Mesenchymal stromal cells inhibit NLRP3 inflammasome activation in a model of Coxsackievirus B3-induced inflammatory cardiomyopathy

Inflammation in myocarditis induces cardiac injury and triggers disease progression to heart failure. NLRP3 inflammasome activation is a newly identified amplifying step in the pathogenesis of myocarditis. We previously have demonstrated that mesenchymal stromal cells (MSC) are cardioprotective in Coxsackievirus B3 (CVB3)-induced myocarditis. In this study, MSC markedly inhibited left ventricular (LV) NOD2, NLRP3, ASC, caspase-1, IL-1β, and IL-18 mRNA expression in CVB3-infected mice. ASC protein expression, essential for NLRP3 inflammasome assembly, increased upon CVB3 infection and was abrogated in MSC-treated mice. Concomitantly, CVB3 infection in vitro induced NOD2 expression, NLRP3 inflammasome activation and IL-1β secretion in HL-1 cells, which was abolished after MSC supplementation. The inhibitory effect of MSC on NLRP3 inflammasome activity in HL-1 cells was partly mediated via secretion of the anti-oxidative protein stanniocalcin-1. Furthermore, MSC application in CVB3-infected mice reduced the percentage of NOD2-, ASC-, p10- and/or IL-1β-positive splenic macrophages, natural killer cells, and dendritic cells. The suppressive effect of MSC on inflammasome activation was associated with normalized expression of prominent regulators of myocardial contractility and fibrosis to levels comparable to control mice. In conclusion, MSC treatment in myocarditis could be a promising strategy limiting the adverse consequences of cardiac and systemic NLRP3 inflammasome activation.

in the innate immune response and is associated with cardiac injury by exacerbation of inflammation and cardiac remodeling 6 . NOD2 activates (NOD)-like receptor pyrin domain-containing 3 (NLRP3) inflammasome and IL-1β processing and secretion 4 , primarily on phagocytic antigen-presenting cells such as macrophages and Bar graphs represent the mean ± SEM of (I) LV caspase 1 activity represented as absorbance at 405 nm and (J) IL-1β protein expression, depicted as pg IL-1ß per µg LV protein, determined in the LV of control, CVB3, control MSC, and CVB3 MSC mice, with n = 5-6/group. dendritic cells (DCs). Activated NLRP3 interacts with the adapter apoptosis-associated speck-like protein containing a C-terminal caspase recruitment domain (ASC), resulting in recruitment of pro-caspase-1 and induction of auto-cleavage of caspase-1 4 . Caspase-1 is known as the inflammatory caspase essential in the maturation of the two highly pro-inflammatory interleukins (IL)−1 family cytokines: IL-1β and IL-18, known to exacerbate the inflammatory responses and induce cardio-depressive effects and cardiac remodeling [7][8][9] . The NLRP3 inflammasome has been identified as a central player in the development of heart diseases. NLRP3 inflammasome activation in resident cardiomyocytes and fibroblasts leads to myocardial dysfunction, while inflammasome inhibition limits inflammation and myocardial fibrosis 10,11 .
CVB3 infection induces NLRP3 inflammasome activation in vitro and in vivo, which inhibition has been shown to significantly alleviate the severity of CVB3-induced myocarditis and to improve cardiac function 12 . The clinical relevance of NOD2 and NLRP3 in CVB3-induced myocarditis follows from recent own findings in CVB3-postive myocarditis patients which show that cardiac NOD2 and NLRP3 expression drops in CVB3-positive patients who eliminated the virus and improved cardiac function over time 13 . Therefore, inhibition of NLRP3 inflammasome activation or IL-1β may be a valuable therapeutic strategy to limit the inflammatory response, which high intensity is a clear negative prognostic marker 14 .
Mesenchymal stromal cells (MSC) are effective immunomodulators of the immune responses [15][16][17] . We have demonstrated that intravenous (i.v.) MSC application has a cardioprotective effect in CVB3-induced and CVB3-infected mice (closed bar) injected with PBS or MSC, expressed as the percentage of total MNCs, with n = 8-9/group. The right panel shows dot plots of NOD2 positive splenocytes of control, CVB3, control MSC, and CVB3 MSC mice, as indicated. (B) Bar graphs represent the mean ± SEM of F4/80 + /NOD2 + in control mice (open bar) and CVB3-infected mice (closed bar) injected with PBS or MSC, expressed as the percentage of total MNCs, with n = 8-9/group and **p < 0.01. The right panel shows dot plots of F4/80 + /NOD2 + -positive splenocytes of control, CVB3, control MSC, and CVB3 MSC mice, as indicated. (C) Bar graphs represent the mean ± SEM of CD49b + /NOD2 + in control mice (open bar) and CVB3-infected mice (closed bar) injected with PBS or MSC, expressed as the percentage of total MNCs, with n = 8-9/group and **p < 0.01. The right panel shows dot plots of CD49b + /NOD2 + -positive splenocytes of control, CVB3, control MSC, and CVB3 MSC mice, as indicated. (D) Bar graphs represent the mean ± SEM of CD11c + /NOD2 + in control mice (open bar) and CVB3-infected mice (closed bar) injected with PBS or MSC, expressed as the percentage of total MNCs, with n = 8-9/group and **p < 0.01. The right panel shows dot plots of CD11c + / NOD2 + -positive splenocytes of control, CVB3, control MSC, and CVB3 MSC mice, as indicated. inflammatory cardiomyopathy since MSC cannot be infected with CVB3, exert anti-viral effects, reduce CVB3-associated cardiomyocyte apoptosis, myocardial fibrosis, inflammation, improve left ventricle (LV) function and induce prominent systemic immunomodulation 18,19 .
Taking into account that the NLRP3 inflammasome is known to play a crucial role in the pathogenesis of CVB3-induced myocarditis and MSC have been shown to inhibit NLRP3 inflammasome activation in macrophages 20 , the aim of the present study was to evaluate the effect of i.v. MSC application on NLRP3 inflammasome activation in murine CVB3-induced myocarditis. We could demonstrate that MSC potently inhibit the NLRP3 inflammasome activation in the heart and mediate systemic immunoregulation via abrogating NLRP3 inflammasome activation and IL-1β secretion in cells of the innate immune system, which could interrupt the inflammatory process amplification and subsequently diminish the adverse cardiac inflammation and dysfunction following myocardial injury.
We have previously demonstrated that MSC exert protective effects in viral myocarditis via release of paracrine factors 18 . Oh et al. 20 have recently shown that MSC efficiently inhibit NLRP3 inflammasome activation in macrophages via secretion of the anti-oxidative protein stanniocalcin (STC-1). Based on this finding and the relevance of oxidative stress in NLRP3 activation 21 , we next investigated the potential contribution of STC-1 in the observed MSC inhibitory effect on CVB3-induced NLRP3 inflammasome activation in HL-1 cells. MSC transfection with STC-1 siRNA decreased STC-1 expression in MSC by 33% (Supplemental Fig. 1A), which was translated in a modest abrogation of the inhibitory effects of MSC on NLRP3 inflammasome activity in HL-1. This follows from the observation that the % of Dil/ASC/IL-1β-positive HL-1 cells was 1.2-fold (p < 0.05) higher in HL-1 cells co-cultured with STC-1 siRNA-transfected MSC compared to HL-1 cells co-cultured with scrambled siRNA-transfected MSC (Supplemental Fig. 1B).
To study whether MSC have an effect on inflammasome activation triggered in a classical manner, we primed HL-1 cells with lipopolysaccharide (LPS) followed by adenosine-triphosphate (ATP) supplementation, which led to an increase in inflammasome activation in HL-1 cells, as evident by a 1.2-fold (p < 0.001) increase in IL-1β-and ASC-double positive HL-1 cells (Supplemental Fig. 2). Supplementation of MSC at the time of LPS priming or at the time of ATP treatment both prominently suppressed the inflammasome formation as evident by a 1.5-fold (p < 0.0001) and 2.6-fold (p < 0.0001) decrease in IL-1β-and ASC-double positive HL-1 cells, respectively (Supplemental Fig. 2).

Mesenchymal stromal cells inhibit Coxsackievirus B3-induced ASC activation in macrophages, natural killer cells, and dendritic cells.
Considering the importance of ASC, which interacts with pro-caspase-1 via a CARD domain resulting in activation and maturation of caspase-1 and production of IL-1β, spleen MNCs were prepared from control, control MSC, CVB3, and CVB3 MSC mice and stained for ASC by flow cytometry. Regulation of the ASC activation was not detected when total splenocytes fraction were evaluated (Fig. 4A). However, specific immune cell subsets showed increased intracellular expression of ASC. ASC activation was induced in macrophages (F4/80), NK cells (CD49b) and DCs (CD11c) upon CVB3 infection as indicated by a 1.7-fold (p < 0.01) higher percentage of ASC in macrophages (F4/80), NK cells (CD49b) and DCs (CD11c) in comparison to control mice. In contrast, CVB3 MSC mice exhibited 1.8-fold (p < 0.01), 1.7-fold (p < 0.01) and 1.7-fold (p < 0.05) lower ASC positive macrophages, NK cells and DCs (Fig. 4B-D).
The associations between cardiac contractility/cardiac fibrosis parameters and NLRP3 infammasome activation is shown by linear regressions (Supplemental Fig. 4).

Discussion
MSC application suppresses NOD2 and NLRP3 inflammasome gene expression and prevents the NLRP3 inflammasome assembly by downregulating the ASC protein expression in the heart of the CVB3-infected mice. Moreover, the present study extends the knowledge about the systemic immunomodulatory effects of MSC in myocarditis, showing that MSC abrogate the expression of NOD2, NLRP3 inflammasome activation and subsequent IL-1β production in macrophages (F4/80), NK cells (CD49b) and DCs (CD11c) of CVB3-infected mice. The suppressive effect of MSC on NOD2 expression and NLRP3 inflammasome activation is translated into normalized expression of important genes of cardiac contractility and fibrosis, which could potently abrogate the development of adverse cardiac dysfunction and remodelling following viral-induced myocardial injury. We suggest that the inhibitory effects of MSC on NLRP3 inflammasome activation is mediated at least partly via suppression of NOD2 expression and via secretion of anti-oxidative STC-1.
NLRP3 inflammasome activation is detected in heart biopsy samples of acute myocarditis patients and postmortem cases of myocarditis of unknown etiology 23 and plays a leading role in the pathogenesis of myocarditis 12 . NLRP3 activation can be due to NLRP3 priming, i.e. induction of NLRP3 mRNA expression involving NF-kB or upstream NOD2, or due to direct activation of the NLRP3 inflammasome via mediators like reactive oxygen species (ROS) and ATP 4 . NOD2 is a cytoplasmatic PRR, which recognizes ssRNA, including CVB3 5 and activates NLRP3 inflammasome and IL-1β processing and secretion 4 . Our recent findings reveal that NOD2 is a major mediator in the pathogenesis of CVB3-induced myocarditis 13 . We showed that NOD2 expression was upregulated in endomyocardial biopsies of CVB3-positive patients. Furthermore, cardiac NOD2 and NLRP3 expression dropped in CVB3-positive patients who eliminated the virus and improved cardiac function over time 13 . The relevance of NOD2 expression in CVB3 myocarditis further followed from NOD2 −/− CVB3 mice which were rescued from the detrimental effects of CVB3. They exhibited a minor inflammatory response, lower cardiac fibrosis and decreased NLRP3 activation. It should be noted however that the NOD2 expression was not increased in patients with a persistence of the cardiotropic viruses parvovirus B19 or human herpesvirus 6 (HHV-6) type B, which are among the most frequently found cardiotropic viruses in endomyocardial biopsies [24][25][26] , whereas its regulation in primarily autoimmune myocarditis still needs to be investigated. Our present findings which indicate a potent inhibitory effect of MSC on cardiac CVB3-induced NOD2 gene expression might therefore only be limited to the pathology of CVB3-induced myocarditis, and not generalized for all myocarditis etiologies. MSC further reduced the gene expression of all NLRP3 inflammasome components and products, which relevance has been shown in different cardiovascular disorders besides myocarditis 11,27 . Only ASC protein level was markedly increased in the heart of CVB3-infected mice and downregulated by MSC, while at this time point, cardiac caspase 1 activity and IL-1ß protein expression remained unchanged upon CVB3 infection and were not affected by MSC application. Interestingly, see supra, caspase 1 and IL-1ß protein expression was already increased in splenic macrophages, NK cells and DCs of those CVB3-infected versus control mice. The observed discrepancy in caspase 1 and IL-1β protein expression in the heart and the spleen at the same time point probably reflects a difference in the basal expression of NLRP3 inflammasome and in the mechanism of inflammasome activation. Tissue resident cells may not need to respond to injurious/infectious agents as promptly as cells of the innate immune response. Indeed, the mechanism of inflammasome regulation has been shown to be tissue-specific as NLRP3 expression in the heart of wildtype mice is lower than that in the spleen 28 . Nevertheless, the upregulated LV protein expression of ASC in CVB3-infected mice indicates already initiation of NLRP3 inflammasome formation 4 in the heart of myocarditis mice, which could be decreased via MSC application. The suppressive effect of MSC on NOD2, NLRP3 inflammasome and IL-1β expression was confirmed in vitro in CVB3-infected cardiomyocytes. MSC supplementation abolished not only the CVB3-induced ASC protein expression and NLRP3 assembly, but also the downstream caspase 1 expression and conversion of pro-IL-1β to active IL-1β in CVB3-infected HL-1 cells.
ROS are a central and common upstream cellular signal triggering NLRP3 inflammasome activation 21 . The intracellular oxidation status has been implicated in the pathogenesis of CVB3 infections 29 . Moreover, Wang et al. 12 showed that the production of ROS is crucial for inflammasome activation in response to CVB3 infection. Importantly, we have previously demonstrated that MSC reduce the CVB3-induced ROS production in cardiomyocytes 18 , whereas Oh et al. 20 recently illustrated that MSC decrease mitochondrial ROS and inhibit LPSand ATP-induced NLRP3 inflammasome activation in macrophages primarily by secreting STC-1. Based on these findings, we wanted to understand if MSC negatively regulate the NLRP3 inflammasome activation in CVB3-infected HL-1 cells in an STC-1-dependent manner. Therefore, we knocked down STC-1 in MSC and subsequently co-cultured them with CVB3-infected HL-1 cardiomyocytes. The partial knockdown of STC-1 in MSC modestly abrogated the inhibitory effect on NLRP3 activation in cardiomyocytes (Supplemental Fig. 1) suggesting that MSC mediate their effect at least partly in an STC-1-dependent manner. Furthermore, we demonstrated that MSC reduce LPS-and ATP-triggered NLRP3 inflammasome activity in HL-1 cardiomyocytes, indicating that the inhibitory effect on the NLRP3 inflammasome is not solely restricted to CVB3 as a trigger, a finding which could also be confirmed in splenic macrophages, NK cells, and DCs. Taken together the discussed findings suggest that MSC suppress NLRP3 inflammasome activation presumably via reduction of NOD2 expression, ROS production and release of paracrine factors, such as the anti-oxidative factor STC-1.
Aside of the direct cardiac injury in CVB3-induced myocarditis 30 , inflammation is the leading component and the dominant mechanism in the pathogenesis of viral myocarditis 1 . MSC have been shown to inhibit NLRP3 inflammasome activation in vitro in macrophages 20 . In the present study, we could demonstrate that MSC mediate an effective systemic immunoregulation in viral myocarditis via reduction of NOD2 expression and abrogation of NLRP3 inflammasome activation in macrophages (F4/80), NK cells (CD49b) and DCs (CD11c). NOD2 activation in inflammatory cells may induces NF-κB and MAPK signalling pathways triggering a cell signaling cascade enhancing the secretion of pro-inflammatory immune factors -TNF-α, IL-6, CCL2, IL-8 -31 , which affect signaling molecules in cardiac residential cells ultimately resulting in cardiac injury. NOD2 induces the production of CC-chemokine ligand 2 (CCL2) and helps to drive the recruitment and priming of innate immune cells, including neutrophils and inflammatory Ly6C hi monocytes 32 . Importantly, we have recently demonstrated that MSC attenuate myocardial inflammation via suppression of the cardiac infiltration of Ly6C hi pro-inflammatory monocytes in CVB3-induced myocarditis 33 . Furthermore, NOD2 supports the recruitment of inflammatory macrophages 34 , known to mediate deteriorating immune overreaction as depletion of macrophages significantly improve both acute and chronic viral myocarditis, further confirming the critical pathological role of macrophages in CVB3-induced myocarditis 35 . Macrophages are the most abundant cells in the inflamed myocardium of CVB3-infected mice and induce the expression of IL-1β, promoting cardiac inflammation, cardiac dilatation, fibrosis and heart failure 36 . Therefore, we assume that the suppressive effect of MSC on NLRP3 inflammasome activation and IL-1β secretion by macrophages could be beneficial in viral myocarditis preventing disease progression to cardiac dilatation and heart failure. DCs infiltrate the myocardium and release a cocktail of proinflammatory cytokines and induce CD4 + T-cell-mediated autoimmune induction in experimental CVB3 myocarditis 37 . MSC inhibit the NLRP3 inflammasome activation and IL-1β release by DCs, which might prevent autoimmunity induction. Given the significant involvement of autoimmune mechanisms in both experimental 38,39 and human 40 myocarditis and the finding that excessive inflammasome activation can cause autoimmune disorders 41 , these inhibitory effects of MSC on inflammasome activation could have major implications on autoimmune reactivity in CVB3 myocarditis which could subsequently lead to an improvement in chronic myocarditis pathology.
The NK cells not only control the virus replication 42 , but also release perforin in CVB3-induced myocarditis 43 . MSC inhibit the NLRP3 inflammasome activation and IL-1β released by NK cells, limiting the long-term NK cells activation and release of cytotoxic factors which could damage myocytes in absence of significant virus titers in the heart tissue.
IL-1β levels strongly correlate with the severity of CVB3-induced myocarditis 12 . MSC treatment in CVB3 mice downregulated LV mRNA expression of IL-1β and IL-18, which both induce cardiodepressive effects in heart failure 10,11 and alter phospholamban expression, a key regulator of cardiac contractility 8 . Blockade of IL-1β or neutralization of IL-18 is an effective cardioprotective approach 10,44 . Therefore, the inhibitory effect of MSC on LV IL-1β and IL-18 gene expression and IL-1β-expressing splenic macrophages, NK cell, and DCs could have beneficial cardiac therapeutic implications.
In agreement with the previously demonstrated anti-fibrotic and cardioprotective effects of MSC, associated with improved LV function in CVB3-infected myocarditis mice as demonstrated via conductance hemodynamic measurments 18,19 , we now show in the same experimental setting that the MSC inhibitory effect on NOD2 and NLRP3 inflammasome activation is associated with an improved expression of markers involved in Ca 2+ regulation (SERCA, phospholamban) and cardiac fibrosis (col3a1, LOX1, LOXL2). Decreased expression levels of SERCA and a lower ratio of SERCA to its endogenous modulator phospholamban in the heart results in decreased contractility 45 , whereas LOX enzymes are key players in extracellular matrix deposition, maturation and cross-linking of collagen fibrils, which leads to myocardial stiffness, left ventricular dysfunction, and heart failure 46,47 . Moreover, we found a strong correlation between LV NLRP3 and phospholamban, col3a1, LOX1, and LOXL2 mRNA expression (Supplemental Fig. 4). These observations further support and clarify that i.v. MSC application improves LV function (contractility, stiffness) in CVB3 myocarditis mice via reducing cardiac fibrosis (collagen and crosslinking) and via modulating the expression of proteins involved in Ca 2+ regulation.
In summary, the present study demonstrates that MSC are potent regulators of the NLRP3 inflammasome in the heart and in innate immune cells. MSC decrease NLRP3 activity, presumably from the combined reduction in NOD2 expression and the decrease in NLRP3 inflammasome activity involving the paracrine factor STC-1 (Fig. 8). Therefore, MSC application could be a promising strategy to hamper the NLRP3 inflammasome activation limiting the adverse consequences of excessive myocardial and systemic inflammation associated with abnormal expression of genes regulators of cardiac contractility and fibrosis.

Methods
Murine Coxsackievirus B3-induced myocarditis and cell application. Eight-week-old male C57BL/6 mice were infected by intraperitoneal injection of 1 × 10 5 plaque forming units (PFU) of CVB3 virus (Nancy strain). Control mice received PBS instead of CVB3. One day after CVB3 virus infection, 1 × 10 6 MSC were i.v. administrated via the tail vein into C57BL/6 mice. All mice were sacrificed on day seven post-CVB3 infection. The LV was harvested and snap-frozen for molecular biology. For flow cytometry analysis, heart, blood, and spleen mononuclear cells (MNCs) were isolated. The investigation was approved by the local ethical committee (Landesamt für Gesundheit und Soziales, Nr: G0094/11) and was performed in accordance with the European principles of laboratory animal care (Directive 2010/63/EU) and German animal protection law.