4F Decreases IRF5 Expression and Activation in Hearts of Tight Skin Mice

Hao Xu,Deron W Jones,Kirkwood A Pritchard,Zhi-Dong Ge,John G Krolikowski,Paul S Pagel,Dorothée Weihrauch,Rajesh Gopalrao Katare

doi:10.1371/journal.pone.0052046

Hao Xu, Deron W Jones + Show 6 more

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https://doi.org/10.1371/journal.pone.0052046

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Abstract

The apoAI mimetic 4F was designed to inhibit atherosclerosis by improving HDL. We reported that treating tight skin (Tsk−/+) mice, a model of systemic sclerosis (SSc), with 4F decreases inflammation and restores angiogenic potential in Tsk−/+ hearts. Interferon regulating factor 5 (IRF5) is important in autoimmunity and apoptosis in immune cells. However, no studies were performed investigating IRF5 in myocardium. We hypothesize that 4F differentially modulates IRF5 expression and activation in Tsk−/+ hearts. Posterior wall thickness was significantly increased in Tsk−/+ compared to C57Bl/6J (control) and Tsk−/+ mice with 4F treatment assessed by echoradiography highlighting reduction of fibrosis in 4F treated Tsk−/+ mice. IRF5 in heart lysates from control and Tsk/+ with and without 4F treatment (sc, 1 mg/kg/d, 6–8 weeks) was determined. Phosphoserine, ubiquitin, ubiquitin K63 on IRF5 were determined on immunoprecipitates of IRF5. Immunofluorescence and TUNEL assays in heart sections were used to determine positive nuclei for IRF5 and apoptosis, respectively. Fluorescence-labeled streptavidin (SA) was used to determine endothelial cell uptake of biotinylated 4F. SA-agarose pulldown and immunoblotting for IRF5 were used to determine 4F binding IRF5 in endothelial cell cytosolic fractions and to confirm biolayer interferometry studies. IRF5 levels in Tsk−/+ hearts were similar to control. 4F treatments decrease IRF5 in Tsk−/+ hearts and decrease phosphoserine and ubiquitin K63 but increase total ubiquitin on IRF5 in Tsk−/+ compared with levels on IRF5 in control hearts. 4F binds IRF5 by mechanisms favoring association over dissociation strong enough to pull down IRF5 from a mixture of endothelial cell cytosolic proteins. IRF5 positive nuclei and apoptotic cells in Tsk−/+ hearts were increased compared with controls. 4F treatments decreased both measurements in Tsk−/+ hearts. IRF5 activation in Tsk−/+ hearts is increased. 4F treatments decrease IRF5 expression and activation in Tsk−/+ hearts by a mechanism related to 4F’s ability to bind IRF5.

Highlights

Scleroderma is an autoimmune disease that is characterized by marked increases in fibrosis of the skin and internal organs in conjunction with enhanced apoptosis [1]
During the course of our studies for our report [1,3], we found the anti-inflammatory effects of 4F on the Tsk2/+ mice to be so profound that it was difficult to attribute all of the effects solely to improvements in high-density lipoprotein (HDL) function
4F treatments markedly decreased Interferon regulating factor 5 (IRF5) expression in Tsk2/+ hearts compared with the levels in hearts isolated from untreated Tsk2/+ mice (Figure 2, lane 3 vs. lane 2)

Summary

Introduction

Scleroderma is an autoimmune disease that is characterized by marked increases in fibrosis of the skin and internal organs in conjunction with enhanced apoptosis [1]. The tight skin (Tsk2/+) mouse is a murine model of systemic scleroderma that replicates many of the features observed in patients suffering from this disease [2]. We showed that Tsk2/+ mice have impaired endothelial vasodilatation and angiogenic responses to VEGF that coincided with marked increases in angiostatin [3]. We showed that the matrix isolated from the hearts of Tsk2/+ mice stimulated endothelial cells (EC) to transition into fibroblasts and assume a more fibrotic phenotype [1]. Our laboratory has observed that do 4F treatments inhibit vascular disease in Tsk2/+

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