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Mucosal intervention with CFA/I fimbriae effectively diminishes CIA by stimulation of TGF-β+ and IL-10+ regulatory CD39+CD4+ T cells (Tregs), resulting in suppression of Th1 and Th17 cells and IL-27. CFA/I also stimulates Tregs expressing p35 and EBI3 subunits simultaneously, suggesting an essential role of IL-35 in CFA/I-mediated protection. We hypothesize CFA/I fimbriae stimulate endogenous IL-35 production to facilitate protection against autoimmune disease. In testing the relevance of endogenous IL-35 for protection, oral delivery of recombinant CFA/I into EBI3-/- mice on day 14 after CIA induction was found ineffective against CIA compared to similarly treated WT mice, as evidenced by the lack of suppression of IL-17 and IFN-γ (the latter increased 5-fold ). CFA/I-treated EBI3-/- mice lacked enhanced CREB phosphorylation that, in turn, failed to stimulate the transcription of CD39 (apyrase). In fact, surface apyrase activity by EBI3-/- CD4+ T cells was uninduced. Because of the lack of induced CD39 expression, IL-10 production was greatly diminished. Clinical improvement of CIA in CFA/I-treated EBI3-/- mice required further intervention with recombinant IL-35, resulting in the induction of CD39+CD4+ T cells, enhanced TGF-β production, and reduction in IFN-γ and IL-17. Thus, in the absence of endogenous IL-35, mucosal CFA/I fimbriae intervention is ineffective against autoimmune arthritis. Supported by AT-004986.

Infection of the joints is the most frequent localized manifestation of brucellosis, which is a common cause of infectious arthritis. However, no experimental murine model of Brucella-induced arthritis has been reported. Here we report that IFN-γ-/- mice develop joint inflammation following oral, nasal, or parenteral infection with B. abortus or B. melitensis. Joints from Brucella-infected IFN-γ-/-, but not wild-type mice, were found to contain extensive inflammatory infiltrates and debris within the joint space which co-localized with brucellae. Osteoarthritis, joint space narrowing, necrosis, soft tissue inflammation, and substantial Brucella burdens were also observed, although antibody or cytokine responses against collagen were not detected. Elevated IL-1β, but not TNF-α, IL-6, nor IL-17, was detected in the joints of Brucella-infected IFN-γ-/- mice. A six-week regimen of rifampicin effectively cleared infection and halted further progression of inflammation, although some symptoms and swelling remained. However, administration of an adenovirus expressing the IL-1receptor antagonist augmented antibiotic resolution of joint swelling by >50%. These results show that the IFN-γ-/- mouse represents a useful model to study the pathogenesis of joint inflammation due to Brucella infection, and that intervention strategies targeting IL-1 can complement antibiotic treatment of Brucella-induced inflammation. Supported by USDA 2007-01612 and USDA 2009-34397-20133.

IL-35, a novel heterodimeric cytokine (IL-12p35 subunit plus Epstein-Barr virus-induced gene 3 [EBI3]), had been reported to expand regulatory CD4+CD25+ T cells and ameliorate CIA in mice. IL-35 was expressed as a single chain polypeptide, and the recombinant cytokine was detected by Western blot using anti-EBI3 or anti-IL-12 mAbs. To assess its activity, BALB/c mice were adoptively transferred with DO11.10 CD4+ T cells, challenged s.c. with OVA in IFA, and treated every day for 5 days with IL-35, resulting in diminished IgG2a and IgG2b anti-OVA Abs, as well as suppressed DTH responses. To exploit IL-35's therapeutic potential, B6 mice were induced with CIA, and upon the onset of disease (day 21), mice received 2.0 μg/mouse every 2 days until day 39. All mice in the control group developed arthritis by day 28 post-induction, whereas none of IL-35-treated mice had any clinical symptoms. Restimulated sorted CD4+ T cells from LNs of protected mice showed decreased IFN-γ and IL-17 production. FACS analysis revealed enhancement of regulatory CD4+ CD25+ T cells, of which, half co-expressed CD39, an ectonucleoside triphosphate diphosphohydrolase. Cell-sorted CD39+ CD4+ T cells from IL-35-treated mice could suppress CII-specific proliferation by CD39- CD4+ T cells via enhanced IL-10 production. These findings suggest the stimulation by IL-35 for the induction of regulatory CD4+ T cells. Supported by NIH R01 AI78938.