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. 2011 Feb;63(2):479-87.
doi: 10.1002/art.30113.

Role of innate immunity in a murine model of histidyl-transfer RNA synthetase (Jo-1)-mediated myositis

Makoto Soejima  1 , Eun Ha Kang, Xinyan Gu, Yasuhiro Katsumata, Paula R Clemens, Dana P Ascherman
Affiliations

Role of innate immunity in a murine model of histidyl-transfer RNA synthetase (Jo-1)-mediated myositis

Makoto Soejima et al. Arthritis Rheum. 2011 Feb.

Abstract

Objective: Previous studies in humans and in animal models support a key role of histidyl-transfer RNA synthetase (HisRS; also known as Jo-1) in the pathogenesis of idiopathic inflammatory myopathy. While most investigations have focused on the ability of HisRS to trigger adaptive immune responses, in vitro studies clearly indicate that HisRS possesses intrinsic chemokine-like properties capable of activating the innate immune system. The purpose of this study was therefore to examine the ability of HisRS to direct innate immune responses in a murine model of myositis.

Methods: Following intramuscular immunization with soluble HisRS in the absence of exogenous adjuvant, selected strains of mice were evaluated at different time points for histopathologic evidence of myositis. Enzyme-linked immunosorbent assay-based assessment of autoantibody formation and carboxyfluorescein succinimidyl ester proliferation studies provided complementary measures of B cell and T cell responses triggered by HisRS immunization.

Results: Compared to appropriate control proteins, a murine HisRS fusion protein induced robust, statistically significant muscle inflammation in multiple congenic strains of C57BL/6 and NOD mice. Time course experiments revealed that this inflammatory response occurred as early as 7 days postimmunization and persisted for up to 7 weeks. Parallel immunization strategies in DO11.10/RAG-2(-/-) and C3H/HeJ (TLR-4(-/-) ) mice indicated that the ability of murine HisRS to drive muscle inflammation was not dependent on B cell receptor or T cell receptor recognition and did not require Toll-like receptor 4 signaling.

Conclusion: Collectively, the findings of these experiments support a model in which HisRS can trigger both innate and adaptive immune responses that culminate in severe muscle inflammation that is the hallmark of idiopathic inflammatory myopathy.

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Figures

Figure 1
Figure 1. Induction of myositis following intramuscular immunization of murine HRS
Panels A–C depict photomicrographs (100×) of hematoxylin and eosin-stained muscle tissue derived from MA/MBP- versus MBP-immunized mice of the indicated strains. Accompanying bar graphs represent the mean severity of muscle inflammation graded (in blinded fashion) on a scale from 0–3, as described in Materials and Methods. Error bars reflect SEM, while p values are derived from Mann U Whitney rank sum analysis. N equals the combined number of mice immunized with the indicated antigen in multiple experiments: C57BL/6 (B6), nMA/MBP=10 and nMBP=10; B6.G7, nMA/MBP=16 and nMBP=9; NOD.Idd3/5, nMA/MBP=17 and nMBP=10. In panel D, photomicrographs (400×) demonstrate immunohistochemical staining of inflammatory infiltrates resulting from MA/MBP immunization of NOD.Idd3/5 mice.
Figure 2
Figure 2. Development of autoantibodies and antigen-specific T cell proliferative responses following intramuscular administration of soluble HRS
A) Dot plots depict relative anti-murine HRS (anti-MJo-1) antibody titers induced by immunization of the designated strains with MA/MBP or MBP. Individual data points represent mean OD450 values (measured by ELISA) generated by combining sera at 1:1000 dilution with wells containing recombinant MJo-1 (0.1 μg/ml). Immunizing antigens are indicated on the x-axis. Error bars are omitted given negligible SEMs. B) Individual histograms represent CFSE dilution of pooled C57BL/6 (B6) Thy1.2+ lymph node cells stimulated by the indicated antigens in 96 hour proliferation assays. While the upper panels correspond to LN-derived T cells obtained from MA/MBP-immunized mice, the lower panels reflect proliferation of LN-derived lymphocytes isolated from MBP-immunized mice. Percentages correspond to the proportion of leftward-shifted, proliferating T cells. No Ag=no antigen added, MBP=Maltose Binding Protein, MA/MBP=amino terminal fragment of murine HRS fused to MBP, ConA=concavalin A.
Figure 3
Figure 3. Development of muscle inflammation at early time points following intramuscular administration of murine HRS
The bar graph in panel A corresponds to mean severity scores of muscle inflammation detected in muscle tissue 7 days following IM immunization of NOD.Idd3/5 mice with the indicated antigens (nMA/MBP=7, nMBP=4). Error bars and p-values are calculated as in Figure 1. While panel B demonstrates lymph node-derived T cell proliferation in response to the indicated antigens, panel C depicts anti-MJo-1 antibody formation promoted by IM immunization with MA/MBP (MA).
Figure 4
Figure 4. Murine HRS induces muscle inflammation in DO11.10/Rag2−/− mice
Photomicrographs (100x) depict representative H&E-stained muscle specimens derived from DO11.10/Rag2−/− mice 17 days following IM immunization of the hamstring musculature with MA/MBP or MBP alone. The accompanying bar graph indicates mean severity scores for mice immunized with MA/MBP (n=5) or MBP (n=5).
Figure 5
Figure 5. Induction of myositis is not dependent on TLR4 signaling
In Panel A, photomicrographs (100×) correspond to H&E-stained muscle tissue obtained from C3H/HeOuJ (WT) versus C3H/HeJ (TLR4−/−) mice 17 days following IM immunization with MA/MBP. The bar graph indicates the relative severity of muscle inflammation resulting from IM immunization with MA/MBP (C3H/HeOuJ (n=10), C3H/HeJ (n=20)) or MBP (C3H/HeJ (n=5)). Immunohistochemical staining (400×) with antibodies targeting CD3, B220, F4/80, CD44, CCR5, and CD11c reveals the relative number as well as activation state of T cells, B cells, macrophages, and dendritic cells comprising inflammatory infiltrates of muscle tissue obtained from MA/MBP-immunized C3H/HeJ mice (Panel B). Finally, the ELISA data in panel C demonstrate relative anti-MJo-1 antibody formation in C3H/HeOuJ versus C3H/HeJ mice immunized with MA/MBP. Data points correspond to mean OD450 values generated by 1:1000 dilutions of sera from individual mice.
Figure 6
Figure 6. Hypothesized role of HRS in the pathogenesis of idiopathic inflammatory myopathy
Combining data from the literature as well as current findings, this schematic demonstrates the putative role of HRS in bridging innate and adaptive immune responses that ultimately result in myocytotoxicity. As shown in the upper left portion of this diagram, muscle damage leads to upregulated surface expression of MHC Class I as well as release of soluble HRS that can recruit immature dendritic cells (iDCs) and naive lymphocytes via CCR5 (innate immunity). Subsequent maturation of DCs that have incorporated HRS allows efficient presentation of HRS peptides in the context of MHC Class II and enhanced co-stimulation, promoting activation of HRS-specific CD4+ T cells (in situ or in draining/regional lymph nodes). This subset of HRS-specific CD4+ T cells can then provide help to autoantibody-producing B cells and CD8+ T cells mediating myocytotoxicity in the adaptive arm of HRS-targeted immune responses.
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