Bidirectional Crosstalk between C5a Receptors and the NLRP3 Inflammasome in Macrophages and Monocytes

Figure 2

C5a suppresses NLRP3 function in macrophages but augments LPS-primed IL-1β production in bone marrow cells. For quantifying IL-1β release, thioglycollate-elicited peritoneal macrophages (a) or total bone marrow cells (b) were harvested, treated with LPS (100 ng/mL) in the absence or copresence of varying C5a concentrations for 4 hours, and then stimulated with ATP (1 mM) for 45 minutes to achieve NLRP3 inflammasome activation. IL-1β levels in cell-free supernatants were determined using ELISA (top panels). qRT-PCR assays (middle and bottom panels) were performed on mRNA isolated from peritoneal macrophages (a) or bone marrow cells (b) 4 hours after culture with or without LPS (100 ng/mL) in the presence or absence of C5a (1,000 ng/mL). ATP treatment was not performed for mRNA quantification experiments. Relative expression levels of inflammasome component genes (indicated as fold change) were normalized to GAPDH expression and calculated using the method. ELISA and qRT-PCR data are shown as mean values ± standard error of the mean. Experiments were performed in triplicate for ≥2 independent experiments, and representative data are shown. Results indicated that C5a attenuates TLR4-mediated macrophage IL-1β production but enhances NLRP3 function in bone marrow cells. Neg. Ctrl. = negative (unstimulated) control.

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