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

Figure 4

C5a augments LPS-primed IL-1β production in bone marrow cells via C5aR1 and p38 signaling but suppresses the NLRP3 pathway in macrophages through PI3K with limited dependency on C5aR1. For signaling inhibition studies (top panels), peritoneal macrophages (a) or bone marrow cells (b) harvested from wild-type mice were pretreated with the indicated concentrations of wortmannin (a) or SB 203580 (b) for 1 hour prior to LPS (100 ng/mL) stimulation (4 hours) in the absence or copresence of C5a (1,000 ng/mL). IL-1β secretion, as quantified by ELISA, was elicited by subsequently treating cells with ATP (1 mM) for 45 minutes. Percentage of IL-1β release was calculated relative to positive control samples (LPS and ATP stimulation in the absence of C5a) for each concentration of inhibitor tested. For studies investigating C5aR1 dependency (bottom panels), peritoneal macrophages (a) or bone marrow cells (b) harvested either from wild-type or mice were treated with LPS (100 ng/mL) in the absence or copresence of C5a (1,000 ng/mL) for 4 hours. ELISA was used to quantify supernatant IL-1β levels following subsequent ATP (1 mM) treatment for 45 minutes. Percent IL-1β release was calculated relative to positive control samples (LPS and ATP treatment in the absence of C5a) for each mouse strain tested. All values are expressed as means ± standard error of the mean. Experiments were performed in triplicate for ≥2 independent experiments, and pooled data are shown. Data indicated that C5a requires p38 and C5aR1 for enhancing NLRP3 function in bone marrow cells but does not fully depend on C5aR1 for suppressing the LPS-primed macrophage IL-1β response, which it regulates via PI3K signaling. Wt = wild type; Pos. Ctrl. = positive control.

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