The Journal of Immunology Monitoring C3aR Expression Using a Floxed tdTomato-C3aR Reporter Knock-in Mouse Katharina M. Quell,*,1 Christian M. Karsten,*,1 Anna Kordowski,* Larissa Nogueira Almeida,* Daria Briukhovetska,* Anna V. Wiese,* Jing Sun,* Fanny Ender,* Konstantina Antoniou,* Torsten Schro¨der,* Inken Schmudde,† Johann L. Berger,† Peter Ko¨nig,† Tillman Vollbrandt,‡ Yves Laumonnier,*,2 and Jo¨rg Ko¨hl*,x,2 C3a exerts multiple biologic functions through activation of its cognate C3a receptor. C32/2 and C3aR2/2 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-C3aR2, 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 Journal of Immunology, 2017, 199: 688–706. xogenous and endogenous threats can activate the com- expression in the brain, but absence in circulating leukocytes (5, 6). plement cascade through canonical and noncanonical The strong pulmonary expression of C3aR was confirmed in mouse E pathways (1–3). Both pathways drive the proteolytic cleav- tissue. In contrast, C3aR expression was absent in the murine brain age of C3 into C3b and the generation of the anaphylatoxin C3a, and in the spleen (10, 11). The strong pulmonary expression of C3aR which exerts a wide range of pro- and anti-inflammatory functions wasassignedtoimmuneandstromalcells(12,13).Amonghuman (4). Most C3a-induced functions require the binding to its cognate pulmonary cells of myeloid origin, eosinophils were reported to C3a receptor, which belongs to the large family of G protein– express C3aR (5, 9). In asthmatic patients, C3aR expression has coupled receptors (5, 6). Binding of C3a to C3aR leads to rapid been reported on endothelial, epithelial, and smooth muscle cells C3aR internalization that depends on phosphorylation of serine and (14). Furthermore, upregulation of C3aR in airway epithelial cells threonine residues at the receptor C terminus (7, 8) and activation of and smooth muscle cells was shown in asthmatics and in experi- pertussis toxin–sensitive G proteins (9). mental allergy models (12, 14). Northern blot analysis revealed strong expression of the human Binding studies, flow cytometric analyses and functional studies C3aR in lung, spleen, small intestine, bone marrow (BM), low level suggest C3aR expression in human neutrophils (15, 16), monocytes *Institute for Systemic Inflammation Research, University of Lubeck,€ Lubeck€ 23562, Ratzeburger Allee 160, Lubeck€ 23562, Germany. E-mail addresses: [email protected] Germany; †Institute of Anatomy, University of Lubeck,€ Lubeck€ 23562, Germany; (J.K.) and [email protected] (Y.L.). ‡Cell Analysis Core Facility, University of Lubeck,€ Lubeck€ 23562, Germany; and x The online version of this article contains supplemental material. Division of Immunobiology, Cincinnati Children’s Hospital Medical Center, Uni- versity of Cincinnati College of Medicine, Cincinnati, OH 45229 Abbreviations used in this article: AF, Alexa Fluor; AM, alveolar macrophage; BAL, 1 bronchoalveolar lavage; BM, bone marrow; BMDC, BM-derived dendritic cell; K.M.Q. and C.M.K. contributed equally to this work. 2+ 2+ BMM, BM-derived macrophage; BV, Brilliant Violet; [Ca ]i, intracellular Ca 2Y.L. and J.K. shared supervision of this work. concentration; CC10, Clara cell protein 10; CD, cluster of differentiation; DC, den- dritic cell; D-PBS, Dulbecco’s PBS; eF, eFluor; HDM, house dust mite; ILC, innate ORCIDs: 0000-0003-3194-4955 (K.M.Q.); 0000-0003-2974-433X (L.N.A.); 0000- lymphoid cell; IRES, internal ribosome entry site; LP, lamina propria; MC, mast 0002-2073-2335 (D.B.); 0000-0001-8103-367X (K.A.). cell; MFI, mean fluorescence intensity; DMFI, change in relative mean fluores- Received for publication March 1, 2017. Accepted for publication May 15, 2017. cence intensity; MHCII, MHC class II; moDC, monocyte-derived DC; PBS20, PBS supplemented with 20% FCS; pDC, plasmacytoid DC; PE, peritoneal; This work was supported by Deutsche Forschungsgemeinschaft Grants IRTG 1911 RBCL, RBC lysis; tdTomato, tandem-dye Tomato; VAT, visceral adipose tissue; (projects A1 and A2 to Y.L., J.K., and P.K., respectively), IRTG 1911 (projects B1 WT, wild-type. and B2 to C.M.K. and J.K.), and KO 1245/3-1 (to J.K.). L.N.A. was supported by the National Council for Technological and Scientific Development/Science without Ó Borders (Brazil). Copyright 2017 by The American Association of Immunologists, Inc. 0022-1767/17/$30.00 Address correspondence and reprint requests to Prof. Jo¨rg Ko¨hl and Dr. Yves Laumonnier, Institute for Systemic Inflammation Research, University of Lubeck,€ www.jimmunol.org/cgi/doi/10.4049/jimmunol.1700318 The Journal of Immunology 689 (16, 17), eosinophils (16, 18, 19), basophils (20, 21), and mast detected using a secondary allophycocyanin-labeled F(ab9)2 anti-goat Ab cells (MCs) (22, 23). The role of C3a as a chemoattractant for (Santa Cruz Biotechnology). human neutrophils is controversial (4, 18). In mice, some reports RBC lysis (RBCL) buffer was prepared by using 155 mM NH4Cl, 10 mM KHCO3, 0.1 mM EDTA (all from Sigma-Aldrich). BSA and an LP dis- suggested the expression of C3aR on neutrophils (24, 25), and sociation kit were purchased from Miltenyi Biotec. Recombinant murine BM-derived dendritic cells (BMDCs) (26). Reports regarding the GM-CSF was from PeproTech. DNase I for total RNA isolation was from expression of C3aR on human T cells are controversial. Earlier Fermentas. Liberase TL was from Roche Diagnostics, and DNase I for cell studies failed to detect C3aR surface expression in naive T cells isolation was from Sigma-Aldrich. FBS, RPMI 1640 medium, and HBSS were all from PAA Laboratories. Dulbecco’s PBS (D-PBS), L-glutamine, (16, 27). More recently, low C3aR surface expression was de- penicillin, and streptomycin were all from Life Technologies. scribed on naive human T cells, but C3aR was upregulated upon cluster of differentiation (CD) 3 and CD28 stimulation (28). Also, Animals + + C3ar1 mRNA expression was reported in naive CD4 Foxp3 C57BL/6 wild-type (WT) mice were obtained from Janvier. BALB/c WT natural regulatory T cells, which was enhanced in response to mice were purchased from the Charles River Laboratories. C3ar12/2, 2 2 2 2 CD3 and CD28 stimulation (29). Another study found intracellular C3ar1 / /C5ar1 / ,andtdTomato-C3ar1fl/fl mice in BALB/c background € C3aR expression in naive human T cells and translocation to the were bred in the animal facility of the University of Lubeck as described (35). All animals were used at 8–12 wk of age and handled in accordance cell surface following CD3 with or without CD46 stimulation with the appropriate institutional and national guidelines. Animals were used (30). In mice, one study reported surface expression of C3aR in for organ removal according to protocols approved by the local authorities of naive T cells (31). the Animal Care and Use Committee (Ministerium fur€ Landwirtschaft, At this point, most of the expression and functional data related Energiewende, Umwelt und la¨ndliche Ra¨ume, Kiel, Germany). The to the C3a/C3aR axis have been obtained with human cells. Direct experimental allergic asthma study and the i.p. injection of thioglycollate were reviewed and approved by the Schleswig-Holstein state authorities evidence for the expression of C3aR in murine immune and stromal (numbers V312-7224.122-39 [37-2/13] and V242-81505/2016 [19-2/2017], cells under steady-state or upon inflammatory conditions is limited, respectively). All experiments were performed by certified personnel. which is mainly due to the lack of C3aR-specific mAbs. Several studies used polyclonal or poorly characterized mAbs raised Induction of house dust mite–mediated experimental allergic against C3aR (13, 32–34). In this study, we report the generation asthma of a floxed tandem-dye Tomato (tdTomato)–C3aR reporter knock- Experimental allergic asthma was induced as previously described (36). in mouse. The reporter strain contains the coding sequence for a Briefly, BALB/c WT and tdTomato-C3ar1fl/fl mice were anesthetized by i. tdTomato-C3aR self-processing polyprotein, flanked by two loxP p. injection with ketamine (Ketavet; Pfizer) and xylazine (Rompun; Bayer) and sensitized intratracheally with 100 mg of house dust mite (HDM) sites. Using this mouse, we evaluated C3aR expression in myeloid extract (Greer Laboratories, lot not. 262538) in 50 ml of PBS on days 0, 7, and lymphoid cells from the circulation and several tissues, in- 14, and 21. Seventy-two hours after the final intratracheal HDM challenge cluding the lung, airways, lamina propria (LP) of the small intestine, on day 21, bronchoalveolar lavage (BAL) fluid and lung tissue were har- spleen, visceral adipose tissue (VAT), and the brain.