C3a exerts multiple biologic functions through activation of its cognate C3a receptor. C3 and C3aR mice have been instrumental in defining important roles of the C3a/C3aR axis in the regulation of acute and chronic inflammatory diseases, including ischemia/reperfusion injury, allergic asthma, autoimmune nephritis, and rheumatoid arthritis. Surprisingly little is known about C3aR expression and function in immune and stromal cells. To close this gap, we generated a floxed tandem-dye Tomato (tdTomato)-C3aR reporter knock-in mouse, which we used to monitor C3aR expression in cells residing in the lung, airways, lamina propria (LP) of the small intestine, brain, visceral adipose tissue, bone marrow (BM), spleen, and the circulation. We found a strong expression of tdTomato-C3aR in the brain, lung, LP, and visceral adipose tissue, whereas it was minor in the spleen, blood, BM, and the airways. Most macrophage and eosinophil populations were tdTomato-C3aR Interestingly, most tissue eosinophils and some macrophage populations expressed C3aR intracellularly. BM-derived dendritic cells (DCs), lung-resident cluster of differentiation (CD) 11b conventional DCs (cDCs) and monocyte-derived DCs, LP CD103, and CD11b cDCs but not pulmonary CD103 cDCs and splenic DCs were tdTomato-C3aR Surprisingly, neither BM, blood, lung neutrophils, nor mast cells expressed C3aR. Similarly, all lymphoid-derived cells were tdTomato-C3aR, except some LP-derived type 3 innate lymphoid cells. Pulmonary and LP-derived epithelial cells expressed at best minor levels of C3aR. In summary, we provide novel insights into the expression pattern of C3aR in mice. The floxed C3aR knock-in mouse will help to reliably track and conditionally delete C3aR expression in experimental models of inflammation.

The biological significance of C5a receptor [(C5aR)2/C5L2], a seven-transmembrane receptor binding C5a and C5adesArg, remains ill-defined. Specific ligation of C5aR2 inhibits C5a-induced ERK1/2 activation, strengthening the view that C5aR2 regulates C5aR1-mediated effector functions. Although C5aR2 and C5aR1 are often coexpressed, a detailed picture of C5aR2 expression in murine cells and tissues is still lacking. To close this gap, we generated a floxed tandem dye (td)Tomato-C5aR2 knock-in mouse that we used to track C5aR2 expression in tissue-residing and circulating immune cells. We found the strongest C5aR2 expression in the brain, bone marrow, and airways. All myeloid-derived cells expressed C5aR2, although with different intensities. C5aR2 expression in blood and tissue neutrophils was strong and homogeneous. Specific ligation of C5aR2 in neutrophils from tdTomato-C5aR2 mice blocked C5a-driven ERK1/2 phosphorylation, demonstrating functionality of C5aR2 in the reporter mice. In contrast to neutrophils, we found tissue-specific differences in C5aR2 expression in eosinophils, macrophages, and dendritic cell subsets. Naive and activated T cells stained negative for C5aR2, whereas B cells from different tissues homogeneously expressed C5aR2. Also, NK cell subsets in blood and spleen strongly expressed C5aR2. Activation of C5aR2 in NK cells suppressed IL-12/IL-18-induced IFN-γ production. Intratracheal IL-33 challenge resulted in decreased C5aR2 expression in pulmonary eosinophils and monocyte-derived dendritic cells. In summary, we provide a detailed map of murine C5aR2 immune cell expression in different tissues under steady-state conditions and upon pulmonary inflammation. The C5aR2 knock-in mouse will help to reliably track and conditionally delete C5aR2 expression in experimental models of inflammation.

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BackgroundFood‐induced anaphylaxis is a serious allergic reaction caused by Fcε‐receptor activation on mast cells (MCs). The exact mechanisms breaking oral tolerance and the effector pathways driving food allergy remain elusive. As complement is activated in food‐induced anaphylaxis, we aimed to assess the role of C5a in disease pathogenesis.MethodsOral antigen‐induced food‐induced anaphylaxis was induced in BALB/c wild‐type (wt) and C5ar1−/− mice. Readouts included diarrhea development, changes in rectal temperature, hematocrit, antigen‐specific serum IgE, MCPT‐1, and intestinal MC numbers, as well as FcεR1‐mediated MC functions including C5a receptor 1 (C5aR1) regulation. Further, histamine‐mediated hypothermia and regulation of endothelial tight junctions were determined.ResultsRepeated oral OVA challenge resulted in diarrhea, hypothermia, increased hematocrit, high OVA‐specific serum IgE, and MCPT‐1 levels in wt mice. Male C5ar1−/− mice were completely whereas female C5ar1−/− mice were partially protected from anaphylaxis development. Serum MCPT‐1 levels were reduced gender‐independent, whereas IgE levels were reduced in male but not in female C5ar1−/− mice. Mechanistically, IgE‐mediated degranulation and IL‐6 production from C5ar1−/− BMMCs of both sexes were significantly reduced. Importantly, FcεR1 cross‐linking strongly upregulated C5aR1 MC expression in vitro and in vivo. Finally, C5ar1−/− male mice were largely protected from histamine‐induced hypovolemic shock, which was associated with protection from histamine‐induced barrier dysfunction in vitro following C5aR targeting.ConclusionsOur findings identify C5aR1 activation as an important driver of IgE‐mediated food allergy through regulation of allergen‐specific IgE production, FcεR1‐mediated MC degranulation, and histamine‐driven effector functions preferentially in male mice.

Background Clinical and experimental analyses have identified a central role for IgE/FcεRI/mast cells in promoting IgE-mediated anaphylaxis. Recent data from human studies suggest that bacterial infections can alter susceptibility to anaphylaxis. Objective We examined the effect of LPS exposure on the induction of IgE-mast cell-(MC) mediated reactions in mice. Methods C57BL/6 WT, TLR-4−/− and IL10−/− mice were exposed to LPS and serum cytokines (TNF and IL-10) were measured. Mice were subsequently treated with anti-IgE and the symptoms of passive IgE-mediated anaphylaxis, MC activation, Ca2+-mobilization and expression of FcεRI on peritoneal MCs were quantitated. Results We show that LPS exposure of C57BL/6 WT mice constrains IgE-MC mediated reactions. LPS-induced suppression of IgE-MC mediated responses was TLR-4-dependent and associated with increased systemic IL-10 levels, decreased surface expression of FcεRI on MCs and loss of sensitivity to IgE activation. Notably, LPS-induced desensitization of MCs was short-term with MC sensitivity to IgE reconstituted within 48 hours which was associated with recapitulation of FcεRI expression on the MCs. Mechanistic analyses revealed a requirement for IL-10 in LPS-mediated decrease in MC FcεRI surface expression. Conclusions & Clinical Relevance Collectively, these studies suggest that LPS-induced IL-10 promotes the down regulation of MC surface FcεRI expression and leads to desensitization of mice to IgE-mediated reactions. These studies indicate that targeting of the LPS-TLR-4-IL-10-pathway maybe used as a therapeutic approach to prevent adverse IgE-mediated reactions.