Heterogeneity of immune cells and their communications unveiled by transcriptome profiling in acute inflammatory lung injury

Background: Acute Respiratory Distress Syndrome (ARDS) or its earlier stage Acute lung injury (ALI), is a worldwide health concern that jeopardizes human well-being. Currently, the treatment strategies to mitigate the incidence and mortality of ARDS are severely restricted. This limitation can be attributed, at least in part, to the substantial variations in immunity observed in individuals with this syndrome. Methods: Bulk and single cell RNA sequencing from ALI mice and single cell RNA sequencing from ARDS patients were analyzed. We utilized the Seurat program package in R and cellmarker 2.0 to cluster and annotate the data. The differential, enrichment, protein interaction, and cell-cell communication analysis were conducted. Results: The mice with ALI caused by pulmonary and extrapulmonary factors demonstrated differential expression including Clec4e, Retnlg, S100a9, Coro1a, and Lars2. We have determined that inflammatory factors have a greater significance in extrapulmonary ALI, while multiple pathways collaborate in the development of pulmonary ALI. Clustering analysis revealed significant heterogeneity in the relative abundance of immune cells in different ALI models. The autocrine action of neutrophils plays a crucial role in pulmonary ALI. Additionally, there was a significant increase in signaling intensity between B cells and M1 macrophages, NKT cells and M1 macrophages in extrapulmonary ALI. The CXCL, CSF3 and MIF, TGFb signaling pathways play a vital role in pulmonary and extrapulmonary ALI, respectively. Moreover, the analysis of human single-cell revealed DCs signaling to monocytes and neutrophils in COVID-19-associated ARDS is stronger compared to sepsis-related ARDS. In sepsis-related ARDS, CD8+ T and Th cells exhibit more prominent signaling to B cell nucleated DCs. Meanwhile, both MIF and CXCL signaling pathways are specific to sepsis-related ARDS. Conclusion: This study has identified specific gene signatures and signaling pathways in animal models and human samples that facilitate the interaction between immune cells, which could be targeted therapeutically in ARDS patients of various etiologies. Overall design: Six to eight weeks old male C57BL/6 mice of SPF grade were fed in the standard animal laboratory at 25 ± 1°C with humidity of 60 ± 5% and 12-hous light/dark cycle, adequate food and water were supplied. The mice were allowed to acclimated to the environment for 1 week prior to the experiment. The mice were randomly divided into control, pulmonary ALI, and extra-pulmonary ALI group. Pulmonary ALI was induced by intratracheal injection of Lipopolysaccharide (LPS) (10 mg/kg, from Pseudomonas aeruginosa 27316, Sigma-Aldrich, USA, L9143) dissolved in 50 ml sterile PBS. Additionally, we used intraperitoneal injection of LPS (25 mg/kg) to construct sepsisrelated ARDS, known as extra-pulmonary ALI.