C5aR2 regulates STING-mediated interferon beta production in human macrophages

The complement system mediates diverse regulatory effects across the immune system. C5aR2, an enigmatic receptor for anaphylatoxin C5a, has been shown to modulate PRR-dependent pro-inflammatory cytokine secretion in human macrophages. However, the specific downstream targets and underlying molecular mechanisms are less defined. In this study, CRISPR-Cas9 was used to generate macrophage models lacking C5aR2, which were used to probe the role of C5aR2 in the context of PRR stimulation. cGAS and STING-induced IFN-β secretion was significantly increased in C5aR2 KO THP-1 cells and C5aR2-edited primary human monocyte-derived macrophages, and expression of STING and IRF3 was increased in C5aR2 KO cell lines, implicating C5aR2 as a regulator of the IFN-β response to cGAS-STING pathway activation. Transcriptomic analysis by RNAseq revealed that nucleic acid sensing and antiviral signalling pathways were significantly up-regulated in C5aR2 KO THP-1 cell lines. These results suggest that C5aR2 is a negative regulator of nucleic acid sensing in macrophages, with potential relevance for viral infection and anti-tumour immunity. PMA-differentiated THP-1 cells (WT, C5aR1 KO or C5aR2 KO) in 75 cm2 flasks were stimulated with culture medium containing 5 µg/mL cAIM(PS)2 Difluor (Rp/Sp), 50 ng/mL C5a or vehicle for 6 hours. Supernatants were harvested for IFN-β ELISA to confirm response to stimulation as a quality control, cells were washed twice with PBS then harvested using TrypLE Express Enzyme. Cells were centrifuged at 300 x g for 5 minutes, the supernatant was removed and the cells were snap-frozen on dry ice. RNASeq was performed by GENEWIZ, comprising total RNA isolation, library preparation with poly(A) selection and 150 bp paired-end sequencing using an HiSeq 2500 (Illumina)