Monocyte-Macrophage Heterogeneity And Its Effect On Inflammatory Lung Injury

Objective: Leukocytes in the hematopoietic system include granulocytes,monocytes,T lymphocytes,B lymphocytes and other types.Among them,monocytes exhibit more pronounced heterogeneity,yet the mechanisms by which these differences in development,differentiation,and function affect innate immunity and disease progression are not clear.The booming development of single-cell sequencing technology in recent years has provided a new perspective for in-depth study of monocytes heterogeneity,and has also provided new ideas for the study of disease mechanisms.This project constructs a single-cell sequencing data analysis framework,and apply it to analyze clinical derived single-cell data of biological infectioninduced sepsis and physicochemical-induced inflammatory lung injury,to reveal the heterogeneity and potential pathogenic mechanisms of circulating and peripheral monocytes in diseases.Methods: In the first part of the work,we constructed a bioinformatics analysis workflow for single-cell sequencing data in the monocyte-macrophage system.This workflow involved several analysis methods,including reference dataset-based cell clustering and subpopulation annotation,differential gene analysis,gene set enrichment analysis,pseudotime trajectory inference,gene set variation analysis,metabolic flux balance analysis,transcriptional regulatory network analysis,and deconvolutionbased cell proportion prediction.To further validate this workflow,we performed meta-synthesis on PBMC transcriptomic profiles of septic patients and severe COVID-19-infected patients from publicly avaliable single-cell sequencing databases,identifing distinct heterogeneous subpopulations of monocytes and macrophages that showed significant differences across clinical groups.Based on determining their abundance and their association with disease prognosis,we described their transcriptional and metabolic pathway characteristics and inferred potential transcriptional regulatory factors.In the second part,the single-cell data analysis pipeline was used to decipher the potential relationship between the transcriptional and functional heterogeneity of monocytes-macrophages and physicochemical-induced inflammatory lung injury.Taking silicosis as an example of a major occupational lung disease,we collected monocytesmacrophages-rich bronchoalveolar lavage fluid in clinical context for single-cell RNA sequencing to identify signaling molecules and pathways in heterogeneous monocytes-macrophages derived from inflammatory lung injury patients.A silica suspension induced inflammatory lung injury mouse model was built via intratracheal instillation to assess the key respiratory parameters variation,lung imaging and pathological changes,as well as inflammatory cytokine levels in the bronchoalveolar lavage fluid.Mouse primary macrophages with various heterogeneity was stimulated by silica,and the expression levels of corresponding inflammatory cytokins were detected to explore the underlying mechanisms.Results: The main findings of the first part of the work are as follows: 1 5 sepsis single-cell RNA-seq datasets including 87 samples were integrated to obtain 25301 gene expression data of 188,941 PBMCs,and 76,788 monocytes and macrophages were clustered into 28 groups.2 CD14+IL1R2+CD163++MARCO++HLA-DRlow monocytesmacrophages with anti-inflammatory property and monocyte-platelet complexes were present specifically in the sepsis group,and the abundance of both cell populations was positively correlated with poor prognosis in sepsis.3 363157 PBMC transcriptomes were analyzed from sepsis and COVID-19 infection single-cell RNA-seq datasets,while CD14+IL1R2+CD163++MARCO++HLA-DRlow monocytes-macrophages and monocyte-platelet complexes also existed in COVID-19 context.4 A subpopulation of MERTK+/PPARG+ was found in the aforementioned monocytes-macrophages with specific transcriptional heterogeneity,which showed higher levels of glycolysis,oxidative phosphorylation,spermidine and spermine metabolism and inositol phosphate metabolism.This subpopulation may represent M2c-like macrophages,which could participate in the phagocytosis of circulating apoptotic cells(a.k.a efferocytosis)in critical conditions.5 M2c-like macrophages may have a gene regulatory network characterized by direct regulation of MERTK and PPARG genes mediated by the transcription factor ZBTB43 activation.6 In terms of cell ontology,there is no apparent relationship between the monocyte-platelet complexes and the abovementioned monocytes with specific transcriptional heterogeneity.The main findings of the second part of the work are as follows: 1 A bronchoalveolar lavage immune landscape of single-cell resolution was generated from silicosis patients and corresponding control donors.Once macrophages re-clustering performed,macrophages with impaired IFN-γ signal was found in silicosis patients.2 Wild-type mice inhaling suspension of silica particles exhibited an increase in respiratory resistance,as well as a decrease in lung compliance,tidal volume,and minute ventilation,their lungs showed significant infiltration of inflammatory cells and fibrosis,while those with IFN-γ receptor knock-out and myeloid-specified IFN-γ receptor knock-out genotype showed worse lesion.3 Wild-type mice inhaling suspension of silica particles exhibited interleukin-1β increase in the bronchoalveolar lavage fluid,which was even more significant in IFN-γ receptor knock-out mice.4 Exogenous IFN-γ reduced the high secretion levels of interleukin-1β in silica stimulated macrophages from wild-type mice,and this effect was blocked in macrophages from IFN-γ receptor knock-out mice.5 Exogenous IFN-γ reduced caspase-1 and interleukin-1β cleavage in silica stimulated macrophages from wild-type mice,and this effect was blocked in macrophages from IFN-γ receptor knock-out mice.6 Lysosomal leakage was found in silica stimulated macrophages by flow cytometry test,which was reduced by IFN-γ pre-treatment.7 IFN-γ reduced lysosomal membrane rupture and the secretion of interleukin-1β in macrophages stimulated by the lysosomal probe L-leucyl-L-leucine methyl ester.Conclusions: Results of the first part suggested that CD14+IL1R2+CD163++MARCO++HLA-DRlow M2 polarized monocytemacrophage population with anti-inflammatory feature associated with disease prognosis existed in the peripheral blood of sep tic and severe COVID-19-infected patients,while M2c-like subpopulation had higher metabolic activity,which may provide new ideas for research on monocyte-macrophage from sepsis.We developed a research methodology to investigate the heterogeneity of monocytes and macrophages at the single-cell resolution.The second part of the study suggested that impaired IFN-γ signaling of pulmonary macrophages was related to the progression of inflammatory lung injury.Activation of IFN-γ signal can inhibit silica-induced lysosomal rupture and subsequent NLRP3 inflammasome activation and assembly,thereby suppressing the release of pro-inflammatory cytokines by macrophages.Targeting the modulation of IFN-γ signaling pathway may provide insights for the prevention and treatment of inflammatory lung injuries such as silicosis.72 pictures,17 tables and 157 references.