CX3CR1 knockout aggravates Coxsackievirus B3-induced myocarditis

Studies on inflammatory disorders elucidated the pivotal role of the CX3CL1/CX3CR1 axis with respect to the pathophysiology and diseases progression. Coxsackievirus B3 (CVB3)-induced myocarditis is associated with severe cardiac inflammation, which may progress to heart failure. We therefore investigated the influence of CX3CR1 ablation in the model of acute myocarditis, which was induced by inoculation with 5x105 plaque forming units of CVB3 (Nancy strain) in either CX3CR1-/- or C57BL6/j (WT) mice. Seven days after infection, myocardial inflammation, remodeling, and titin expression and phosphorylation were examined by immunohistochemistry, real-time PCR and Pro-Q diamond stain. Cardiac function was assessed by tip catheter. Compared to WT CVB3 mice, CX3CR1-/- CVB3 mice exhibited enhanced left ventricular expression of inflammatory cytokines and chemokines, which was associated with an increase of immune cell infiltration/presence. This shift towards a pro-inflammatory immune response further resulted in increased cardiac fibrosis and cardiomyocyte apoptosis, which was reflected by an impaired cardiac function in CX3CR1-/- CVB3 compared to WT CVB3 mice. These findings demonstrate a cardioprotective role of CX3CR1 in CVB3-infected mice and indicate the relevance of the CX3CL1/CX3CR1 system in CVB3-induced myocarditis.


Introduction
Viral myocarditis is a cardiac disorder characterized by cardiac inflammation, which is often caused by cardiotropic viruses like Coxsackievirus group B type 3 (CVB3) and can progress to dilated cardiomyopathy (DCM) and congestive heart failure [1]. With respect to CVB3, a direct cardiomyocyte injury and subsequent long-term inflammatory reaction belong to the discussed mechanisms involved in the CVB3-induced pathogenesis [2,3]. However, the exact pathogenesis of CVB3-induced myocarditis needs still a better understanding [4] to find efficient therapeutic options counteracting the virus-induced inflammatory response [5].
Chemokine-induced migration of inflammatory cells plays a crucial role during cardiac inflammation [6][7][8]. Among the chemokine super-family, fractalkine (CX3CL1) exists in two distinct forms. The membrane-bound form serves as an adhesion protein. The soluble molecule has chemoattractant properties and is proteolytically cleaved from the cell membraneanchored form of fractalkine [9,10]. Cellular sources of CX3CL1 include endothelial cells, epithelial cells, dendritic cells, macrophages and cardiomyocytes [9,11]. Both chemotaxis and adhesion are mediated by the G-protein-coupled receptor CX3CR1 [12], which is mainly expressed on natural killer cells, some T cell populations, dendritic cells and monocytes [10]. Interestingly, soluble CX3CL1 attracts natural killer cells, T cells, and dendritic cells and inhibits the function of the monocyte chemoattractant protein-1 (MCP-1) [10,13].
In previous studies, the CX3CL1/CX3CR1 system has been shown to be involved in the pathophysiology of cardiovascular disorders including heart failure [14,15] and inflammatory cardiomyopathy [9]. CX3CL1/CX3CR1 is also of relevance in the pathogenesis of other inflammatory disorders such as glomerulonephritis [16], rheumatoid arthritis [17] and cardiac allograft rejection [18]. Fractalkine and its receptor CX3CR1 have been shown to exert detrimental effects, since neutralization of the chemokine improved cardiac function after myocardial infarction [19] and inhibition of the respective receptor reduced atherosclerosis in mice [20]. Besides these findings, there are also data indicating a protective role of CX3CR1 since the loss of this receptor results in higher liver fibrosis in a model of hepatic fibrosis [21] and increased accumulation of inflammatory monocytes in gliomagenesis [22]. Abovementioned findings indicate the complexity of the CX3CL1/CX3CR1 system.
Since CX3CR1 is involved in inflammatory disorders, cardiovascular diseases and viral infection, and due to the lack of data regarding the role of CX3CR1 in viral experimental myocarditis, we aimed to investigate the pathophysiological role of CX3CR1 in experimental CVB3-induced acute myocarditis.

Induction of myocarditis and hemodynamic measurements
Six week-old male C57BL6/j mice (further named as WT; provided by the Forschungseinrichtung für experimentelle Medizin (FEM), Berlin, Germany) and CX3CR1 deficient mice (further named as CX3CR1 -/-, provided by I. Hamann and C. Infante-Duarte [23]) were randomly divided into 4 groups (n = 7-12 per group). The experimental groups consisted of WT, WT CVB3, CX3CR1 -/-, and CX3CR1 -/-CVB3. Mice were either treated with 5×10 5 plaque forming units of CVB3 (Nancy strain) or with saline. One week after infection, all animals were anesthetized (Urethane 0.8-1.2 g/kg i.p., Sigma, Germany; Buprenorphin 0.05 mg/kg i.p. Essex Pharma, Germany), intubated, and artificially ventilated with a rodent ventilator type 7025 (Ugo Basile, Comerio VA, Italy). Via a tip catheter (1.2F) system, indices of systolic and diastolic left ventricular function were recorded as described previously [2,24]. After determination of the cardiac function, mice were euthanized by cervical dislocation and the left ventricle (LV) and spleen were excised, immediately snap frozen in liquid nitrogen and stored at -80˚C for molecular biology and immunohistological analyses. This investigation conforms to the European principles of laboratory animal care (Directive 2010/63/EU) and was approved by the local ethical committee (Landesamt für Gesundheit und Soziales, Berlin, Nr: G0313/09). Mice were kept under standard conditions (1212 h light-dark cycle, 20-24˚C), with unlimited access to water and food. After CVB3 infection, mice were daily monitored and scored according the following criteria: 0 = no disease; 1 = weight loss; 2 = reduced activity; 3 = apathy and severe signs of heart failure.

RNA isolation, cDNA synthesis and gene expression analysis
For total RNA isolation from murine tissue, the TRIzol 1 (Life Technologies GmbH, Darmstadt, Germany) method was used followed by cDNA synthesis with the High Capacity Kit (Life Technologies GmbH). To assess the relative mRNA expression of the target genes in murine LVs and spleens, real-time PCR was performed on a QuantStudioTM 6 Flex (Life Technologies) using gene expression assays from Life Technologies ( Table 1). All data were normalized to the housekeeping genes 18S as endogenous controls (Table 1) and are expressed using the 2 -ΔCt formula followed by a normalization to the WT group, which was set as 1. CVB3 mRNA expression was detected using the forward primer and the reverse primer at a final concentration of 60 ng/μl and a FAM-labelled MGB probe at a final concentration of 5 pM (TIB Molbiol, Berlin Germany; Table 1).

All-titin phosphorylation by Pro-Q Diamond stain
The composition as well as the phosphorylation state of the cardiac titin isoform N2B was determined using Pro-Q Diamond phospho-protein stain as described elsewhere [26]. To preserve the endogenous phosphorylation state of the proteins, frozen tissues from LV mouse hearts were solubilized and treated as described for titin isoform separation. The gels were stained for 1 h with Pro-Q Diamond phosphoprotein stain. Fixation, washing and de-staining were performed according to the manufacturer's guidelines. To assess total protein content, gels were stained overnight with SYPRO Ruby. Staining was visualized using the LAS-4000 Image Reader (460 nm/605 nm Ex/Em; 2 s illumination) and signals were analyzed with Multi Gauge V3.2 and AIDA software.

Statistical analysis
Statistical analysis was performed using GraphPad Prism 5.0 software (GraphPad Software, La Jolla, CA). Data are expressed as the mean ± SEM. Ordinary one-way ANOVA was performed to compare parametric data, whereas non-parametric data were compared with Kruskal-Wallis. Both analyses were followed by a post hoc test. Differences were considered statistically significant at a value of p<0.05.

Discussion
In the present study, we could demonstrate that CX3CR1 plays a critical role in the pathogenesis of CVB3-induced myocarditis. This follows from the findings that CX3CR1 -/-CVB3 mice exhibited an exacerbated CVB3-induced myocarditis as shown by a higher LV MCP-1 and CCR2 expression, more cardiac infiltrates, and higher cytokine levels versus CVB3-infected WT mice. In addition, cardiac fibrosis, apoptosis and CVB3 presence were more pronounced and titin was dysregulated in CX3CR1 -/-CVB3 mice compared to WT CVB3 mice.
Although the exact pathogenesis of CVB3-induced myocarditis is still a matter of debate [4], chemokine-induced cardiac migration of inflammatory mononuclear cells is considered to be the initial step [7]. Nevertheless, the role of the chemokine receptor CX3CR1 has not been unraveled yet in experimental CVB3-induced myocarditis. Accumulating evidence demonstrates that the CX3CL1/CX3CR1 axis plays a role in cardiac disorders. CVB3-positive inflammatory cardiomyopathy patients showed induced CX3CL1 expression in endomyocardial biopsies and increased CX3CR1 expression in peripheral blood mononuclear cells [9]. This finding encouraged us to examine the role of CX3CR1 in CVB3-induced myocarditis.
In agreement with the abovementioned findings in CVB3-positive inflammatory cardiomyopathy patients [9], we observed increased CX3CL1 mRNA and protein expression levels in the LV of CVB3-infected versus control mice. Since CX3CL1 is expressed by CD68 and CD11b monocytes/macrophages and cardiac fibroblasts (S1 Fig), the upregulation in LV CX3CL1 expression in CVB3-infected mice can partly be explained by the increase in monocytes/macrophages (Fig 2) and cardiac fibroblast presence (Fig 5) in myocarditis mice. Conform to the systemic increased CX3CR1 expression in peripheral blood mononuclear cells of CVB3-positive inflammatory cardiomyopathy patients [9], an upregulation in CX3CR1 mRNA expression was found in the spleen of CVB3-infected myocarditis compared to control mice. However, CX3CR1 LV mRNA levels were unchanged among CVB3 and control mice. Furthermore, we showed an increased expression of the chemokine MCP-1 and its receptor CCR2 in cardiac and splenic tissue of CVB3-induced myocarditis versus control mice. This is in further harmony with the MCP-1 findings in EMBs and peripheral blood mononuclear cells of CVB3-positive inflammatory cardiomyopathy patients [9]. Both MCP-1 and CCR2 play a pivotal in the CVB3-induced myocarditis pathogenesis [7]. In addition, the expression of Ly6C-positive monocytes was also increased in the LVs of CVB3-infected mice. Interestingly, CX3CR1 -/-CVB3 mice exhibited higher LV MCP-1 and CCR2 expression levels, but no . Bar graphs represent the mean ± SEM. Statistical analysis was performed by One-way ANOVA or the Kruskal-Wallis test. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 with n = 7-12 per group.
https://doi.org/10.1371/journal.pone.0182643.g002 CX3CR1 CVB3-induced myocarditis further increase in LV CX3CL1 expression compared to WT CVB3 mice. Furthermore, the increased LV MCP-1 and CCR-2 levels were also accompanied by enhanced Ly6C expression and a higher presence of CD68 positive cells. The data concerning LV MCP-1 and Ly6C are consistent with other findings where loss of CX3CR1 increased the accumulation of inflammatory monocytes [22], whereas the expression of CCR2 and CD68 positive cells in CX3CR1 -/mice has not been elucidated before. Additionally, we observed an induction of the adhesion molecules VCAM-1 and ICAM-1 in the LV, which is in harmony with the increased levels of MCP-1 and CCR2, which are triggers of these two adhesion molecules [34].
Basically, these observations suggest that under experimental CVB3 conditions, the infiltration of pro-inflammatory monocytes dominates over the invasion of anti-inflammatory
https://doi.org/10.1371/journal.pone.0182643.g003   . The composition as well as the phosphorylation state of the cardiac titin isoform N2B was determined using Pro-Q Diamond phospho-protein stain. In contrast to N2B phosphorylation the cardiac titin isoform composition is different in CX3CR1 -/-CVB3 mice to WT CVB3 mice, respectively. Bar graphs represent the mean ± SEM. Statistical analysis was performed by One-way ANOVA or the Kruskal-Wallis test. *p<0.05, ***p<0.001 with n = 7-12 per group.
In harmony with the increased inflammatory cell presence in CX3CR1 -/-CVB3 versus WT CVB3 mice, we demonstrated enhanced LV cytokine expression including IL-6, IL-1β, TNF-α and IFN-γ in CX3CR1 -/-CVB3 compared to WT CVB3 mice. This is in alignment with other findings, where the loss of CX3CR1 -/led to enhanced IL-6, IL-1β, TNF-α, and IFN-γ levels and where IL-1β is stated as a canonical inducer of MCP-1 [22,35]. Additionally, the enhanced presence of CD3+ and CD4+ lymphocytes in CX3CR1 -/-CVB3 mice may also account for the enhanced inflammatory mediator levels.
Besides the detrimental CVB3-induced inflammatory effect, also oxidative stress leads to an aggravation of myocardial injury [37] and thus exacerbates inflammation [38]. No changes were found in the expression of NOX1 and NOX4, which comprise two of the seven NADPH oxidases members [39] and are a major source of ROS [40]. However, iNOS and eNOS, the two main members of the NO family synthases [41], were differently expressed in CX3CR1 -/-CVB3 mice compared to WT CVB3 animals: whereas eNOS was downregulated in CX3CR1 -/-  After inoculation with CVB3 CX3CR1 -/mice show declined heart function as indicated by (A) reduced max. LV pressure (LVP max ), (B) reduced LV contractility (dP/dt max ) and (C) decreased LV relaxation (dP/dt min ). Bar graphs represent the mean ± SEM. Statistical analysis was performed by One-way ANOVA or the Kruskal-Wallis test. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 with n = 7-12 per group.
https://doi.org/10.1371/journal.pone.0182643.g008 CX3CR1 CVB3-induced myocarditis CVB3 versus WT CVB3 mice, iNOS expression was increased in those mice. This suggests a higher oxidative stress in the LV of CVB3 mice deficient for CX3CR1 compared to WT CVB3 mice, which might underlie the increased viral presence in CX3CR1 -/-CVB3 versus WT CVB3 mice since oxidative stress and subsequent cardiomyocyte apoptosis contribute to viral progeny release [2]. Along with this hypothesis, the Bax/Bcl2 ratio, an important marker for cardiomyocyte apoptosis as consistently shown by Spillmann et al. [29], Leri et al. [30,31], as well as by Van Linthout et al. [32], was higher in CX3CR1 -/-CVB3 mice compared to WT CVB3 mice.
The more pronounced cardiac fibrosis in CVB3-infected CX3CR1 -/versus CVB3 WT mice, as reflected by an enhancement of pro-fibrotic TGF-β1 [27] and collagen I and III expression, is in line with mouse CX3CR1 -/models of liver or kidney fibrosis [42,43]. Since TGF-β is produced by infiltrating monocytes and T cells [44,45], these findings suggest that the enhanced infiltration of monocytes and T cells in CX3CR1 -/-CVB3 mice underlie the raised expression of TGF-β, leading to more myofibroblasts and thus more collagen I and III in CX3CR1 -/-CVB3 versus WT CVB3 mice, further corroborating the link between cardiac inflammation and cardiac fibrosis [27,46].
The giant myofilament titin influences cardiac contractility and relaxation [28]. Two main titin isoforms exist: N2B and N2BA, of which among others impaired N2B phosphorylation leads to cardiac dysfunction [26]. The present study revealed no changes between WT CVB3 and CX3CR1 -/-CVB3 mice in the phosphorylation state of N2B, while we could demonstrate a lower N2B titin isoform expression in CX3CR1 -/-CVB3 mice. Mutations in the N2B isoform have been shown to be associated with DCM and hypertrophic CM in humans [47], and targeted deletion of the N2B region in mice resulted in diastolic dysfunction [48]. Based on these data, we assume that besides the accumulation of collagens, the lower N2B isoform expression might explain the reduced cardiac function, as displayed by a reduction of the systolic and diastolic parameters dP/dt max , and dP/dt min in CX3CR1 -/mice compared to the WT infected animals. A further explanation for the impaired cardiac function in CX3CR1 -/-CVB3 mice versus WT CVB3 mice might be the increased apoptosis of cardiomyocytes, as suggested by the higher Bax/Bcl-2 ratio in CX3CR1 -/-CVB3 versus WT CVB3 mice [29]. Overall, cardiac function was determined with a tip catheter. In contrast to a non-invasive method as echocardiography, this method only allows the measurement at a single time point, which is hence a limitation of the study.

Conclusion
Our observations allow us to conclude that CX3CR1 emerges to be cardioprotective in the pathogenesis of CVB3-induced myocarditis since CX3CR1 -/mice were more susceptible to CVB3 infection as WT animals. A functional CX3CR1 seems to be critical for the control of MCP-1/CCR2-mediated inflammatory cell invasion and the related inflammatory response, apoptosis, and CVB3 presence. Nevertheless, other studies demonstrated a controversial role of CX3CR1, which emphasizes the complexity of the CX3CL1/CX3CR1 system and its potential difference depending on the pathogenic disorder. Finally, we conclude that the modulation of the CX3CL1/CX3CR1 axis could be a new important target in the treatment of CVB3-induced myocarditis.