The role of the anaphylatoxin C5a in intestinal immune responses

Antigen presenting cells (APCs) exert key roles in the regulation of mucosal immunity of the intestine. They are integral for the maintenance of mucosal tolerance and drive immune responses to protect the intestine from pathogens. The molecular mechanisms underlying this dual role of intestinal APCs in the regulation of tolerance and immunity remain elusive. Activation of pattern recognition receptors such as the Toll-like receptors on APCs by commensal bacteria or by pathogens regulates intestinal immunity by balancing effector and regulatory CD4 T cell responses. Intestinal inflammation often results in complement activation and release of the anaphylatoxins (ATs) C3a and C5a. Our preliminary data show that AT receptors are expressed on intestinal APCs; yet it is unclear whether such expression is modulated in response to intestinal inflammation. In addition to regulation of APC functions in peritoneum, lung and spleen by TLR-dependent and independent pathways our preliminary data suggest that C5a suppresses TLR2-driven IL-12 production from lamina propria (LP) DCs but enhances IL-10 production from LP macrophages. Further, we found that LP CX3CR1+ mononuclear phagocytes were reduced in C5aR-/- mice. Thus, we propose that cross-talk between C5aR receptors and pattern recognition receptor (PRRs) also occurs in intestinal APCs regulating innate and adaptive mucosal immune responses. This proposal aims at delineating the molecular mechanisms underlying such crosstalk, its impact on mucosal tolerance and the development of inflammatory bowel disease.

The anaphylatoxins C3a and C5a and their cognate anaphylatoxin receptors play important roles in the activation and regulation of innate immune cells. While a body of data has accumulated showing critical roles of C5a-mediated activation of C5aR and C5aR2 on macrophages and dendritic cells (DC) in the airway and the lung for the development of pulmonary allergy, less is known about anaphylatoxin receptor expression in intestinal phagocytes and DCs and the functional impact of their activation. We generated floxed anaphylatoxin receptor reporter mice that we used to monitor C3aR, C5aR1 and C5aR2 expression in phagocytes and conventional DCs (cDC) from the lamina propria (LP) of the small intestine. We found that CD11b+F4/80+MHCII+ phagocytes strongly expressed all three anaphylatoxin receptors. In contrast, C5aR1 was absent in CD103+ or CD11bhi cDCs from LP and C5aR2 was only marginally expressed in such cDC subsets. About 10% of CD103+ and 20% of CD11bhi cDCs expressed C3aR. Given that several subsets of antigen-presenting cells in the LP express anaphylatoxin receptors, we determined the potential impact of the C5a/C5aR1 axis on the composition and diversity of the gut microbiota. Our results demonstrate a significant influence of the C5ar1 genotype on numerous aspects of microbial community composition and structure at several different anatomical locations, in addition to influences of sex at some locations. This includes differences in the abundance of Firmicutes and Proteobacteria in the cecum and colon and Actinobacteria in the jejunum. Alpha diversity analyses revealed corresponding differences in evenness (Shannon index) with respect to the C5ar1 genotype in the jejunum. Further, beta diversity analyses also showed significant differences between genotypes in the cecum. Taken together, we uncovered a novel role for the C5a/C5aR1 axis on microbiota composition and diversity of the intestine. Food allergy is associated with changes in the intestinal microbiota or dysbiosis early in life. Further, decreased bacterial diversity is one important factor that predisposes to food allergy. Our findings regarding the impact of the C5a/C5aR1 axis on microbiota composition and diversity of the intestine suggest that the C5a/C5aR1 axis may have an impact on the development of food allergy. So far, the involvement of the C5a/C5aR1 axis in the development of food allergy has not been assessed. Thus, we determined the role of C5a for the development of food allergy in vivo and in vitro. We subjected C5ar1-/- and wild-type (wt) BALB/c mice to oral ovalbumin (OVA)- induced anaphylaxis. We observed that 78% of wt but only 9% of C5ar1-/- mice suffered from diarrhea and hypothermia after the 7th oral OVA challenge. Importantly, peritoneal MCs strongly expressed C5aR1 after the 7th oral OVA treatment. OVA-specific serum IgE and MCPT-1 levels were significantly decreased in C5aR1-deficient as compared with wt mice. To directly examine the impact of the C5a/C5aR1 axis on MC function, we generated bone marrow-derived MCs (BMMCs) and tested their activation and degranulation potency in response to FcεR1 cross-linking. Degranulation of C5ar1-/- BMMCs was reduced by 50% and IL-6 production was decreased by 35% as compared with wt cells. Also, FcεR1 cross-linking markedly upregulated C5aR1 MC expression. Further, in contrast to wt mice, C5ar1-/- mice were largely protected from histamine-induced temperature drop in vivo. In line with this finding, C5aR-targeting protected human endothelial cells from histamine-induced barrier dysfunction and disruption of tight junction proteins in vitro. Our findings identify a critical role of the C5a/C5aR1 axis in IgE-mediated oral antigen-induced anaphylaxis. C5aR1 may serve as a novel therapeutic target to suppress the inflammatory response in food-induced anaphylaxis.