Complement C5a Receptor Signaling in Macrophages Enhances Trained Immunity Through mTOR Pathway Activation

Abstract

Complement C5a receptor (C5aR) signaling in immune cells has various functions, inducing inflammatory or anti-inflammatory responses based on the type of ligand present. The Co1 peptide (SFHQLPARSRPLP) has been reported to activate C5aR signaling in dendritic cells. We investigated the effect of C5aR signaling via the Co1 peptide on macrophages. In peritoneal macrophages, the interaction between C5aR and the Co1 peptide activated the mTOR pathway, resulting in the production of pro-inflammatory cytokines. Considering the close associations of mTOR signaling with IL-6 and TNF-α in macrophage training, our findings indicate that the Co1 peptide amplifies β-glucan-induced trained immunity. Overall, this research highlights a previously underappreciated aspect of C5aR signaling in trained immunity, and posits that the Co1 peptide is a potentially effective immunomodulator for enhancing trained immunity.

Keywords: Adjuvants, immunogenic; C5a receptor; Immunomodulator; Inflammation; Trained immunity.

Figure 1. Activation of Akt/mTOR signaling pathway in macrophages by C5aR-Co1 peptide signaling. (A) RAW264.7 cells were treated with each indicated molecule for 15 min. The panel displays array data and densitometry results for p-AKT, p-mTOR, p-P70S6, and p-RPS6 in RAW264.7 cell lysates. Bar graph shows the mean of replicated spots in the array for indicated molecules. Data are representative of three independent experiments. (B) RAW264.7 cells were preincubated with or without rapamycin for 1 h, followed by treatment with each indicated molecule. Bar graph shows the level of p-S6 normalized to total S6 protein. Data are representative of three independent experiments. (C) The schematic shows our hypothesis that C5aR-Co1 peptide signaling activates the mTOR signaling pathway.

Figure 2. C5aR-Co1 peptide signaling induces in vitro peritoneal macrophage training via the mTOR pathway. (A) Schematic diagram of the in vitro model strategy for inducing trained immunity. Bar graph shows the levels of the indicated cytokines in supernatants. Data are presented as means ± standard errors; p-values were analyzed by ordinary one-way ANOVA in Prism; Data are representative of three independent experiments. (B) Schematic diagram of the β-glucan training pathway and our hypothesis that C5aR-Co1 peptide signaling induces cytokines via the mTOR signaling pathway.

^*p<0.05, ^**p<0.01, ^***p<0.0005, ^****p<0.0001.

Figure 3. Enhancement of β-glucan-induced macrophage training by C5aR-Co1 signaling. Schematic diagram illustrates the in vivo model strategy for inducing trained immunity. Serum was collected 60 min after LPS challenge. Bar graph shows the levels of the indicated cytokines in serum. Data are presented as means ± standard errors; p-values were analyzed by ordinary one-way ANOVA in Prism.