kinase (CSK) mutants, and 3) T cells from mice that contain a CSK transgene susceptible to chemical inhibition. Our results indicated that LAIR-1 engagement by collagen or by complement C1q (C1Q, which contains a collagen-like domain) inhibits TCR signaling by decreasing the phosphorylation of key components in the canonical T-cell signaling pathway, including LCK proto-oncogene SRC family tyrosine kinase (LCK), LYN proto-oncogene SRC family tyrosine kinase (LYN), ζ chain of T-cell receptor-associated protein kinase 70 (ZAP-70), and three mitogen-activated protein kinases (extracellular signal-regulated kinase, c-Jun N-terminal kinase 1/2, and p38). The intracellular region of LAIR-1 contains two immunoreceptor tyrosine-based inhibition motifs that are both phosphorylated by LAIR-1 activation, and immunoprecipitation experiments revealed that Tyr-251 in LAIR-1 binds CSK. Using CRISPR/Cas9-mediated genome editing, we demonstrate that CSK is essential for the LAIR-1-induced inhibition of the human TCR signal transduction. T cells from mice that expressed a PP1 analog-sensitive form of CSK (CskAS) corroborated these findings, and we also found that Tyr-251 is critical for LAIR-1's inhibitory function. We propose that LAIR-1 activation may be a strategy for controlling inflammation and may offer a potential therapeutic approach for managing autoimmune diseases.

1,25-Dihydroxyvitamin D3 and 20-Hydroxyvitamin D3 Upregulate LAIR-1 and Attenuate Collagen Induced Arthritis.

Vitamin D plays a crucial role in regulation of the immune response. However, treatment of autoimmune diseases with 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] doses sufficient to be effective is prohibitive due to its calcemic and toxic effects. We use the collagen-induced arthritis (CIA) model to analyze the efficacy of the noncalcemic analog of vitamin D, 20S-hydroxyvitamin D3 [20S(OH)D3], as well as 1,25(OH)2D3, to attenuate arthritis and explore a potential mechanism of action. Mice fed a diet deficient in vitamin D developed a more severe arthritis characterized by enhanced secretion of T cell inflammatory cytokines, compared to mice fed a normal diet. The T cell inflammatory cytokines were effectively suppressed, however, by culture of the cells with 20S(OH)D3. Interestingly, one of the consequences of culture with 1,25(OH)2D3 or 20S(OH)D3, was upregulation of the natural inhibitory receptor leukocyte associated immunoglobulin-like receptor-1 (LAIR-1 or CD305). Polyclonal antibodies which activate LAIR-1 were also capable of attenuating arthritis. Moreover, oral therapy with active forms of vitamin D suppressed arthritis in LAIR-1 sufficient DR1 mice, but were ineffective in LAIR-1-/- deficient mice. Taken together, these data show that the effect of vitamin D on inflammation is at least, in part, mediated by LAIR-1 and that non-calcemic 20S(OH)D3 may be a promising therapeutic agent for the treatment of autoimmune diseases such as Rheumatoid Arthritis.

Modulation by 17,20S(OH)2pD of Fibrosis-Related Mediators in Dermal Fibroblast Lines from Healthy Donors and from Patients with Systemic Sclerosis.

We previously demonstrated that the non-calcemic pregnacalciferol (pD) analog 17,20S (OH)2pD suppressed TGF-ß1-induced type I collagen production in cultured normal human dermal fibroblasts. In the present studies, we examined fibroblasts cultured from the lesional skin of patients with systemic sclerosis (scleroderma (SSc)) and assessed the effects of 17,20S(OH)2pD on fibrosis-related mediators. Dermal fibroblast lines were established from skin biopsies from patients with SSc and healthy controls. Fibroblasts were cultured with either 17,20S(OH)2pD or 1,25(OH)2D3 (positive control) with/without TGF-ß1 stimulation and extracted for protein and/or mRNA for collagen synthesis and mediators of fibrosis (MMP-1, TIMP-1, PAI-1, BMP-7, PGES, GLI1, and GLI2). 1 7,20S(OH)2pD (similar to 1,25(OH)2D3) significantly suppressed net total collagen production in TGF-ß1-stimulated normal donor fibroblast cultures and in cultures of SSc dermal fibroblasts. 17,20S(OH)2pD (similar to 1,25(OH)2D3) also increased MMP-1, BMP-7, and PGES and decreased TIMP-1 and PAI1 expression in SSc fibroblasts. Although 17,20S(OH)2pD had no effect on Gli1 or Gli2 in SSc fibroblasts, it increased Gli2 expression when cultured with TGF-ß1 in normal fibroblasts. These studies demonstrated that 17,20S(OH)2pD modulates mediators of fibrosis to favor the reduction of fibrosis and may offer new noncalcemic secosteroidal therapeutic approaches for treating SSc and fibrosis.

17,20S(OH)2pD Can Prevent the Development of Skin Fibrosis in the Bleomycin-Induced Scleroderma Mouse Model.

Systemic sclerosis (SSc; scleroderma) is a chronic fibrotic disease involving TGF-ß1. Low serum vitamin D (vit D) correlates with the degree of fibrosis and expression of TGF-ß1. This study was designed to determine whether the noncalcemic vit D analog, 17,20S(OH)2pD, suppresses fibrosis and mediators of the TGF-ß1 pathway in the bleomycin (BLM) model of fibrosis. Fibrosis was induced into the skin of female C57BL/6 mice by repeated injections of BLM (50 µg/100 µL) subcutaneously. Mice received daily oral gavage with either vehicle (propylene glycol) or 17,20S(OH)2pD using 5, 15, or 30 µg/kg for 21 days. The injected skin was biopsied; analyzed histologically; examined for total collagen by Sircol; and examined for mRNA expression of MMP-13, BMP-7, MCP-1, Gli1, and Gli2 by TR-PCR. Spleen was analyzed for lymphocytes using flow cytometry. Serum was analyzed for cytokines using a multiplexed ELISA. Results showed that all three doses of 17,20S(OH)2pD suppressed net total collagen production, dermal thickness, and total collagen content in the BLM fibrosis model. 17,20S(OH)2pD also increased MMP-13 expression, decreased MCP-1 and Gli-2 expression in vivo, and suppressed serum levels of IL-13, TNF-α, IL-6, IL-10, IL-17, and IL-12p70. In summary, 17,20S(OH)2pD modulates the mediators of fibrosis in vivo and suppresses total collagen production and dermal thickness. This antifibrotic property of 17,20S(OH)2pD offers new therapeutic approaches for fibrotic disorders.

The Role of Leukocyte-Associated Ig-like Receptor-1 in Suppressing Collagen-Induced Arthritis.

Several observations implicate a critical role for T cell dysregulation as a central problem in rheumatoid arthritis. We investigated a mechanism for suppressing T cell activation by stimulating a natural inhibitory receptor called leukocyte-associated Ig-like receptor-1 (LAIR-1). The collagen-induced arthritis (CIA) model and DR-1 transgenic mice were used to study the importance of LAIR-1 in autoimmune arthritis. Splenocytes from wild-type or LAIR-1-/- mice were stimulated with soluble anti-CD3 Ab in the presence or absence of α1(II) and supernatants were collected for cytokine analysis. B6.DR1 mice were immunized with type II collagen/CFA to induce arthritis and were treated with either the stimulatory mAb to LAIR-1 or a hamster IgG control. Finally, B6.DR1/LAIR-1-/- and B6.DR1/LAIR-1+/+ mice were challenged for CIA and mean severity scores were recorded thrice weekly. Using splenocytes or purified CD4+ cells that were sufficient in LAIR-1, CD3-induced cytokine secretion was significantly suppressed in the presence of collagen, whereas LAIR-1-deficient splenocytes had no attenuation. Treatment with a stimulatory mAb to LAIR-1 also significantly attenuated CIA in the LAIR+/+ mice. When B6.DR1/LAIR-1-/- mice were immunized with type II collagen they developed more severe arthritis and had a greater percentage of affected limbs than the wild-type mice. These data demonstrate that collagen can suppress the T cell cytokine response through the action of LAIR-1. Treatment with stimulating LAIR-1 Abs suppresses CIA whereas B6.DR1/LAIR-1-/- mice develop more severe arthritis than wild-type controls. These data suggest that LAIR-1 may be a potential therapeutic target for suppressing rheumatoid arthritis.

The role of Syk in peripheral T cells.

The aim of this study was to understand how Syk affects peripheral T cell function. T cells from Syk-/- chimeric mice and DR1 Sykfl/fl CD4cre conditional mice gave strong CD3-induced Th1, Th2, and Th17 cytokine responses. However, an altered peptide ligand (APL) of human CII (256-276) with two substitutions (F263N, E266D), also called A12, elicited only Th2 cytokine responses from Sykfl/fl T cells but not Sykfl/fl-CD4cre T cells. Western blots revealed a marked increase in the phosphorylation of Syk, JNK and p38 upon A12/DR1 activation in WT or Sykfl/fl T cells but not in Sykfl/flCD4-cre cells. We demonstrate that Syk is required for the APL- induction of suppressive cytokines. Chemical Syk inhibitors blocked activation of GATA-3 by peptide A12/DR1. In conclusion, this study provides novel insights into the role that Syk plays in directing T cell activity, and may shape therapeutic approaches for autoimmune diseases.

Characterization of the Syk-Dependent T Cell Signaling Response to an Altered Peptide.

Rheumatoid arthritis is an autoimmune disorder characterized by T cell dysregulation. We have shown that an altered peptide ligand (A9) activates T cells to use an alternate signaling pathway that is dependent on FcRγ and spleen tyrosine kinase, resulting in downregulation of inflammation. In the experiments described in this study, we have attempted to determine the molecular basis of this paradox. Three major Src family kinases found in T cells (Lck, Fyn, and Lyn) were tested for activation following stimulation by A9/I-Aq Unexpectedly we found they are not required for T cell functions induced by A9/I-Aq, nor are they required for APL stimulation of cytokines. On the other hand, the induction of the second messenger inositol trisphosphate and the mobilization of calcium are clearly triggered by the APL A9/I-Aq stimulation and are required for cytokine production, albeit the cytokines induced are different from those produced after activation of the canonical pathway. DBA/1 mice doubly deficient in IL-4 and IL-10 were used to confirm that these two cytokines are important for the APL-induced attenuation of arthritis. These studies provide a basis for exploring the effectiveness of analog peptides and the inhibitory T cells they induce as therapeutic tools for autoimmune arthritis.

Peptide ligand structure and I-Aq binding avidity influence T cell signaling pathway utilization.

Factors that drive T cells to signal through differing pathways remain unclear. We have shown that an altered peptide ligand (A9) activates T cells to utilize an alternate signaling pathway which is dependent upon FcRγ and Syk. However, it remains unknown whether the affinity of peptide binding to MHC drives this selection. To answer this question we developed a panel of peptides designed so that amino acids interacting with the p6 and p9 predicted MHC binding pockets were altered. Analogs were tested for binding to I-A(q) using a competitive binding assay and selected analogs were administered to arthritic mice. Using the collagen-induced arthritis (CIA) model, arthritis severity was correlated with T cell cytokine production and molecular T cell signaling responses. We establish that reduced affinity of interaction with the MHC correlates with T cell signaling through the alternative pathway, leading ultimately to secretion of suppressive cytokines and attenuation of arthritis.

Optimizing oral immune tolerance to Type II collagen in patients with rheumatoid arthritis: The importance of dose, Interfering medication and Genetics.

OBJECTIVES: Oral immune tolerance (OT) is a complex process with unknown genetic regulation. Our aim is to explore possible genetic control of OT in patients with rheumatoid arthritis (RA). METHODS: RA patients with increased interferon γ production invitro when their isolated peripheral blood mononuclear cells (PBMC) were cultured with type II bovine collagen α1 chain [α1 (II)] were enrolled in this study and were randomly assigned to the "Low dose" type II collagen (CII) group (30 µg/day for 10 weeks, followed by 50 µg/day for 10 weeks, followed by 70 µg/day for 10 weeks) or "High dose" CII group (90 µg/day for 10 weeks, followed by 110 µg/day for 10 weeks, followed by 130 µg/day for 10 weeks). Heparinized blood was obtained at baseline and after each of the 10 weeks treatment for analysis of the invitro production of IFNγ by their PBMC stimulated by α1(II) . Single nucleotide polymorphism (SNP) analysis of the responders and non-responders to oral CII was conducted using GeneChip Mapping 10 K 2.0 Array. RESULTS: The SNP A-15,737 was found to associate with the ability of CII to suppress IFNγ production by α1(CII)-stimulated RA PBMC. The potential for SNP A-15,737 to associate with the OT response for patients with another autoimmune disease [OT induced by oral type I bovine collagen (CI) in patients with diffuse cutaneous systemic sclersodid (dsSSc)] was also explored. CONCLUSIONS: The ROT1 region plays a role in the control of IFNγ production after oral dosing of auto-antigens, thereby determining if oral tolerance to that antigen will develop.

Molecular basis for T cell response induced by altered peptide ligand of type II collagen.

Mounting evidence from animal models has demonstrated that alterations in peptide-MHC interactions with the T cell receptor (TCR) can lead to dramatically different T cell outcomes. We have developed an altered peptide ligand of type II collagen, referred to as A9, which differentially regulates TCR signaling in murine T cells leading to suppression of arthritis in the experimental model of collagen-induced arthritis. This study delineates the T cell signaling pathway used by T cells stimulated by the A9·I-A(q) complex. We have found that T cells activated by A9 bypass the requirement for Zap-70 and CD3-ζ and signal via FcRγ and Syk. Using collagen-specific T cell hybridomas engineered to overexpress either Syk, Zap-70, TCR-FcRγ, or CD3-ζ, we demonstrate that A9·I-A(q) preferentially activates FcRγ/Syk but not CD3-ζ/Zap-70. Moreover, a genetic absence of Syk or FcRγ significantly reduces the altered peptide ligand induction of the nuclear factor GATA3. By dissecting the molecular mechanism of A9-induced T cell signaling we have defined a new alternate pathway that is dependent upon FcRγ and Syk to secrete immunoregulatory cytokines. Given the interest in using Syk inhibitors to treat patients with rheumatoid arthritis, understanding this pathway may be critical for the proper application of this therapy.

A 9-year-old boy with chronic urticaria and progressive spondyloarthritis.

A 9-year-old African American boy presented with chronic urticaria and progressive spondyloarthritis. Laboratory tests were abnormal for persistently positive antinuclear antibodies and undetectable total hemolytic complement (CH50) despite normal levels of complement C2, C3, and C4. Ultimately, further testing revealed an underlying deficiency in the immune system that may be associated with autoimmune disease and thus have a bearing on the patient's urticaria and spondyloarthritis. This is a unique presentation of a rare disease and underscores the importance of evaluating for systemic disease in the workup of chronic urticaria.

Variation of sexual dimorphism and asymmetry in disease expression of inflammatory arthritis among laboratory mouse models with different genomic backgrounds.

Sex difference has shown in the arthritis diseases in human population and animal models. We investigate how the sex and symmetry vary among mouse models with different genomic backgrounds. Disease data of sex and limbs accumulated in the past more than two decades from four unique populations of murine arthritis models were analyzed. They are (1) interleukin-1 receptor antagonist (IL-1ra) deficient mice under Balb/c background (Balb/c KO); (2) Mice with collagen II induced arthritis under DBA/1 background; (3) Mice with collagen II induced arthritis under C57BL/6 (B6) background and (4) A F2 generation population created by Balb/c KO X DBA/1 KO. Our data shows that there is a great variation in sexual dimorphism for arthritis incidence and severity of arthritis in mice harboring specific genetic modifications. For a F2 population, the incidence of arthritis was 57.1% in female mice and 75.6% in male mice. There was a difference in severity related to sex in two populations: B6.DR1/ B6.DR4 (P < 0.001) and F2 (P = 0.023) There was no difference Balb/c parental strain or in collagen-induced arthritis (CIA) in DBA/1 mice. Among these populations, the right hindlimbs are significantly higher than the scores for the left hindlimbs in males (P < 0.05). However, when examining disease expression using the collagen induced arthritis model with DBA/1 mice, sex-dimorphism did not reach statistical significance, while left hindlimbs showed a tendency toward greater disease expression over the right. Sexual dimorphism in disease expression in mouse models is strain and genomic background dependent. It sets an alarm that potential variation in sexual dimorphism among different racial and ethnic groups in human populations may exist. It is important to not only include both sexes and but also pay attention to possible variations caused by disease expression and response to treatment in all the studies of arthritis in animal models and human populations.

T cells stimulated with an analog peptide of type II collagen require the Fc receptor γ-chain to secrete interleukin-4 and suppress autoimmune arthritis in mice.

OBJECTIVE: To explore the characteristics of the T cell population that responds to an analog peptide (A9) of type II collagen and regulates autoimmunity, using the collagen-induced arthritis (CIA) model. METHODS: Analog peptide A9 is a 26-amino acid peptide analogous to the sequence of a segment of type II collagen (CII245-270) but with substitutions at amino acid positions 260 (alanine for isoleucine), 261 (hydroxyproline for alanine), and 263 (asparagine for phenylalanine). We previously showed that A9 profoundly suppressed CIA and immune responses to type II collagen. In order to determine the mechanism of suppression, we used transgenic mice whose T cells express a type II collagen-specific receptor (T cell receptor) and performed passive cell transfer experiments. RESULTS: The results demonstrated that suppression of CIA by A9 is dependent on T cells. Using multiparameter flow cytometry, we determined that the cells responsible for suppression were CD4+ and expressed high levels of Fcε receptor Iγ chain (FcRγ). To establish the significance of this finding, we obtained mice genetically deficient in FcRγ in order to perform passive transfer experiments. The resulting FcRγ-/- CD4+ T cells, when primed by culture with A9, could not transfer the suppression of arthritis or secrete cytokines in response to A9. CONCLUSION: Taken together, the results of this study suggest that the suppression of arthritis and the Th2 cytokine profile elicited by A9 is dependent on the presence of FcRγ in T cells. These findings are novel and may have therapeutic potential for patients with autoimmune arthritis.

Beta-caryophyllene prevents the defects in trabecular bone caused by Vitamin D deficiency through pathways instated by increased expression of klotho.

AIMS: This study investigated the effects of ß-caryophyllene (BCP) on protecting bone from vitamin D deficiency in mice fed on a diet either lacking (D-) or containing (D+) vitamin D. METHODS: A total of 40 female mice were assigned to four treatment groups (n = 10/group): D+ diet with propylene glycol control, D+ diet with BCP, D-deficient diet with control, and D-deficient diet with BCP. The D+ diet is a commercial basal diet, while the D-deficient diet contains 0.47% calcium, 0.3% phosphorus, and no vitamin D. All the mice were housed in conditions without ultraviolet light. Bone properties were evaluated by X-ray micro-CT. Serum levels of klotho were measured by enzyme-linked immunosorbent assay. RESULTS: Under these conditions, the D-deficient diet enhanced the length of femur and tibia bones (p < 0.050), and increased bone volume (BV; p < 0.010) and trabecular bone volume fraction (BV/TV; p < 0.010) compared to D+ diet. With a diet containing BCP, the mice exhibited higher BV and bone mineral density (BMD; p < 0.050) than control group. The trabecular and cortical bone were also affected by vitamin D and BCP. In addition, inclusion of dietary BCP improved the serum concentrations of klotho (p < 0.050). In mice, klotho regulates the expression level of cannabinoid type 2 receptor (Cnr2) and fibroblast growth factor 23 (Fgf23) through CD300a. In humans, data suggest that klotho is connected to BMD. The expression of klotho is also associated with bone markers. CONCLUSION: These data indicate that BCP enhances the serum level of klotho, leading to improved bone properties and mineralization in an experimental mouse model.Cite this article: Bone Joint Res 2022;11(8):528-540.

20S-Hydroxyvitamin D3, a Secosteroid Produced in Humans, Is Anti-Inflammatory and Inhibits Murine Autoimmune Arthritis.

The ability to use large doses of vitamin D3 (D3) to chronically treat autoimmune diseases such as rheumatoid arthritis (RA) is prohibitive due to its calcemic effect which can damage vital organs. Cytochrome P450scc (CYP11A1) is able to convert D3 into the noncalcemic analog 20S-hydroxyvitamin D3 [20S(OH)D3]. We demonstrate that 20S(OH)D3 markedly suppresses clinical signs of arthritis and joint damage in a mouse model of RA. Furthermore, treatment with 20S(OH)D3 reduces lymphocyte subsets such as CD4+ T cells and CD19+ B cells leading to a significant reduction in inflammatory cytokines. The ratio of T reg cells (CD4+CD25+Foxp3+ T cells) to CD3+CD4+ T cells is increased while there is a decrease in critical complement-fixing anti-CII antibodies. Since pro-inflammatory cytokines and antibodies against type II collagen ordinarily lead to destruction of cartilage and bone, their decline explains why arthritis is attenuated by 20(OH) D3. These results provide a basis for further consideration of 20S(OH)D3 as a potential treatment for RA and other autoimmune disorders.

T cell receptor signaling induced by an analog peptide of type II collagen requires activation of Syk.

We have previously described an analog peptide of type II collagen (CII) that can suppress collagen-induced arthritis (CIA). This analog peptide represents CII(245-270), the immunodominant epitope of CII, but with substitutions at 260, 261, and 263 - CII(245-270) (A(260), B(261), and N(263)) (A9). To elucidate the mechanisms responsible for suppression, we used mice transgenic for a collagen-specific T cell receptor (TCR). When we found that APCs pulsed with A9 failed to induce T cell phosphorylation of TCR-zeta and ZAP-70, we explored alternative signaling pathways. We determined that A9 instead induced phosphorylation of spleen tyrosine kinase (Syk). The importance of Syk was confirmed by the use of chemical Syk inhibitors, which blocked both cytokine secretion and activation of GATA-3 mediated by peptide A9. In summary, T cells use an alternative pathway in response to A9 that involves Syk. This novel T cell pathway may represent an important means for altering T cell phenotypes.

Ameliorating effects of Gö6976, a pharmacological agent that inhibits protein kinase D, on collagen-induced arthritis.

Toll-like receptor (TLR) signaling can contribute to the pathogenesis of arthritis. Disruption of TLR signaling at early stages of arthritis might thereby provide an opportunity to halt the disease progression and ameliorate outcomes. We previously found that Gö6976 inhibits TLR-mediated cytokine production in human and mouse macrophages by inhibiting TLR-dependent activation of protein kinase D1 (PKD1), and that PKD1 is essential for proinflammatory responses mediated by MyD88-dependent TLRs. In this study, we investigated whether PKD1 contributes to TLR-mediated proinflammatory responses in human synovial cells, and whether Gö6976 treatment can suppress the development and progression of type II collagen (CII)-induced arthritis (CIA) in mouse. We found that TLR/IL-1R ligands induced activation of PKD1 in human fibroblast-like synoviocytes (HFLS). TLR/IL-1R-induced expression of cytokines/chemokines was substantially inhibited in Gö6976-treated HFLS and PKD1-knockdown HFLS. In addition, serum levels of anti-CII IgG antibodies, and the incidence and severity of arthritis after CII immunization were significantly reduced in mice treated daily with Gö6976. Synergistic effects of T-cell receptor and TLR, as well as TLR alone, on spleen cell proliferation and cytokine production were significantly inhibited in the presence of Gö6976. Our results suggest a possibility that ameliorating effects of Gö6976 on CIA may be due to its ability to inhibit TLR/IL-1R-activated PKD1, which might play an important role in proinflammatory responses in arthritis, and that PKD1 could be a therapeutic target for inflammatory arthritis.

An altered peptide ligand of type II collagen suppresses autoimmune arthritis.

On the basis of the hypothesis that immunity to type II collagen (CII) contributes to joint inflammation, our goal is to develop an immunotherapy capable of selectively blocking immunity to a particular autoantigen without interfering with the beneficial functions of the immune system. CII is the major protein component of articular cartilage and autoimmunity to CII is strongly associated with rheumatoid arthritis in man. Our laboratory has previously identified a region of type II collagen (CII), CII245-270 that contains a prominent T-cell epitope in the immune response to CII. Residues critical to the I-Aq-restricted presentation of this determinant have been characterized. When synthetic analog peptides were developed that contain site-directed substitutions in critical positions, we found that that CII245-270 (A260, B261, N263) (A9), profoundly suppressed collagen-induced arthritis. When DBA/1 mice were coimmunized with CII and the analog peptide, the incidence and severity of arthritis was greatly reduced concordant with the humoral immune responses to CII. Moreover, the suppression could be transferred with A9-immune spleen cells and was accompanied by a Th2-type cytokine profile. When we compared T-cell signals in response to A9 to those of wild-type (WT) peptide, we found that APCs prepulsed with WT peptide induced strong phosphorylation of both TCR zeta chain and Zap-70, while A9 did not. Since T cells clearly respond to A9 with cytokine secretion, we hypothesize that A9 induces an alternate signaling pathway and we speculate that this pathway involves phosphorylation of Syk, a kinase ordinarily utilized by B cells. Activation of this alternative pathway is a novel observation and may represent an important means by which the phenotype of the responding T cell is altered. Elucidation of the mechanism by which A9 prevents arthritis may lead to development of novel immunotherapeutic approaches to antigen specific treatment of autoimmunity.