Primate Monocytes - CD14, CD16 - Ziegler-Heitbrock

Integrated single-cell transcriptomics and in vivo investigation reveal the role of ANXA1 in intestinal barrier dysfunction during heat stroke by mediating neutrophil-monocyte interaction and regulating the TLR4/ERK/NF-kappaB pathway.

Abstract

Heat stroke (HS) causes high mortality via multiple organ dysfunction syndrome, with intestinal barrier dysfunction as an early trigger of systemic inflammation. However, the roles of annexin A1 (ANXA1) in HS-induced intestinal barrier dysfunction remain unclear. Therefore, this study investigated the roles of ANXA1 in HS-induced intestinal barrier dysfunction through integrating single-cell transcriptomics and in vivo experiments. The single-cell RNA sequencing (scRNA-seq) data were used to analyze the cell subpopulation changes between healthy controls and HS patients, as well as between the inflammatory sites and non-inflammatory sites in inflammatory bowel disease. In vivo, the H&E staining, flow cytometry, ELISA, and Western blot were used to determine the pathological injuries, the proportion of neutrophils and monocytes, inflammatory cytokine levels, and related protein expression levels in the colon tissue. Immunofluorescence co-localization analysis was performed to detect the interaction between ANXA1 and immune cells. The scRNA-seq data showed that the proportion of neutrophil and CD14+Mono subpopulations was increased in HS patients. Concurrently, the ANXA1 expression was significantly upregulated in multiple immune cell subpopulations, including the CD14+Mono, CD16+Mono, mDC, MDSC, and neutrophil subpopulations. Cell-cell communication analysis further demonstrated that the ANXA1-formyl peptide receptor 2 (FPR2) ligand-receptor pairs were the dominant mode of interaction during HS. Importantly, there was partial overlap between cell populations expressing ANXA1 and barrier genes. In addition, ANXA1 was positively associated with M2 monocyte phenotype in both inflammatory and non-inflammatory sites. After establishing the HS model, there were some alterations in the colon tissue, including the exacerbated pathological injuries, upregulated ANXA1 and FPR2 protein expression levels, downregulated ZO-1 and occludin protein expression levels, increased inflammatory cytokine levels, and activated TLR4/ERK/NF-kappaB pathway. Immunofluorescence co-localization analysis revealed that the mean density of ANXA1, CD14+, and Ly6G in the colon tissue of HS mice was significantly elevated compared with the control group, and the co-localization of ANXA1 with CD14+ and ANXA1 with Ly6G was enhanced. The treatment of Boc1 led to a dramatic reduction in ANXA1 mean density, a further increase in CD14+ and Ly6G mean density, and a reduction in the co-localization of ANXA1 with CD14+ and Ly6G. Apart from reversing the ANXA1 and FPR2 protein expression levels, inhibiting ANXA1 aggravated the damaging effects of HS on the colon tissue. In conclusion, ANXA1 protected against HS-induced intestinal barrier dysfunction by regulating neutrophil-monocyte interaction and inhibiting TLR4/ERK/NF-kappaB, with ANXA1-FPR2 as a key axis, which offers a novel target and strategy for the clinical treatment of HS-induced intestinal barrier dysfunction.