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Abstract Number: 1778

Physical Interactions Between Histidyl-tRNA Synthetase and Endogenous/Exogenous Alarmins Enhance Immunogenicity In a Model Of Antigen-Induced Myositis

Irina Fernandez1, Lisa Harlow2, William Ridgway3 and Dana P. Ascherman4, 1Rheumatology, University of Miami, Miami, FL, 2Rheumatology and Clinical Immunology, University of Miami Miller School of Medicine, Miami, FL, 3Immunology, Allergy and Rheumatology, University of Cincinnati, Cincinnati, OH, 4Medicine/Rheumatology, University of Miami Miller School of Medicine, Miami, FL

Meeting: 2013 ACR/ARHP Annual Meeting

Keywords: autoantigens, innate immunity, myositis and toll-like receptors

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Session Information

Session Title: Muscle Biology, Myositis and Myopathies I: Insights into Mechanisms of the Idiopathic Inflammatory Myopathies

Session Type: Abstract Submissions (ACR)

Objective:  To assess the biological interplay between histidyl-tRNA synthetase (HRS) and different endogenous/exogenous ligands capable of generating signals through MyD88-dependent receptor systems in idiopathic inflammatory myopathy.

Background/Purpose:  Recent immunohistochemical and array-based studies of human muscle tissue implicate Toll-like receptor (TLR)- and other MyD88-dependent signaling pathways in the pathogenesis of idiopathic inflammatory myopathy.  Our model of HRS-induced myositis replicates many of these observations, demonstrating that intramuscular immunization of mice with bacterially produced HRS fusion proteins preferentially triggers muscle inflammation (relative to other bacterially generated control proteins) via partially redundant MyD88-dependent signaling cascades.  However, the biophysical properties endowing HRS with this unique signaling capacity remain undefined.

Methods:   Binding interactions between eukaryotically expressed HRS and ligands of TLR2 (FSL-1), TLR4 (LPS), or RAGE (HMGB1) were assessed via standard solid phase ELISA following co-incubation of soluble HRS or TLR/RAGE ligands with defined concentrations of pre-bound substrate antigen.  As an alternative method for detection of HRS-TLR ligand complex formation, HRS and specified ligands were pre-incubated in solution for 2 hours prior to plating in BSA-blocked ELISA wells and subsequent probing with anti-HRS serum.  The ability of these complexes to signal via defined TLRs was assessed through stimulation of TLR-transfected HEK23 cells and measurement of resulting IL-8 production.

Results:  ELISA-based experiments demonstrated that both human and murine HRS expressed in a eukaryotic system bound HMGB1 in dose-dependent fashion.  Similarly, the TLR2 ligand FSL-1 formed complexes with baculovirus-expressed HRS that inhibited subsequent binding of HRS to BSA-blocked ELISA wells.  LPS also readily formed complexes with baculovirus-expressed HRS, as evidenced by diminished ELISA-based binding of these complexes relative to HRS alone.  Comparative ELISAs employing a biotinylated LPS/streptavidin-HRP probe system and various substrate antigens demonstrated that HRS bound LPS with 10-100 fold greater affinity/aviditiy than did ovalbumin or control autoantigens such as baculovirus-expressed Ro52.  Corresponding to these heightened interactions with the TLR ligands LPS and FSL-1, complexes of HRS-LPS and HRS-FSL-1 synergistically activated HEK293 cells transfected with TLR4 and TLR2, respectively—producing levels of IL-8 which significantly exceeded those induced by HRS or ligand alone.

Conclusion: HRS preferentially binds various ligands of innate immune receptors with high affinity, resulting in synergistic activation of TLR2- and TLR4-mediated signaling pathways that likely contribute to human idiopathic inflammatory myopathy as well as our model of HRS-induced myositis.  Of note, these findings support a more general paradigm in which autoantigens such as HRS bind endogenous/exogenous alarmins to activate innate immune signaling cascades that enhance immunogenicity and prime subsequent adaptive autoimmune responses.


Disclosure:

I. Fernandez,
None;

L. Harlow,
None;

W. Ridgway,
None;

D. P. Ascherman,
None.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/physical-interactions-between-histidyl-trna-synthetase-and-endogenousexogenous-alarmins-enhance-immunogenicity-in-a-model-of-antigen-induced-myositis/

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