Single-Cell Profiling Identifies Cell Subsets with Inflammatory Makers Expression in Colon of Heterogeneous Nuclear Ribonucleoprotein (hnRNPI) Knockout Mice

Abstract

ObjectivesPreviously we found that intestinal-epithelial-cell-specific-hnRNPI knockout (KO) upregulates pro-inflammatory cytokines expression in colon, suggesting a potential link between hnRNPI KO and colonic immunity. Moreover, high protein diets were found to inhibit the inflammatory responses in KO mice, mechanism remains unclear. Hence, this current study is aimed to profile cell types in colon of hnRNPI wildtype (WT) and KO mice, characterize differences on cellular population, and identify cell types mostly responsible for the cytokine expression. MethodsIn this study, whole colon tissues from WT and KO mice (n = 3) were collected for scRNA-seq. Single cells were filtered by mitochondria percentage and UMI counts. 31,798 cells were used for profiling. Uniform manifold approximation and projection (UMAP) was adopted for dimension reduction and graph clustering, proportion of each cell subtype was normalized to cell density. Gene expression of specific pro-inflammatory markers were calculated by Loupe Browser (10X Genomics). ResultsA total of 25 clusters were sorted to the following cell types: B-cell, endothelial, epithelial, erythrocytes, fibroblast, innate lymphoid cell (ILC), mast, monocytes, myeloid and T-cell. Comparing to WT, proportion of tuft cells is increased (P = 0.08) in KO. As for immune cells, total T-cell (P = 0.1), especially non-cytotoxic T-cell (P < 0.05), as well as ILC (P < 0.01) and naïve B-cell (P = 0.09) all increased in KO. Previously identified cytokines that were downregulated by the addition of dietary protein in KO showed associations to specific cell type clusters. Specifically, IL1β mostly expressed in myeloid cells, IL6 mostly in mast cells, Ccl2 mostly in myeloid and mast cells while Cxcl1 expressed evenly in different clusters. ConclusionsIn summary, this current study reports that hnRNPI knockout significantly affects the cellular population in colon, regulating pro-inflammatory responses by recruiting non-cytotoxic T-cell, naïve B-cell and ILC. Dietary protein may inhibit inflammation trigged by knockout of hnRNPI via modulations of cytokine expression in mast and myeloid cells. Funding SourcesUSDA Cooperative State Research, Education and Extension Service (Hatch project numbers # ILLU-971,351 and ILLU-698,923), and the Office of the Vice Chancellor for Research in University of Illinois at Urbana-Champaign.