Single‐Cell RNA‐Seq Profiling Identifies L1 Cell Adhesion Molecule (L1cam) as a Mediator in Colonic Tuft Cells ‐ Innate Lymphoid Cell (ILCs) Signaling in the Hnrnp I Knockout Mice
Authors: Xu Guanying B. , Pan Yuan‐Xiang , Mei Wenyan , Chen Hong 
Published: May 2022

ObjectivesPreviously we found that ablation of heterogeneous nuclear ribonucleoprotein I, (Hnrnp I) in intestinal epithelial cells (IECs) in colon significantly upregulates inflammatory cytokines and chemokines expression and increases number of tuft cells and innate lymphoid cells (ILCs, including ILC1, ILC2 and ILC3) in colon. The objective of this current study is to investigate the cell‐cell communications and to identify the mediators involved in the colon of Hnrnp I knockout (KO) mice. We hypothesize that signal transduction between colonic tuft cells and ILCs stimulates cytokine expression in the knockout mice.MethodsSingle‐cell RNAseq libraries (n = 3) were prepared from total RNA extracted from colon using Chromium Single‐Cell Gene Expression NextGem (v3.1, 10X Genomics). Analyses from raw counts were performed using the Seurat (v3.2.0) R package using default parameters. A computational cell calling algorithm was used to identify cell types using two up‐to‐date annotated mouse cell datasets. Analyses of cell‐cell communications and identification of significant pathways were performed using CellChat (v1.1.3). Immunofluorescence staining of colon tissue was performed with anti‐DCAMKL1 antibody (Abcam, ab31704). Colon cytokine levels were examined using cytokine 32‐plex array (Eve Technologies) using total protein extracted from colon tissues.ResultsImmunofluorescence staining of DCAMKL1, a tuft cell marker, confirms that the number of Tuft cells in colon (normalized to number of crypt) is significantly increased in Hnrnp I KO mice comparing to Hnrnp I wildtype (WT). Results from CellChat suggest communications between tuft cells and immune cells (ILCs, cytotoxic T cells, helper T cells, active lymphocytes and myeloid progenitors) are mainly through L1 Cell Adhesion Molecule (L1cam) – Integrins interaction, which is KO‐specific because the ablation of Hnrnp I results in the expression of L1cam in tuft cells in KO mice. Expression of integrin αv and β3 subunits (Itgav and Itgb3) are also higher in ILCs in the KO mice. NF‐κB subunits P65, and NF‐κB pathway target genes P21 and c‐Myc, also show higher expression levels in ILCs in KO mice comparing to WT. Cytokines assay results show that IL4, IL6 and IL13 all significantly increased, while IFNγ unchanged, and IL17 decreased in colon in KO mice comparing to WT.ConclusionsOur results demonstrate an Hnrnp I KO‐specific communication in colon between tuft cells and ILCs via L1cam‐integrin αv and β3 subunits interaction. The interaction further activates NF‐κB signaling in ILCs, regulating cell cycle of ILCs. The activation of ILCs further triggers immune responses in KO mice as indicated by increased proinflammatory cytokine expressions. Our results also suggest that among ILC1, ILC2 and ILC3, ILC2 is the major cell type involved in this communication because those increased cytokines – IL4, IL6 and IL13 observed in KO mice are all effectors secreted by mature ILC2.