Protease-activated receptor 4 protects mice from Coxsackievirus B3 and H1N1 influenza A virus infection
Introduction
Protease-activated receptors (PARs) are a family of 4 G-protein coupled receptors (PAR1-4) [1], [2]. Serine proteases and matrix metalloproteinases (MMPs), including immune cell-derived proteases and coagulation proteases, can either activate or inactivate PARs depending on the cell type and post-translational modification of the PAR [3]. For instance, thrombin activates both PAR1 and PAR4 as well as human PAR3. Human platelets express PAR1 and PAR4, whereas PAR4 is the functional thrombin receptor on mouse platelets since they lack PAR1 [4], [5]. A PAR1 inhibitor is FDA-approved to reduce platelet activation in patients in combination with standard anti-platelet dual therapy [6]. However, chronic PAR1 inhibition can increase the risk of hemorrhagic strokes potentially due to a protective role of PAR1 on vascular integrity [6].
We and others showed that PARs are modulators of antiviral responses to infections with the RNA viruses Coxsackievirus B3 (CVB3) and H1N1 influenza A virus (H1N1 IAV) [3], [7], [8], [9], [10]. For instance, we and others reported that PAR1 enhances and PAR2 inhibits toll-like receptor (TLR3) dependent anti-viral immune responses [7], [8], [9], [11], [12]. Furthermore, we found that thrombin inhibition reduced immune responses to a TLR3 agonist in mice and increased CVB3 myocarditis [8], [11]. In line with our findings, a recent study showed that thrombin inhibition reduced TLR3-dependent responses in human bronchial epithelial cells [12].
Importantly, there are some conflicting data regarding the contribution of PARs to the outcome in viral infection of the lung [1]. We observed that PAR1-deficiency increased H1N1 infection of mice [8], whereas another group showed a protection in PAR1−/− mice after IAV infection [10]. In addition, we and others found that PAR2−/− mice were protected from H1N1 infection [9], [13]. However, another group found that PAR2−/− mice exhibited more severe disease after H1N1 IAV infection [14]. The reasons for these opposing data are unclear and are the object of ongoing investigations.
Recent studies have focused on PAR4 as a druggable target to reduce platelet activation and thrombosis [15], [16]. Like PAR1, many cells other than platelets express PAR4, including cardiac myocytes (CMs) and immune cells [3], [17]. Interestingly, two studies have suggested that PAR4 antagonism might promote cardiac protection by inhibiting cardiomyocyte apoptosis and pathologic platelet activation in ischemia reperfusion injury [15], [17]. Furthermore, a recent study showed that repeated administration of a PAR4 inhibitor (pepducin P4pal-10) in wild-type (WT) mice daily between day 2 and 4 after infection resulted in reduced H1N1 IAV infection-mediated lung inflammation and improved survival [18].
In the study here, we used global PAR4 deficient mice, to investigate the role of PAR4 in two different RNA virus infections of the heart and lung.
Section snippets
Mice
Adult PAR4−/− (ΔPAR4) and WT (PAR4+/+) mice on the C57Bl/6 background were used for this study [5]. All animal experiments were performed in accordance with the guidelines of the animal care and use committee of the UNC at Chapel Hill and complies with National Institutes of Health guidelines.
In vitro experiments
Bone marrow-derived macrophages (BMM) were generated from WT and ΔPAR4 mouse bone marrow using the L929-conditioned media method [11]. Mouse embryonic fibroblasts (MEFs) and murine CMs were isolated from
PAR4 activation increases CXCL10 expression in BMMs and MEFs
In previous studies, we showed that TLR3-depdendent CXCL10 expression in response to the dsRNA mimetic poly I:C was enhanced by co-stimulation of PAR1 on macrophages and cardiac fibroblasts [8], [11]. Here, we investigated if PAR4 activation also enhances poly I:C induced CXCL10 expression in BMMs and MEFs. First, we analyzed poly I:C induction of CXCL10 protein expression in BMMs derived from WT and ΔPAR4 mice. Poly I:C but not the PAR4 activating protease thrombin induced CXCL10 protein
Discussion
In this study, we used global PAR4 deficient mice to investigate the role of PAR4 in two human disease relevant virus infection models CVB3 myocarditis and H1N1 IAV infection. We observed that PAR4 protects the heart and lung against CVB3 and H1N1 IAV infection, respectively. As previously shown for PAR1, we observed that PAR4 enhanced TLR3:IRF3-dependent IFN responses by increasing IFNβ and CXCL10 expression in vivo and in vitro. We think that PAR1 and PAR4 use a common pathway in positively
Study limitations
We cannot finally exclude the potential contribution of PAR4 on platelets in H1N1 IAV or CVB3 infection. The use of a conditional PAR4 knock-out mouse will help to analyze the cell-specific contribution of PAR4 in various diseases models including H1N1 IAV infection and CVB3 myocarditis. Importantly, additional studies with PAR4 antagonists in WT and ΔPAR4 mice are needed.
Grant support
The study was supported by the NIH to N. Mackman (HL119523) and S. Antoniak (HL142799).
Declaration of Competing Interest
None.
Acknowledgment
We want to thank Ying Zhang for excellent technical assistance.
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