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.

C5a drives airway constriction and inflammation during the effector phase of allergic asthma, mainly through the activation of C5a receptor 1 (C5aR1). Yet, C5aR1 expression on myeloid and lymphoid cells during the allergic effector phase is ill-defined. Recently, we generated and characterized a floxed green fluorescent protein (GFP)-C5aR1 knock-in mouse. Here, we used this reporter strain to monitor C5aR1 expression in airway, pulmonary and lymph node cells during the effector phase of OVA-driven allergic asthma. C5aR1 reporter and wildtype mice developed a similar allergic phenotype with comparable airway resistance, mucus production, eosinophilic/neutrophilic airway inflammation and Th2/Th17 cytokine production. During the allergic effector phase, C5aR1 expression increased in lung tissue eosinophils but decreased in airway and pulmonary macrophages as well as in pulmonary CD11b+ conventional dendritic cells (cDCs) and monocyte-derived DCs (moDCs). Surprisingly, expression in neutrophils was not affected. Of note, moDCs but not CD11b+ cDCs from mediastinal lymph nodes (mLN) expressed less C5aR1 than DCs residing in the lung after OVA challenge. Finally, neither CD103+ cDCs nor cells of the lymphoid lineage such as Th2 or Th17-differentiated CD4+ T cells, B cells or type 2 innate lymphoid cells (ILC2) expressed C5aR1 under allergic conditions. Our findings demonstrate a complex regulation pattern of C5aR1 in the airways, lung tissue and mLN of mice, suggesting that the C5a/C5aR1 axis controls airway constriction and inflammation through activation of myeloid cells in all three compartments in an experimental model of allergic asthma.

Conventional dendritic cells (cDC) are necessary and sufficient to drive mixed maladaptive Th2/Th17 immune responses toward aeroallergens in experimental allergy models. Previous studies suggest that the anaphylatoxin C3a promotes, whereas C5a protects from the development of maladaptive immunity during allergen sensitization. However, only limited evidence exists that such effects are directly mediated through anaphylatoxin-receptor signaling in cDCs. In this study, we assessed the impact of C3a and C5a on cDC-mediated induction pulmonary allergy by adoptively transferring house dust mite (HDM)–pulsed bone marrow–derived DCs (BMDC) from wild-type (WT) C3aR−/−, C5aR1−/−, or C3aR−/−/C5aR1−/− into WT mice. Transfer of HDM-pulsed WT BMDCs promoted a strong asthmatic phenotype characterized by marked airway resistance, strong Th2 cytokine, and mucus production, as well as mixed eosinophilic and neurophilic airway inflammation. Surprisingly, C3aR−/− cDCs induced a strong allergic phenotype, but no IL-17A production, whereas HDM-pulsed C5aR1−/− cDCs failed to drive pulmonary allergy. Transfer of C3aR−/−/C5aR1−/− cDCs resulted in a slightly reduced allergic phenotype associated with increased IFN-γ production. Mechanistically, C3aR and C5aR1 signaling is required for IL-23 production from HDM-pulsed BMDCs in vitro. Furthermore, C3aR−/− BMDCs produced less IL-1β. The mechanisms underlying the failure of C5aR1−/− BMDCs to induce experimental allergy include a reduced capability to migrate into the lung tissue and a decreased potency to direct pulmonary homing of effector T cells. Thus, we uncovered a crucial role for C5a, but only a minor role for C3a in BMDC-mediated pulmonary allergy, suggesting that BMDCs inappropriately reflect the impact of complement on lung cDC-mediated allergic asthma development.