Arthritis & Rheumatism, Volume 63,
November 2011 Abstract Supplement
Abstracts of the American College of
Rheumatology/Association of Rheumatology Health Professionals
Annual Scientific Meeting
Chicago, Illinois November 4-9, 2011.
Role of MyD88-Dependent Toll-Like Receptor Signaling in a Murine Model of Histidyl-tRNA Synthetase-Induced Myositis.
Harlow1, Lisa, Fernandez1, Irina, Zang1, Yunjuan, Soejima2, Makoto, Greidinger3, Eric L., Ascherman3, Dana P.
In previous work, we have demonstrated that IM immunization with Jo-1 induces exuberant muscle inflammation in multiple strains of mice. This phenotype does not require Jo-1-specific TCR recognition and can also occur in the absence of functional TLR4 signaling, suggesting the contribution of other non-TLR4-mediated signaling pathways of the innate immune system.
To determine the contribution of toll-like receptor signaling in a murine model of myositis induced by intramuscular immunization with histidyl-tRNA synthetase (Jo-1)
To examine the potential role of innate signaling pathways beyond TLR4, we immunized B6.MyD88-/- and B6.TLR2-/- mice with a recombinant amino terminal fragment of murine Jo-1 fused to maltose binding protein (MA/MBP=amino acids 1151 of murine Jo-1 linked to maltose binding protein) in the absence of exogenous adjuvant. At Day 17, we obtained quadriceps and hamstring muscle from euthanized mice for subsequent analysis of lymphocytic infiltrates in hematoxylin/eosin stained specimens. As a complement to this in vivo modeling, we assessed the capacity of our recombinant murine Jo-1 fragment and appropriate control proteins to activate TLR2 and TLR4 signaling through in vitro stimulation of TLR2 transfected 293 cells and Limulus Amebocyte Lysate testing, respectively.
MA/MBP immunization of B6.MyD88-/- mice (n=7) failed to replicate the muscle inflammation previously demonstrated in parental C57BL/6 mice, implicating MyD88-dependent TLR signaling in this model system of Jo-1-induced myositis. In vitro assessment of Limulus Amebocyte Lysate activation demonstrated that recombinant Jo-1 proteins/fragments possess endotoxin-like activity (log order greater activity than MBP or the control autoantigen, 70 kDa RNP) capable of activating TLR4. At the same time, in vitro stimulation of TLR2 transfected 293 cells with different versions of recombinant Jo-1 revealed antigen-specific induction of IL-8 secretion relative to MBP and other recombinant autoantigens, supporting a potential role for this alternative MyD88-dependent signaling pathway in modulating the muscle inflammation detectable in the setting of functional TLR4 deficiency. Strikingly, however, IM immunization of B6.TLR2-/- mice (n=10) with MA/MBP yielded robust lymphocytic infiltration of muscle tissue relative to that generated in C57BL/6 WT micefindings that paralleled the relationship between muscle inflammation induced in TLR4-deficient C3H/HeJ and WT C3H/HeOuJ mice.
Collectively, these results demonstrate that our model of Jo-1-induced myositis requires the TLR adaptor molecule MyD88, but is not absolutely dependent on either TLR2- or TLR4-mediated signaling cascades. Future assessment of TLR2-/-/TLR4-/- double knockout mice will further clarify the apparent functional redundancy of these TLR systems (suggested by in vitro stimulation assays) and allow us to rule out the involvement of other, less likely MyD88-dependent TLR signaling pathways.
To cite this abstract, please use the following information:
Harlow, Lisa, Fernandez, Irina, Zang, Yunjuan, Soejima, Makoto, Greidinger, Eric L., Ascherman, Dana P.; Role of MyD88-Dependent Toll-Like Receptor Signaling in a Murine Model of Histidyl-tRNA Synthetase-Induced Myositis. [abstract]. Arthritis Rheum 2011;63 Suppl 10 :823