Recruitment of CCR5+ inflammatory monocytes in pulmonary tissue contributes to acute lung injury

Fig. 1. Increased proportion of monocytes in lung tissue in the DHCA group. A Representative flow cytometry of monocytes in rat lung tissue showed an increased proportion of monocytes in the DHCA group. SIRPα (CD172α) is a specific marker for rat monocytes. Numbers in the flow plot correspond to the percentage of the parent population. B Bar graph showed the proportion of monocytes among cells in rat lung tissue (n = 5). C, D Compared to the Sham group, immunofluorescence staining for CD172α showed a significant increase in the number of puncta in the DHCA 6 h group (Scale bar = 50 μm). Green, CD172α-positive cells; blue, DAPI-stained nuclei. Quantitative analysis is shown in (C; n = 5). *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Fig. 2. DHCA-induced lung injury was attenuated after clodronate was given intravenously 24 h before surgery. Twenty SD rats were randomly divided into 4 groups: Sham group, DHCA group, Clo-Lip group and PBS-Lip serving as the placebo for a control group. Rats in the Clo-Lip group experienced standar DHCA intervention 24 h after i.v. injection of Clo-Lip (n = 5 independent experiments). A Flow cytometry of monocytes in lung tissue from rats undergoing the DHCA procedure showed a decreased proportion of monocytes 24 h after PBS or Clo-Lip injection (n = 5 rats per group). B Bar graph showing the proportion of monocytes among leucocytes and that preoperative administration of Clo-Lip significantly reduced the proportion of monocytes in lung tissue 6 h after DHCA. C, D Compared to the DHCA group, immunofluorescence staining for CD172α showed a significant decrease in the number of puncta in the Clo-Lip group (Scale bar = 50 μm). Green, CD172α-positive cells; blue, DAPI-stained nuclei. Quantitative analysis is shown in (C; n = 5). E Representative haematoxylin and eosin staining images in DHCA 6 h group and Clo-Lip group. The DHCA group rats exhibited prominent alveolar inflammatory cells…

Fig. 3. Heterogeneous changes in monocytes in the lung tissue of the Sham and DHCA groups. A Single-cell RNA sequencing results showing changes in mononuclear subpopulations in lung tissue after DHCA surgery: monocytes were classified into 4 clusters according to CD43 expression: clusters 1 and 4 were predominant in the Sham group; clusters 2 and 3 were predominant in the DHCA group. B Heatmap of 4 monocyte subpopulations. C Cluster 2 of CD43low monocytes in the DHCA group showed a pro-inflammatory phenotype with high expression of CCR5, S100a8, S100a9, Nos2, Vegfa, and IL-1β. D, E Gene Ontology (GO) enrichment analysis. F Cell communication network. G Genes closely related to leucocyte migration, macrophage activation and other inflammation-related signalling pathways were significantly upregulated in monocytes from the DHCA group compared to the control group.

Fig. 4. Intravenous application of MVC half an hour before DHCA significantly reduces CCR5 expression in rat lung tissue. A Gating strategy for CD45+CD172α+CCR5+ monocytes. B Representative flow cytometry of CCR5+ monocytes showed a decrease in the MVC group compared to the DHCA group. Numbers in the flow plot correspond to the percentage of CCR5+ monocytes in the CD45+ population. C Bar graph showed the proportion of CCR5+ monocytes among leucocytes in rat lung tissue (n = 5). D, E Compared to the DHCA group, immunofluorescence staining of CD172α and CCR5 co-staining showed a significant reduction in the number of puncta in the MVC group (Scale bar = 50 μm). Green, CD172α-positive cells; red, CCR5-positive cells; blue, DAPI-stained nuclei. Quantitative analysis is shown in (E; n = 5). F, G The expression level of CCR5 was significantly reduced after administration of MVC. Quantitative analysis is shown in (G; n = 5). *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Fig. 5. DHCA-induced lung injury was attenuated by MVC. A Representative haematoxylin and eosin staining images in DHCA 6 h group and MVC group. Compared with the DHCA group, the degree of lung injury in MVC group decreased (Scale bar = 100 μm). B Inhibition of CCR5 expression significantly attenuates lung injury score after DHCA. C Total alveolar protein concentration was significantly reduced in the MVC group compared to the DHCA group. D Lung wet/dry weight ratio also decreased in the MVC group. E The levels of inflammatory factors in BALF and lung tissue were significantly decreased when MVC group compared with the DHCA group. *, p < 0.05; **, p < 0.01; ***, p < 0.001. F, G The expression levels of autophagy-related proteins were significantly reduced after administration of MVC. Quantitative analysis is shown in (G; n = 5). *, p < 0.05; **, p < 0.01; ***, p < 0.001.