Blockade of C5aR1 resets M1 via gut microbiota-mediated PFKM stabilization in a TLR5-dependent manner

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Background: Targeting C5aR1 modulates the function of infiltrated immune cells including tumor-associated macrophages (TAMs). The gut microbiome plays a pivotal role in colorectal cancer (CRC) tumorigenesis and development through TAM education. However, whether and how the gut flora is involved in C5aR1 inhibition-mediated TAMs remains unclear. Methods: Genetic deletion C5ar1 or pharmacological inhibition of C5aR1 using anti-C5aR1 antibodies or PMX-53, in the presence or absence of deletion antibodies, were employed to investigate the impact and mechanism by which C5aR1 inhibition regulates polarization of tumor-associated macrophages through modulation of gut microbiota composition. The effects of C5aR1 inhibition in tumor growth were evaluated in MC-38 tumor bearing mice treated with or without the indicated compounds. The infiltrated immune cells and the polarization of TAMs was measured by flow cytometry. The glycolysis was examined by metabolic assays. Microarray analysis was utilized to assess the gene profiling. The levels of key enzymes in glycolysis were determined by Western blotting and quantitative PCR. The composition of gut microbiota was investigated using bacterial 16S rRNA sequencing. The relative expression of C5aR1 in TAMs from CRC patients was assessed using single-cell RNA-seq data from SCDVA and multiplexed immunofluorescence staining, respectively. Results: C5aR1 inhibition elevated the concentration of serum IL-22 and the mRNA levels of its downstream target genes encoded antimicrobial peptides (AMPs), leading to gut microbiota modulation and flagellin releasement, which contributed to M1 polarization. In addition, our data revealed that high levels of C5aR1 in TAMs predicted poor prognosis. Conclusion: C5aR1 inhibition reduced CRC growth via resetting M1 by AKT2 activation-mediated PFKM stabilization in a TLR5-dependent manner, which relied on IL-22-regulated gut flora.