Pediatric and adult dilated cardiomyopathy represent distinct pathological entities

Pediatric dilated cardiomyopathy (DCM) is the most common indication for heart transplantation in children. Despite similar genetic etiologies, medications routinely used in adult heart failure patients do not improve outcomes in the pediatric population. The mechanistic basis for these observations is unknown. We hypothesized that pediatric and adult DCM comprise distinct pathological entities, in that children do not undergo adverse remodeling, the target of adult heart failure therapies. To test this hypothesis, we examined LV specimens obtained from pediatric and adult donor controls and DCM patients. Consistent with the established pathophysiology of adult heart failure, adults with DCM displayed marked cardiomyocyte hypertrophy and myocardial fibrosis compared with donor controls. In contrast, pediatric DCM specimens demonstrated minimal cardiomyocyte hypertrophy and myocardial fibrosis compared with both age-matched controls and adults with DCM. Strikingly, RNA sequencing uncovered divergent gene expression profiles in pediatric and adult patients, including enrichment of transcripts associated with adverse remodeling and innate immune activation in adult DCM specimens. Collectively, these findings reveal that pediatric and adult DCM represent distinct pathological entities, provide a mechanistic basis to explain why children fail to respond to adult heart failure therapies, and suggest the need to develop new approaches for pediatric DCM

Immunomodulatory role of non-neuronal cholinergic signaling in myocardial injury

Whereas prior studies have demonstrated an important immunomodulatory role for the neuronal cholinergic system in the heart, the role of the non-neuronal cholinergic system is not well understood. To address the immunomodulatory role of the non-neuronal cholinergic system in the heart we used a previously validated diphtheria toxin (DT)-induced cardiomyocyte ablation model (Rosa26-DTMlc2v-Cre mice). DT-injected Rosa26-DTMlc2v-Cre mice were treated with diluent or Pyridostigmine Bromide (PYR), a reversible cholinesterase inhibitor. PYR treatment resulted in increased survival and decreased numbers of MHC-IIlowCCR2+ macrophages in DT-injected Rosa26-DTMlc2v-Cre mice compared to diluent treated Rosa26-DTMlc2v-Cre mice. Importantly, the expression of CCL2/7 mRNA and protein was reduced in the hearts of PYR-treated mice. Backcrossing Rosa26-DTMlc2v-Cre mice with a transgenic mouse line (Chat-ChR2) that constitutively overexpresses the vesicular acetylcholine transporter (VAChT) resulted in decreased expression of Ccl2/7 mRNA and decreased numbers of CD68+ cells in DT-injured Rosa26-DTMlc2v-Cre/Chat-ChR2 mouse hearts, consistent with the pharmacologic studies with PYR. In vitro studies with cultures of LPS-stimulated peritoneal macrophages revealed a concentration-dependent reduction in CCL2 secretion following stimulation with ACh, nicotine and muscarine. Viewed together, these findings reveal a previously unappreciated immunomodulatory role for the non-neuronal cholinergic system in regulating homeostatic responses in the heart following tissue injury

Modulation of subsets of cardiac B lymphocytes improves cardiac function after acute injury

Despite the long-standing recognition that the immune response to acute myocardial injury contributes to adverse left ventricular (LV) remodeling, it has not been possible to effectively target this clinically. Using 2 different in vivo models of acute myocardial injury, we show that pirfenidone confers beneficial effects in the murine heart through an unexpected mechanism that depends on cardiac B lymphocytes. Naive hearts contained a large population of CD19+CD11b-CD23-CD21-IgD+IgMlo lymphocytes, and 2 smaller populations of CD19+CD11b+ B1a and B1b cells. In response to tissue injury, there was an increase in neutrophils, monocytes, macrophages, as well as an increase in CD19+ CD11b- B lymphocytes. Treatment with pirfenidone had no effect on the number of neutrophils, monocytes, or macrophages, but decreased CD19+CD11b- lymphocytes. B cell depletion abrogated the beneficial effects of pirfenidone. In vitro studies demonstrated that stimulation with lipopolysaccharide and extracts from necrotic cells activated CD19+ lymphocytes through a TIRAP-dependent pathway. Treatment with pirfenidone attenuated this activation of B cells. These findings reveal a previously unappreciated complexity of myocardial B lymphocytes within the inflammatory infiltrate triggered by cardiac injury and suggest that pirfenidone exerts beneficial effects in the heart through a unique mechanism that involves modulation of cardiac B lymphocytes

Network analysis of large-scale ImmGen and Tabula Muris datasets highlights metabolic diversity of tissue mononuclear phagocytes

The diversity of mononuclear phagocyte (MNP) subpopulations across tissues is one of the key physiological characteristics of the immune system. Here, we focus on understanding the metabolic variability of MNPs through metabolic network analysis applied to three large-scale transcriptional datasets: we introduce (1) an ImmGen MNP open-source dataset of 337 samples across 26 tissues; (2) a myeloid subset of ImmGen Phase I dataset (202 MNP samples); and (3) a myeloid mouse single-cell RNA sequencing (scRNA-seq) dataset (51,364 cells) assembled based on Tabula Muris Senis. To analyze such large-scale datasets, we develop a network-based computational approach, genes and metabolites (GAM) clustering, for unbiased identification of the key metabolic subnetworks based on transcriptional profiles. We define 9 metabolic subnetworks that encapsulate the metabolic differences within MNP from 38 different tissues. Obtained modules reveal that cholesterol synthesis appears particularly active within the migratory dendritic cells, while glutathione synthesis is essential for cysteinyl leukotriene production by peritoneal and lung macrophages

Local Modulation and Measurement of Macrophage-Derived Bioactive Proteins from Implanted Biomaterials in Rat

Fibrosis around the implanted medical devices is a severe problem that can plague long-term device reliability. Activation of macrophage phenotype (macrophage polarization) has emerged as a new and possible means for reducing fibrosis in the fields of biomaterials and regenerative medicine. Macrophages are phagocytic cells that respond to microenvironmental cues that direct their phenotype. Macrophage activation has been widely studied in mouse and human in the context of tumor biology, yet little information is available regarding how macrophage activation could be used in a biomaterials context. Further, rats rather than mice are the common subjects in biomaterials experiments. A significant need is to determine if directed macrophage activation can be achieved in a biomaterials context. Cultured rat-derived macrophages (NR8383, peritoneal and spleen) were tested with modulators (dexamethasone, LPS, IL-4 and IL10) to ensure activation towards the expected phenotype. Phenotypic characterization involved gene expression assays (ARG2, CD163, CD206, NOS2, IL-12), protein analysis (IL-6, IL-10, TNF), and flow cytometry (for receptors CD86, CD163, CD206, MHC II). The successful in vitro immunomodulation suggested the feasibility of modulation in vivo. Microdialysis has been used for cytokine sampling (from various tissues including rat mammary tissue for Leptin, CCL2 and IL-6; procedures developed in this work), but can also be employed for modulator delivery. To achieve in vivo immunomodulation, microdialysis experiments were performed to locally deliver either IL-4 or IL-10. No significant differences in dialysate cytokine levels (CCL2, IL-1& and IL-6) or histological sections (H&E and Masson\u27s trichrome) of tissue surrounding the probe were observed between control and treatment groups. However, gene expression analysis of tissue to which IL-4 was delivered suggests down-regulation in CCL2 and IL-6. To enhance modulator amount and surface area, a polyvinyl alcohol (PVA) sponge model was employed. The IL-4 impregnated sponges had reduced IL-1 concentrations compared to controls. However, no significant differences in gene expression were observed. For IL-10, down regulation of CCL2 (also at protein level), IL-1, IL-12 and NOS2 genes was observed. This initial work in macrophage activation for biomaterials in rats provides a fundamental framework for future work in this fiel

TFEB activation in macrophages attenuates postmyocardial infarction ventricular dysfunction independently of ATG5-mediated autophagy

Lysosomes are at the epicenter of cellular processes critical for inflammasome activation in macrophages. Inflammasome activation and IL-1β secretion are implicated in myocardial infarction (MI) and resultant heart failure; however, little is known about how macrophage lysosomes regulate these processes. In mice subjected to cardiac ischemia/reperfusion (IR) injury and humans with ischemic cardiomyopathy, we observed evidence of lysosomal impairment in macrophages. Inducible macrophage-specific overexpression of transcription factor EB (TFEB), a master regulator of lysosome biogenesis (Mϕ-TFEB), attenuated postinfarction remodeling, decreased abundance of proinflammatory macrophages, and reduced levels of myocardial IL-1β compared with controls. Surprisingly, neither inflammasome suppression nor Mϕ-TFEB-mediated attenuation of postinfarction myocardial dysfunction required intact ATG5-dependent macroautophagy (hereafter termed autophagy ). RNA-seq of flow-sorted macrophages postinfarction revealed that Mϕ-TFEB upregulated key targets involved in lysosomal lipid metabolism. Specifically, inhibition of the TFEB target, lysosomal acid lipase, in vivo abrogated the beneficial effect of Mϕ-TFEB on postinfarction ventricular function. Thus, TFEB reprograms macrophage lysosomal lipid metabolism to attenuate remodeling after IR, suggesting an alternative paradigm whereby lysosome function affects inflammation