Protease-activated receptor-2 regulates the innate immune response to viral infection in a coxsackievirus B3-induced myocarditis

Abstract

Objectives: This study sought to evaluate the role of protease-activated receptor-2 (PAR2) in coxsackievirus B3 (CVB3)-induced myocarditis.

Background: An infection with CVB3 leads to myocarditis. PAR2 modulates the innate immune response. Toll-like receptor-3 (TLR3) is crucial for the innate immune response by inducing the expression of the antiviral cytokine interferon-beta (IFNβ).

Methods: To induce myocarditis, wild-type (wt) and PAR2 knockout (ko) mice were infected with 10(5) plaque-forming units CVB3. Mice underwent hemodynamic measurements with a 1.2-F microconductance catheter. Wt and PAR2ko hearts and cardiac cells were analyzed for viral replication and immune response with plaque assay, quantitative polymerase chain reaction, Western blot, and immunohistochemistry.

Results: Compared with wt mice, PAR2ko mice and cardiomyocytes exhibited a reduced viral load and developed no myocarditis after infection with CVB3. Hearts and cardiac fibroblasts from PAR2ko mice expressed higher basal levels of IFNβ than wt mice did. Treatment with CVB3 and polyinosinic:polycytidylic acid led to higher IFNβ expression in PAR2ko than in wt fibroblasts and reduced virus replication in PAR2ko fibroblasts was abrogated by neutralizing IFNβ antibody. Overexpression of PAR2 reduced the basal IFNβ expression. Moreover, a direct interaction between PAR2 and Toll-like receptor 3 was observed. PAR2 expression in endomyocardial biopsies of patients with nonischemic cardiomyopathy was positively correlated with myocardial inflammation and negatively with IFNβ expression and left ventricular ejection fraction.

Conclusions: PAR2 negatively regulates the innate immune response to CVB3 infection and contributes to myocardial dysfunction. The antagonism of PAR2 is of therapeutic interest to strengthen the antiviral response after an infection with a cardiotropic virus.

Keywords: AP; CAR; CCL; CD; CVB3; DAF; DMEM; Dulbecco modified Eagle medium; FBS; HL; IFNβ; IFNγ; IL; LVEF; P-Stat1; PAR2; PBS; PCR; TLR; TNFα; Toll-like receptor; Toll-like receptor 3; activating peptide; chemokine ligand; cluster of differentiation; coxsackievirus B3; coxsackievirus-adenovirus receptor; decay-accelerating receptor; double-stranded ribonucleic acid; dsRNA; fetal bovine serum; human leukocyte; interferon beta; interferon gamma; interferon-beta; interleukin; knockout; ko; left ventricular ejection fraction; mRNA; messenger ribonucleic acid; mock plasmid; myocarditis; p.i.; pVitro; phosphate-buffered saline; phospho-signal transducer and activator of transcription-1; poly(I:C); polyinosinic: polycytidylic acid; polymerase chain reaction; post-infection; protease-activated receptor 2; protease-activated receptor-2; tumor necrosis factor-alpha; wild type; wt.

Figure 1. Virus Distribution and Replication in wt and PAR2ko Mice at 2, 4, and 8 Days p.i. With CVB3

(A) The viral genome in pancreas, (B) liver, and (C) spleen was analyzed with quantitative polymerase chain reaction (qPCR) at 2 and 4 days (d) post-infection (p.i.). (D) Plaque assays of wild type (wt) and protease-activated receptor 2 (PAR2) knockout (ko) hearts at 4 and 8 days p.i. Each symbol represents the coxsackievirus B3 (CVB3) messenger ribonucleic acid (mRNA) expression and plaque forming units (pfu) from an individual mouse. (E) In situ hybridization of CVB3 plus strand genome of wt and PAR2ko mice at 4 and 8 day p.i. (F) In situ hybridization of the minus or replicating strand in hearts at 8 days p.i. (E, F) There are 6 to 8 mice for each time point. Scale bar = 100 μm. *p < 0.05 for PAR2ko versus respective wt.

Figure 2. Up-Regulation of IFNβ Expression and IFNβ-Related Pathways in PAR2ko Mice

Interferon-beta (IFNβ) expression from infected and control (co) (A) pancreas, (B) livers, and (C) hearts measured by qPCR. (A, B) Expression levels are fold to wt at 2 days p.i. (mean was set to 1). (C) Expression levels are fold to wt co (mean was set to 1). Data are mean ± SEM (n = 6 to 8 mice/group). *p < 0.05 for PAR2ko versus wt, †p < 0.05 for PAR2ko versus co mice of the respective genotype and p value according to nonparametric Brunner modeling of longitudinal data. (D) Representative Western blot from infected and co hearts shows phospho-signal transducer and activator of transcription-1 (P-Stat1) and beta-actin (β-actin) at 8 days p.i. Abbreviations as in Figure 1.

Figure 3. Less Immune Cell Infiltration and Cytokine Expression in PAR2ko Hearts Post-Infection

Hematoxylin and eosin staining of wt and PAR2ko cardiac tissue from co mice and mice at 8 days p.i. (A) Arrows indicate immune cell infiltration and tissue damage after CVB3 infection (scale bar = 100 μm). (B) Quantification of staining for CD68+ and (C) CD3+ cells at 8 days p.i. (D to I) Expression of (D) TNFα, (E) IL6, (F) IL1β, (G) CCL2, (H) CCL5, and (I) IFNγ. Expression levels are fold to wt co (mean was set to 1). Data are mean ± SEM (n = 6 to 8 mice/group). *p < 0.05 for PAR2ko versus wt and p value according to nonparametric Brunner modeling of longitudinal data. CCL = chemokine ligand; CD = cluster of differentiation; IL = interleukin; TNF = tumor necrosis factor; other abbreviations as in Figures 1 and 2.

Figure 4. PAR2 Reduces TLR3-Dependent IFNβ and Stat1 Phosphorylation in Cardiac Fibroblasts

(A) IFNβ expression from wt and PAR2ko cardiac fibroblasts infected with 1 multiplicity of infection CVB3 and (B) stimulated with 10 μg/ml polyinosinic:polycytidylic acid (poly[I:C]) for 6 h. (C) CCL5 expression from wt and PAR2ko cardiac fibroblasts stimulated with 10 μg/ml poly(I:C) for 6 h. Expression levels are fold to wt co (mean was set to 1) (n = 3, performed in duplicates). *p < 0.05 for PAR2ko versus wt. †p < 0.05 for PAR2ko versus co cells of the respective genotype and p value according to nonparametric Brunner modeling of longitudinal data. (D to F) Representative Western blots show phosphorylation for Stat1 in wt and PAR2ko fibroblasts (D) before and (E) after poly(I:C) stimulation for 6 h and (F) 24 h (n = 3). Gapdh = glycerinaldehyd-3-phosphat-dehydrogenase; TLR = Toll-like receptor; other abbreviations as in Figures 1 and 2.

Figure 5. PAR2 Reduces TLR3-Dependent IFNβ and Stat1 Phosphorylation in Cardiac Fibroblasts

(A) IFNβ expression in mock plasmid (pVitro)- and PAR2pVitro-transfected PAR2ko fibroblasts. Data are fold to pVitro (mean was set to 1). Data are mean ± SEM (n = 3, performed in duplicates). *p < 0.05 for PAR2ko versus wt. (B) Representative Western blots for phosphorylation of Stat1 in pVitro- and PAR2pVitro-transfected PAR2ko fibroblasts before and after stimulation with 200 μmol/l activating peptide (AP) and/or 10 μg/ml poly(I:C) for 2 and 24 h (n = 3). (C, D) pVitro- or PAR2pVitro-transfected mouse cardiomyocytic cell line (HL)-1 were stimulated with 200 μmol/l AP for 1 h. Representative Western blots show (C) TLR3 and (D) TLR4 after PAR2-immunoprecipitation. Densitometry of (C) TLR3 and (D) TLR4 bands is presented as fold of unstimulated control cells ± SEM. *p < 0.05 (n = 3, performed in duplicates). Abbreviations as in Figures 1, 2, and 4.

Figure 6. Cardiac PAR2 Expression in Patients With Cardiomyopathy Is Associated With Increased Cardiac Inflammation, Decreased IFNβ Expression, and Reduced LVEF

(A) Differences in myocardial IFNβ expression and (B to D) CD3+, leukocyte function-associated antigen (LFA)+ and CD45+ cells dependent on low and high myocardial PAR2 expression. (E, F) Relations between myocardial PAR2 expression and left ventricular ejection fraction (LVEF) or (F) left ventricular end-diastolic diameter (LVEDD). Data are expressed as mean ± SEM. *p < 0.05 low versus high PAR2 expression. Abbreviations as in Figures 1 and 3.