Abstract 314: D-4F Decreases Myocardial Inflammation in a Model of Murine Scleroderma by Decreasing Interferon Regulating Factor-5 Levels

Abstract

D-4F, an ApoAI mimetic, has potent anti-inflammatory properties. It is reported to alter intracellular signaling cascades such as the Jak/Stat pathway which many consider to play an important role in autoimmunity. Interferon regulating factor 5 (IRF5) is a transcription factor that has recently been shown to serve as a critical switch in controlling macrophage phenotype. Interestingly, IRF5 is downstream of Jak/Stat. Previously, we showed that D-4F decreases inflammation and fibrosis in the myocardium of tight skin mice (Tsk -/+ ), an established murine model of scleroderma. As D-4F alters Jak/Stat signaling we hypothesize D-4F may inhibit IRF5 to decrease inflammation and fibrosis in the hearts of Tsk -/+ mice. To test this hypothesis we performed 1) western blot analysis for IRF5 on myocardium of C57Bl/6J mice, Tsk -/+ mice, and Tsk -/+ mice treated with D4F; 2) immunoblots (IBs) for phosphoserine and ubiquitin on immunopreciptiates (IPs) for IRF5; 3) immunohistochemistry on myocardial sections for IRF5; and, determined D-4F interactions with IRF5 by biolayer interferometry (BLI). Our studies reveal that IRF5 levels in Tsk -/+ myocardium were virtually the same as in the myocardium of C57Bl/6J mice and that D-4F treatments reduced IRF5 protein levels in the hearts of Tsk -/+ mice by 70 % (p<0.002, n=3). IPs/IBs studies showed that IRF5 phosphoserine levels, an index of activation, in Tsk -/+ mice were essentially the same as in C57BL/6J mice. D-4F treatments however, markedly reduced phosphoserine levels on IRF5 in Tsk -/+ myocardium (∼50%, p<0.002, n=3). In addition, IRF5 ubiquination, which is required for transport into the nucleus, was increased in Tsk -/+ mice by ∼3-fold with no signs of degradation, compared to the levels in C57BL/6J mice. In contrast, D-4F decreased IRF5 ubiquitination in Tsk -/+ mice by ∼50% with signs of degradation. Interestingly, BLI studies suggested that D-4F binds to IRF5 irreversibly. Taken together these data suggest D-4F decreases myocardial inflammation and fibrosis by decreasing total IRF5 protein and IRF5 activation.