Absence of NLRP3 Inflammasome in Hematopoietic Cells Reduces Adverse Remodeling After Experimental Myocardial Infarction.

An inflammatory response is required for tissue healing after a myocardial infarction (MI), but the process must be balanced to prevent maladaptive remodeling. This study shows that improved survival and cardiac function following MI, in mice deficient for the NLRP3 inflammasome, can be recapitulated in wild-type mice receiving bone marrow from Nlrp3 -/- mice. This suggests that NLRP3 activation in hematopoietic cells infiltrating in the myocardium increases mortality and late ventricular remodeling. Our data should encourage performing clinical trials directly targeting NLRP3 inflammasome and their inflammatory cytokines (interleukin-1ß and -18) in MI patients.

NLRP3 Inflammasome Promotes Myocardial Remodeling During Diet-Induced Obesity.

Background: Obesity is an increasingly prevalent metabolic disorder in the modern world and is associated with structural and functional changes in the heart. The NLRP3 inflammasome is an innate immune sensor that can be activated in response to endogenous danger signals and triggers activation of interleukin (IL)-1ß and IL-18. Increasing evidence points to the involvement of the NLRP3 inflammasome in obesity-induced inflammation and insulin resistance, and we hypothesized that it also could play a role in the development of obesity induced cardiac alterations. Methods and Results: WT, Nlrp3-/-, and ASC-/- (Pycard-/-) male mice were exposed to high fat diet (HFD; 60 cal% fat) or control diet for 52 weeks. Cardiac structure and function were evaluated by echocardiography and magnetic resonance imaging, respectively. Whereas, NLRP3 and ASC deficiency did not affect the cardiac hypertrophic response to obesity, it was preventive against left ventricle concentric remodeling and impairment of diastolic function. Furthermore, whereas NLRP3 and ASC deficiency attenuated systemic inflammation in HFD fed mice; long-term HFD did not induce significant cardiac fibrosis or inflammation, suggesting that the beneficial effects of NLRP3 inflammasome deficiency on myocardial remodeling at least partly reflect systemic mechanisms. Nlrp3 and ASC (Pycard) deficient mice were also protected against obesity-induced systemic metabolic dysregulation, as well as lipid accumulation and impaired insulin signaling in hepatic and cardiac tissues. Conclusions: Our data indicate that the NLRP3 inflammasome modulates cardiac concentric remodeling in obesity through effects on systemic inflammation and metabolic disturbances, with effect on insulin signaling as a potential mediator within the myocardium.

Mammalian Target of Rapamycin (mTOR) and the Proteasome Attenuates IL-1ß Expression in Primary Mouse Cardiac Fibroblasts.

Background: IL-1ß is a highly potent pro-inflammatory cytokine and its secretion is tightly regulated. Inactive pro-IL-1ß is transcribed in response to innate immune receptors activating NFκB. If tissue damage occurs, danger signals released from necrotic cells, such as ATP, can activate NLRP3-inflammasomes (multiprotein complexes consisting of NLRP3, ASC, and active caspase-1) which cleaves and activates pro-IL-1ß. NLRP3 activation also depends on NEK7 and mitochondrial ROS-production. Thus, IL-1ß secretion may be regulated at the level of each involved component. We have previously shown that NLRP3-dependent IL-1ß release can be induced in cardiac fibroblasts by pro-inflammatory stimuli. However, anti-inflammatory mechanisms targeting IL-1ß release in cardiac cells have not been investigated. mTOR is a key regulator of protein metabolism, including autophagy and proteasome activity. In this study we explored whether autophagy or proteasomal degradation are regulators of NLRP3 inflammasome activation and IL-1ß release from cardiac fibroblasts. Methods and Results: Serum starvation selectively reduced LPS/ATP-induced IL-1ß secretion from cardiac fibroblasts. However, no other inflammasome components, nor mitochondrial mass, were affected. The mTOR inhibitor rapamycin restored pro-IL-1ß protein levels as well as LPS/ATP-induced IL-1ß release from serum starved cells. However, neither serum starvation nor rapamycin induced autophagy in cardiac fibroblasts. Conversely, chloroquine and bafilomycin A (inhibitors of autophagy) and betulinic acid (a proteasome activator) effectively reduced LPS-induced pro-IL-1ß protein levels. Key findings were reinvestigated in human monocyte-derived macrophages. Conclusion: In cardiac fibroblasts, mTOR inhibition selectively favors pro-IL-1ß synthesis while proteasomal degradation and not autophagy is the major catabolic anti-inflammatory mechanism for degradation of this cytokine.

Toll-Like Receptor 9 Promotes Survival in SERCA2a KO Heart Failure Mice.

Aim. Inflammation is important in heart failure (HF). The role of the immune receptor toll-like receptor 9 (TLR9) in HF is not understood and not investigated in diastolic HF. We investigated the role of TLR9 in a murine diastolic HF model caused by cardiomyocyte SERCA2a excision. Methods and Results. We crossed SERCA2a KO and TLR9 KO mice to generate four mouse lines. Tamoxifen-induced cardiomyocyte SERCA2a gene excision was carried out in mice, causing diastolic HF. After 7.6 weeks, cardiac functions and dimensions were analyzed by echocardiography and heart tissues were processed. HF mice depleted of TLR9 demonstrated reduced survival compared to SERC2a KO mice, with a median life expectancy of 58 days compared to 63 days. Both HF groups displayed increased left atrium size, lung weight, fetal gene expressions, monocyte/macrophage infiltration, and fibrosis. However, there were no significant differences between the groups. Conclusion. In mice with SERCA2a KO-induced diastolic HF, the absence of TLR9 reduced median life expectancy. The cause remains elusive, as all investigated HF parameters were unaltered. Still, these findings support a salutary role of TLR9 in some subsets of HF conditions and underline the importance for future studies on the mechanisms of TLR9 in diastolic HF.

Wnt5a is elevated in heart failure and affects cardiac fibroblast function.

Wnt signaling is dysregulated in heart failure (HF) and may promote cardiac hypertrophy, fibrosis, and inflammation. Blocking the Wnt ligand Wnt5a prevents HF in animal models. However, the role of Wnt5a in human HF and its functions in cardiac cells remain unclear. Here, we investigated Wnt5a regulation in HF patients and its effects on primary mouse and human cardiac fibroblasts. Serum Wnt5a was elevated in HF patients and associated with hemodynamic, neurohormonal, and clinical measures of disease severity. In failing human hearts, Wnt5a protein correlated with interleukin (IL)-6 and tissue inhibitor of metalloproteinase (TIMP)-1. Wnt5a messenger RNA (mRNA) levels were markedly upregulated in failing myocardium and both mRNA and protein levels declined following left ventricular assist device therapy. In primary mouse and human cardiac fibroblasts, recombinant Wnt5a dose-dependently upregulated mRNA and protein release of IL-6 and TIMP-1. Wnt5a did not affect ß-catenin levels, but activated extracellular signal-regulated kinase 1/2 (ERK1/2) signaling. Importantly, inhibition of ERK1/2 activation attenuated Wnt5a-induced release of IL-6 and TIMP-1. In conclusion, our results show that Wnt5a is elevated in the serum and myocardium of HF patients and is associated with measures of progressive HF. Wnt5a induces IL-6 and TIMP-1 in cardiac fibroblasts, which might promote myocardial inflammation and fibrosis, and thereby contribute to HF progression. KEY MESSAGES: • Wnt5a is elevated in serum and myocardium of HF patients and is associated with measures of progressive HF. • In cardiac fibroblasts, Wnt5a upregulates interleukin (IL)-6 and tissue inhibitor of metalloproteinase (TIMP)-1 through the ERK pathway. • Wnt5a-mediated effects might promote myocardial inflammation and fibrosis, and thereby contribute to HF progression.

NEIL3-Dependent Regulation of Cardiac Fibroblast Proliferation Prevents Myocardial Rupture.

Myocardial infarction (MI) triggers a reparative response involving fibroblast proliferation and differentiation driving extracellular matrix modulation necessary to form a stabilizing scar. Recently, it was shown that a genetic variant of the base excision repair enzyme NEIL3 was associated with increased risk of MI in humans. Here, we report elevated myocardial NEIL3 expression in heart failure patients and marked myocardial upregulation of Neil3 after MI in mice, especially in a fibroblast-enriched cell fraction. Neil3-/- mice show increased mortality after MI caused by myocardial rupture. Genome-wide analysis of 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) reveals changes in the cardiac epigenome, including in genes related to the post-MI transcriptional response. Differentially methylated genes are enriched in pathways related to proliferation and myofibroblast differentiation. Accordingly, Neil3-/- ruptured hearts show increased proliferation of fibroblasts and myofibroblasts. We propose that NEIL3-dependent modulation of DNA methylation regulates cardiac fibroblast proliferation and thereby affects extracellular matrix modulation after MI.

Palmitate promotes inflammatory responses and cellular senescence in cardiac fibroblasts.

Palmitate triggers inflammatory responses in several cell types, but its effects on cardiac fibroblasts are at present unknown. The aims of the study were to (1) assess the potential of palmitate to promote inflammatory signaling in cardiac fibroblasts through TLR4 and the NLRP3 inflammasome and (2) characterize the cellular phenotype of cardiac fibroblasts exposed to palmitate. We examined whether palmitate induces inflammatory responses in cardiac fibroblasts from WT, NLRP3-/- and ASC-/-mice (C57BL/6 background). Exposure to palmitate caused production of TNF, IL-6 and CXCL2 via TLR4 activation. NLRP3 inflammasomes are activated in a two-step manner. Whereas palmitate did not prime the NLRP3 inflammasome, it induced activation in LPS-primed cardiac fibroblasts as indicated by IL-1ß, IL-18 production and NLRP3-ASC co-localization. Palmitate-induced NLRP3 inflammasome activation in LPS-primed cardiac fibroblasts was associated with reduced AMPK activity, mitochondrial reactive oxygen species production and mitochondrial dysfunction. The cardiac fibroblast phenotype caused by palmitate, in an LPS and NLRP3 independent manner, was characterized by decreased cellular proliferation, contractility, collagen and MMP-2 expression, as well as increased senescence-associated ß-galactosidase activity, and consistent with a state of cellular senescence. This study establishes that in vitro palmitate exposure of cardiac fibroblasts provides inflammatory responses via TLR4 and NLRP3 inflammasome activation. Palmitate also modulates cardiac fibroblast functionality, in a NLRP3 independent manner, resulting in a phenotype related to cellular senescence. These effects of palmitate could be of importance for myocardial dysfunction in obese and diabetic patients.

Sustained Toll-Like Receptor 9 Activation Promotes Systemic and Cardiac Inflammation, and Aggravates Diastolic Heart Failure in SERCA2a KO Mice.

AIM: Cardiac inflammation is important in the pathogenesis of heart failure. However, the consequence of systemic inflammation on concomitant established heart failure, and in particular diastolic heart failure, is less explored. Here we investigated the impact of systemic inflammation, caused by sustained Toll-like receptor 9 activation, on established diastolic heart failure. METHODS AND RESULTS: Diastolic heart failure was established in 8-10 week old cardiomyocyte specific, inducible SERCA2a knock out (i.e., SERCA2a KO) C57Bl/6J mice. Four weeks after conditional KO, mice were randomized to receive Toll-like receptor 9 agonist (CpG B; 2µg/g body weight) or PBS every third day. After additional four weeks, echocardiography, phase contrast magnetic resonance imaging, histology, flow cytometry, and cardiac RNA analyses were performed. A subgroup was followed, registering morbidity and death. Non-heart failure control groups treated with CpG B or PBS served as controls. Our main findings were: (i) Toll-like receptor 9 activation (CpG B) reduced life expectancy in SERCA2a KO mice compared to PBS treated SERCA2a KO mice. (ii) Diastolic function was lower in SERCA2a KO mice with Toll-like receptor 9 activation. (iii) Toll-like receptor 9 stimulated SERCA2a KO mice also had increased cardiac and systemic inflammation. CONCLUSION: Sustained activation of Toll-like receptor 9 causes cardiac and systemic inflammation, and deterioration of SERCA2a depletion-mediated diastolic heart failure.

Toll-like receptor 9 mediated responses in cardiac fibroblasts.

Altered cardiac Toll-like receptor 9 (TLR9) signaling is important in several experimental cardiovascular disorders. These studies have predominantly focused on cardiac myocytes or the heart as a whole. Cardiac fibroblasts have recently been attributed increasing significance in mediating inflammatory signaling. However, putative TLR9-signaling through cardiac fibroblasts remains non-investigated. Thus, our aim was to explore TLR9-signaling in cardiac fibroblasts and investigate the consequence of such receptor activity on classical cardiac fibroblast cellular functions. Cultivated murine cardiac fibroblasts were stimulated with different TLR9 agonists (CpG A, B and C) and assayed for the secretion of inflammatory cytokines (tumor necrosis factor α [TNFα], CXCL2 and interferon α/ß). Expression of functional cardiac fibroblast TLR9 was proven as stimulation with CpG B and -C caused significant CXCL2 and TNFα-release. These responses were TLR9-specific as complete inhibition of receptor-stimulated responses was achieved by co-treatment with a TLR9-antagonist (ODN 2088) or chloroquine diphosphate. TLR9-stimulated responses were also found more potent in cardiac fibroblasts when compared with classical innate immune cells. Stimulation of cardiac fibroblasts TLR9 was also found to attenuate migration and proliferation, but did not influence myofibroblast differentiation in vitro. Finally, results from in vivo TLR9-stimulation with subsequent fractionation of specific cardiac cell-types (cardiac myocytes, CD45+ cells, CD31+ cells and cardiac fibroblast-enriched cell-fractions) corroborated our in vitro data and provided evidence of differentiated cell-specific cardiac responses. Thus, we conclude that cardiac fibroblast may constitute a significant TLR9 responder cell within the myocardium and, further, that such receptor activity may impact important cardiac fibroblast cellular functions.

Toll-like receptor 9-activation during onset of myocardial ischemia does not influence infarct extension.

AIM: Myocardial infarction (MI) remains a major cause of death and disability worldwide, despite available reperfusion therapies. Inflammatory signaling is considered nodal in defining final infarct size. Activation of the innate immune receptor toll-like receptors (TLR) 9 prior to ischemia and reperfusion (I/R) reduces infarct size, but the consequence of TLR9 activation timed to the onset of ischemia is not known. METHODS AND RESULTS: The TLR9-agonist; CpG B was injected i.p. in C57BL/6 mice immediately after induction of ischemia (30 minutes). Final infarct size, as well as area-at-risk, was measured after 24 hours of reperfusion. CpG B injection resulted in a significant increase in circulating granulocytes and monocytes both in sham and I/R mice. Paradoxically, clear evidence of reduced cardiac infiltration of both monocytes and granulocytes could be demonstrated in I/R mice treated with CpG B (immunocytochemistry, myeloperoxidase activity and mRNA expression patterns). In addition, systemic TLR9 activation elicited significant alterations of cardiac inflammatory genes. Despite these biochemical and cellular changes, there was no difference in infarct size between vehicle and CpG B treated I/R mice. CONCLUSION: Systemic TLR9-stimulation upon onset of ischemia and subsequent reperfusion does not alter final infarct size despite causing clear alterations of both systemic and cardiac inflammatory parameters. Our results question the clinical usefulness of TLR9 activation during cardiac I/R.

The NLRP3 inflammasome is up-regulated in cardiac fibroblasts and mediates myocardial ischaemia-reperfusion injury.

AIMS: Nucleotide-binding oligomerization domain-Like Receptor with a Pyrin domain 3 (NLRP3) is considered necessary for initiating a profound sterile inflammatory response. NLRP3 forms multi-protein complexes with Apoptosis-associated Speck-like protein containing a Caspase recruitment domain (ASC) and Caspase-1, which activate pro-interleukin-1ß (IL-1ß) and pro-IL-18. The role of NLRP3 in cardiac cells is not known. Thus, we investigated the expression and function of NLRP3 during myocardial ischaemia. METHODS AND RESULTS: Myocardial infarction (MI) was induced in adult C57BL/6 mice and Wistar rats by ligation of the coronary artery. A marked increase in NLRP3, IL-1ß, and IL-18 mRNA expression was found in the left ventricle after MI, primarily located to myocardial fibroblasts. In vitro studies in cells from adult mice showed that myocardial fibroblasts released IL-1ß and IL-18 when primed with lipopolysaccharide and subsequently exposed to the danger signal adenosine triphosphate, a molecule released after tissue damage during MI. When hearts were isolated from NLRP3-deficient mice, perfused and subjected to global ischaemia and reperfusion, a marked improvement of cardiac function and reduction of hypoxic damage was found compared with wild-type hearts. This was not observed in ASC-deficient hearts, potentially reflecting a protective role of other ASC-dependent inflammasomes or inflammasome-independent effects of NLRP3. CONCLUSION: This study shows that the NLRP3 inflammasome is up-regulated in myocardial fibroblasts post-MI, and may be a significant contributor to infarct size development during ischaemia-reperfusion.