Fig 2: Macrophage/monocyte specific gene regulatory landscape in experimental anti-GBM cGN in Tlr4flox/flox and Tlr4fl/fl/LysM-cre mice. A) Heatmap of cell type-specific top ranked TFs regulons in each macrophage/monocyte cluster, as inferred by SCENIC algorithm. Enrichment score scale is shown on the right-side legend. B) UMAP depiction of cell-type specific top regulon activity (“high-pink/red,” “low-dark/blue”) and TF gene expression (brown scale) of exemplary regulons for Jun (Cluster 0 pro-inflammatory monocyte), Ets2 (Cluster1 neutrophil-like monocyte), Nr4a1 (Cluster7 anti-inflammatory macrophage), Gata3 (Cluster9 moDC), Mafb (Cluster 3 and 4 M2 macrophage). Metacore analysis revealed top GRN reconstruction in C) Cluster 0, D) Cluster 1, and E) Cluster 7. Unbiased regulatory networks are generated based on shortest direct interactions algorithms via MetaCore analytical program according to the high confidential DEGs (p < 0.01) found in each macrophage/monocyte subclusters. Involved DEGs from the cell type-specific data set are highlighted by a solid circle at the upper right-hand side whose relative expression level is represented by different intensity in red color.

Fig 3: Identification of the TLR4-dependent novel Cluster 7 (Nr4a1+ Ear2+ ) anti-inflammatory macrophage phenotype in anti-GBM cGN in Tlr4flox/flox and Tlr4fl/fl/LysM-cre mice. A) Flow cytometry analysis for PBMC at day 7 post experimental anti-GBM cGN. For upper panel: total CD11b+ CD68+ cells, lower panel: F4/80+ NUR77 (NR4A1)+ macrophages. B) Flow cytometry analysis for kidney single cell suspension, for upper panel: total CD11b+ myeloid cells, lower panel: F4/80+ NUR77/NR4A1+ macrophages. C) Statistical data of (A) and (B). Data represent the mean ± SEM for groups of at least three mice. **P < 0.01 versus corresponding Tlr4flox/flox . D) Representative immunofluorescence images of EAR2 (red), F4/80 (green), and Hoechst 33342 (blue) in the kidney and spleen of normal control and at day 7 and day 14 post anti-GBM cGN in Tlr4flox/flox and Tlr4fl/fl/LysM-cre mice. Arrows demonstrate Cluster 7 macrophage phenotype by co-expressing EAR2 (red) and F4/80 (green). E) Nr4a1 and Ear2 gene expression in normal kidney and kidney at day 7 and day 14 post anti-GBM cGN. Data represent the mean ± SEM for groups of at least three mice. **P < 0.01, ****P < 0.0001 versus Normal; NS: no significant difference, ##P < 0.01, ###P < 0.001 versus corresponding Tlr4flox/flox .