The Emerging Role of B Lymphocytes in Cardiovascular Disease.

B cells are traditionally known for their ability to produce antibodies in the context of adaptive immune responses. However, over the last decade B cells have been increasingly recognized as modulators of both adaptive and innate immune responses, as well as players in an important role in the pathogenesis of a variety of human diseases. Here, after briefly summarizing our current understanding of B cell biology, we present a systematic review of the literature from both animal models and human studies that highlight the important role that B lymphocytes play in cardiac and vascular disease. While many aspects of B cell biology in the vasculature and, to an even greater extent, in the heart remain unclear, B cells are emerging as key regulators of cardiovascular adaptation to injury.

Myocardial Immune Cells: The Basis of Cardiac Immunology.

The mammalian heart is characterized by the presence of striated myocytes, which allow continuous rhythmic contraction from early embryonic development until the last moments of life. However, the myocardium contains a significant contingent of leukocytes from every major class. This leukocyte pool includes both resident and nonresident immune cells. Over recent decades, it has become increasingly apparent that the heart is intimately sensitive to immune signaling and that myocardial leukocytes exhibit an array of critical functions, both in homeostasis and in the context of cardiac adaptation to injury. Here, we systematically review current knowledge of all major leukocyte classes in the heart, discussing their functions in health and disease. We also highlight the connection between the myocardium, immune cells, lymphoid organs, and both local and systemic immune responses.

Rilonacept use in lupus pericarditis.

OBJECTIVES: Lupus pericarditis affects 22% of patients with systemic lupus erythematosus (SLE), is associated with worse outcomes, and often requires immunosuppression. Rilonacept is an interleukin-1 receptor antagonist approved for the treatment of recurrent idiopathic pericarditis, but its efficacy in lupus pericarditis is unknown. Here, we report the efficacy of rilonacept in a case series of patients with lupus pericarditis. METHODS: We describe a case series of 4 patients with refractory lupus pericarditis treated with rilonacept in the Johns Hopkins Lupus Center. All patients met the 2012 SLICC criteria for SLE. Refractory lupus pericarditis was defined as recurring or persistent typical pericardial pain symptoms despite standard-of-care treatment including at least one immunosuppressant. RESULTS: Four patients with refractory pericarditis were included. All patients were women, age ranged 26-44 years, 2 patients reported White, 1 Black, and 1 Hispanic ethnicity. Extra-pericardial SLE manifestations were heterogeneous among patients. Only 1 of 3 patient had elevated CRP (not measured in one). Two patients were previously treated with anakinra with initial response, but pericarditis redeveloped in both. Rilonacept led to complete resolution of pericardial symptoms in 3 patients, and partial resolution (40%) in 1, within 2 weeks. CONCLUSIONS: Rilonacept successfully treated lupus pericarditis in this case series. Rilonacept should be considered for the treatment of lupus pericarditis.

The Role of B Cells in Cardiomyopathy and Heart Failure.

PURPOSE OF REVIEW: To summarize the current knowledge on the role that B lymphocytes play in heart failure. RECENT FINDINGS: Several studies from murine models have shown that B cells modulate cardiac adaptation to injury and ultimately affect the degree of cardiac dysfunction after acute ischemic damage. In addition, a B cell-modulating small molecule was recently shown to have beneficial effects in humans with heart failure with preserved ejection fraction. B lymphocytes are specialized immune cells present in all jawed vertebrates. They are characteristically known for their ability to produce antibodies, but they have other functions and are important players in virtually all forms of immune responses. A growing body of evidence indicates that B cells are intimately connected with the heart and that B cell dysregulation might play a role in the pathogenesis and progression of both heart failure with reduced ejection fraction and heart failure with preserved ejection fraction. B cells are therefore gathering attention as potential targets for the development of novel immunomodulatory-based treatments for heart failure.

B cells modulate the expression of MHC-II on cardiac CCR2- macrophages.

The uninjured murine heart contains a heterogeneous population of macrophages with disparate ontogenies and functions. These macrophages are often associated with blood vessels and can be subclassified based on the expression of CC chemokine receptor 2 (CCR2) and major histocompatibility complex class II (MHC-II). The biological cues that modulate these macrophage pool subpopulations have not been completely identified. It has been recently shown that a sub-population of circulating naïve B cells adheres to the myocardial microvasculature. We hypothesized that B cells might modulate the phenotype of myocardial macrophages. To test this hypothesis, we analyzed both the relative location of B cells and macrophages in myocardial histological section and the prevalence of myocardial macrophage subsets in hearts from B cell-deficient mice (µMT) and mice depleted of B cells through administration of an anti-CD20 antibody. We found that B cells pause in the microvasculature in proximity of macrophages and modulate the number of myocardial CCR2-MHC-IIhigh cells. Through in vitro studies we found that this is likely the result of a paracrine effect of B cells on the expression of MHC-II in CCR2- cells. These results reveal an unexpected relationship between B cells and resident macrophages and, highlighting a direct intramyocardial effect of circulating B cells, challenge the currently held belief that naïve recirculating B lymphocytes merely shuttle between lymphoid stations.

Reduced Apolipoprotein M and Adverse Outcomes Across the Spectrum of Human Heart Failure.

BACKGROUND: Apo (apolipoprotein) M mediates the physical interaction between high-density lipoprotein (HDL) particles and sphingosine-1-phosphate (S1P). Apo M exerts anti-inflammatory and cardioprotective effects in animal models. METHODS: In a subset of PHFS (Penn Heart Failure Study) participants (n=297), we measured apo M by Enzyme-Linked ImmunoSorbent Assay (ELISA). We also measured total S1P by liquid chromatography-mass spectrometry and isolated HDL particles to test the association between apo M and HDL-associated S1P. We confirmed the relationship between apo M and outcomes using modified aptamer-based apo M measurements among 2170 adults in the PHFS and 2 independent cohorts: the Washington University Heart Failure Registry (n=173) and a subset of TOPCAT (Treatment of Preserved Cardiac Function Heart Failure With an Aldosterone Antagonist Trial; n=218). Last, we examined the relationship between apo M and ≈5000 other proteins (SomaScan assay) to identify biological pathways associated with apo M in heart failure. RESULTS: In the PHFS, apo M was inversely associated with the risk of death (standardized hazard ratio, 0.56 [95% CI, 0.51-0.61]; P<0.0001) and the composite of death/ventricular assist device implantation/heart transplantation (standardized hazard ratio, 0.62 [95% CI, 0.58-0.67]; P<0.0001). This relationship was independent of HDL cholesterol or apo AI levels. Apo M remained associated with death (hazard ratio, 0.78 [95% CI, 0.69-0.88]; P<0.0001) and the composite of death/ventricular assist device/heart transplantation (hazard ratio, 0.85 [95% CI, 0.76-0.94]; P=0.001) in models that adjusted for multiple confounders. This association was present in both heart failure with reduced and preserved ejection fraction and was replicated in the Washington University cohort and a cohort with heart failure with preserved ejection fraction only (TOPCAT). The S1P and apo M content of isolated HDL particles strongly correlated (R=0.81, P<0.0001). The top canonical pathways associated with apo M were inflammation (negative association), the coagulation system (negative association), and liver X receptor/retinoid X receptor activation (positive association). The relationship with inflammation was validated with multiple inflammatory markers measured with independent assays. CONCLUSIONS: Reduced circulating apo M is independently associated with adverse outcomes across the spectrum of human heart failure. Further research is needed to assess whether the apo M/S1P axis is a suitable therapeutic target in heart failure.

Pirfenidone is a cardioprotective drug: Mechanisms of action and preclinical evidence.

Myocardial fibrosis is an endogenous response to different cardiac insults that may become maladaptive over time and contribute to the onset and progression of heart failure (HF). Fibrosis is a direct and indirect target of established HF therapies, namely inhibitors of the renin-angiotensin-aldosterone system, but its resilience to therapy warrants a search for novel, more targeted approaches to myocardial fibrosis. Pirfenidone is a drug approved for idiopathic pulmonary fibrosis, a severe form of idiopathic interstitial pneumonias. Pirfenidone is a small synthetic molecule with high oral bioavailability, exerting an antifibrotic activity, but also anti-oxidant and anti-inflammatory effects. These effects have been attributed to the inhibition of several growth factors (in particular transforming growth factor-ß, but also platelet-derived growth factor and beta fibroblast growth factor), matrix metalloproteinases, and pro-inflammatory mediators (such as interleukin-1ß and tumour necrosis factor-α), and possibly also an improvement of mitochondrial function and modulation of lymphocyte activation. Given the activation of similar profibrotic pathways in lung and heart disease, the crucial role of fibrosis in several cardiac disorders, and the wide spectrum of activity of pirfenidone, this drug has been evaluated with interest as a potential treatment for cardiac disorders. In animal studies, pirfenidone has shown cardioprotective effects across different species and in a variety of models of cardiomyopathy. In the present review we summarize the pharmacological characteristics of pirfenidone and the data from animal studies supporting its cardioprotective effects.

Biomechanical forces promote embryonic haematopoiesis.

Biomechanical forces are emerging as critical regulators of embryogenesis, particularly in the developing cardiovascular system. After initiation of the heartbeat in vertebrates, cells lining the ventral aspect of the dorsal aorta, the placental vessels, and the umbilical and vitelline arteries initiate expression of the transcription factor Runx1 (refs 3-5), a master regulator of haematopoiesis, and give rise to haematopoietic cells. It remains unknown whether the biomechanical forces imposed on the vascular wall at this developmental stage act as a determinant of haematopoietic potential. Here, using mouse embryonic stem cells differentiated in vitro, we show that fluid shear stress increases the expression of Runx1 in CD41(+)c-Kit(+) haematopoietic progenitor cells, concomitantly augmenting their haematopoietic colony-forming potential. Moreover, we find that shear stress increases haematopoietic colony-forming potential and expression of haematopoietic markers in the para-aortic splanchnopleura/aorta-gonads-mesonephros of mouse embryos and that abrogation of nitric oxide, a mediator of shear-stress-induced signalling, compromises haematopoietic potential in vitro and in vivo. Collectively, these data reveal a critical role for biomechanical forces in haematopoietic development.

B cell-mediated antigen presentation promotes adverse cardiac remodeling in chronic heart failure.

Cardiovascular disease remains the leading cause of death worldwide. A primary driver of cardiovascular mortality is ischemic heart failure, a form of cardiac dysfunction that can develop in patients who survive myocardial infarction. Acute cardiac damage triggers robust changes in the spleen with rapid migration of immune cells from the spleen to the heart. Activating this "cardio-splenic" axis contributes to progressive cardiac dysfunction. The cardio-splenic axis has, therefore, been identified as a promising therapeutic target to prevent or treat heart failure. However, our understanding of the precise mechanisms by which specific immune cells contribute to adverse cardiac remodeling within the cardio-splenic axis remains limited. Here, we show that splenic B cells contribute to the development of heart failure via MHC II-mediated antigen presentation. We found that the adoptive transfer of splenic B cells from mice with ischemic heart failure promoted adverse cardiac remodeling and splenic inflammatory changes in naïve recipient mice. Based on single-cell RNA sequencing analysis of splenic B cells from mice with ischemic heart failure, we hypothesized that B cells contributed to adverse cardiac remodeling through antigen presentation by MHC II molecules. This mechanism was confirmed using transgenic mice with B cell-specific MHC II deletion, and by analyzing circulating B cells from humans who experienced myocardial infarction. Our results broaden our understanding of B lymphocyte biology, reshape current models of immune activation in response to myocardial injury, and point towards MHC II-mediated signaling in B cells as a novel and specific therapeutic target in chronic heart failure.

B cell-mediated antigen presentation promotes adverse cardiac remodeling in chronic heart failure.

Cardiovascular disease remains the leading cause of death worldwide. A primary driver of cardiovascular mortality is ischemic heart failure, a form of cardiac dysfunction that can develop in patients who survive myocardial infarction. Acute cardiac damage triggers robust changes in the spleen with rapid migration of immune cells from the spleen to the heart. Activating this "cardio-splenic" axis contributes to progressive cardiac dysfunction. The cardio-splenic axis has, therefore, been identified as a promising therapeutic target to prevent or treat heart failure. However, our understanding of the precise mechanisms by which specific immune cells contribute to adverse cardiac remodeling within the cardio-splenic axis remains limited. Here, we show that splenic B cells contribute to the development of heart failure via MHC II-mediated antigen presentation. We found that the adoptive transfer of splenic B cells from mice with ischemic heart failure promoted adverse cardiac remodeling and splenic inflammatory changes in naïve recipient mice. Based on single-cell RNA sequencing analysis of splenic B cells from mice with ischemic heart failure, we hypothesized that B cells contributed to adverse cardiac remodeling through antigen presentation by MHC II molecules. This mechanism was confirmed using transgenic mice with B cell-specific MHC II deletion, and by analyzing circulating B cells from humans who experienced myocardial infarction. Our results broaden our understanding of B lymphocyte biology, reshape current models of immune activation in response to myocardial injury, and point towards MHC II-mediated signaling in B cells as a novel and specific therapeutic target in chronic heart failure.

Myocardial B cells have specific gene expression and predicted interactions in dilated cardiomyopathy and arrhythmogenic right ventricular cardiomyopathy.

Introduction: Growing evidence from animal models indicates that the myocardium hosts a population of B cells that play a role in the development of cardiomyopathy. However, there is minimal data on human myocardial B cells in the context of cardiomyopathy. Methods: We integrated single-cell and single-nuclei datasets from 45 healthy human hearts, 70 hearts with dilated cardiomyopathy (DCM), and 8 hearts with arrhythmogenic right ventricular cardiomyopathy (ARVC). Interactions between B cells and other cell types were investigated using the CellChat Package. Differential gene expression analysis comparing B cells across conditions was performed using DESeq2. Pathway analysis was performed using Ingenuity, KEGG, and GO pathways analysis. Results: We identified 1,100 B cells, including naive B cells and plasma cells. Cells showed an extensive network of interactions within the healthy myocardium that included outgoing signaling to macrophages, T cells, endothelial cells, and pericytes, and incoming signaling from endothelial cells, pericytes, and fibroblasts. This niche relied on ECM-receptor, contact, and paracrine interactions; and changed significantly in the context of cardiomyopathy, displaying disease-specific features. Differential gene expression analysis showed that in the context of DCM both naive and plasma B cells upregulated several pathways related to immune activation, including upregulation of oxidative phosphorylation, upregulation of leukocyte extravasation, and, in naive B cells, antigen presentation. Discussion: The human myocardium contains naive B cells and plasma cells, integrated into a diverse and dynamic niche that has distinctive features in healthy, DCM, and ARVC. Naive myocardial-associated B cells likely contribute to the pathogenesis of human DCM.

A Machine Learning-Derived Score to Effectively Identify Heart Failure With Preserved Ejection Fraction.

Background: The diagnosis of heart failure with preserved ejection fraction (HFpEF) in the clinical setting remains challenging, especially in patients with obesity. Objectives: This study aimed to identify novel predictors of HFpEF well suited for patients with obesity. Methods: We performed a retrospective analysis of a well-characterized cohort of patients with obesity with HFpEF (n = 404; mean body mass index [BMI] 36.6 kg/m2) and controls (n = 67). We used the machine learning algorithm Gradient Boosting Machine to analyze the association of various parameters with the diagnosis of HFpEF and subsequently created a multivariate logistic model for the diagnosis. Results: Gradient Boosting Machine identified BMI, estimated glomerular filtration rate, left ventricular mass index, and left atrial to left ventricular volume ratio as the strongest predictors of HFpEF. These variables were used to build a model that identified HFpEF with a sensitivity of 0.83, a specificity of 0.82, and an area under the curve (AUC) of 0.88. Internal validation of the model with optimism-adjusted AUC showed an AUC of 0.87. Within the studied cohort, the novel score outperformed the H2FPEF score (AUC: 0.88 vs 0.74; P < 0.001). Conclusions: In a HFpEF cohort with obesity, BMI, estimated glomerular filtration rate, left ventricular mass index, and left atrial to left ventricular volume ratio most correlated with the identification of HFpEF, and a score based on these variables (HFpEF-JH score) outperformed the currently used H2PEF score. Further validation of this novel score is warranted, as it may facilitate improved diagnostic accuracy of HFpEF, particularly in patients with obesity.

Myocardial B cells have specific gene expression and predicted interactions in Dilated Cardiomyopathy and Arrhythmogenic Right Ventricular Cardiomyopathy.

Introduction: Growing evidence from animal models indicates that the myocardium hosts a population of B cells that play a role in the development of cardiomyopathy. However, there is minimal data on human myocardial B cells in the context of cardiomyopathy. Methods: We integrated single-cell and single-nuclei datasets from 45 healthy human hearts, 70 hearts with dilated cardiomyopathy (DCM), and 8 hearts with Arrhythmogenic Right Ventricular Cardiomyopathy (ARVC). Interactions between B cells and other cell types were investigated using the CellChat Package. Differential gene expression analysis comparing B cells across conditions was performed using DESeq2. Pathway analysis was performed using Ingenuity, KEGG, and GO pathways analysis. Results: We identified 1,100 B cells, including naive B cells and plasma cells. B cells showed an extensive network of interactions within the healthy myocardium that included outgoing signaling to macrophages, T cells, endothelial cells, and pericytes, and incoming signaling from endothelial cells, pericytes, and fibroblasts. This niche relied on ECM-receptor, contact, and paracrine interaction; and changed significantly in the context of cardiomyopathy, displaying disease-specific features. Differential gene expression analysis showed that in the context of DCM both naive and plasma B cells upregulated several pathways related to immune activation, including upregulation of oxidative phosphorylation, upregulation of leukocyte extravasation, and, in naive B cells, antigen presentation. Discussion: The human myocardium contains naive B cells and plasma cells, integrated into a diverse and dynamic niche that has distinctive features in healthy myocardium, DCM, and ARVC. Naive myocardial-associated B cells likely contribute to the pathogenesis of human DCM.

RelA-mediated signaling connects adaptation to chronic cardiomyocyte stress with myocardial and systemic inflammation in the ADCY8 model of accelerated aging.

Mice with cardiac-specific overexpression of adenylyl cyclase (AC) type 8 (TGAC8) are under a constant state of severe myocardial stress. They have a remarkable ability to adapt to this stress, but they eventually develop accelerated cardiac aging and experience reduced longevity. We have previously demonstrated through bioinformatics that constitutive adenylyl cyclase activation in TGAC8 mice is associated with the activation of inflammation-related signaling pathways. However, the immune response associated with chronic myocardial stress in the TGAC8 mouse remains unexplored. Here we demonstrate that chronic activation of adenylyl cyclase in cardiomyocytes of TGAC8 mice results in activation of cell-autonomous RelA-mediated NF-κB signaling. This is associated with non-cell-autonomous activation of proinflammatory and age-associated signaling in myocardial endothelial cells and myocardial smooth muscle cells, expansion of myocardial immune cells, increase in serum levels of inflammatory cytokines, and changes in the size or composition of lymphoid organs. All these changes precede the appearance of cardiac fibrosis. We provide evidence indicating that RelA activation in cardiomyocytes with chronic activation of adenylyl cyclase is mediated by calcium-protein Kinase A (PKA) signaling. Using a model of chronic cardiomyocyte stress and accelerated aging, we highlight a novel, calcium/PKA/RelA-dependent connection between cardiomyocyte stress, myocardial inflammation, and systemic inflammation. These findings suggest that RelA-mediated signaling in cardiomyocytes might be an adaptive response to stress that, when chronically activated, ultimately contributes to both cardiac and systemic aging.

The vascular origin of hematopoietic cells.

More than a century ago, several embryologists described sites of hematopoietic activity in the vascular wall of mid-gestation vertebrate embryos, and postulated the transient existence of a blood generating endothelium during ontogeny. This hypothesis gained significant attention in the 1970s when orthotopic transplantation experiments between quail and chick embryos revealed specific vascular areas as the site of the origin of definitive hematopoiesis. However, the vascular origin of hematopoietic precursors remained elusive and controversial for decades. Only recently, multiple experimental approaches have clearly documented that during vertebrate development definitive hematopoietic precursors arise from a subset of vascular endothelial cells. Interestingly, this differentiation is promoted by the intravascular fluid mechanical forces generated by the establishment of blood flow upon the initiation of heartbeat, and it is therefore connected with cardiovascular development in several critical aspects. In this review we present our current understanding of the relationship between vascular and definitive hematopoietic development through an historical analysis of the scientific evidence produced in this area of investigation.

A murine model of Trypanosoma brucei-induced myocarditis and cardiac dysfunction.

Trypanosoma brucei is a protozoan parasite that causes human and animal African trypanosomiases (HAT and AAT). Cardiac symptoms are commonly reported in HAT patients, and intracardiac parasites with accompanying myocarditis have been observed in both natural hosts and animal models of T. brucei infection. However, despite the importance of T. brucei as a cause of cardiac dysfunction and the dramatic socioeconomic impact of African trypanosomiases in sub-Saharan Africa, there are currently no reproducible murine models of T. brucei-associated cardiomyopathy. We present the first clinically relevant, reproducible murine model of cardiac dysfunction in chronic T. brucei infection. Similar to humans, mice showed histological evidence of myocarditis and elevation of serum NT-proBNP. Serum NT-proBNP levels were elevated prior to the development of severe ventricular dysfunction. On flow cytometry, myocarditis was associated with an increase of most myocardial immune cell populations, including multiple T cell and macrophage subsets, corroborating the notion that T. brucei-associated cardiac damage is an immune-mediated event. This novel mouse model represents a powerful and practical tool to investigate the pathogenesis of T. brucei-mediated heart damage and support the development of therapeutic options for T. brucei-associated cardiac disease.

Necrotic cardiac myocytes skew macrophage polarization towards a classically activated phenotype.

Necrotic and dying cells release damage-associated molecular patterns (DAMPs) that can initiate sterile inflammatory responses in the heart. Although macrophages are essential for myocardial repair and regeneration, the effect of DAMPs on macrophage activation remains unclear. To address this gap in knowledge we studied the effect of necrotic cardiac myocyte extracts on primary peritoneal macrophage (PPM) cultures in vitro. We first performed unbiased transcriptomic profiling with RNA-sequencing of PPMs cultured for up to 72 hours in the presence and absence of: 1) necrotic cell extracts (NCEs) from necrotic cardiac myocytes in order to mimic the release of DAMPs; 2) lipopolysaccharide (LPS), which is known to polarize macrophages towards a classically activated phenotype and 3) Interleukin-4 (IL-4), which is known to promote polarization of macrophages towards an alternatively activated phenotype. NCEs provoke changes in differential gene expression (DEGs) that had considerable overlap with LPS-induced changes, suggesting that NCEs promote macrophage polarization towards a classically activated phenotype. Treating NCEs with proteinase-K abolished the effects of NCEs on macrophage activation, whereas NCE treatment with DNase and RNase did not affect macrophage activation. Stimulation of macrophage cultures with NCEs and LPS resulted in a significant increase in macrophage phagocytosis and interleukin-1ß secretion, whereas treatment with IL-4 had no significant effect on phagocytosis and interleukin-1ß. Taken together, our findings suggest that proteins released from necrotic cardiac myocytes are sufficient to skew the polarization of macrophages towards a classically activated phenotype.

Cardiomyocyte-specific adenylyl cyclase type-8 overexpression induces activation of RelA together with myocardial and systemic inflammation.

Background: Mice with cardiac-specific overexpression of adenylyl cyclase (AC) type 8 (TG AC8 ) are under a constant state of severe myocardial stress. They have a remarkable ability to adapt to this stress, but they eventually develop accelerated cardiac aging and experience reduced longevity. Results: Here we demonstrate that activation of ACVIII in cardiomyocytes results in cell-autonomous RelA-mediated NF-κB signaling. This is associated with non-cell-autonomous activation of proinflammatory and age-associated signaling in myocardial endothelial cells and myocardial smooth muscle cells, expansion of myocardial immune cells, increase in serum levels of inflammatory cytokines, and changes in the size or composition of lymphoid organs. These changes precede the appearance of cardiac fibrosis. We provide evidence indicating that ACVIII-driven RelA activation in cardiomyocytes is mediated by calcium-Protein Kinase A (PKA) signaling. Conclusions: Using a model of chronic cardiomyocyte stress and accelerated aging we highlight a novel, PKA/RelA-dependent connection between cardiomyocyte stress, myocardial para-inflammation and systemic inflammation. These findings point to RelA-mediated signaling in cardiomyocytes and inter-organ communication between the heart and lymphoid organs as novel potential therapeutic targets to reduce age-associated myocardial deterioration.