Single cell transcriptomics of bone marrow derived macrophages reveals Ccl5 as a biomarker of direct IFNAR-independent responses to DNA sensing.

Introduction: The type I interferon (IFN) response is an innate immune program that mediates anti-viral, anti-cancer, auto-immune, auto-inflammatory, and sterile injury responses. Bone marrow derived macrophages (BMDMs) are commonly used to model macrophage type I IFN responses, but the use of bulk measurement techniques obscures underlying cellular heterogeneity. This is particularly important for the IFN response to immune stimulatory double-stranded DNA (dsDNA) because it elicits overlapping direct and indirect responses, the latter of which depend on type I IFN cytokines signaling via the IFN alpha receptor (IFNAR) to upregulate expression of interferon stimulated genes (ISGs). Single cell transcriptomics has emerged as a powerful tool for revealing functional variability within cell populations. Methods: Here, we use single cell RNA-Seq to examine BMDM heterogeneity at steady state and after immune-stimulatory DNA stimulation, with or without IFNAR-dependent amplification. Results: We find that many macrophages express ISGs after DNA stimulation. We also find that a subset of macrophages express ISGs even if IFNAR is inhibited, suggesting that they are direct responders. Analysis of this subset reveals Ccl5 to be an IFNAR-independent marker gene of direct DNA sensing cells. Discussion: Our studies provide a method for studying direct responders to IFN-inducing stimuli and demonstrate the importance of characterizing BMDM models of innate immune responses with single cell resolution.

Neutrophils incite and macrophages avert electrical storm after myocardial infarction.

Sudden cardiac death, arising from abnormal electrical conduction, occurs frequently in patients with coronary heart disease. Myocardial ischemia simultaneously induces arrhythmia and massive myocardial leukocyte changes. In this study, we optimized a mouse model in which hypokalemia combined with myocardial infarction triggered spontaneous ventricular tachycardia in ambulatory mice, and we showed that major leukocyte subsets have opposing effects on cardiac conduction. Neutrophils increased ventricular tachycardia via lipocalin-2 in mice, whereas neutrophilia associated with ventricular tachycardia in patients. In contrast, macrophages protected against arrhythmia. Depleting recruited macrophages in Ccr2 -/- mice or all macrophage subsets with Csf1 receptor inhibition increased both ventricular tachycardia and fibrillation. Higher arrhythmia burden and mortality in Cd36 -/- and Mertk -/- mice, viewed together with reduced mitochondrial integrity and accelerated cardiomyocyte death in the absence of macrophages, indicated that receptor-mediated phagocytosis protects against lethal electrical storm. Thus, modulation of leukocyte function provides a potential therapeutic pathway for reducing the risk of sudden cardiac death.

NOD-like receptor protein 3 activation causes spontaneous inflammation and fibrosis that mimics human NASH.

BACKGROUND AND AIMS: The NOD-like receptor protein 3 (NLRP3) inflammasome is a central contributor to human acute and chronic liver disease, yet the molecular and cellular mechanisms by which its activation precipitates injury remain incompletely understood. Here, we present single cell transcriptomic profiling of livers from a global transgenic tamoxifen-inducible constitutively activated Nlrp3A350V mutant mouse, and we investigate the changes in parenchymal and nonparenchymal liver cell gene expression that accompany inflammation and fibrosis. APPROACH AND RESULTS: Our results demonstrate that NLRP3 activation causes chronic extramedullary myelopoiesis marked by myeloid progenitors that differentiate into proinflammatory neutrophils, monocytes, and monocyte-derived macrophages. We observed prominent neutrophil infiltrates with increased Ly6gHI and Ly6gINT cells exhibiting transcriptomic signatures of granulopoiesis typically found in the bone marrow. This was accompanied by a marked increase in Ly6cHI monocytes differentiating into monocyte-derived macrophages that express transcriptional programs similar to macrophages of NASH models. NLRP3 activation also down-regulated metabolic pathways in hepatocytes and shifted hepatic stellate cells toward an activated profibrotic state based on expression of collagen and extracellular matrix regulatory genes. CONCLUSIONS: These results define the single cell transcriptomes underlying hepatic inflammation and fibrosis precipitated by NLRP3 activation. Clinically, our data support the notion that NLRP3-induced mechanisms should be explored as therapeutic target in NASH-like inflammation.

SiglecF(HI) Marks Late-Stage Neutrophils of the Infarcted Heart: A Single-Cell Transcriptomic Analysis of Neutrophil Diversification.

Background Neutrophils are thought to be short-lived first responders to tissue injuries such as myocardial infarction (MI), but little is known about their diversification or dynamics. Methods and Results We permanently ligated the left anterior descending coronary arteries of mice and performed single-cell RNA sequencing and analysis of >28 000 neutrophil transcriptomes isolated from the heart, peripheral blood, and bone marrow of mice on days 1 to 4 after MI or at steady-state. Unsupervised clustering of cardiac neutrophils revealed 5 major subsets, 3 of which originated in the bone marrow, including a late-emerging granulocyte expressing SiglecF, a marker classically used to define eosinophils. SiglecFHI neutrophils represented ≈25% of neutrophils on day 1 and grew to account for >50% of neutrophils by day 4 post-MI. Validation studies using quantitative polymerase chain reaction of fluorescent-activated cell sorter sorted Ly6G+SiglecFHI and Ly6G+SiglecFLO neutrophils confirmed the distinct nature of these populations. To confirm that the cells were neutrophils rather than eosinophils, we infarcted GATA-deficient mice (∆dblGATA) and observed similar quantities of infiltrating Ly6G+SiglecFHI cells despite marked reductions of conventional eosinophils. In contrast to other neutrophil subsets, Ly6G+SiglecFHI neutrophils expressed high levels of Myc-regulated genes, which are associated with longevity and are consistent with the persistence of this population on day 4 after MI. Conclusions Overall, our data provide a spatial and temporal atlas of neutrophil specialization in response to MI and reveal a dynamic proinflammatory cardiac Ly6G+SigF+(Myc+NFÏ°B+) neutrophil that has been overlooked because of negative selection.

Macrophage calcium reporter mice reveal immune cell communication in vitro and in vivo.

Cell communication underlies emergent functions in diverse cell types and tissues. Recent evidence suggests that macrophages are organized in communicating networks, but new tools are needed to quantitatively characterize the resulting cellular conversations. Here, we infer cell communication from spatiotemporal correlations of intracellular calcium dynamics that are non-destructively imaged across cell populations expressing genetically encoded calcium indicators. We describe a hematopoietic calcium reporter mouse (Csf1rCreGCaMP5fl) and a computational analysis pipeline for inferring communication between reporter cells based on "excess synchrony." We observed signals suggestive of cell communication in macrophages treated with immune-stimulatory DNA in vitro and tumor-associated immune cells imaged in a dorsal window chamber model in vivo. Together, the methods described here expand the toolkit for discovery of cell communication events in macrophages and other immune cells.

The myeloid type I interferon response to myocardial infarction begins in bone marrow and is regulated by Nrf2-activated macrophages.

Sterile tissue injury is thought to locally activate innate immune responses via damage-associated molecular patterns (DAMPs). Whether innate immune pathways are remotely activated remains relatively unexplored. Here, by analyzing ~145,000 single-cell transcriptomes at steady state and after myocardial infarction (MI) in mice and humans, we show that the type I interferon (IFN) response, characterized by expression of IFN-stimulated genes (ISGs), begins far from the site of injury, in neutrophil and monocyte progenitors within the bone marrow. In the peripheral blood of patients, we observed defined subsets of ISG-expressing neutrophils and monocytes. In the bone marrow and blood of mice, ISG expression was detected in neutrophils and monocytes and their progenitors, intensified with maturation at steady-state and after MI, and was controlled by Tet2 and Irf3 transcriptional regulators. Within the infarcted heart, ISG-expressing cells were negatively regulated by Nrf2 activation in Ccr2- steady-state cardiac macrophages. Our results show that IFN signaling begins in the bone marrow, implicate multiple transcriptional regulators (Tet2, Irf3, and Nrf2) in governing ISG expression, and provide a clinical biomarker (ISG score) for studying IFN signaling in patients.

Tissue-Specific Macrophage Responses to Remote Injury Impact the Outcome of Subsequent Local Immune Challenge.

Myocardial infarction, stroke, and sepsis trigger systemic inflammation and organism-wide complications that are difficult to manage. Here, we examined the contribution of macrophages residing in vital organs to the systemic response after these injuries. We generated a comprehensive catalog of changes in macrophage number, origin, and gene expression in the heart, brain, liver, kidney, and lung of mice with myocardial infarction, stroke, or sepsis. Predominantly fueled by heightened local proliferation, tissue macrophage numbers increased systemically. Macrophages in the same organ responded similarly to different injuries by altering expression of tissue-specific gene sets. Preceding myocardial infarction improved survival of subsequent pneumonia due to enhanced bacterial clearance, which was caused by IFNÉ£ priming of alveolar macrophages. Conversely, EGF receptor signaling in macrophages exacerbated inflammatory lung injury. Our data suggest that local injury activates macrophages in remote organs and that targeting macrophages could improve resilience against systemic complications following myocardial infarction, stroke, and sepsis.