Immune Contribution to Heart Failure and Therapeutic Opportunities

Myocarditis or inflammation of the heart muscle is a leading cause of sudden death from heart failure in children and adults under the age of 50, and can progress to dilated cardiomyopathy (DCM) necessitating a heart transplant. Viral infections are the most common cause of myocarditis in developed countries. A number of viruses are known to cause myocarditis including coxsackieviruses, influenza, parvovirus B19, HIV, hepatitis C virus and the virus that causes COVID-19, SARS-CoV-2. Men have a higher incidence and severity of myocarditis and DCM compared to women. Sex hormones are known to contribute to increasing inflammation in animal models of viral myocarditis with testosterone increasing disease while estrogen is protective. Cardiac inflammation predicts worse outcome in patients with suspected myocarditis or acute DCM. TLR4 mRNA expression leading to IL-1β production is higher in patients with biopsy-proven myocarditis/DCM than controls and strongly correlates with enteroviral RNA levels in the heart. We showed previously that TLR4/IL-1β increases myocarditis, that men and male mice with myocarditis have more TLR4+CD11b+ cardiac immune cells compared to females, and CD11b cells are increased by testosterone during CVB3 myocarditis. In patients TLR4 increases IL-1β while TLR2 increases IL-6, IL-23, and TGFβ1 driving Th17 immune responses resulting in progression to DCM and heart failiure, but only in male mice and men with myocarditis.

Women with early-stage breast cancer are at excess risk of cardiovascular disease (CVD) due to therapy-induced direct and indirect perturbations across the entire cardiovascular system. CVD events such as myocardial infarction (MI) induce a systemic inflammatory response that accelerates underlying atherosclerosis. Whether an AMI-induced systemic response affects cancer progression is not known.
Methods: In a prospective case cohort study, we evaluated the relationship between a new onset, post-diagnosis CVD event (e.g., MI, stroke, heart failure) and recurrence in 1724 patients with early- stage breast cancer. To assess causality, we tested the effects of surgically-induced MI on cancer progression and metastasis in mouse models of breast cancer.

Results: A new onset CVD event was associated with increased risk of recurrence (HR: 1.59; 95% CI:
1.23 to 2.05) and cancer-specific mortality (HR: 1.60, 95% CI: 1.15–2.22) compared to patients not experiencing an event. In preclinical models, surgically-induced MI significantly accelerated tumor growth compared to sham surgery controls (p<0.001), as well as metastatic burden (p<0.05). MI epigenetically reprogrammed Ly6Chi monocytes in the bone marrow to an immunosuppressive phenotype that was maintained at the transcriptional level in monocytes in both the circulation and tumor. In parallel, MI increased circulating Ly6Chi monocytes and recruitment to tumors and depletion of these cells abrogated MI- induced tumor growth.

Heart transplant is the definitive treatment for patients with end-stage heart failure but is limited by donor availability and immunological complications related to acute rejection. Current immunosuppression targets recipient immune populations but increases life-threatening infections and malignancies. An alternative is to target donor immune cells and pathways.

Several distinct immune cell types reside within the donor heart. Recent studies have shown surprising diversity amongst cardiac macrophage populations. The mouse and human heart contain two populations of macrophages that can be distinguished based on expression of C-C chemokine receptor 2 (CCR2). CCR2+ macrophages are derived from adult hematopoietic progenitors and participate in inflammatory responses. CCR2- macrophages are derived from embryonic hematopoietic progenitors and promote tissue repair. Elucidating the respective roles and interactions between these donor myeloid populations is likely to yield mechanistic information regarding the pathogenesis of allograft inflammation and rejection.

Donor CCR2- and donor CCR2+ macrophages have opposing roles in heart transplant. Depletion of donor CCR2- macrophages accelerates allograft rejection whereas depletion of donor CCR2+ macrophages extend allograft survival. The protective effects of donor CCR2- macrophages are in part due to direct interactions with donor CCR2+ macrophages while pro-rejection CCR2+ macrophages signal through a MyD88 pathway.

Transglutaminase-2 (TG2) is a reliable M2 macrophage marker; however, its role in macrophage biology is poorly understood. We studied the role of TG2 in macrophage function, using mice with myeloid cell-specific loss of TG2 (MyTG2KO) in a model of myocardial infarction (MI) and in vitro assays. TG2 was highly expressed in spleen, liver and lung macrophages, and was markedly upregulated in M2-like macrophages infiltrating the infarcted heart. In contrast, infarct neutrophils expressed low levels of TG2. MyTG2KO mice had no baseline defects. Myeloid cell-specific TG2 loss did not affect post-MI systolic dysfunction, but improved diastolic function and reduced atrial size. TG2KO and TG2 fl/fl infarct macrophages had comparable inflammatory gene levels, but markedly increased synthesis of MMPs and TIMPs and of the anti-fibrotic mediators decorin and collagen V, suggesting a matrix-remodeling phenotype. Consistent with the in vivo findings, isolated bone marrow (BM) TG2KO macrophages had increased secretion of decorin and collagen V. RNAseq comparing the transcriptomic profile of TGF-b-stimulated TG2KO and WT BM macrophages suggested differential expression of genes associated with cell surface interactions, focal adhesion formation, and receptor tyrosine kinase signaling. In summary, TG2 expression is induced in M2-like infarct macrophages and mediates diastolic dysfunction, possibly through effects on signaling pathways that promote an anti-fibrotic matrix-remodeling program.

Innate immunity is a critical regulator of cardiac development, heart failure progression, and myocardial tissue repair. Within these contexts, functionally and ontologically distinct macrophage populations reside within the myocardium and are essential determinants of outcomes following tissue injury or during chronic disease. Under steady state conditions the human and mouse heart contains two distinct macrophage populations that can be distinguished based on the expression of C-C Chemokine Receptor 2 (CCR2). CCR2- macrophages are derived from extra-embryonic progenitors that establish residency within the heart during development and are maintained independent of monocyte input. CCR2- macrophages display minimal inflammatory potential and are essential for coronary development and growth, neonatal heart regeneration, and adaptive cardiac remodeling. In contrast, CCR2+ macrophages are derived from definitive monocyte progenitors and are maintained through a combination of local proliferation and gradual monocyte recruitment. CCR2+ macrophages are activated in response to cardiomyocyte cell death and generate robust levels of inflammatory cytokines, chemokines, and oxidative products via a MYD88 and STAT5 dependent pathways. Through these mechanisms, CCR2+ macrophages orchestrate neutrophil and monocyte recruitment, instruct monocytes to acquire inflammatory cell fates, regulate regulatory T cell trafficking, and contribute to heart failure pathogenesis through infarct expansion and adverse left ventricular remodeling. Collectively, these studies have established the importance of macrophage heterogeneity and identified CCR2+ macrophages as a therapeutic target for heart failure.

Inflammation is associated with cardiac remodeling and heart failure, but how it is initiated in response to non-ischemic interventions in the absence of cell death is not known. I will discuss evidence that the activation of CaMKIIδ in cardiomyocytes initiates inflammatory responses that lead to adverse remodeling. Our conclusions are based on studies with CaMKIIδ transgenic mice, and mice in which CaMKIIδ is selectively deleted from cardiomyocytes to generate cardiac specific knockout (CKO) mice. We show that Angiotensin II (AngII) or transverse aortic constriction (TAC) induce rapid and robust increases in pro-inflammatory chemokine and cytokine gene expression resulting from activation of NFkB. In CKO mice the ability of AngII and TAC to induce NFkB activation and pro-inflammatory gene expression are markedly attenuated. Inflammatory gene mRNA expression occurs within the cardiomyocyte (CM) compartment since isolated CMs from TAC hearts show robust CaMKδ dependent increases in gene expression at early times whereas inflammatory gene mRNAs are not increased in the non-myocyte fraction containing immune cells and fibroblasts. Priming of the NLRP3 inflammasome, assessed by measuring IL-1β, IL-18 and NLRP3 mRNA levels and inflammasome activation assessed via caspase-1 activity and IL-18 cleavage, are also increased in CM at 3 dayTAC in CTL and diminished in CaMKII KO hearts. NLRP3 activation is mediated in part through increased mitochondrial ROS. Macrophages (CD68+) accumulate in the heart in response to AngII or TAC treatment and this is diminished in CKO and inhibited by blocking MCP-1 signaling or inflammasome activation. Fibrosis is also attenuated by these interventions and in the CKO heart. Ventricular dilation and contractile dysfunction assessed by echocardiography at day 42 post TAC are diminished in the CKO as well as by inhibition of CaMKII, NFkB or inflammasome signaling in the first one or two weeks after TAC. Systemic inflammation assessed as serum amyloid is also increased by TAC and diminished when cardiomyocyte CaMKIIδ is deleted. We conclude that CaMKIIδ transduces hormonal and other non-ischemic signals to initiate inflammatory responses within cardiomyocytes which then drive subsequent immune cell recruitment, fibrosis and adverse remodeling.

Fibrosis is observed in nearly every form of myocardial disease. Interventions that effectively target fibrosis remain limited. We have explored the use of redirected T cell immunotherapy to specifically target pathological cardiac fibrosis in mice. Through expression analysis of the gene signatures of cardiac fibroblasts obtained from healthy and diseased human hearts, we found an endogenous target of cardiac fibroblasts-fibroblast activation protein (FAP). Adoptive transfer of T cells that express a chimeric antigen receptor against FAP results in a significant reduction in cardiac fibrosis and restoration of function after injury in mice. These results provide proof-of-principle for the development of immunotherapeutic drugs for the treatment of cardiac disease.

Despite the well-established association between T cell-mediated inflammation and non-ischemic heart failure (HF), the specific mechanisms triggering T cell activation and the antigens involved are poorly understood. This seminar will discuss evidence from experimental animal models demonstrating a causal role for T cells, primarily IFNy secreting Th1 cells infiltrated in the heart, in promoting adverse cardiac remodeling and dysfunction. I will present new evidence that support T cell receptor (TCR) recognition of cardiac neoantigens induced by myocardial oxidative stress using the mouse model of transverse aortic constriction (TAC), and the presence of oxidative stress modified cardiac proteins in the human failing heart. Using a number of immunological, cellular and molecular approaches and reporter mice for T cell activation, we identified a limited repertoire of activated CD4+ T cell clonotypes in the heart associated with cardiac dysfunction. Heart CD4+ T cell activation and systolic dysfunction were reduced when using the anti-oxidant TEMPOL and specific scavengers of Isolevuglandulins (Iso-LG) modified cardiac proteins, formed in response to TAC that function as cardiac neoantigens for T cell activation. Our results provide novel mechanistic insights to therapeutically ameliorate cardiac dysfunction by dampening CD4+ T cell responses.

The observation that heart failure with reduced ejection fraction is associated with elevated circulating levels of pro-inflammatory cytokines opened a new area of research that has revealed a potentially important role for the immune system in the pathogenesis of heart failure. However, until the publication in 2019 of the CANTOS trial findings on heart failure outcomes, all attempts to target inflammation in the heart failure setting in phase III clinical trials resulted in neutral effects or worsening of clinical outcomes. This lack of positive results in turn prompted questions on whether inflammation is a cause or consequence of heart failure. This seminar will summarize the latest developments in our understanding of the role of the innate and adaptive immune systems in the pathogenesis of heart failure, and will highlight the results of phase III clinical trials of therapies targeting inflammatory processes in the heart failure setting, such as anti-inflammatory and immunomodulatory strategies. The most recent of these studies, the CANTOS trial, raises the exciting possibility that, in the foreseeable future, we might be able to identify those patients with heart failure who have a cardio-inflammatory phenotype and will thus benefit from therapies targeting inflammation.