Fig 2: NLRP3 mediates M1 polarization in prorenin-treated microglia. A Analysis of the expression profile of CD11b by flow cytometry confirming that the purity of isolated primary microglia was > 94%. B Representative fluorescence-activated cell sorting plots showing the M1 (CD86+) and M2 (CD206+) phenotypes by flow cytometry in Ctrl, prorenin, and prorenin + MCC950-treated microglia. C Statistical data showing the proportions of M1/M2 phenotypes in Ctrl, prorenin, and prorenin + MCC950-treated microglia. D qRT-PCR analysis showing that the pro-inflammatory factors TNF-α and IL-β are increased in prorenin-treated microglia, while co-treatment with prorenin and MCC950, an NLRP3 inhibitor, decreases the release of TNF-α and IL-β. Data represent the mean ± SEM; n = 6; *P < 0.05 vs Ctrl; #P < 0.05 vs prorenin.

Fig 3: LARRPM repressed M2 macrophage infiltration. a Schematic diagram of in vitro co-culture system of phorbol-12-myristate-13-acetate (PMA)-stimulated human monocyte line THP-1 cells and LUAD cells. b THP-1 cells were subjected to transwell migration assays towards A549 cells with LARRPM overexpression or A549 control cells. Scale bar: 100 µm. c THP-1 cells were subjected to transwell migration assays towards HCC827 cells with LARRPM depletion or HCC827 control cells. Scale bar: 100 µm. d Expression of M1 and M2 polarization markers in THP-1 cells co-cultured with A649 cells with LARRPM overexpression or A549 control cells was detected by qRT-PCR. e Expression of M1 and M2 polarization markers in THP-1 cells co-cultured with HCC827 cells with LARRPM depleted or HCC827 control cells was detected by qRT-PCR. f, g CD163 (f) and CD206 (g) IF staining in THP-1 cells co-cultured with A549 cells with LARRPM overexpression or A549 control cells, or THP-1 cells co-cultured with HCC827 cells with LARRPM depleted or HCC827 control cells. Results are shown as the mean ± SD based on three independent experiments. Asterisks indicate a significant difference at *P < 0.05, **P < 0.01, ***P < 0.001, by Student’s t-test (b, d) or one-way ANOVA followed by Dunnett's multiple comparisons test (c, e). ARG1 Arginase 1, IL interleukin, TNF tumor necrosis factor

Fig 4: CAF-derived C-6-S induces M2 macrophage polarization and causes immune exclusion. A, IF staining showing C-6-S in ECM and CD206 in BMDMs colcultured with CAFs in a 3D system. Nuclei are shown in blue (DAPI), CD206 (M2-type marker) is in green, and C-6-S is in red. Scale bar: 50 μm. B, The correlation between CS metabolism ssGSEA scores and M1-type markers (left), M2-type markers (right) in TCGA MSS colorectal cancer cohort. Mouse PMs (C) and human colorectal cancer TAMs (D) were treated with various concentrations of C-6-S. Flow cytometry detected the CD11c−CD206+ or CD86−CD163+ M2 macrophages. Bar graphs or boxplot show the summarized data. Student t test was used to calculate P values. Mouse PMs with or without CAF cocultivation and CS antagonists (Surfen and Ch-ABC) were detected with flow cytometry (E and F) and BMDMs were detected with qPCR (G). H–K, CT26 tumor–bearing mice were randomized to control or Surfen group (n = 5 for each group). Tumor growth curves from day 0–21, and P value by Student t test on day 21 (H). Representative IHC images of C-6-S, CD8, and CD206 in each group on day 16 (J), along with statistical plots (I). Scale bar: 200 μm. On day 16 (top) and day 23 (bottom), macrophages isolated from each group were analyzed for CD11c, CD206, and PD-L1 expression, and the M1/M2 type and PD-L1+ proportions were summarized (K). Statistics were calculated with 3 (D and G), 4 (C), or 5 (E) samples. Student t test was performed in C–E, G, I, and K. Data are presented as mean ± SD. ****, P < 0.0001; ***, P < 0.001; **, P < 0.01; *, P < 0.05; ns, P > 0.05.

Fig 5: Subpopulations of M2 macrophage in glomeruli of PMN patients. M2a macrophages were positive for CD206 by immunochemistry, M2b macrophages were positive for CD86, and M2c macrophages were positive for CD163. The arrows point to positive cells.