Single-cell RNA Sequencing Reveals Heterogeneity Amongst Immune Cell Subpopulations in LPS-Induced ALI Mice treated with I3C

Abstract

Abstract Traditionally, macrophage M1-vs-M2 dichotomy have remained fundamentally unchanged since their identification. The oversimplification of immune cell subpopulations using flow cytometric analysis fails to capture the full heterogeneity of these cells in response to disease state and treatment. However, single-cell RNA sequencing (sc-RNAseq) serves as a diagnostic tool that clusters cells based on the whole transcriptomics of individual cells, without the limitation of surface markers and transcriptional factors. In the current study, we aimed to better understand the immunomodulatory mechanism of indole-3-carbinol (I3C) on cell state. To that end, 5mg/kg of LPS was intranasally injected in C57BL/6 mice to induce ALI, and the mice were treated with 80mg/kg I3C three hours after disease induction. After 72 hours mice were euthanized. Whole lung cells were utilized for flow cytometry and sc-RNAseq. Strikingly, the traditional M1-vs-M2 separation was inadequate in classifying all macrophage clusters. LPS+I3C treated-mice had a decrease in Isg15, Ccl5, and Ly6c2 expression in M1 cluster; genes linked to inflammation. Also, the expression of monocyte chemoattractant genes (Cxcl2 and Cxcl3), arginine-associated transport gene (Slc7ac), and major histocompatibility complex (MHC) genes (H2-Ab1 and H2-K1) in M2 cluster was decreased in the diseased group following I3C treatment. In conclusion, several alterations in gene expression in myeloid immune cell subpopulations were identified following treatment with indole-3-carbinol. Thus, shedding light on several new macrophage subpopulations not identified using traditional flow cytometric analysis may offer novel mechanistic insights into disease induction.