King, K. R. | Search bioRxiv

bioRxiv2021

Single-Cell Transcriptomic Analysis of Livers During NLRP3 Inflammasome Activation Reveals a Novel Immune Niche

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 non-parenchymal liver cell gene expression that accompany inflammation and fibrosis. Our results demonstrate that NLRP3 activation causes chronic extramedullary myelopoiesis marked by an increase in proliferating myeloid progenitors that differentiate into neutrophils, monocytes, and monocyte-derived macrophages, results that were corroborated by flow cytometry and histological staining. 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 Cd11bHITim4HIClec4fHI macrophages that express proinflammatory transcriptional programs similar to macrophages of non-alcoholic steatohepatitis (NASH) models. NLRP3 activation also downregulated metabolic pathways in hepatocytes and shifted hepatic stellate cells towards an activated pro-fibrotic state based on expression of collagen and extracellular matrix (ECM) regulatory genes. These results, which highlight abundant neutrophils and extramedullary granulopoiesis define an inflamed and fibrotic hepatic single cell microenvironment, precipitated solely by NLRP3 activation. Clinically, our data support the notion that neutrophils and NLRP3 should be explored as therapeutic targets in NASH-like inflammation.

immunology↗

bioRxiv2021

Csf1r-GCaMP5 Reporter Mice Reveal Immune Cell Communication in Vitro and in Vivo

Interconnected cells are responsible for emergent functions ranging from cognition in the brain to cyclic contraction in the heart. In electrically excitable cells, methods for studying cell communication are highly advanced, but in non-excitable cells, generalized methods for studying cell communication are less mature. Immune cells have generally been classified as non-excitable cells with diverse pathophysiologic roles that span every tissue in the body, yet little is known about their interconnectedness because assays are destructive and have low temporal resolution. In this work, we hypothesize that non-excitable immune cells are functionally interconnected in previously unrecognized cell communication networks. To test the hypothesis, we created a hematopoietic calcium reporter mouse (Csf1r-Cre x GCaMP5) and non-destructively quantified the spatiotemporal dynamics of intracellular calcium in vitro and in vivo. In vitro, bone marrow derived macrophages calcium reporters reveal that fatal immune stimulatory DNA-sensing induces rapid intercellular communication to neighboring cells. In vivo, using intravital microscopy through a dorsal window chamber in the context of MC38-H2B-mCherry tumors, Csf1r-GCaMP5 reporters exhibit spatiotemporal dynamics consistent with cell communication. We present a theoretical framework and analysis pipeline for identifying spatiotemporal locations of "excess synchrony" of calcium spiking as a means of inferring previously unrecognized cell communication events. Together, these methods provide a toolkit for investigating known and as-yet-undiscovered cell communication events in vitro and in vivo.

immunology↗

bioRxiv2020

Bedscales: A non-contact adherence-independent multi-person sensor for longitudinal physiologic monitoring in the home bed

Home health monitoring technologies promise to improve care and reduce costs, yet they are limited by the need for adherence to self-monitoring, usage of an app, or application of a wearable. While implantable sensors overcome the adherence barrier, they are expensive and require invasive procedures. Here, we describe a non-invasive, non-contact, adherence-independent sensor, that when placed beneath the legs of a patients home bed, longitudinally monitors total body weight, detailed respiratory signals, and ballistocardiograms for months, without requiring any active patient participation. Accompanying algorithms demix weight and respiratory signals when the bed is shared by a partner or a pet. Validation studies during overnight clinical sleep studies exhibit quantitative equivalence to commercial sensors and allow discrimination of obstructive and central sleep apneas. In-home studies discriminate atrial fibrillation from normal sinus rhythm. To demonstrate real-world feasibility, we performed 3 months of continuous in-home monitoring in a patient with heart failure as he awaited and recovered from coronary artery bypass surgery. By overcoming the adherence barrier, Bedscales has the potential to create a multidimensional picture of chronic disease, learn signatures of impending hospitalization, and enable optimization of care in the home. DisclosuresDrs. King and Coleman and Nicholas Harrington are inventors on a patent application describing the Bedscales technology. Dr. Kevin R. King discloses consulting relationships with Bristol Myers Squibb and Astrazeneca, Medimmune and is founder of Nightingale Labs. Dr. Deepak L. Bhatt discloses the following relationships - Advisory Board: Cardax, Cereno Scientific, Elsevier Practice Update Cardiology, Medscape Cardiology, PhaseBio, Regado Biosciences; Board of Directors: Boston VA Research Institute, Society of Cardiovascular Patient Care, TobeSoft; Chair: American Heart Association Quality Oversight Committee; Data Monitoring Committees: Baim Institute for Clinical Research (formerly Harvard Clinical Research Institute, for the PORTICO trial, funded by St. Jude Medical, now Abbott), Cleveland Clinic (including for the ExCEED trial, funded by Edwards), Duke Clinical Research Institute, Mayo Clinic, Mount Sinai School of Medicine (for the ENVISAGE trial, funded by Daiichi Sankyo), Population Health Research Institute; Honoraria: American College of Cardiology (Senior Associate Editor, Clinical Trials and News, ACC.org; Vice-Chair, ACC Accreditation Committee), Baim Institute for Clinical Research (formerly Harvard Clinical Research Institute; RE-DUAL PCI clinical trial steering committee funded by Boehringer Ingelheim; AEGIS-II executive committee funded by CSL Behring), Belvoir Publications (Editor in Chief, Harvard Heart Letter), Duke Clinical Research Institute (clinical trial steering committees, including for the PRONOUNCE trial, funded by Ferring Pharmaceuticals), HMP Global (Editor in Chief, Journal of Invasive Cardiology), Journal of the American College of Cardiology (Guest Editor; Associate Editor), Medtelligence/ReachMD (CME steering committees), Population Health Research Institute (for the COMPASS operations committee, publications committee, steering committee, and USA national co-leader, funded by Bayer), Slack Publications (Chief Medical Editor, Cardiology Todays Intervention), Society of Cardiovascular Patient Care (Secretary/Treasurer), WebMD (CME steering committees); Other: Clinical Cardiology (Deputy Editor), NCDR-ACTION Registry Steering Committee (Chair), VA CART Research and Publications Committee (Chair); Research Funding: Abbott, Afimmune, Amarin, Amgen, AstraZeneca, Bayer, Boehringer Ingelheim, Bristol-Myers Squibb, Chiesi, CSL Behring, Eisai, Ethicon, Ferring Pharmaceuticals, Forest Laboratories, Fractyl, Idorsia, Ironwood, Ischemix, Lilly, Medtronic, PhaseBio, Pfizer, PLx Pharma, Regeneron, Roche, Sanofi Aventis, Synaptic, The Medicines Company; Royalties: Elsevier (Editor, Cardiovascular Intervention: A Companion to Braunwalds Heart Disease); Site Co-Investigator: Biotronik, Boston Scientific, CSI, St. Jude Medical (now Abbott), Svelte; Trustee: American College of Cardiology; Unfunded Research: FlowCo, Merck, Novo Nordisk, Takeda. Dr. Akshay S. Desai discloses the following relationships - Research grants to Brigham and Womens Hospital to support clinical trial activities from Alnylam, AstraZeneca, and Novartis; Consulting fees from Abbott, Alnylam, AstraZeneca, Biofourmis, Boehringer-Ingelheim, Boston Scientific, Merck, Novartis, Relypsa, Regeneron. Dr. Owens reports consulting fees from Novartis, and research grants to UCSD from Snoozeal, Nitto Denko, and Masimo.

bioengineering↗