Sex-specific signaling through Toll-Like Receptors 2 and 4 contributes to survival outcome of Coxsackievirus B3 infection in C57Bl/6 mice

Coxsackievirus B3 (CVB3) induces myocarditis, an inflammatory heart disease, which affects men more than women. Toll-like receptor (TLR) signaling has been shown to determine the severity of CVB3-induced myocarditis. No direct role for signaling through TLR2 had been shown in myocarditis although published studies show that cardiac myosin is an endogenous TLR2 ligand and stimulates pro-inflammatory cytokine expression by dendritic cells in vitro. The goal of this study is to determine which TLRs show differential expression in CVB3 infected mice corresponding to male susceptibility and female resistance in this disease. Male and female C57Bl/6 mice were infected with 102 PFU CVB3 and killed on day 3 or 6 post infection. Hearts were evaluated for virus titer, myocardial inflammation, and TLR mRNA expression by PCR array and microarray analysis. Splenic lymphocytes only were evaluated by flow cytometry for the number of TLR+/CD3+, TLR+/CD4+, TLR+F4/80+ and TLR+/CD11c+ subpopulations and the mean fluorescence intensity to assess upregulation of TLR expression on these cells. Mice were additionally treated with PAM3CSK4 (TLR2 agonist) or ultrapure LPS (TLR4 agonist) on the same day as CVB3 infection or 3 days post infection to confirm their role in myocarditis susceptibility. Despite equivalent viral titers, male C57Bl/6 mice develop more severe myocarditis than females by day 6 after infection. Microarray analysis shows a differential expression of TLR2 at day 3 with female mice having higher levels of TLR2 gene expression compared to males. Disease severity correlates to greater TLR4 protein expression on splenic lymphocytes in male mice 3 days after infection while resistance in females correlates to preferential TLR2 expression, especially in spleen lymphocytes. Treating male mice with PAM reduced mortality from 55% in control CVB3 infected animals to 10%. Treating female mice with LPS increased mortality from 0% in control infected animals to 60%. CVB3 infection causes an up-regulation of TLR2 in female and of TLR4 in male mice and this differential expression between the sexes contributes to disease resistance of females and susceptibility of males. While previous reports demonstrated a pathogenic role for TLR4 this is the first report that TLR2 is preferentially up-regulated in CVB3 infected female mice or that signaling through this TLR directly causes myocarditis resistance.