Fig. 4: C5aR1 inhibition reset M1 polarization relies on modulating gut microbiota.

A Serum samples were obtained from WT and C5ar1^-/- mice bearing MC-38 cells and subjected to examine the concentration of cytokines (n = 5/group). B Flagellin levels in the serum of WT and C5ar1^-/- mice with or without tumors were examined by ELISA (before tumor-bearing, n = 8; after tumor-bearing, n = 11 including 3 samples of a pilot experiment). C WT mice bearing MC-38 cells were injected with isotype control Ab or anti-C5aR1 Ab, and serum samples were obtained and subjected to ELISA to examine flagellin levels (n = 12 including 4 samples of the pilot experiment). D Shannon and Simpson diversities were assessed (n = 5). E principal coordinate analysis (PCoA) on the OUT level was examined (n = 5). Grey, WT; Blue, C5ar1^-/-. F The differences between taxonomic or functional trees in two different groups (n = 5). G Wilcoxon rank-sum test bar plot on Genus level (n = 5). H The phenotype of mobile element initial for bacterial fitness and pathogenicity using the BugBase tool (n = 5). D–H The data shown are representative of one of two independent experiments. I The relative abundances of Parasutterella, Bifidobacterium, B.fragilisf, and Butyricicoccus were determined by real-time PCR (n = 8). The data shown represent a single experiment. J C5ar1^+/+ and C5ar1^-/- mice were treated with ABX or normal water 3 weeks before tumor inoculation. The tumor growth and tumor weight were examined (n = 10). K MC-38 tumor growth in WT mice undergoing i.p. PMX-53 (1 mg/kg) with or without bacterial cocktail before tumor inoculation (ABX treated C5ar1^-/- mice, n = 6; others, n = 10/group). L C5ar1^+/+ and C5ar1^-/- mice were housed alone or co-housed for 2 weeks prior to tumor inoculation. The tumor growth and tumor weight were examined (n = 14). M The numbers of F4/80⁺CD86⁺ TAMs were counted in tumor sections by Immunofluorescence staining (n = 3 tumor/group, 3 slide/tumor, 4 filed/slide). M The data shown represent a single experiment. N The concentrations of NO were assessed (n = 3). O Relative mRNA levels of the indicated genes were assessed by real-time RT-PCR (n = 3). M–O SI, single breeding, CO, co-cohoused breeding. P Relative mRNA levels of the Tnfa were assessed by real-time RT-PCR (n = 3). CO co-cohoused breeding, F flagellin. *P < 0.05; **P < 0.01; ***P < 0.001.