979 - TIR-Domain-Containing Adaptor-Inducing Interferon-β (TRIF)-Dependent Toll-Like Receptor 4 Signaling Mediates Antibody Class Switching in a Mouse Model of Histidyl-tRNA Synthetase-Induced Myositis

Monday, November 7, 2011: 9:00 AM-6:00 PM
Hall F2 - Poster Hall (McCormick Place West)
Lisa Harlow, University of Miami Miller School of Medicine, Miami, FL, Makoto Soejima, University of Pittsburgh, Pittsburgh, PA and Dana P. Ascherman, University of Miami, Miami, FL
Presentation Number: 979

Objective:  To determine the role of toll-like receptor (TLR) signaling in histidyl-tRNA synthetase (Jo-1)-induced autoantibody formation and class switching

Background/Purpose:  In our previously published model of myositis generated by intramuscular (IM) immunization with a recombinant fragment of murine Jo-1 fused to Maltose Binding Protein (MA/MBP), we determined that induction of IgG anti-Jo-1 autoantibodies does not require the addition of exogenous adjuvant, but is dependent on TLR4 signaling.  While this observation highlighted the potential role of innate immune pathways in directing the production of IgG autoantibodies, the relative contribution of TLR4 signaling to antibody formation versus class switching remained undefined.

Method:  We used standard solid phase ELISA to determine IgM and/or IgG anti-Jo-1 antibody responses at various time points following IM immunization of different mouse strains with a recombinant amino-terminal fragment of murine Jo-1 fused to Maltose Binding Protein (MA/MBP).  Mouse strains used for this analysis included C3H/HeOuJ (WT), C3H/HeJ (TLR4 loss of function mutation), C57BL/6 (B6), B6.MyD88-/-, and B6.Ticam1 (TRIF loss of function mutation).  

Result:  Relative to post-immunization sera derived from C3H/HeOuJ (WT) mice, sera obtained from C3H/HeJ (functional TLR4 knockout) mice following IM immunization with MA/MBP demonstrated markedly reduced titers (5-40 fold) of IgG anti-Jo-1 autoantibodies.  Although this finding implicated TLR4 signaling in anti-Jo-1 autoantibody formation/class switching, MA/MBP-immunized B6.MyD88-/- mice surprisingly demonstrated equivalent levels of anti-IgG Jo-1 autoantibodies relative to their WT counterparts, C57BL/6 (with some differences in the ratio of IgG1/IgG2a).  In contrast, IM immunization of B6.Ticam1 (deficient TRIF signaling) mice with MA/MBP yielded markedly reduced titers of IgG anti-Jo-1 autoantibodies (similar to C3H/HeJ mice), implicating the TRIF-dependent arm of TLR4 signaling in the generation of IgG anti-Jo-1 autoantibodies.  To clarify the role of TRIF-mediated TLR4 signaling in autoantibody formation versus class switching, we assessed the induction of IgM and IgG anti-Jo-1 antibodies at Days 4, 7, and 11 following IM immunization of C3H/HeJ and C3H/HeOuJ mice.  These experiments demonstrated that C3H/HeJ mice produced equivalent levels of anti-Jo-1 IgM compared to C3H/HeOuJ mice at early time points following MA/MBP immunization, but failed to generate significant titers of class switched anti-Jo-1 autoantibodies. 

Conclusion:  IM immunization with recombinant murine Jo-1 in the absence of exogenous adjuvant induces antigen-specific, class switched autoantibodies.  This humoral immune response is dependent on TRIF-mediated TLR4 signals regulating IgG class switching rather than IgM antibody formation.  With relevance to the study of autoimmunity as well as vaccine biology, these findings provide insight regarding the contribution of innate immune signaling pathways to the generation of adaptive humoral immune responses. 

Keywords: autoantibodies and myositis

Disclosure: L. Harlow, None; M. Soejima, None; D. P. Ascherman, None.