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

Abstract

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.