Immune Mechanisms and Therapeutic Advances in Heart Failure

Autoimmune Valvular Carditis

Although rheumatic diseases are commonly associated with pain and swelling in the joints, they can also cause inflammation in the heart, blood vessels, and valves, putting rheumatic disease patients at an increased risk of cardiovascular disease (CVD). Bryce Binstadt, a pediatric rheumatologist, studies the pathogenesis of autoimmune diseases with a particular emphasis on immune-mediated cardiovascular disease. In examining valvular carditis, Binstadt has used a K/BxN T-cell receptor (TCR) transgenic mouse that models arthritis. Sectioned hearts from these arthritic mice have valvular carditis, inflamed mitral valves, and valve fibrosis. “We think this a very nice model to investigate mechanisms driving valvular carditis in the setting of an autoantibody-mediated disease,” said Binstadt. With single-cell RNA (scRNA) sequencing, Binstadt and his colleagues  looked at cell composition and infiltration of the cardiac valves. They found immune cells infiltrating the valves, and apparent domination of macrophages.

For both single producing and double producing cells for IL-6 and TNF, blockage of CD47 leads to a reduction in inflammatory cytokine production.

Binstadt’s team also characterized a CX3CR1 knockout mouse, which had valve thickness values closer to those of wild type mice. They concluded that depletion of CD301b+ macrophages was key to normalize the valve thickness and that together, the combination of CX3CR1+ and CD301b+ macrophages are critical in cardiac pathogenesis because the macrophages produce IL-6, IL-13, and TNF cytokines. Blocking these cytokines prevents carditis and reduces mitral valve thickness. Blocking the CD47 receptor, which is expressed on apoptotic cells, prevents and treats valve fibrosis by inhibiting macrophage TNF, IL-6, and IL-13 production. After defining these targetable pathways that drive inflammation and fibrosis of the cardiovascular system in rheumatic diseases, Binstadt believes CD47 could be a potential therapeutic target to reduce valvular carditis.

Inflammasome Signaling in the Development of Atrial Fibrillation

Atrial fibrillation (AF), which affects 33.5 million people worldwide, is the most common cause of cardiac arrhythmia and increases the risk of stroke or heart failure. AF patients are known to have enhanced inflammatory response. Na Li’s lab focuses on whether inflammation plays a causal role in AF and the relevance of nucleotide-binding domain, leucine-rich-containing family, pyrin-domain-containing-3 (NLRP3) inflammasome, which is expressed predominantly in macrophages and is upregulated in AF patients. Li’s lab uses a cardiomyocyte-specific knock-in mouse line with constitutively active NLRP3. These mice with active NLRP3 have ectopic firing, premature atrial contraction, and AERP shortening. NLRP3 also promotes electric remodeling in the heart; because Kir currents are much larger, they have increased incidences of inducible AF and increased calcium sparks. The inflammasomes promote structural remodeling characterized by enlarged hearts, increased fibrosis, and elevated fibrosis markers. “This data suggests that CaMKIIδ and NLRP3 could form a forward feedback loop that promotes the arrhythmogenic calcium release,” said Li. Because obesity increases the risk of AF, these results are more pronounced in an obesity mouse model. Li raised the question of whether blocking inflammasomes could prevent AF. In mice, NLRP3 inhibition prevents abnormal calcium release, electric remodeling, and AERP shortening. Overall, these results indicate that inhibition of NLRP3 reduces susceptibility to AF and that it could be a viable therapeutic target for AF prevention.

Sex Differences in Viral Causes of Cardiac Inflammation Including COVID-19

Studies have shown that rates of cardiovascular disease are higher in men compared to women. This is due to differences in the basic physiology and heart function between the sexes. DeLisa Fairweather cautioned that when performing scientific experiments, endocrine disruptions can occur based on how cells are plated and the conditions in which they are cultured. These disruptions can alter the expression of genes and experimental outcomes. For human trials, sex, as well as age, should be considered. In fact, mitochondria can cause the cells of men and women to age differently. Like many disorders, inflammatory diseases differ in incidences by sex. Estrogen activates B cells, which in turn increases antibodies. Since estrogen levels vary during a woman’s life, the immune response changes over her lifespan as well.

Fairweather studies myocarditis, or inflammation of the heart, which can be caused by viral infections, including SARS-CoV-2. Although not well studied, men seem to develop dilated cardiomyopathy (DCM) and have worse recovery than women. Using  Coxsackievirus, Fairweather created a mouse model where male mice develop greater inflammation and dilation than female mice. But what is causing these differences? CD11b+ immune cells are elevated in male mice during myocarditis, while females have more B cells and T regulatory cells.  The increase in CD11b+ in males is also true in patients.  Testosterone increases myocardial inflammation, whereas estradiol prevents myocardial inflammation.

ACE2 is located on the X chromosome, and its expression differs by sex. In women, X inactivation is an important factor that influences what happens in disease.

Recently, she has been involved in studies on convalescent plasma safety for use in COVID-19 patients. There are many cardiovascular complications associated with COVID-19, one being myocarditis. ACE2 is on the X chromosome and is expressed more in men, which may explain why men are more susceptible to COVID-19 disease progression. Overall, Fairweather’s research highlights the importance of understanding differences between basic physiology and immune responses in men and women, which can eventually lead to individualized medicine strategies.

Clonal Hematopoiesis and Atherosclerotic Cardiovascular Disease

As we age, we gather more and more mutations in our genes. Siddhartha Jaiswal’s lab analyzes premalignant mutations related to clonal hematopoiesis and aging. He described Clonal Hematopoiesis of Indeterminate Potential (CHIP) as clonal mutations that are common with aging and are associated with increased risk of malignancy, blood cancer, and incidences of coronary heart disease. The two most commonly mutated genes are both enzymes involved in DNA methylation. Jaiswal believes CHIP is either a marker of aging or is causally associated with cardiovascular disease. To find out, he used a common Ldlr knockout mouse model of atherosclerosis. He then depleted the mouse endogenous bone marrow and replaced it with bone marrow from TET2 mutant mice. After a high cholesterol diet, the mice had substantially increased atherosclerotic lesion size with loss of one or two TET2 copies in the bone marrow. “This does seem to be a very robust finding in mice and is presumptive evidence of causality,” said Jaiswal. “At least some of these mutations are indeed causal for accelerated atherosclerosis.” Mouse and patient data, as well as human genetics data, all point favorably toward an anti-inflammatory blockade being helpful as an intervention for CHIP-associated CVD.

Myocardial infarction accelerates breast cancer through innate immune programming

Graeme Koelwyn studied the links between breast cancer and cardiovascular disease while working in Katheryn Moore’s lab at New York University. While breast cancer patients are at higher risk for cardiovascular disease, patients who have had a cardiac event have an increased risk of breast cancer or another cardiovascular event. Koelwyn noted that myocardial infarctions (MI) accelerate tumor growth in multiple mouse models. But how does this occur? MI alters the tumor immune landscape, which increases the number of cells present that accelerate tumor growth. This depends on Ly6Chi monocyte recruitment to the tumor. However, with depletion of Ly6Chi monocytes, antitumoral T cell balance is restored. Koelwyn believes MI-driven immunosuppression is epigenetically imprinted in the bone marrow.  Mice with bone marrow transplants have more monocytes; MI accelerates increased bone marrow hematopoiesis and increases systemic circulation, with more Ly6Chi monocyte cells accumulating in the tumor environment.

The Roles of Donor CCR2+ and CCR2- Macrophages After Heart Transplantation

Although there are many immunosuppressive methods for heart transplants, some patients still reject transplantation, and the roles of donor antigen presenting cells are unknown. The heart contains distinct macrophage populations with different origins and functions, including CCR2+ and CCR2- macrophages. Benjamin Kopecky, a cardiology fellow, utilized a heterotopic allogenic mouse model with a heart transplant to recapitulate human acute cellular rejection. He depleted either CCR2+ or CCR2- macrophages and found that the donor macrophages modulate allograft rejection or survival. CCR2- macrophages protect the allograft, whereas, in their absence, the recipient has more cellular rejection. Kopecky monitored where the donor and recipient macrophages were dispersed over time. He hypothesized that the CCR2- macrophages might modulate the CCR2+ macrophages. He also saw that donor CCR2+ macrophages are myD88- dependent and regulated through this signaling pathway. Overall, Kopecky believes modulation of donor cardiac macrophages is sufficient to suppress rejection and extend allograft survival.

Transglutaminase 2 (TG2) in regulation of macrophage phenotype in homeostasis and disease

Arti Shinde, of Albert Einstein College of Medicine, studies the role of transglutaminase 2 (TG2) regulation of macrophages in infarcted hearts.  TG2 is a ubiquitously-expressed, stress-activated protein with a wide range of functions; it is upregulated in failing and remodeling hearts. The protein is found in cardiomyocytes, vascular cells, fibroblasts, and macrophages. Shinde assesses whether TG2 is critical for macrophage function or if it is just a marker. In wild-type mice, TG2 is highly expressed in macrophages, and after myocardial infarction, TG2 is upregulated and localized in Arg1+ M2 cells.

MyTG2 absence in macrophages reduces mortality in mice. Myeloid cell specific TG2 loss improved ventricular compliance post MI.

In contrast, TG2 knockout mice have reduced mortality, normal LV systolic function, no change in ejection fraction, and attenuated diastolic disfunction compared to the wild-type mice. TG2 loss also reduces IL-1beta expression levels, but not inflammatory chemokines such as IL6 and TGF-beta. Shinde believes TG2 changes in matrix remodeling in macrophages and that this could be the reason for improved diastolic function and reduction in scar stiffness in the KO mice. Additionally, she showed that TG2 acts as an intracellular membrane-localized protein that restrains activation of matrix remodeling.

Leveraging Macrophage Diversity in Heart Failure

CCR2+ and CCR2- play distinct roles in the heart. CCR2- macrophages are essential for cardiac regeneration, coronary development, AV node conduction, and adaptive tissue remodeling. Importantly, they also suppress inflammation. CCR2+ macrophages, found in the myocardium, are activated following myocardial injury to initiate myocardial inflammation. When CCR2+ macrophages are depleted, infarcts are smaller, with less remodeling and less monocyte and neutrophil recruitment. When CCR2- macrophages are depleted, the opposite occurs. From patient images, Kory Lavine noted that CCR2+ macrophages accumulate in the diseased heart, mostly in the infarct, and subsets of these macrophages express inflammation mediators.

Mice with depletion of CCR2- cells prior to myocardial infarction have increased infarct size compared to control mice. Mice with CCR2+ cell depletion have the opposite effect and restored function.

CCL17 is a chemokine that signals exclusively through CCR4. It is expressed in macrophages and dendritic cells recruited to the injured heart. Lavine showed that CCL17 confers protection through a T-regulatory cell-dependent mechanism, and that this process can be reversed. The suppression of fibrosis is dependent on T-regulatory cells. Ultimately, his work showcases the significance of macrophage heterogeneity and the value of CCR2+ macrophages as a therapeutic target for heart failure.

Igniting the Flame of Inflammation through Cardiomyocyte CaMKII and Inflammasome Activation

Joan Heller Brown’s lab is interested in cell growth regulation mechanisms and the role of G-protein coupled receptors (GPCRs) in disease progression. Heller Brown and her colleagues have extensively studied the role of calcium/calmodulin-dependent protein kinase II (CAMKII) and inflammasome activation in the heart. In cardiac specific CAMKII delta (CaMKIIδ) overexpressing transgenic mice, hearts are large, dilated, and very arrhythmia prone. They have many heart failure features, including phosphorylation of RyR2 and increased calcium leak. Heller Brown’s lab at UCSD used a knockout mouse model to understand the role of CaMKIIδ, the predominant isoform in the heart, in inflammation and heart failure. After transverse aortic constriction (TAC), these mice had similar cell size, heart weight, and body weight compared to the wild-type mice. Heller Brown concluded that CAMKII is not required for the development of pathologic hypertrophy. She believes that CaMKIIδ activation in cardiomyocytes triggers inflammation and leads to the progression of heart failure.  “We see robust inflammation in a cell death-independent way as an early response to pressure overload (TAC), angiotensin II infusion, and isoproterenol infusion,” said Heller Brown. In summary, CaMKIIδ transduces non-ischemic signals to create inflammatory responses that ultimately drive immune cell recruitment, fibrosis, and heart failure.

Immunotherapy for Cardiac Disease

Jonathan Epstein’s research focuses on cardiac fibrosis and the therapeutic potential for immune-mediated modulation of scarring. His proposition uses the same scientific approach that CAR T cells use to target cancer cells. Epstein hopes to use CAR T cells to treat cardiac cells by engineering T cells with modified receptors. Using a mouse line expressing an artificial antigen, ovalbumin peptide (OVA), T cell treatment significantly reduced cardiac fibrosis with a partial rescue of cardiac hypertrophy. As a result of this data, Epstein examined human cardiac fibroblast gene expression. In patients who had transplants, he looked for genes in the diseased hearts from fibroblasts and found Fibroblast Activation Protein (FAP). FAP is a marker of activated fibroblasts and expressed by fibroblasts of human HCM and DCM LV myocardium. It is a cell surface glycoprotein expressed during embryogenesis but not found in adult tissues unless injured or cancerous. FAP was seen in heart epicardium one-week post-myocardial infarction and in mouse hearts after Ang/PE treatment.

Mice injected with FAP CAR T cells had reduced cardiac fibrosis and a turnover of extracellular matrix with time.

He then used engineered T cells against FAP and treated the mice at one and two weeks. Unlike control hearts, injured hearts had a lot of FAP CAR T cells populating the scar, indicating that FAP CAR T therapy can reduce cardiac fibrosis. Epstein is optimistic about the future of this technology. “I hope that one day we can create a whole array of CAR T cells engineered against different subsets of activated fibroblasts that might be important in different diseases.” The FAP CAR T cells in mice showed mild and reversible cardiac toxicities and inflammation. So far, FAP CAR T cells have been used in humans with limited reported toxicities. They may play a role in treating skeletal and cardiac muscles, specifically for patients with Duchenne muscular dystrophy and potentially for COVID-19 patients. Eventually, Epstein hopes to make CAR T cells in vivo with lipid nanoparticles to reduce disease burden with a short-term T cell therapy that would only have short-term risks.

T Cell Immune Responses and Cardiac Remodeling

Pilar Alcaide’s lab studies adaptive immunity in diverse inflammatory settings, with a particular focus on heart failure. In examining whether T cells are major contributors to heart failure, Alcaide has shown that T cells from non-ischemic heart failure patients adhere more to activated endothelium. She used a transverse aortic construction model of pressure overload (TAC) to induce heart failure. CD4+ T cells infiltrate the hearts of mice with TAC. However, T cell deficient mice are protected from TAC induced cardiac remodeling. CXCR3+ CD3+ T cells are recruited to the left ventricle in cardiac pressure overload. After identifying cardiac cell populations with FACS, Alcaide learned that CXCL9/CXCL10 production occurs in cardiac myeloid cells and fibroblasts in response to TAC. CXCR3 deficient mice are protected from TAC; they do not have heart infiltrated CD4+ T cells and do not develop cardiac fibrosis in response to TAC. The lab’s overall findings were that CXCR3+ Th1 cells are recruited to the heart in an LFA1-ICAM1 dependent manner by myeloid and CFB released CXCL9 and CXCL10. Currently, Alcaide is studying how T cells are activated through the T cell receptor in heart failure and the role of ROS induced Isolevuglandins (IsoLGs) modified cardiac neoantigens.

Keynote: Reappraising the Role of Inflammation in Heart Failure

In his keynote address, cardiologist Douglas Mann examined how research on inflammation in heart failure has transitioned from the laboratory to the bedside over the past few decades. In the 1990s, researchers noted that tumor necrosis factor was elevated in patients with heart failure. Proinflammatory cytokines were sufficient to cause myocyte hypertrophy, degradation of the matrix, and indirectly stimulate fibrosis. Mann and his colleagues hypothesized that the biological properties of inflammatory mediators could stimulate disease progression in HF. At the time, there was an increase in studies looking at cytokines and cardiomyopathy, many of which ultimately revealed that various cytokines upregulate the genes involved in the progression of HF.

But by 2001, more than one clinical trial studying these therapies was halted. For example, the ATTACH trial using Infliximab, a targeted anti-cytokine therapy, went straight to Phase II without Phase I studies, giving patients varying infusions of the drug. Unfortunately, the drug’s high dosage had an unpredicted effect—significant worsening of HF and death. It was noted that serum TNF levels went up for patients after each infusion. However, after the infusions, the TNF levels stayed elevated long-term, indicating sustained inflammation. The ATTACH trial was one of several attempts to target inflammation in heart failure that ended with neutral results or worsening clinical outcomes.

Progression of studies looking at cytokines and cardiomyopathy from 1990-2000. Experimental findings showed that cytokines and chemokines were sufficient to upregulate genes associated with the progression of heart failure.

However, this class of research returned to the spotlight when Bruce Beutler, Jules Hoffman, and Ralph Steinman won the 2011 Nobel Prize in Physiology or Medicine for their work on innate and adaptive immunity. More researchers have shifted their focus to the potentially significant role of the immune system in the pathogenesis of heart failure. Mann pointed to the importance of the recent CANTOS trial, which showed that the use of canakinumab favorably affected all HF endpoints if there was evidence of inflammation. “This was the first direct study that showed you could target inflammation,” said Mann.

It’s important to note that cardiomyopathies are inherently inflammatory, as they all involve tissue injury and inflammation trying to regain homeostasis. If he could envision the future methods and techniques for his “translational toolbox,” Mann hopes for a method to detect pathophysiological inflammation, a way to look at distinct biomarkers, and improved molecular imaging techniques. He’s confident that the surge of interest in inflammation research will lead to favorable outcomes.  “There’s hope for the future,” said Mann, “that one day we will see trials that are directly targeting inflammation as the primary therapy in advancing or improving outcomes for patients afflicted with cardiovascular disease.”