1-Methyl-tryptophan synergizes with methotrexate to alleviate arthritis in a mouse model of arthritis.

Rheumatoid arthritis (RA) is an inflammatory autoimmune disease with no known cure. Current strategies to treat RA, including methotrexate (MTX), target the later inflammatory stage of disease. Recently, we showed that inhibiting indoleamine-2,3-dioxygenase (IDO) with 1-methyl-tryptophan (1MT) targets autoantibodies and cytokines that drive the initiation of the autoimmune response. Therefore, we hypothesized that combining 1MT with MTX would target both the initiation and chronic inflammatory phases of the autoimmune response and be an effective co-therapeutic strategy for arthritis. To test this, we used K/BxN mice, a pre-clinical model of arthritis that develops joint-specific inflammation with many characteristics of human RA. Mice were treated with 1MT, MTX, alone or in combination, and followed for arthritis, autoantibodies, and inflammatory cytokines. Both 1MT and MTX were able to partially inhibit arthritis when used individually; however, combining MTX + 1MT was significantly more effective than either treatment alone at delaying the onset and alleviating the severity of joint inflammation. We went on to show that combination of MTX + 1MT did not lower inflammatory cytokine or autoantibody levels, nor could the synergistic co-therapeutic effect be reversed by the adenosine receptor antagonist theophylline or be mimicked by inhibition of polyamine synthesis. However, supplementation with folinic acid did reverse the synergistic co-therapeutic effect, demonstrating that, in the K/BxN model, MTX synergizes with 1MT by blocking folate metabolism. These data suggest that pharmacological inhibition of IDO with 1MT is a potential candidate for use in combination with MTX to increase its efficacy in the treatment of RA

The regulation of anti -double -stranded DNA B cells in healthy and autoimmune mice

A hallmark of systemic lupus erythematosus is the presence of anti-double-stranded (ds) DNA Abs that are absent in healthy individuals. To identify the mechanisms involved in the regulation of anti-dsDNA B cells in non-autoimmune mice and the steps leading to the production of these Abs in autoimmune mice, we have compared the phenotype and localization of anti-dsDNA B cells in autoimmune-prone (MRL+/+, MRL-lpr/lpr , and bcl-2 Tg) mice with that in non-autoimmune-prone (BALB/c) mice. To increase the frequency of anti-DNA B cells so that they can be identified and tracked, we have utilized a H-chain Tg that can pair with endogenous L-chains to generate anti-single-stranded (ss) DNA, anti-dsDNA, and non-DNA B cells, allowing us to study the regulation of anti-dsDNA B cells in the context of a diverse B cell repertoire. We have found that anti-dsDNA B cells are actively regulated in BALB/c mice as indicated by the lack of their Ig in the serum, their developmental arrest, increased turnover rate, and accumulation at the T/B interface of the splenic follicle. In the MRL genetic background, anti-dsDNA B cells are no longer developmentally arrested, suggesting an intrinsic B cell defect conferred by MRL background genes. With intact Fas, they continue to exhibit follicular exclusion; however, in the presence of the lpr/lpr mutation, anti-dsDNA B cells are now present in the follicle and their Ig becomes present in the serum. These data suggest that MRL mice are defective in maintaining the developmental arrest of autoreactive B cells and indicate a role for Fas in restricting entry into the follicle. In contrast, the presence of a bcl-2 Tg increased the lifespan of anti-dsDNA B cells, but did not alter the other features of tolerance. This suggests that the serum anti-dsDNA Abs present in bcl-2 Tg mice were not due to a breakdown in central tolerance, as was found for MRL-lpr/lpr mice. Instead, we provide evidence that these Abs originate from B cells that have transited a GC, in that they are somatically mutated and clonally expanded. Together these data directly show that a breakdown in the regulation of anti-dsDNA B cells can occur at two levels: in the generation of the primary repertoire in the BM or in the formation of the modified repertoire during GC maturation

The regulation of anti -double -stranded DNA B cells in healthy and autoimmune mice

A hallmark of systemic lupus erythematosus is the presence of anti-double-stranded (ds) DNA Abs that are absent in healthy individuals. To identify the mechanisms involved in the regulation of anti-dsDNA B cells in non-autoimmune mice and the steps leading to the production of these Abs in autoimmune mice, we have compared the phenotype and localization of anti-dsDNA B cells in autoimmune-prone (MRL+/+, MRL-lpr/lpr , and bcl-2 Tg) mice with that in non-autoimmune-prone (BALB/c) mice. To increase the frequency of anti-DNA B cells so that they can be identified and tracked, we have utilized a H-chain Tg that can pair with endogenous L-chains to generate anti-single-stranded (ss) DNA, anti-dsDNA, and non-DNA B cells, allowing us to study the regulation of anti-dsDNA B cells in the context of a diverse B cell repertoire. We have found that anti-dsDNA B cells are actively regulated in BALB/c mice as indicated by the lack of their Ig in the serum, their developmental arrest, increased turnover rate, and accumulation at the T/B interface of the splenic follicle. In the MRL genetic background, anti-dsDNA B cells are no longer developmentally arrested, suggesting an intrinsic B cell defect conferred by MRL background genes. With intact Fas, they continue to exhibit follicular exclusion; however, in the presence of the lpr/lpr mutation, anti-dsDNA B cells are now present in the follicle and their Ig becomes present in the serum. These data suggest that MRL mice are defective in maintaining the developmental arrest of autoreactive B cells and indicate a role for Fas in restricting entry into the follicle. In contrast, the presence of a bcl-2 Tg increased the lifespan of anti-dsDNA B cells, but did not alter the other features of tolerance. This suggests that the serum anti-dsDNA Abs present in bcl-2 Tg mice were not due to a breakdown in central tolerance, as was found for MRL-lpr/lpr mice. Instead, we provide evidence that these Abs originate from B cells that have transited a GC, in that they are somatically mutated and clonally expanded. Together these data directly show that a breakdown in the regulation of anti-dsDNA B cells can occur at two levels: in the generation of the primary repertoire in the BM or in the formation of the modified repertoire during GC maturation

RhoB blockade selectively inhibits autoantibody production in autoimmune models of rheumatoid arthritis and lupus

During the development of autoimmune disease, a switch occurs in the antibody repertoire of B cells so that the production of pathogenic rather than non-pathogenic autoantibodies is enabled. However, there is limited knowledge concerning how this pivotal step occurs. Here, we present genetic and pharmacological evidence of a positive modifier function for the vesicular small GTPase RhoB in specifically mediating the generation of pathogenic autoantibodies and disease progression in the K/BxN preclinical mouse model of inflammatory arthritis. Genetic deletion of RhoB abolished the production of pathogenic autoantibodies and ablated joint inflammation in the model. Similarly, administration of a novel RhoB-targeted monoclonal antibody was sufficient to ablate autoantibody production and joint inflammation. In the MRL/lpr mouse model of systemic lupus erythematosus (SLE), another established preclinical model of autoimmune disease associated with autoantibody production, administration of the anti-RhoB antibody also reduced serum levels of anti-dsDNA antibodies. Notably, the therapeutic effects of RhoB blockade reflected a selective deficiency in response to self-antigens, insofar as RhoB-deficient mice and mice treated with anti-RhoB immunoglobulin (Ig) both mounted comparable productive antibody responses after immunization with a model foreign antigen. Overall, our results highlight a newly identified function for RhoB in supporting the specific production of pathogenic autoantibodies, and offer a preclinical proof of concept for use of anti-RhoB Ig as a disease-selective therapy to treat autoimmune disorders driven by pathogenic autoantibodies