Elsevier

Cellular Immunology

Volume 344, October 2019, 103949
Cellular Immunology

Protease-activated receptor 4 protects mice from Coxsackievirus B3 and H1N1 influenza A virus infection

https://doi.org/10.1016/j.cellimm.2019.103949Get rights and content

Highlights

  • Protease-activated receptor 4 (PAR4) reduces Coxsackievirus B3 (CVB3) myocarditis and H1N1 influenza A virus (IAV) infection.

  • PAR4−/− mice exhibited increased virus load and reduced innate immune response in the heart after CVB3 infection.

  • PAR4−/− mice exhibit more severe myocarditis and heart function impairment.

  • PAR4−/− mice exhibited more pronounced cellular and cytokine immune responses in the lung after IAV infection.

  • More severe H1N1 IAV infection in PAR4−/− mice led to more lung injury and death compared to WT mice.

Abstract

PAR4 is expressed by a variety of cells, including platelets, cardiac, lung and immune cells. We investigated the contribution of PAR4 to viral infections of the heart and lung. Toll-like receptor (TLR) 3-dependent immune responses were analyzed after co-stimulation of PAR4 in murine bone-marrow derived macrophages, embryonic fibroblasts and embryonic cardiomyocytes. In addition, we analyzed Coxsackievirus B3 (CVB3) or H1N1 influenza A virus (H1N1 IAV) infection of PAR4−/− (ΔPAR4) and wild-type (WT) mice. Lastly, we investigated the effect of platelet inhibition on H1N1 IAV infection. In vitro experiments revealed that PAR4 stimulation enhances the expression of TLR3-dependent CXCL10 expression and decreases TLR3-dependent NFκB-mediated proinflammatory gene expression. Furthermore, CVB3-infected ΔPAR4 mice exhibited a decreased anti-viral response and increased viral genomes in the heart leading to more pronounced CVB3 myocarditis compared to WT mice. Similarly, H1N1 IAV-infected ΔPAR4 mice had increased immune cell numbers and inflammatory mediators in the lung, and increased mortality compared with infected WT controls. The study showed that PAR4 protects mice from viral infections of the heart and lung.

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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      The role of platelets in this pathology is interesting, as the inhibition of thrombin-mediated activation by argatroban or PAR4 antagonism, the principal thrombin receptor on mouse platelets, leads to improved outcomes following severe influenza infection [173]. However, when mice were infected with mild to moderate Influenza infection, a global deficiency of PAR4 worsens disease suggesting PAR4 may be protective during influenza infection [174]. This creates an interesting dichotomy whereby the same receptor is both protective and pathogenic depending on the viral load.

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