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.

Inferring cell communication using single-cell calcium spatiotemporal dynamics.

There are a limited number of experimental tools for non-destructively discovering cell communication events in vitro and in vivo. Here, using tissue-specific genetically encoded calcium indicator (GECI) mice, we describe a protocol for preprocessing GECI fluorescence time-series measured by live cell imaging or intravital microscopy, detecting peaks of single-cell calcium fluorescence transients, and inferring putative cell communication events from peak synchrony. For complete details on the use and execution of this protocol, please refer to Taghdiri et al. (2021).

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.

Nocturnal Respiratory Rate Dynamics Enable Early Recognition of Impending Hospitalizations.

The days and weeks preceding hospitalization are poorly understood because they transpire before patients are seen in conventional clinical care settings. Home health sensors offer opportunities to learn signatures of impending hospitalizations and facilitate early interventions, however the relevant biomarkers are unknown. Nocturnal respiratory rate (NRR) is an activity-independent biomarker that can be measured by adherence-independent sensors in the home bed. Here, we report automated longitudinal monitoring of NRR dynamics in a cohort of high-risk recently hospitalized patients using non-contact mechanical sensors under patients' home beds. Since the distribution of nocturnal respiratory rates in populations is not well defined, we first quantified it in 2,000 overnight sleep studies from the NHLBI Sleep Heart Health Study. This revealed that interpatient variability was significantly greater than intrapatient variability (NRR variances of 11.7 brpm2 and 5.2 brpm2 respectively, n=1,844,110 epochs), which motivated the use of patient-specific references when monitoring longitudinally. We then performed adherence-independent longitudinal monitoring in the home beds of 34 high-risk patients and collected raw waveforms (sampled at 80 Hz) and derived quantitative NRR statistics and dynamics across 3,403 patient-nights (n= 4,326,167 epochs). We observed 23 hospitalizations for diverse causes (a 30-day hospitalization rate of 20%). Hospitalized patients had significantly greater NRR deviations from baseline compared to those who were not hospitalized (NRR variances of 3.78 brpm2 and 0.84 brpm2 respectively, n= 2,920 nights). These deviations were concentrated prior to the clinical event, suggesting that NRR can identify impending hospitalizations. We analyzed alarm threshold tradeoffs and demonstrated that nominal values would detect 11 of the 23 clinical events while only alarming 2 times in non-hospitalized patients. Taken together, our data demonstrate that NRR dynamics change days to weeks in advance of hospitalizations, with longer prodromes associating with volume overload and heart failure, and shorter prodromes associating with acute infections (pneumonia, septic shock, and covid-19), inflammation (diverticulitis), and GI bleeding. In summary, adherence-independent longitudinal NRR monitoring has potential to facilitate early recognition and management of pre-symptomatic disease.

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.

Low mortality in SARS-CoV-2 infected heart transplant recipients at a single center.

Immunosuppressed heart transplant (HT) recipients are thought to be at higher risk of infection and mortality from SARS-CoV-2 infection coronavirus disease 2019 (COVID-19); however, evidence guiding management of HT patients are limited. Retrospective search of electronic health records from February 2020 to February 2021, identified 28 HT recipients out of 400 followed by UC San Diego who tested positive for SARS-CoV-2. Patient demographics, COVID-19 directed therapies, hospital course and outcomes were compared to control HT recipients who tested negative for SARS-CoV-2 during the same period (n = 80). Among 28 HT recipients who tested positive for SARS-CoV-2, 15 were admitted to the hospital and 13 were monitored closely as outpatients. Among inpatients, five developed severe illness and two died (7% mortality). Nine patients were treated with remdesivir, and four received dexamethasone and remdesivir. Two outpatients received neutralizing monoclonal antibody therapy and one outpatient received dexamethasone for persistent dyspnea. Immunosuppressed HT recipients, especially Hispanic patients and patients with higher body mass index, were at greater risk of infection and mortality from COVID-19 than the general population. Use of remdesivir and dexamethasone may have improved outcomes in our HT recipients compared to HT recipients at other centers.

Passive longitudinal weight and cardiopulmonary monitoring in the home bed.

Home health monitoring has the potential to improve outpatient management of chronic cardiopulmonary diseases such as heart failure. However, it is often limited by the need for adherence to self-measurement, charging and self-application of wearables, or usage of apps. Here, we describe a non-contact, adherence-independent sensor, that when placed beneath the legs of a patient's home bed, longitudinally monitors total body weight, detailed respiratory signals, and ballistocardiograms for months, without requiring any active patient participation. Accompanying algorithms separate weight and respiratory signals when the bed is shared by a partner or a pet. Validation studies demonstrate quantitative equivalence to commercial sensors during overnight sleep studies. The feasibility of detecting obstructive and central apneas, cardiopulmonary coupling, and the hemodynamic consequences of non-sustained ventricular tachycardia is also established. Real-world durability is demonstrated by 3 months of in-home monitoring in an example patient with heart failure and ischemic cardiomyopathy as he recovers from coronary artery bypass grafting surgery. BedScales is the first sensor to measure adherence-independent total body weight as well as longitudinal cardiopulmonary physiology. As such, it has the potential to create a multidimensional picture of chronic disease, learn signatures of impending hospitalization, and enable optimization of care in the home.

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 Power of Single-Cell Analysis for the Study of Liver Pathobiology.

Single cell transcriptomics has emerged as a powerful lens through which to study the molecular diversity of complex tissues such as the liver, during health and disease, both in animal models and in humans. The earliest gene expression methods measured bulk tissue RNA, but the results were often confusing because they derived from the combined transcriptomes of many different cell types in unknown proportions. To better delineate cell-type-specific expression, investigators developed cell isolation, purification, and sorting protocols, yet still, the RNA derived from ensembles of cells obscured recognition of cellular heterogeneity. Profiling transcriptomes at the single-cell level has opened the door to analyses that were not possible in the past. In this review, we discuss the evolution of single cell transcriptomics and how it has been applied for the study of liver physiology and pathobiology to date.

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.

Hepatic gap junctions amplify alcohol liver injury by propagating cGAS-mediated IRF3 activation.

Alcohol-related liver disease (ALD) accounts for the majority of cirrhosis and liver-related deaths worldwide. Activation of IFN-regulatory factor (IRF3) initiates alcohol-induced hepatocyte apoptosis, which fuels a robust secondary inflammatory response that drives ALD. The dominant molecular mechanism by which alcohol activates IRF3 and the pathways that amplify inflammatory signals in ALD remains unknown. Here we show that cytoplasmic sensor cyclic guanosine monophosphate-adenosine monophosphate (AMP) synthase (cGAS) drives IRF3 activation in both alcohol-injured hepatocytes and the neighboring parenchyma via a gap junction intercellular communication pathway. Hepatic RNA-seq analysis of patients with a wide spectrum of ALD revealed that expression of the cGAS-IRF3 pathway correlated positively with disease severity. Alcohol-fed mice demonstrated increased hepatic expression of the cGAS-IRF3 pathway. Mice genetically deficient in cGAS and IRF3 were protected against ALD. Ablation of cGAS in hepatocytes only phenocopied this hepatoprotection, highlighting the critical role of hepatocytes in fueling the cGAS-IRF3 response to alcohol. We identified connexin 32 (Cx32), the predominant hepatic gap junction, as a critical regulator of spreading cGAS-driven IRF3 activation through the liver parenchyma. Disruption of Cx32 in ALD impaired IRF3-stimulated gene expression, resulting in decreased hepatic injury despite an increase in hepatic steatosis. Taken together, these results identify cGAS and Cx32 as key factors in ALD pathogenesis and as potential therapeutic targets for hepatoprotection.

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.

A Miniaturized, Programmable Pacemaker for Long-Term Studies in the Mouse.

RATIONALE: Cardiac pacing is a critical technology for the treatment of arrhythmia and heart failure. The impact of specific pacing strategies on myocardial function is an area of intense research and high clinical significance. Mouse models have proven extremely useful for probing mechanisms of heart disease, but there is currently no reliable technology for long-term pacing in the mouse. OBJECTIVE: We sought to develop a device for long-term pacing studies in mice. We evaluated the device for (1) treating third-degree atrioventricular block after macrophage depletion, (2) ventricular pacing-induced cardiomyopathy, and (3) high-rate atrial pacing. METHODS AND RESULTS: We developed a mouse pacemaker by refashioning a 26 mm×6.7 mm clinical device powered by a miniaturized, highly efficient battery. The electrode was fitted with a single flexible lead, and custom software extended the pacing rate to up to 1200 bpm. The wirelessly programmable device was implanted in the dorsal subcutaneous space of 39 mice. The tunneled lead was passed through a left thoracotomy incision and attached to the epicardial surface of the apex (for ventricular pacing) or the left atrium (for atrial pacing). Mice tolerated the implantation and both long-term atrial and ventricular pacing over weeks. We then validated the pacemaker's suitability for the treatment of atrioventricular block after macrophage depletion in Cd11b DTR mice. Ventricular pacing increased the heart rate from 313±59 to 550 bpm ( P<0.05). In addition, we characterized tachypacing-induced cardiomyopathy in mice. Four weeks of ventricular pacing resulted in reduced left ventricular function, fibrosis, and an increased number of cardiac leukocytes and endothelial activation. Finally, we demonstrated the feasibility of chronic atrial pacing at 1200 bpm. CONCLUSIONS: Long-term pacing with a fully implantable, programmable, and battery-powered device enables previously impossible investigations of arrhythmia and heart failure in the mouse.

IRF3 and type I interferons fuel a fatal response to myocardial infarction.

Interferon regulatory factor 3 (IRF3) and type I interferons (IFNs) protect against infections and cancer, but excessive IRF3 activation and type I IFN production cause autoinflammatory conditions such as Aicardi-Goutières syndrome and STING-associated vasculopathy of infancy (SAVI). Myocardial infarction (MI) elicits inflammation, but the dominant molecular drivers of MI-associated inflammation remain unclear. Here we show that ischemic cell death and uptake of cell debris by macrophages in the heart fuel a fatal response to MI by activating IRF3 and type I IFN production. In mice, single-cell RNA-seq analysis of 4,215 leukocytes isolated from infarcted and non-infarcted hearts showed that MI provokes activation of an IRF3-interferon axis in a distinct population of interferon-inducible cells (IFNICs) that were classified as cardiac macrophages. Mice genetically deficient in cyclic GMP-AMP synthase (cGAS), its adaptor STING, IRF3, or the type I IFN receptor IFNAR exhibited impaired interferon-stimulated gene (ISG) expression and, in the case of mice deficient in IRF3 or IFNAR, improved survival after MI as compared to controls. Interruption of IRF3-dependent signaling resulted in decreased cardiac expression of inflammatory cytokines and chemokines and decreased inflammatory cell infiltration of the heart, as well as in attenuated ventricular dilation and improved cardiac function. Similarly, treatment of mice with an IFNAR-neutralizing antibody after MI ablated the interferon response and improved left ventricular dysfunction and survival. These results identify IRF3 and the type I IFN response as a potential therapeutic target for post-MI cardioprotection.

Stunning neutrophils changes the forecast: No more showers.

Metoprolol inhibits neutrophil-platelet interactions and reduces microvascular obstructions after acute myocardial infarction.

Macrophages Facilitate Electrical Conduction in the Heart.

Organ-specific functions of tissue-resident macrophages in the steady-state heart are unknown. Here, we show that cardiac macrophages facilitate electrical conduction through the distal atrioventricular node, where conducting cells densely intersperse with elongated macrophages expressing connexin 43. When coupled to spontaneously beating cardiomyocytes via connexin-43-containing gap junctions, cardiac macrophages have a negative resting membrane potential and depolarize in synchrony with cardiomyocytes. Conversely, macrophages render the resting membrane potential of cardiomyocytes more positive and, according to computational modeling, accelerate their repolarization. Photostimulation of channelrhodopsin-2-expressing macrophages improves atrioventricular conduction, whereas conditional deletion of connexin 43 in macrophages and congenital lack of macrophages delay atrioventricular conduction. In the Cd11bDTR mouse, macrophage ablation induces progressive atrioventricular block. These observations implicate macrophages in normal and aberrant cardiac conduction.

Squeeze-E squeeze-E.

Adding an electrical pulse to a microfluidic device that squeezes cells through narrow channels efficiently delivers DNA to the nucleus.