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. We identified vested for tdTomato-C3ar and C3aR expression. + various tdTomato-C3aR cell populations. They were evaluated for Induction and collection of thioglycollate-elicited peritoneal C3aR surface expression using commercially available anti-C3aR macrophages Abs, tested for their specificity using cells from C3aR-deficient fl/fl mice. This approach provided detailed insights into the C3aR BALB/c WT and tdTomato-C3ar1 mice were injected i.p. with 1 ml of 3% thioglycollate medium (BD Biosciences). Seventy-two hours later, peritoneal expression pattern in immune and stromal cells under steady-state (PE) cell exudates were collected using 5 ml of ice-cold PBS. Subsequently, conditions. cells were identified as F4/80+ cells by flow cytometry and used for the assessment of C3a-mediated mobilization of intracellular Ca2+. Materials and Methods Generation of the floxed tdTomato-C3aR knock-in reporter Reagents mouse strain Monoclonal allophycocyanin-Cy7–labeled Ab against CD11b (M1/70), The tdTomato-C3aR knock-in mice were generated by gene targeting. A PerCP-Cy5.5–labeled Ab against CD8a (53-6.7), allophycocyanin or scheme of the targeting strategy is shown in Fig. 1A. The targeting vector, V450-labeled Ab against Ly6G (1A8), V450-labeled Ab against CD45R the embryonic stem cells, and the targeted mice were generated by Ozgene (RA3-6B2), FITC-labeled Ab against CD90.2 (30-H12), allophycocyanin- (Perth, Australia). Our strategy was to insert tdTomato in frame with the H7–labeled Ab against CD19 (1D3), and Brilliant Violet (BV)421–labeled coding sequence of C3ar1 and simultaneously flank the tdTomato-C3ar1 Ab against Siglec-F (E50-2440) and CD43 (1G10) were all purchased cassette with two loci of loxP sites. A construct that was the precursor to from BD Biosciences. PerCP-Cy5.5–labeled Ab against Ly6C (RB6-8C5); the final targeting construct was generated by the sequential cloning of six allophycocyanin-labeled Ab against CD115 (AFS98), CD11c (N418), cloned fragments into a final recipient plasmid housing a neomycin se- NK1.1 (PK136), CD4 (GK1.5), and CD11b (M1/70); PE-Cy7–labeled Ab lectable marker cassette. Primers used to amplify the fragments also en- against CD25 (PC61.5) and CD4 (RM4-5); eFluor (eF)450–labeled Ab coded the restriction enzyme sites required for the assembly of the fragments against CD19 (1D3), CD3e (145-2C11), and CD49b (DX5); Alexa Fluor generated. All fragments were housed in the Surf2 vector backbone (AF)488–labeled Ab against CD3 (17A2); allophycocyanin-eF780–labeled (Ozgene). The first fragment was a 3.1-kb fragment encoding the 39 half of Ab against MHC class II (MHCII) (M5/144.15.2); BV510-labeled Ab the 39 homology arm. This fragment was amplified from BALB/c genomic against CD11b (M1/70); BV711-labeled Ab against CD64 (X54-5/7.1); DNA using primers 1471_46 (59-CTAAACTGTGCATCCTAGGTTCAAC- BV421-labeled Ab against F4/80 (BM8); and PerCP-Cy5.5–labeled Ab TTC-39) and 1471_56 (59-TAAGCATTGGTAATTCGAAGCAGGCTGTA- against CD103 (2E7) were all purchased from BioLegend. PerCP- AAGTCTTGGCATGATGTG-39). The second fragment was a 3.1-kb fragment Cy5.5–labeled Ab against CD3 (17A2), CD5 (53-73), CD27 (LG.7F9), encoding the 59 half of the 39 homology arm. This fragment was amplified NK1.1 (PK136), TCR-b (H57-597), and CD11b (M1/70); allophycocya- from BALB/c genomic DNA using primers 1471_47 (59-TAAGCATTG- nin-eF780–labeled Ab against CD11c (N418), B220 (RA3-6B2), and GTAATTCGAATGACCGTCACATAACCCAGGGATGGA-39) and 1471_57 CD49b (DX5); eF450-labeled Ab against CD25 (PC61.5) and CD317 (59-CTAATTAATTAAGGGAACCTAAGCAGAGTTCTCA-39). The third (ebio129c); FITC-labeled Ab against CD11c (N418); AF700-labeled Ab fragment was a 1.4-kb fragment encoding the floxed region of exon 2 of against CD11b (M1/70); PE-Cy5–labeled Ab against CD127 (A7R34); and C3ar1. This fragment was amplified from BALB/c genomic DNA using PE-Cy7 IgM (11/41) were purchased from eBioscience (Affymetrix). For primers 1471_42 (59-TAAGCATTGGTAATTAATTAACAATTGATAA- surface and intracellular staining, a rat unlabeled C3aR-specific Ab (14D4) CTTCGTATAGCATACATTATACGAAGTTATAGGGCCACATCTTCA- was purchased from Hycult Biotech. Binding of this Ab was detected CACATCTGTACT-39) and 1471_52 (59-CTAAGCGGCCGCCACCATG- using a secondary allophycocyanin-labeled F(ab9)2 anti-rat Ab (Cell Sig- GAGTCTTTCGATGCTGACAC-39) where primer 1471_42 also incorporated naling Technology). Additionally, we used a goat unlabeled C3aR-specific the loxP sequence. The fourth fragment was a 0.6-kb fragment encoding an Ab (D20) from Santa Cruz Biotechnology, the binding of which was internal ribosomal entry site (IRES). This fragment was obtained from one of 690 C3aR EXPRESSION IN A tdTomato-C3aR KNOCK-IN MOUSE

Ozgene’s proprietary cloning vectors. The fifth fragment was a 1.4-kb mediastinal lymph nodes, and the Peyer’s patches from the small intestine fragment encoding tdTomato and was obtained from one of Ozgene’s pro- was performed by mechanical disruption using a cell strainer (40 mm prietary cloning vectors. The sixth fragment was a 3.0-kb fragment encoding nylon; BD Biosciences) and a plunger of a 5-ml syringe (BD Biosciences). the 59 homology arm. This fragment was amplified from BALB/c genomic The cell strainer was flushed three times with 5 ml of PBS. Cells were then DNA using primers 1471_45 (59-CTAACTGATAGACAGTTTCAGGTCT- incubated in RBCL buffer for 3 min and finally washed with PBS. LP cell ATGC-39) and 1471_55 (59-TAAGCATTGGTAACCAAATGTCCCAGCT- suspension was obtained using an LP dissociation kit (Miltenyi Biotec), TCCATTTA-39). The vector backbone into which this amplicon was introduced following the manufacturer’s recommendation. Briefly, the small intestine contained the neomycin selection cassette. The six fragments were assem- was removed from the mice and quickly freshly collected and cleared from bled sequentially as follows: fragment 1 was excised by digestion with BstBI feces, residual fat, and Peyer’s patches. Then, they were cut longitudinally and ligated into fragment 2 that had been digested with the same enzyme. and washed once with HBSS supplemented with EDTA (5 mM) and once Combined fragments 1 and 2 were excised from the resulting construct by with HBSS only. The tissue was digested using the manufacturer’s enzyme digestion with PacI and ligated into fragment 3, which had been digested mixture in HBSS with Ca2+ and Mg2+ for 30 min at 37˚C and homogenized with the same enzyme. Combined fragments 1, 2, and 3 were excised from with the gentleMACS dissociator (Miltenyi Biotec). After the last washing the resulting construct by digestion with NotI and ligated into fragment 4, step, the cell number was determined using a Neubauer chamber, after which had been digested with the same enzyme. Combined fragments 1, 2, 3, which the cells were resuspended in PBS containing 1% BSA. The via- and 4 were excised from the resulting construct by digestion with FseI and bility of the collected cells was determined by trypan blue exclusion or a ligated into fragment 5, which had been digested with the same enzyme. Live/Dead cell viability assay (eBioscience). Small intestine epithelial Combined fragments 1, 2, 3, 4, and 5 were excised from the resulting con- cells were obtained by incubating freshly isolated tissue, cleared from struct by digestion with AscI and ligated into fragment 6, which had been feces, residual fat, and Peyer’s patches, in HBSS supplemented with EDTA digested with the same enzyme, resulting in the vector 1471_pTV. To con- (5 mM) for 30 min. Cell suspension was cleared from clumps through a struct the final targeting vector, 1471_pTV was digested with MfeI and KpnI 100-mm cell strainer before blocking. Brain tissue was collected, sliced to excise the region encoding tdTomato_IRES_exon2_loxP. A synthetic frag- into pieces, and digested with Liberase TL (0.1 mg/ml) and DNase I ment (Blue Heron Biotech) encoding tdTomato_P2A_exon2_loxP digested (0.1 mg/ml) for 30 min at 37˚C. Brain homogenates were passed through a with MfeI and KpnI was ligated into 1741_pTV in place of the excised 40-mm cell strainer and the cell suspension was washed and fractionated fragment. We replaced the IRES by the porcine teschovirus-1 2A (P2A) be- on a 30–75% discontinuous Percoll gradient (GE Healthcare) for 30 min at cause of its short length, high cleavage efficiency, and the stoichiometric ex- 1350 3 g without brake. The interphase-containing cells were collected, pression of multiple proteins flanking the 2A peptide (37, 38). The resulting washed with D-PBS, and the cellular composition was analyzed. targeting construct encoded a 6.2-kb 59 homology arm and a 3.0-kb 39 ho- mology arm flanking the floxed coding region of exon 2 where tdTomato_P2A Flow cytometry and cell sorting had been knocked in at the ATG of exon 2. Phenotypic characterization and determination of tdTomato-C3aR ex- Genotyping of floxed tdTomato-C3aR knock-in mouse pression of tissue and circulating cells was performed using a BD FACSAria III cell sorter (BD Biosciences) and a MoFlo Legacy (Beckman Coulter). For genotyping, we used ear biopsies. Extraction of DNA from tissue was The markers and gating strategies used to identify the myeloid, lymphoid, performed using the KAPAExpress extraction kit from Peqlab Biotechnologie and stromal cells in the different tissues and the circulation are summarized following the manufacturer’s instructions. To amplify the different DNA in Supplemental Fig. 3C–H and in the text. Flow cytometric data were fragments, we used following primers: GK342, 59-AACAACAGAAGTA- analyzed using FlowJo 10 (Tree Star). GGGAGGTGTAA-39; GK45, 59-TCCCAATAGACAAGTGAGACCAA-39 (both from Eurofins Scientific). The PCRs were run using the following Determination of surface and intracellular C3aR expression in conditions: 95˚C for 3 min, followed by 35 cycles at 95˚C for 15 s, 62˚C for WT, C3aR-deficient, and tdTomato-C3aR cells by flow cytometry 15 s, and 72˚C for 60 s, followed by 72˚C for 120 s. Then, the samples were loaded to a 1.0% sodium borate agarose gel. Amplification products were All cells that stained positive for tdTomato-C3aR were also assessed for detected by GelRed staining (Biotrend). C3aR expression on the cell surface using two different C3aR-specific Abs (clones 14D4 and D20). In some cells, we also determined intracellular Cell preparations from different organs expression of C3aR. For surface staining with the rat C3aR-specific mAb 14D4 (Hycult Biotech), cells were first incubated with PBS supplemented Mice were killed by cervical dislocation under anesthesia or after CO2 with 20% FCS (PBS20) for 30 min at 4˚C, after which the cells were stained exposure and the heart was perfused with 10 ml of ice-cold PBS before with the Ab in PBS20 for 30 min at 4˚C. After washing with PBS20, cells organ removal. For BM preparation, femurs and tibias were removed, were stained with an F(ab9)2 anti-rat allophycocyanin Ab (Cell Signaling placed in PBS on ice, and subsequently flushed with PBS. RBCs were Technology). After three washes with PBS20, cells were resuspended in removed by incubating the cells in RBCL buffer for 3 min. The reaction PBS/BSA containing Fc Block (eBioscience) and stained for cell-specific was terminated by addition of a large volume of PBS. BMDCs were markers. The surface staining with a goat polyclonal C3aR-specific Ab generated as described (39). Briefly, BM cells were differentiated for 9 d + + (D20) was done in PBS/BSA buffer, together with BMM-specific Abs (F4/80 with GM-CSF (20 ng/ml); BMDCs were defined as CD11c CD11b and CD11b) in the presence of Fc Block. After washing with PBS/BSA, MHCIIhiCD1152 cells. To obtain BM-derived plasmacytoid DCs (pDCs), cells were stained with an F(ab9)2 anti-goat allophycocyanin Ab (Santa BM cells were differentiated for 9 d in the presence of Flt3L (200 ng/ml). Cruz Biotechnology). For intracellular staining, cells from WT, C3ar12/2 To generate BM-derived macrophages (BMMs), we cultured BM cells for or tdTomato-C3ar1fl/fl mice were first fixed in 1.5% paraformaldehyde, 6 d in the presence of L929 cell–conditioned medium (40). Alternatively, then resuspended in a saponin (0.2%) buffer containg 20% FCS instead of BM cells were cultured in GM-CSF (20 ng/ml) for 9 d and identified as + + + lo PBS20. After 30 min, cells were stained with the 14D14 Ab in saponin CD11c CD11b CD115 MHC BMMs as described (41). For pulmonary (0.2%) buffer containg 20% FCS instead of PBS20. After washing with cell preparation, lungs were harvested, digested using 0.25 mg/ml Liberase PBS20, cells were stained with an F(ab9)2 anti-rat allophycocyanin Ab TL and 0.5 mg/ml DNase I in pure RPMI 1640 medium for 45 min at (Cell Signaling Technology). After three washes with PBS20, cells were 37˚C, and single-cell suspensions were prepared as described (42). For resuspended in PBS/BSA containing Fc Block (eBioscience) and stained VAT cell preparations, perigonadal fat tissue was harvested, taking care not for cell-specific markers. C3aR surface and intracellular expression was to remove the gonads, and minced into small pieces. The tissue was measured by flow cytometry. digested using 0.25 mg/ml Liberase TL and 0.5 mg/ml DNase I in pure RPMI 1640 medium for 45 min at 37˚C. The cell suspension was filtered Isolation and in vitro activation of spleen-derived CD4+ T cells through a nylon mesh (40 mm) and washed with 10 ml of RPMI 1640 medium complemented with 10% FCS containing 0.5 mg/ml DNase I. The spleen was harvested and dissociated by mechanical disaggregation. Cells were centrifugated at 350 3 g for 10 min at 4˚C, resuspended in After RBCL, cells were counted and whole spleen cells were used for PBS, transferred into a fresh 15-ml tube, and washed once more. BAL fluid in vitro T cell activation. To stimulate CD4+ T cells, a 96-well cell culture samples were obtained as described (39). To collect cells from the PE plate (Greiner Bio-One International) was coated with unconjugated anti- cavity, mice were lavaged with 5 ml of ice-cold PBS. Collected cells were CD3 (17A2; eBioscience) in 150 ml of PBS at a final concentration of washed once with PBS. RBCs were removed by incubating the cells in 2 mg/ml. The plate was kept at 37˚C, 5% CO2 for at least 2 h before RBCL buffer for 3 min and then washed with PBS as described (42). Blood discarding the coating solution and adding the cell suspension. T cells were was collected by cardiac puncture and immediately transferred into tubes cultured in presence of 10 mg/ml anti-CD28 (37.51; eBioscience) for 5 d in containing 10 mM EDTA to prevent coagulation. Diluted blood samples complete RPMI 1640 culture medium supplemented with 10% FBS, 2 mM were incubated several times in RBCL buffer to remove RBCs and then L-glutamine, 100 U/ml penicillin, and 100 mg/ml streptomycin. Cells were washed with PBS as described (42). Isolation of cells from the spleen, subsequently analyzed by flow cytometry for tdTomato-C3aR expression. The Journal of Immunology 691

RNA isolation and PCR (TBS plus 0.1% Tween 20), the membrane was incubated with an anti-goat IgG-HRP (1:2500 in TBS plus 5% Rockland Immunochemicals Blotto low- T and B cells as well as neutrophils from different compartments were fat dry milk plus 0.1% Tween 20; Santa Cruz Biotechnology) for 1 h at room sorted using a BD FACSAria III or a MoFlo Legacy cell sorter. Total RNAwas temperature. After washing, detection was performed using the Immun-Star isolated using TRIzol reagent (Thermo Fisher Scientific) according to the WesternC kit (Bio-Rad Laboratories) and Fusion SL (Vilbert Lourmat). manufacturer’s instructions. After DNAse I (Fermentas) treatment of the RNA, reverse transcription reaction was performed using a RevertAid first- C3aR internalization assay strand cDNA synthesis kit (Thermo Fisher Scientific). PCR was done using 3 7 fl/fl the following primers: b-actin,59-GCACCACACCTTCTACAATGAG-39 PE cells (1 10 cells/ml) from WT or tdTomato-C3ar1 mice were and 59-AAATAGCACAGCCTGGATAGCAAC-39, C3ar1,59-TCGATGCT- resuspended in PBS and allowed to rest for 5 min at 37˚C. Cells were left GACACCAATTCAA-39 and 59-TCCCAATAGACAAGTGAGACCAA-39. untreated or stimulated either with 1, 10, and 100 nM C3a (Hycult Biotech) The temperature profiles were 95˚C for 3 min, followed by 34 cycles at 95˚C for 3 min at 37˚C, or with 10 nM for 1, 3, and 9 min at 37˚C. The reaction for 30 s, 57˚C for both targets for 30 s, and 72˚C for 30 s. Amplification was stopped by addition of paraformaldehyde (1.5% final concentration) and products were separated using a 2% sodium borate agarose gel. Amplifica- cells were immediately placed on ice. After 30 min fixation, cells were ad- tion products were detected by GelRed staining (Biotrend). ditionally blocked for 30 min with PBS supplemented with PBS20, and then stained first with anti-C3aR mAb (clone 14D4; Hycult) in PBS20 for 30 min Lung slice preparation and immunofluorescence microscopy at 4˚C. After washing with PBS20, cells were stained with an F(ab9)2 anti-rat allophycocyanin Ab (Cell Signaling Technology). After three washes with WT and tdTomato-C3ar1fl/fl mice were sacrificed by isoflurane inhalation PBS20, they were resuspended in PBS/BSA containing Fc Block (eBioscience) (Baxter), and blood was removed from pulmonary vasculature by perfu- and stained with an F4/80-specific Ab. Expression of C3aR in F4/80+ cells was sion with 5 ml of 37˚C HEPES-Ringer buffer (10 mM HEPES, 136.4 mM determined by flow cytometry. NaCl, 5.6 mM KCl, 1 mM MgCl2, 2.2 mM, CaCl2, 11 mM glucose [pH 7.4]) containing 300 ml of heparin-natrium (25000; Ratiopharm) via the C3a-mediated increase in intracellular calcium right ventricle. Then, the airways were filled via the cannulated trachea The C3a-induced increase in intracellular Ca2+ concentration ([Ca2+] )was with 3% low melting point agarose (Bio-Rad Laboratories) dissolved in i determined, as described (42, 43), using thioglycollate-elicited F4/80+ PE HEPES-Ringer buffer. The lungs were removed en bloc and transferred macrophages. Briefly, cells were collected in 5 ml of PBS and loaded with into ice-cold HEPES-Ringer buffer to solidify the agarose. The lungs were 5mMoftheCa2+-sensitive fluorophore Fluo-4AM (Molecular Probes) ac- cut as precision-cut lung slices into 300-mm-thick slices using a Vibratome cording to the manufacturer’s recommendations. Cells were stained with (VT1200S; Leica). For fixation and freezing, slices were placed in 1% F4/80 for 20 min in PBS/BSA. F4/80hi cells were then analyzed on the LSR paraformaldehyde (Sigma-Aldrich), then washed three times in D-PBS and II flow cytometer. The background signal was recorded for 30 s. Then, C3a incubated overnight with 20% D(+)-saccharose (Carl Roth) and frozen (37 nM) was added, and recording continued for another 90 s. The increase at 220˚C until further use. in [Ca2+] was calculated by assessment of the maximal Ca2+ peak using the Immunohistochemical stainings were performed in 24-well plates (tissue i kinetic plug-in tool of the FlowJo software (version 9; Tree Star, Ashland, culture OrPlate; Orange Scientific) in the dark to protect the fluorophores OR). The mean background signal recorded during the 30 s was subtracted from light. Precision-cut lung slices were defrosted at room temperature on from the Ca2+ peak signal to determine the final change in relative mean a shaker and washed three times in 1 ml of TBS for 10 min. Primary fluorescence intensity (DMFI). Alternatively, thioglycollate-elicited PE antibodies were diluted in 500 ml of TBS and incubated on a shaker macrophages were allowed to adhere on glass coverslips for 2 h before overnight at room temperature. The next day, the slices were washed three loading with Fluo-4AM as indicated. Glass slides were washed and the assay times in TBS for 10 min. The slices were transferred to an object slide was performed in PBS. Images were obtained using the Olympus FV 1000 (Superfrost; Menzel Gla¨ser), dried carefully, and coverslipped with confocal microscope (Olympus, Germany) with a 340 objective. Image Mowiol as mounting medium (Mowiol 4-88 [Ho¨chst], 200 mM Tris buffer analysis and capturing was performed using the FluoView 2.1c software. [pH 8.5; Roth], glycerin [Merck]). Images were acquired with an LSM 710 confocal laser-scanning microscope (Carl Zeiss) with a 320/0.5 M27 Statistical analysis objective and an immersion oil objective 340/1.30 oil differential inter- ference contrast M27 (Carl Zeiss). Emitted light was detected by three Statistical analysis was performed using GraphPad Prism (version 5.0c; wavelength-separated photomultiplier tubes at 505–534, 555–593, and GraphPad Software). Statistical differences between groups were assessed 657–723 nm. Zen 2011 (Carl Zeiss) was used as acquisition software. by Student t test. Comparisons involving multiple groups were first ana- Image processing was conducted using Imaris software (Bitplane). mAbs lyzed by ANOVA. In case of significant differences (p , 0.05), all groups used for staining were AF647-labeled Ab against Siglec-F (E50-2440) were post hoc compared pairwise by a Tukey test. A p value ,0.05 was from BD Biosciences, AF488-labeled Ab against CD11c (N418), and considered significant (*p , 0.05, **p , 0.01, ***p , 0.001). AF647-labeled Ab against Clara cell protein 10 (CC10).

Assessment of pulmonary tdTomato-C3aR expression by Results two-photon microscopy Generation and initial evaluation of the floxed tdTomato-C3aR knock-in mouse Imaging of fixed lung slices was performed using the TriM Scope II mul- tiphoton microscope (LaVision BioTec) equipped with a XLUMPLFL 320 We aimed at generating a tdTomato-C3aR reporter knock-in mouse W/0.95 water immersion objective (Olympus). Images were acquired at 740 that allows for conditional deletion of the C3ar1 gene. Therefore, we and 1100 nm performed by a Mai Tai HP (Spectra-Physics) and an InSight cloned a tdTomato cassette directly upstream of the 59 end of exon DeepSee (Spectra-Physics). Emitted light was detected by three wavelength- separated photomultiplier tubes (Hamamatsu) at 435–495, 495–560, and 2oftheC3ar1 gene. To allow for conditional deletion of the C3ar1 .560 nm. Imspector Pro (LaVision BioTec) was used as acquisition gene, we placed two loxP sites upstream of the tdTomato coding software. Image processing was conducted using Imaris software (Bit- sequence and downstream of exon 2 of the C3ar1 gene (Fig. 1A). We plane) and ImageJ (National Institutes of Health). obtained several embryonic stem cells that incorporated tdTomato and the two loxP sites into the C3ar1 gene locus, one of which was Western immunoblot successfully used to produce heterozygous (tdTomato-C3ar1fl/+)and Sorted BM neutrophil and PE macrophage lysates from WT, tdTomato- fl/fl fl/fl 2/2 2/2 homozygous (tdTomato-C3ar1 ) floxed tdTomato-C3aR knock-in C3ar1 , and C3ar1 /C5ar1 animals were obtained by resuspending mice (Fig. 1B). A first screen of tdTomato-C3aR tissue expression the cells in Laemmli buffer preheated at 95˚C. Lysates were homogenized through a needle and heated at 95˚C before loading. Cell lysates were sep- revealed a strong expression in the brain, lung, LP of the small intes- arated by SDS-PAGE according to standard procedures using a Mini-Protean tine, and VAT (Fig. 1C). In contrast, the expression of tdTomato-C3aR TGX precast gradient gel 4–12% (Bio-Rad Laboratories). Proteins were in the spleen, blood, BM, and the airways was at best minor (Fig. 1D). transferred onto Trans-Blot nitrocellulose membrane (Bio-Rad Laboratories) using a Trans-Blot SD system (Bio-Rad Laboratories). Western blot analysis tdTomato-C3aR is expressed in neutrophils upon inflammatory was performed according to standard procedures. Briefly, after blocking, the conditions but not at steady-state membrane was incubated overnight with a polyclonal Ab raised against + C3aR (clone D20; Santa Cruz Biotechnology) in TBS plus 5% Blotto low-fat In previous studies, C3aR expression has been found in Gr-1 cells dry milk (Rockland Immunochemicals) plus 0.1% Tween 20. After washing that have been considered as neutrophils (32, 44). However, Gr-1 692 C3aR EXPRESSION IN A tdTomato-C3aR KNOCK-IN MOUSE

FIGURE 1. Generation and initial characterization of the floxed tdTomato-C3aR knock-in mouse. (A) Schematic of the gene-targeting strategy. WT exon 2ofC3ar1 was replaced with a cassette encoding a fusion protein comprising tdTomato-C3aR flanked with loxP sites by homologous recombination. (B) PCR-based phenotyping of WT, heterozygous tdTomato-C3ar1fl/+, and homozygous tdTomato-C3ar1fl/fl mice. The primer combination GK342: GK45 amplifies a 400-bp DNA fragment in BALB/c WT and tdTomato-C3aRfl/+ mice and a 2000-bp DNA fragment in tdTomato-C3ar1fl/fl mice. The arrows point toward the amplified PCR fragments. (C and D) Flow cytometric analysis of the tdTomato signal in (C) brain, lung, VAT, and LP of the small intestine; and (D) spleen, BAL, blood, and BM from WT and tdTomato-C3ar1fl/fl mice. is not only expressed on neutrophils but broadly on other immune or C57BL/6 BM neutrophils with C3aR-specific mAb 14D4 were cells, including eosinophils, monocytes, DCs, T cells, NK cells, negative (Supplemental Fig. 1A). Previously, it was reported that and NKT cells. High expression of Ly6G can be used to reliably Gr-1+ cells express C3aR based on Western immunoblot analysis identify neutrophils (45). Therefore, we first assessed the ex- and by flow cytometry (24). Re-evaluating the Ab used in that study pression of tdTomato-C3aR in Ly6Ghi neutrophils from BM, (D20), we found no C3aR surface staining in BM neutrophils blood, and lung tissue using tdTomato as a surrogate for C3aR ex- (Supplemental Fig. 1A) but a band in Western blot analysis that also pression. Surprisingly, neither BM, blood, nor lung neutrophils appeared in BM neutrophils isolated from C3ar12/2C5ar12/2 mice expressed C3aR (Fig. 2A). Also, C3aR surface staining of BALB/c (Supplemental Fig. 1B). To exclude a neutrophil-specific proteolysis The Journal of Immunology 693 of the protein, we also determined the mRNA expression of C3ar1 staining in permeabilized LP and brain macrophages precluded in WT and tdTomato-C3ar1fl/fl mice. We found no C3ar1 transcripts intracellular assessment of C3aR in such cells. In summary, our in Ly6G+-sorted blood- and lung-derived neutrophils and a very data demonstrate that only F4/80+Siglec-F2 macrophages ex- faint band in Ly6G+ BM neutrophils. In contrast, C3ar1 mRNA was press C3aR and that their C3aR surface expression is hetero- strongly expressed in GM-CSF–differentiated BMDCs/BMMs used geneous in the investigated organs. as positive control (Supplemental Fig. 1C). These data demonstrate that mouse neutrophils in the circulation and in pulmonary tissue do Most eosinophils express C3aR intracellularly not express C3aR, whereas C3aR is at best marginally expressed in The BM and VATare considered reservoirs of eosinophils (51, 52). BM neutrophils at the mRNA level. We found that BM eosinophils do not express tdTomato-C3aR A recent study showed that C3aR in neutrophils is upregulated (Fig. 4A). In contrast, eosinophils from blood, lung, LP, and under proinflammatory conditions (46). The C3a/C3aR plays an VAT showed a clear tdTomato signal (Fig. 4B–E). The tdTomato- important role in the effector phase of experimental allergic C3aR expression was strongest in VAT and lung tissue eosino- asthma (14, 47, 48). Thus, we analyzed dTomato-C3aR expression phils. PE MCs did not express a tdTomato-C3aR signal (Fig. 4F). in airway and lung neutrophils in HDM-driven allergic asthma. To correlate the tdTomato-C3aR with C3aR surface expression, Seventy-two hours after the final HDM challenge, we observed a we stained all tdTomato+ eosinophils with the C3aR Ab. Surpris- marked and significant increase in neutrophil numbers in the air- ingly, we found no surface expression of C3aR on almost all eo- ways of HDM-treated mice as compared with PBS controls sinophil populations (Fig. 4B, 4C, 4E), except eosinophils from the (Fig. 2B). Such neutrophils showed a slight but significant in- LP of the small intestine (Fig. 4D). However, after permeabilization, crease in the tdTomato-C3aR signal when compared with WT all tdTomato+ eosinophil populations and their WT counterparts mice. However, we found no C3aR surface expression in WT or stained C3aR+, whereas eosinophils from C3ar12/2 mice stained tdTomato-C3aR neutrophils (Fig. 2C). Similarly, lung neutro- negative (Fig. 4B, 4C, 4E). Thus, lung and VAT eosinophils as well phils showed an increase in the tdTomato-C3aR signal, but no as circulating eosinophils express the C3aR intracellularly, whereas C3aR surface expression in WT or tdTomato-C3aR neutrophils LP eosinophils express the C3aR on the cell surface. (Fig. 2D). Thus, airway and lung tissue neutrophils upregulate C3aR expression during the effector phase of HDM-driven al- Mucosa-associated but not lymphoid-associated DCs express lergic asthma. tdTomato-C3aR

2 + During the past years, several DC subsets in lymphoid and non- Siglec-F but not Siglec-F macrophages express lymphoid compartments have been defined (53). Currently, three tdTomato-C3aR distinct DC subsets can be distinguished comprising conventional F4/80 is considered a key lineage marker for macrophages (49). DCs (cDCs), inflammatory monocyte-derived DCs (moDCs), and Alveolar and tissue-associated alveolar macrophages (AMs) are plasmacytoid DCs (pDCs). In mice, cDCs are further subdivided characterized by the expression of Siglec-F (50). Surprisingly, into CD11b+ and CD8-like subsets. Following this differentiation airway and tissue-associated AMs did not express the tdTomato scheme, CD11b+ cDCs comprise splenic CD11b+CD82 DCs and signal (Supplemental Fig. 2A, 2B). Furthermore, we found no the pulmonary CD11b+ cDCs, whereas the CD8a-like cDCs subset tdTomato signal in Siglec-F+CD11c+ AMs by confocal micros- is composed of splenic CD8a+CD11b2 and pulmonary CD103+ copy (Supplemental Fig. 2C). In line with this finding, we found CD11b2 cDCs (54). At steady-state, lungs harbor a minor popula- no C3ar1 mRNA expression in AMs as determined by RT-PCR tion of moDCs, which increases under inflammatory conditions (55). (Supplemental Fig. 2D). In contrast, F4/80+Siglec-F2 macrophages Similarly, in the LP of the small intestine, CD11b+ and CD103+ differentiated with M-CSF (Fig. 3A) or GM-CSF from BM cells cDCs subpopulations have been described (56). In contrast, DCs in (Fig. 3B) or PE macrophages (Fig. 3C) as well as macrophages the VAT are poorly defined. We found an F4/802Siglec-F2MHCII+ from the LP of the small intestine (Fig. 3D) or from VAT (Fig. 3E) CD11c+CD11b+ population in the VAT that we consider CD11b+ showed a strong tdTomato signal. Similarly, brain macrophages cDCs but no CD8-like cDC equivalent (data not shown). Finally, we (Fig. 3F) were homogeneously positive for tdTomato-C3aR. In observed a mixed BMDC/BMM population, as recently described contrast, M-CSF–derived BMMs (Fig. 3A), VAT macrophages (Fig. by Helft et al. (41), with 20–30% CD11c+CD11b+CD1152MHCII+ 3E), and microglia cells can be divided into C3aR+ and C3aR2 BMDCs and 70–80% CD11c+CD11b+CD115+ BMMs, when BM subpopulations. cells were cultured in GM-CSF–supplemented medium. + 2 In the lung, only CD11b cDCs and moDCs showed a tdTomato Organ-specific C3aR expression in Siglec-F macrophages signal, whereas CD103+ cDCs and pDCs did not (Fig. 5A, 5E). In To determine whether tdTomato-C3aR expression correlates with contrast, both splenic DC subsets stained negative for the tdTomato C3aR protein expression, WT and tdTomato+ macrophages were signal (Supplemental Fig. 3A). Furthermore, we detected a strong stained with C3aR-specific mAb 14D4. The C3aR specificity was tdTomato-C3aR signal in GM-CSF–derived BMDCs (Fig. 5B). In verified by flow cytometry using C3aR-deficient cells as control the LP of the small intestine, CD11b+ and CD103+ cDCs both and by immunofluorescence microscopy using PE macrophages expressed tdTomato-C3aR (Fig. 5C), although the expression level and GM-CSF–differentiated BMMs/BMDCs from WT and was somewhat higher in the CD11b+ cDC population. The VAT DC C3ar12/2 mice (Supplemental Fig. 2E). The tdTomato-C3aR ex- subset showed no tdTomato signal (Fig. 5D). pression matched the C3aR surface expression using mAb 14D4 In the next step, we assessed tdTomato-C3aR expression in in some but not all macrophage populations. PE macrophages different tissue pDCs and Flt3L-induced pDCs. The pDCs are and M-CSF–derived BMMs showed a strong C3aR surface ex- strong producers of IFN-a upon viral infection (57) and have been pression (Fig. 3A, 3C). Furthermore, GM-CSF BMMs and brain identified in the spleen (58), lung (59), LP, Peyer’s patches (60), microglia expressed C3aR at their surface, although at lower and in the adipose tissue (61). Additionally, 10–20% pDCs differ- levels (Fig. 3B, 3F). In contrast, brain, LP, and VAT macro- entiate from BM cells in response to Flt3L stimulation (62). Using phages stained C3aR2 (Fig. 3D–F), suggesting intracellular lo- CD11b, CD11c, B220, mPDCA-1, and Siglec-H as markers, we calization. Indeed, we found C3aR expression in permeabilized identified pDCs in all tissues outlined above using the gating VAT macrophages (Fig. 3E). Unfortunately, high background strategies provided in Supplemental Fig. 3C–H. Among the different 694 C3aR EXPRESSION IN A tdTomato-C3aR KNOCK-IN MOUSE

FIGURE 2. Low level of tdTomato-C3aR expression in airway and tissue neutrophils during the effector phase of HDM-driven allergic asthma. Flow cytometric assessment of tdTomato-C3aR expression in different neutrophil populations is shown. The gray histograms show the WT signal; the black lines correspond to the signal in the tdTomato-C3ar1fl/fl mice. In case of C3aR surface expression, the dashed lines depict the staining of cells from C3ar12/2 mice. (A) Flow cytometric analysis of the tdTomato signal in Ly6G+ neutrophils from BM, blood, and lung. (B) Strong influx of neutrophils in the airways during the allergic effector phase. BAL fluid cells were first gated as Siglec-F2CD11c2 cells (left histogram). Within the population, neutrophils were identified as Ly6G+ cells (right histogram). The graph on the right shows the number of airway neutrophils in the indicated groups. Values shown are the mean 6 SEM; n = 4 PBS groups and n = 6 HDM groups. Statistical differences between groups were assessed by a Student t test. (C) tdTomato-C3aR (left) and C3aR surface (right) expression in BAL neutrophils. Values shown are the mean 6 SEM; n = 3 for tdTomato-C3aR expression in WT and tdTomato- C3ar1fl/fl mice; n = 4–6 for C3aR surface expression in WT (open symbols) and tdTomato-C3ar1fl/fl (closed symbols) mice treated with PBS (circles) or HDM (squares). Statistical differences between groups were assessed by Student t test. (D) tdTomato-C3aR and C3aR surface expression in lung tissue neutrophils. Shown is the gating strategy to identify Ly6G+ neutrophils in lung tissue (two histograms upper row), tdTomato-C3aR expression (lower left histogram and graph), and surface C3aR expression (lower right histogrgam and graph). Values shown are the mean 6 SEM; n = 3 for tdTomato expression in WT and tdTomato-C3ar1fl/fl mice; n = 4–6 for C3aR surface expression in WT (open symbols) and tdTomato-C3ar1fl/fl (closed symbols) mice treated with PBS (circles) or HDM (squares). Statistical differences between groups were assessed by Student t test. *p , 0.05, **p , 0.01. The Journal of Immunology 695

FIGURE 3. Siglec-F2F4/80+ macrophages express dTomato-C3aR and C3aR. Flow cytometric assessment of tdTomato-C3aR expression in different macrophage populations. The gray histograms show the WT signal; the black lines correspond to the signal in the tdTomato-C3ar1fl/fl mice. In case of C3aR surface expression, the dashed lines depict the staining of cells from C3ar12/2 mice. (A–F) tdTomato-C3aR and C3aR expression in (A) M-CSF–derived F4/80+CD11b+ BM macrophages (M-CSF BMM); (B) GM-CSF–derived BM macrophage population (GM-CSF BMM) that was identified as CD115+ MHCIIlo cells within the CD11c+CD11b+ cell population; (C) F4/80+CD11b+ PE macrophages; (D) Lineage2CD11c+CD11b+MHCII+F4/80+ macrophages from the LP of the small intestine; (E) Siglec-F2F4/80+CD11b+ macrophages from VAT; and (F) CD45hiF4/80+CD11b+ macrophages and CD45int microglia. tissue-residing pDCs tested, only a small fraction of the LP pDCs CD11b+ intracellular panel). By confocal microscopy, the tdTomato stained positive for tdTomato-C3aR (Fig. 5E). signal colocalized with some but not all CD11b+CD11c+ cells (Supplemental Fig. 3B), confirming that most tdTomato-C3aR+ Tissue-specific C3aR expression in mucosa-associated cDCs cells in the airways were resident moDCs. In the small intestine, and pDCs LP CD11b+ cDCs showed a clear surface expression of C3aR, Counterstaining of pulmonary tdTomato+ DCs with mAb 14D4 whereas C3aR expression in CD103+ cDCs was very weak (Fig. showed that moDCs but not CD11b+ cDCs (Fig. 5A) express C3aR 5C). Finally, we found weak C3aR surface expression in GM- at their cell surface. However, CD11b+ cDCs express C3aR intra- CSF–derived BMDCs (Fig. 5B). Similarly, we counterstained cellularly in low amounts as shown in permeabilized cells (Fig. 5A, tdTomato-C3aR+ pDCs and found that LP pDCs express C3aR at 696 C3aR EXPRESSION IN A tdTomato-C3aR KNOCK-IN MOUSE

FIGURE 4. Circulating and most tissue-re- siding eosinophils express tdTomato-C3aR in- tracellularly. Flow cytometric determination of tdTomato-C3aR expression in eosinophils is shown. The gray histograms show the WT signal; the black lines correspond to the signal in the tdTomato-C3ar1fl/fl mice. In case of C3aR surface or intracellular expression, the dashed lines depict the staining of cells from C3ar12/2 mice. (A–F) tdTomato-C3aR and/or C3aR ex- pression in Siglec-F+ (A) BM, (B) blood, (C) lung, (D) LP lineage2CD11c+CD11b+MHCIIlo F4/80lo,(E) VAT F4/80int eosinophils, and (F) FcεRI+ST2+ PE MCs.

the cell surface (Fig. 5E). From all DC populations tested, moDCs including the spleen, lymph nodes and Peyer’s patches, as well as were the DC population with the strongest C3aR surface expres- the lung. Additionally, we assessed C3aR expression in B-1 cell sion. In summary, we demonstrate that small intestinal LP cDCs subsets residing in the PE cavity (64). We found no tdTomato- and pDCs, lung moDCs, and GM-CSF–derived BMDCs express C3aR+ B or T cells in the circulation or any of the tissues under C3aR. Furthermore, pulmonary CD11b+ cDCs show low intracel- steady-state conditions (Fig. 6A–E, 6G–I). In line with this find- lular C3aR expression. In contrast, neither CD11b+ splenic DCs ing, we observed no C3ar1 mRNA expression in splenic T and nor pulmonary and splenic CD8-like cDCs express C3aR. B cells (Supplemental Fig. 4). Next, we stimulated naive splenic T cells using anti-CD3 and anti-CD28 Abs. We found a strong A subset of type 3 innate lymphoid cells but not B and T cells proliferative response of the T cells (data not shown). However, express tdTomato-C3aR this treatment did not result in the induction of tdTomato-C3aR The expression of the anaphylatoxin receptors by murine lymphoid expression in the T cells (Fig. 6F). cells is controversial (29–31, 42, 63). In this study, we used the In the lung and intestine, innate lymphoid cells (ILCs) play tdTomato-C3aR reporter mouse to monitor C3aR expression in B important roles in the regulation of tolerance (65). Three different and T cells in the circulation, different secondary lymphoid organs ILC subsets have been described: group 1 includes ILC1 and NK The Journal of Immunology 697

FIGURE 5. Mucosal surface-associated DCs express tdTomato-C3aR and C3aR. Flow cytometric assessment of tdTomato-C3aR and C3aR surface or intracellular expression in different DC subpopulations. The gray histograms show the WT signal; the black lines correspond to the signal in the tdTomato- C3ar1fl/fl mice. In case of C3aR surface or intracellular expression, the dashed lines depict the staining of cells from C3ar12/2 mice. (A) tdTomato-C3aR and C3aR expression in different pulmonary DC subsets. After excluding Siglec-F+ eosinophils and AMs as well as lineage+ cells (CD19, CD49b, CD3e, Ly6G), DCs were identified as CD11c+MHCII+ cells. In the DC population, CD103+ cDCs and CD11b+ DCs could be distinguished. The latter population was further subdivided into CD11b+ cDCs and moDCs. (B) tdTomato-C3aR and C3aR expression in GM-CSF–derived BMDCs that were identified as CD1152MHCIIhi cells within the CD11c+CD11b+ population. (C) tdTomato-C3aR and C3aR expression in CD11c+CD11b2MHCII+CD103+ and CD11c+ CD11b+CD103loF4/802 DCs of the LP. (D) tdTomato-C3aR expression in F4/802Siglec-F2CD11c+MHCII+ DCs from VAT. (E) tdTomato-C3aR ex- pression in different pDC populations. In LP pDCs, C3aR surface staining was also performed. The gating strategy is depicted in Supplemental Fig. 3C–H. cells, group 2 consists of ILC2, and group 3 consists of ILC3 (65). intestine (66). Splenic NK cells did not stain positive for tdTomato- NK cells and ILC2 reside in the lung. NK cells are also found in C3aR (Fig. 7A). In the lung, we observed low numbers of NK the spleen. ILC2 are further recruited upon activation by alarmins cells and ILC2 but neither ILC1 nor ILC3 (Fig. 7B, 7C), both of (65). ILC3 are not present in the lung but, together with NK and which stained negative for tdTomato-C3aR. In contrast to the ILC2,canbefoundinhighnumbersintheLPofthesmall lung, LP of the small intestine contained some tdTomato-C3aR+ 698 C3aR EXPRESSION IN A tdTomato-C3aR KNOCK-IN MOUSE

FIGURE 6. Circulating and tissue-resident B and T lymphocytes do not express tdTomato-C3aR. Flow cytometric assessment of tdTomato-C3aR ex- pression in different B and T lymphocyte populations is shown. The gray histograms show the WT signal; the black lines correspond to the signal in the tdTomato-C3ar1fl/fl mice. tdTomato-C3aR expression in B (A–D) and T lymphocytes (E–H) from the circulation and different tissues is shown. tdTomato- C3aR expression in (A) splenic CD19+B220+ B lymphocytes, (B) circulating CD19+ B cells, and (C) PE B1 lymphocytes is shown. After exclusion of CD11bhi macrophages, B-1 lymphocytes were identified as CD19+IgM+CD43+ cells. They were further divided into CD5+ B-1a and CD52 B-1b lym- phocytes. (D) tdTomato-C3aR expression in CD19+B220+ B lymphocytes from mediastinal lymph nodes. (E and F) tdTomato-C3aR expression in (E) naive splenic CD49b2CD3+ T lymphocytes or in (F) T lymphocytes stimulated in vitro for 5 d in the presence of CD3- and CD28-specific Abs. (G–I) tdTomato- C3aR expression in CD3+CD4+ T lymphocytes from (G) blood, (H) mediastinal lymph nodes, and (I) lung tissue. mLN, mediastinal lymph node.

ILCs. Whereas NK, ILC1 (Fig. 7D), and ILC2 (Fig. 7E) were surface staining of ILC3 using the C3aR-specific mAb 14D4 was tdTomato-C3aR2, a subset of lineage2CD90.2+CD252CD127+ negative, suggesting that C3aR is either expressed intracellularly ILC3 cells showed a positive tdTomato signal (Fig. 7E). C3aR or that the sensitivity of flow cytometry is too low to detect its The Journal of Immunology 699 expression (Fig. 7E). In summary, our results show that most ILCs intracellular calcium follows the same pattern in WT and tdTomato- do not express C3aR at steady-state. Interestingly, a subgroup of C3ar1fl/fl macrophages, suggesting that the C3aR in tdTomato- LP ILC3 expresses low numbers of C3aR. C3ar1fl/fl mice is functional.

A small subset of intestinal epithelial cells, but not lung Discussion epithelial cells, expresses tdTomato-C3aR Although the mouse C3aR was cloned 20 y ago (11), a detailed The expression of C3aR was reported in steady-state lung epi- understanding of C3aR expression in tissue-residing immune and thelium (13). To explore the expression of the tdTomato reporter in stromal cells is still lacking (9). In this study, we generated a the different lung epithelial populations, we examined them by two- knock-in reporter mouse strain, designed to express a fluorescent photon and confocal microscopy. Examination of 300-mm-thick tdTomato complex and C3aR. To avoid interference with C3aR sections of WT and tdTomato-C3aRfl/fl lungs by two-photon mi- signal transduction, we added the tdTomato sequence to the 59 end croscopy revealed several tdTomato-C3aR+ cells in the bronchial of C3ar1, because structural data have shown that the N-terminal compartment of the airways but only a vey small number of part of the C3aR does not bind C3a (6, 70). In contrast, the tdTomato-C3aR+ cells in the alveolar compartment (Fig. 8A). C-terminal part of the C3aR harbors important phosphorylation Confocal microscopy showed that tdTomato-C3aR+ cells in the sites, conserved in human and mice that drive receptor internali- airways were not epithelial nonciliated bronchiolar secretory cells zation and signal transduction (7). Furthermore, N-terminal ex- (Clara cells), because the tdTomato signal did not colocalize with tension of C5aR1 with GFP did not affect C5aR1 expression and the CC10 molecule (67) (Fig. 8B). Consistent with the very low function (42) whereas C-terminal GFP fusion resulted in a block numbers of tdTomato-C3aR+ cells in the alveolar compartment of C5aR1 translocation from the cytoplasm to the cell surface (Fig.8A),CD452autofluorecence+MHCII+ cells, which corre- (63). In agreement with these observations, the C3aR protein from spond to the alveolar type II cells (68), stained negative for tdTomato-C3ar1fl/fl mice was correctly targeted to the cell surface tdTomato-C3aR (Fig. 8C). Finally, we found that a small group and fully functional as shown by equivalent C3aR internalization and within the epithelial cells from the small intestine expresses calcium mobilization in PE macrophages from tdTomato-C3ar1fl/fl tdTomato-C3aR (Fig. 8D). andWTmiceinresponsetoC3a. Previous studies showed differences in C3ar1 mRNA expression C3a induces similar C3aR internalization and intracellular fl/fl patterns between mice and humans. In the former, the mRNA was calcium mobilization in WT and tdTomato-C3ar1 mice detected mostly in the heart and the lung, and to a lesser extent in To assess whether the genomic construct of tdTomato with C3aR the kidney, liver, skeletal muscles, and adipose tissue, but not in alters the functionality of the C3aR, we compared functional re- the brain and the spleen (10, 11). In humans, C3AR1 mRNA was sponses of both receptors side by side. Stimulation of C3aR with found in large amounts in lung, spleen, small intestine, peripheral C3a results in rapid internalization (7, 69). As we found strong blood leukocytes, s.c. adipose tissue, and at low levels in the C3aR surface expression in PE macrophages, we used this cell kidney and heart but not in the brain (5, 6, 71). Our data obtained population to determine C3a-driven C3aR internalization. PE with the tdTomato-C3aR knock-in mouse confirmed the presence macrophages were identified in PE lavage as F4/80+C3aR+ cells. of C3aR expression in the lung and VAT. Surprisingly, in contrast WT and tdTomato-C3ar1fl/fl macrophages express similar levels of to previous mRNA studies, we observed a strong tdTomato signal C3aR at their surface (Fig. 9A). When we incubated WT and in the brain. Until now, C3ar1 mRNA expression had been evaluated tdTomato-C3ar1fl/fl macrophages with increasing concentrations of solely at the level of the whole mouse brain (10, 11). Differences C3a, we observed a rapid and similar dose-dependent decrease of in the isolation procedure may account for the different expression C3aR surface expression in WT and tdTomato-C3ar1fl/fl macro- pattern observed in these studies as compared with results ob- phages. The maximum of C3aR internalization was reached 3 min tained with the tdTomato-C3aR reporter mouse. However, in hu- after C3a stimulation using a concentration of 10 nM C3a mans, the low C3aR expression in whole brain neglects high C3AR1 (Fig. 9B), although most of the fluorescence signal disappeared mRNA expression in defined regions and underrepresented cell within 1 min, suggesting a rapid internalization of the C3aR upon populations of the brain (5). Furthermore, we found tdTomato- C3a stimulation. Between 1 and 3 min after C3a administration, C3aR expression in the LP of the small intestine, whereas the the fluorescence signal further declined although at a much lower number of tdTomato-C3aR+ cells in the spleen was at best minor. pace. Eventually, the normalized C3aR MFI decreased to 30% of Also, the number of tdTomato-C3aR+ cells in BAL, blood, and BM unstimulated controls (Fig. 9C). was very low. The cell-specific determination of C3aR expression 2+ Furthermore, we determined C3a-induced mobilization of [Ca ]i revealed that cells of the myeloid lineage such as macrophages, in thioglycollate-elicited PE macrophages from WT and tdTomato- eosinophils, and DCs and in much lower frequency ILC3 from the C3ar1fl/fl mice. Stimulation of F4/80+ macrophages with C3a small intestine but not other ILCs, B or T lymphocytes express this 2+ resulted in a transient increase in [Ca ]i in cells from WT and anaphylatoxin receptor. tdTomato-C3ar1fl/fl mice (Fig. 9D). The amplitude of the response, In agreement with our observations that only a few tdTomato- measured as the DMFI, was similar in WT and tdTomato-C3ar1fl/fl C3aR+ cells were present in the BM and in strong contrast to macrophages. In line with the flow cytometric analysis, micro- human data (5, 16, 69, 72), we did not observe tdTomato-C3aR+ 2+ scopic assessment of the C3a-mediated change in [Ca ]i con- neutrophils in that compartment. Furthermore, we observed at best firmed the rapid and transient influx of calcium into macrophages a very minor expression of C3ar1 mRNA in sorted BM neutro- from WT and tdTomato-C3ar1fl/fl macrophages. We found a peak phils from WT or tdTomato-C3ar1fl/fl mice. In agreement with this 2+ of [Ca ]i 6 s after C3a stimulation (Fig. 9E). In contrast, we finding, we found no surface or intracellular C3aR expression in 2+ observed no change in [Ca ]i in thioglycollate-elicited PE mac- WT BM neutrophils. In the literature, direct evidence for C3aR rophages from C3ar12/2C5ar12/2 mice upon C3a challenge (data expression in mouse neutrophils is scarce. In a study in which Gr- not shown), either by flow cytometry or by confocal microscopy. 1 was used to identify neutrophils (24), the authors described In conclusion, our findings demonstrate that the C3aR expression C3aR surface and intracellular C3aR expression using the D20 in tdTomato-C3ar1fl/fl and WT macrophages is similar. Further- Ab. Using this Ab, we found no C3aR expression in Ly6G+ more, C3a-mediated internalization of C3aR and mobilization of neutrophils, suggesting that C3aR expression in Gr-1+ cells 700 C3aR EXPRESSION IN A tdTomato-C3aR KNOCK-IN MOUSE

FIGURE 7. Expression of tdTomato-C3aR in ILC3 but not in ILC1, NK, or ILC2 from LP of the small intestine. Flow cytometric evaluation of tdTomato-C3aR expression in group 1–3 ILCs is shown. The gray histograms show the WT signal; the black lines correspond to the signal in the tdTomato- C3ar1fl/fl mice. In case of C3aR surface expression, the dashed lines depict the staining of cells from C3ar12/2 mice. tdTomato-C3aR expression in (A) splenic NK cells, (B) pulmonary NK cells, (C) pulmonary ILC2, (D) CD127+CD49b2 ILC1 and CD1272CD49b+ NK cells from the LP, and (E) CD90.2+ CD25+CD127+ ILC2 and CD90.2+CD252CD127+ ILC3 from the LP of the small intestine is shown. resulted from other cells that express Gr-1 such as monocytes (45, suggesting either intracellularlocalizationofC3aRoravery 73). Furthermore, our data suggest that the D20 Ab recognizes a low level of surface expression that is below the detection limit protein that is also present in the absence of C3aR expression. In of the assay. line with our findings, two other reports found no C3aR expression Our data show that the major cell population expressing C3aR in BM neutrophils (33, 46). Interestingly, C3aR was upregulated under steady-state conditions are macrophages. In agreement with in response to LPS in low- and high-density neutrophils in a small earlier studies (74, 75), we found that all PE macrophages from intestinal tumor model and promoted neutrophil extracellular trap WT and tdTomato-C3ar1fl/fl mice express C3aR at the cell surface. formation (46). In agreement with these observations, we found Our data also confirm C3aR expression in VAT macrophages (76), upregulation of tdTomato-C3aR expression in airway and tissue microglia (77, 78), and in BMMs (17, 24). Additionally, we are neutrophils during the effector phase of HDM-driven allergic reporting, to our knowledge for the first time, the expression of asthma. However, C3aR was not expressed at the cell surface, C3aR in macrophages of the brain and the LP of the small The Journal of Immunology 701

FIGURE 8. Minor tdTomato-C3aR expression in epithelial cells from the small intestine. (A) Assessment of tdTomato-C3aR expression in the lung and the airways of WT and tdTomato-C3ar1fl/fl mice by two-photon microscopy (original magnification 320). Shown is the fluorescence signal of tdTomato- C3aR (red) from autofluorescent connective tissue cells of the airways (green) and from the alveolar epithelial cells (turquoise). (B) Determination of the spatial localization of tdTomato-C3aR expression in lung tissue by confocal microscopy (original magnification 340). Lung sections from tdTomato- C3ar1fl/fl mice were stained with CC10-specific Ab to identify Clara cells. (C) tdTomato-C3aR expression in CD452autofluorescence+MHCII+ type II alveolar epithelial cells. The gray histogram shows the WT signal; the black line corresponds to the signal in cells from tdTomato-C3ar1fl/fl mice. (D) tdTomato-C3aR expression in CD452CD32autofluorescence+ epithelial cells from the LP of the small intestine. Shown is the signal obtained in cells from WT and tdTomato-C3ar1fl/fl mice. The panel on the right quantifies the tdTomato-C3aR signal in n = 4–5 mice from each group. *p , 0.05. intestine, although C3aR is not expressed at the cell surface. In- subsets, comprising AMs residing in the alveolar space and a terestingly, we observed an inverse correlation between the ex- sessile fraction of AMs, attached to the alveolar epithelium via pression of C3aR and Siglec-F in macrophages. Indeed, all F4/80+ connexin 43 (80). Our data show that both populations stain Siglec-F2 macrophages were tdTomato-C3aR+, whereas Siglec-F+ negative for tdTomato-C3aR, confirming a previous report show- macrophages stained negative. Siglec-F is a C-type lectin, whose ing that pulmonary leukocytes, but not AMs, express C3aR (34). function is poorly understood. Recently, the pulmonary glyco- In addition to macrophages, we found that eosinophils from protein Muc5b was identified as a ligand for Siglec-F that induces different organs express tdTomato-C3aR and C3aR. Earlier studies apoptosis of eosinophils (79). Of note, the C3a/C3aR axis has showed that C3a is expressed on human eosinophils (81), triggers been reported to drive the production of a similar mucin, Muc5a, reactive oxygen species and calcium flux, and is chemotactic for from Clara cells (12). The AM population can be divided into two human blood eosinophils (18–20, 82). Our data demonstrate that 702 C3aR EXPRESSION IN A tdTomato-C3aR KNOCK-IN MOUSE

FIGURE 9. C3a drives rapid C3aR internalization and mobilization of intracellular calcium in PE macrophages from WT and tdTomato-C3ar1fl/fl mice. (A) Determination of C3aR surface expression in F4/80+ PE macrophages from WT (gray histogram) and tdTomato-C3ar1fl/fl mice (black line) by flow cytometry. PE macrophages from C3ar12/2 mice (dashed line) served as controls. Data show representative histograms of the C3aR signal in unstimulated PE macrophages as compared with 1, 10, and 100 nM C3a stimulation for 3 min at 37˚C. (B) Comparison of C3aR surface expression in PE macrophages from WT and tdTomato-C3ar1fl/fl mice in response to stimulation with increasing concentrations of C3a (1, 10, 100 nM) for 3 min at 37˚C. Shown is the DMFI of C3aR staining, which is defined as the MFI obtained by C3aR staining of cells from WT or tdTomato-C3ar1fl/fl mice corrected by the C3aR staining obtained with PE macrophages from C3aR-deficient mice. Values shown are the mean 6 SEM; n = 6 per group. (C) Comparison of C3aR surface expression before as well as 1, 3, and 9 min after stimulation with 10 nM C3a at 37˚C. Shown is the DMFI of C3aR staining. Values shown are the mean 6 2+ + SEM; n = 5–6 per group. (D) C3a-mediated increase of [Ca ]i in F4/80 thioglycollate-elicited PE macrophages from WT mice (gray line), tdTomato- C3ar1fl/fl mice (black line), or C3ar12/2C5ar12/2 (dashed line) mice. Shown is the DMFI of the fluorescence peak. Values shown are the mean 6 SEM; n = 2+ 7 per group. Statistical differences between groups were assessed by a Student t test. (E) Micrcoscopic evaluation of the C3a-mediated change of [Ca ]i in thioglycollate-elicited PE macrophages from WT and tdTomato-C3ar1fl/fl mice. Adherent thioglycollate-elicited PE macrophages from both groups were loaded with Fluo4-AM and challenged with C3a (37 nM). The images show the fluorescence emission in the FITC (Fluo4 emission) and PE channels (tdTomato), as wells as from polarized light before and 6 s after C3a stimulation (340 objective). Scale bar, 10 mm. Data are representative of three independent experiments. *p , 0.05, **p , 0.01, ***p , 0.001. The Journal of Immunology 703 blood eosinophils express tdTomato-C3aR. Similarly, we dem- data provide no evidence for C3aR expression on naive alveolar or onstrate intracellular C3aR expression in pulmonary and VAT airway epithelial cells. Thus, C3aR expression in the lung is re- eosinophils. Only eosinophils from the LP of the small intestine stricted to myeloid cells. This does not exclude that C3aR might expressed C3aR on the cell surface. Eosinophils from the BM, be induced on epithelial cells under inflammatory conditions. the site of eosinophil development and differentiation (51), Similar to the lung, most epithelial cells in the intestine were stained C3aR2. BM eosinophils are immature and require activa- tdTomato-C3aR2. Only a minor fraction expressed the C3aR. tion by IL-5 to egress and home to tissues via the bloodstream (51). Finally, we assessed the expression of the C3aR in lymphoid Our observation that eosinophils start to express C3aR intracellu- cells, which is still controversial. In previous studies, the ex- larly in the blood, which is enhanced in the lung and the VAT and pression of C3aR was described in activated human T lymphocytes already translocates to the cell surface in LP eosinophils, suggests (27, 100), but not in B lymphocytes (27). However, more recent that C3aR may also play important roles in eosinophil activation. studies reported that C3aR is expressed intracellularly by naive Furthermore, the intestine (51) and the VAT (52) have been de- and in vitro–activated human T lymphocytes (30). Furthermore, scribed as retention sites for eosinophils, indicating that C3aR may several studies reported weak expression of both C3aR and play a role in their retention, at least in the LP. C5aR1 in naive and activated mouse T lymphocytes (31) and an Our data confirm a previous report (83) demonstrating that important role for the development of regulatory T cells (29, PE MCs do not express C3aR at steady-state, although func- 101). However, we and others have failed in detecting C5aR1 in tional studies suggest that C3a activates MCs from skin and the B, T, and NKT cells using a GFP-C5aR1 reporter mouse at small intestine (84, 85). In line with this notion, variable and steady-state or upon activation (42, 63). Our data obtained with complex C3aR expression has been shown in human MCs. the tdTomato-C3aR knock-in mouse demonstrate no C3aR ex- Human skin MCs (23, 86), CD34+-derived human MCs (87), pression in naive B and T lymphocytes. Also, stimulation of the human MC line HMC-1 (88), and LAD1 and 2 (89) have naive T lymphocytes with anti-CD3/CD28 mAbs did not induce been reported to express C3aR (87) whereas BM-derived human C3aR expression. In addition to B and T lymphocytes, we MCs do not (90). assessed C3aR expression in ILCs, which play important roles The third population largely expressing tdTomato-C3aR was in mucosal immunology (65). ILCs are divided into three groups DCs. In vitro–generated DC populations of human (17, 91, 92) and containing ILC1 and NK cells (group 1), ILC2 (group 2), and murine origin have been reported to express C3aR (26, 73). We ILC3 (group 3). We found no tdTomato-C3aR signal in group 1 found that GM-CSF–differentiated BM cells, which contained and2ILCs.Incontrast,weobservedthatasmallfractionof macrophages and DCs (41), were tdTomato-C3aR+ and expressed ILC3 in the LP of the small intestine expressed tdTomato-C3aR. C3aR at the cell surface. In tissue, we observed that some pul- Transcriptome analysis revealed that ILC3 are a heterogeneous monary and LP-resident DCs express tdTomato-C3aR and C3aR population of cells that produce several cytokines, including at the surface, that is, moDCs and CD11b+ cDCs in the lung as IL17-A/F and IL-22 in response to myeloid cell–derived IL-1b well as CD103+, CD11b+ cDCs, and pDCs in the LP of the small and IL-23 (102). intestine. At this point, the function of C3aR on mucosal cDCs Finally, we demonstrate that the C3aR in tdTomato-C3ar1fl/fl and pDCs is unclear. Importantly, LP CD103+ cDCs produce knock-in mice is fully functional. Using WT and the tdTomato- retinoic acid and TGF-b, both of which are important for the C3aR PE macrophages, we show that C3a triggers C3aR inter- generation of Foxp3+ regulatory T cells (93). Also, they drive the nalization. The concentration of C3a, needed to drive C3aR differentiation of IgA-producing plasma cells, whereas CD11b+ internalization, was similar to that reported for human cells (69), cDCs promote Th17 cell differentiation (94). In the lung, CD11b+ but the maximum internalization in mouse cells already occurred cDCs are critical drivers of Th2/Th17 maladaptive immune in response to 10 nM C3a as compared with 100 nM using human response in allergic asthma (50, 55). Previous studies have granulocytes. Similarly, we observed a more rapid internalization shown that C3aR signaling in BMDCs is important for Ag up- of C3aR compared with human cells (69), where most of the C3aR take and T cell activation (26), indicating that the C3a/C3aR signal was lost only 10 min after C3a stimulation. Additionally, axis in tissue DCs may have similar functions. Lymphoid tissue we observed a similar rapid and transient increase in intracellular DCs and most pDC populations stained negative for tdTomato- calcium concentration in thioglycollate-elicited PE macrophages C3aR. from WT and tdTomato-C3ar1fl/fl. Interestingly, some subsets of macrophages, DCs, and most of Collectively, our findings provide detailed and novel insights the tissue eosinophils expressed C3aR intracellularly. Recent into steady-state C3aR expression in myeloid, lymphoid, and studies already reported in human cells the intracellular activation pulmonary parenchymal cells. Our data clearly demonstrate a of the C3/C3a/C3aR signaling axis, either through the conversion heterogeneous C3aR expression pattern in macrophages, eosin- of C3 into C3a/C3b in vesicles where C3aR resides (30), or through ophils, and DCs, depending on tissue distribution and the cellular the activation of NLRP3 inflammasome in the cytosol leading to subsets within such tissues. Furthermore, our results show spatial the secretion of IL-1b by such cells (95). Importantly, IL-1b plays heterogeneity of C3aR expression, that is, intracellular and an important role in the regulation of the experimental allergic surface expression. Finally, we provide evidence that naive asthma phenotype through its impact on Th2/Th17 immune re- neutrophils, B and T lymphocytes, most ILCs, as well as pul- sponses (96). In line with this notion, human eosinophils secrete monary epithelial cells do not express C3aR. The tdTomato-C3aR IL-1b and drive the production of IL-17A by CD4+ T cells (97). will prove useful to track C3aR expression and cell-specifically The expression of C3aR in lung parenchymal cells is unclear. delete C3aR in experimental models of allergic asthma (14, 73), Using RNA hybridization of lung sections (13) or immunohisto- emphysema (103), ischemia/reperfusion injury (104), cancer chemical C3aR staining with polyclonal Abs, the expression of (46), inflammatory neurologic (105) and renal diseases (106), as C3aR was reported on bronchial (13, 98, 99), alveolar epithelial well as infections (84, 107) among others, where the C3a/C3aR cells and on smooth muscle cells (12, 13). In contrast, another axis has been demonstrated to play important roles. It will also study demonstrated C3aR expression solely on leukocytes using be instrumental to define the role of C3aR in VGF peptide TLQP- C3aR-specific mAb 14D4 for surface staining (34). In line with 21–mediated functions, as this peptide can also bind to C3aR, at this observation, our confocal microscopy and flow cytometric least in rodents (108). 704 C3aR EXPRESSION IN A tdTomato-C3aR KNOCK-IN MOUSE

Acknowledgments 23. Nilsson, G., M. Johnell, C. H. Hammer, H. L. Tiffany, K. Nilsson, D. D. Metcalfe, A. Siegbahn, and P. M. Murphy. 1996. C3a and C5a are We thank E. Strerath, G. Ko¨hl, D. Theil, L. Pohl, and C. Lehmann for chemotaxins for human mast cells and act through distinct receptors via excellent technical assistance. a pertussis toxin-sensitive signal transduction pathway. J. Immunol. 157: 1693–1698. 24. Chen, N. J., C. Mirtsos, D. Suh, Y. C. Lu, W. J. Lin, C. McKerlie, T. Lee, Disclosures H. Baribault, H. Tian, and W. C. Yeh. 2007. C5L2 is critical for the biological The authors have no financial conflicts of interest. activities of the anaphylatoxins C5a and C3a. Nature 446: 203–207. 25. Wu, M. C., F. H. Brennan, J. P. Lynch, S. Mantovani, S. Phipps, R. A. Wetsel, M. J. Ruitenberg, S. M. Taylor, and T. M. Woodruff. 2013. The receptor for complement component C3a mediates protection from intestinal ischemia- References reperfusion injuries by inhibiting neutrophil mobilization. Proc. Natl. Acad. 1. Klos, A., A. J. Tenner, K. O. Johswich, R. R. Ager, E. S. Reis, and J. Ko¨hl. Sci. USA 110: 9439–9444. 2009. The role of the anaphylatoxins in health and disease. Mol. Immunol. 46: 26. Li, K., K. J. Anderson, Q. Peng, A. Noble, B. Lu, A. P. Kelly, N. Wang, 2753–2766. S. H. Sacks, and W. Zhou. 2008. Cyclic AMP plays a critical role in C3a- 2. Kolev, M., G. Le Friec, and C. Kemper. 2014. Complement—tapping into new receptor-mediated regulation of dendritic cells in antigen uptake and T-cell sites and effector systems. Nat. Rev. Immunol. 14: 811–820. stimulation. Blood 112: 5084–5094. 3. Verschoor, A., C. M. Karsten, S. P. Broadley, Y. Laumonnier, and J. Ko¨hl. 2016. 27. Werfel, T., K. Kirchhoff, M. Wittmann, G. Begemann, A. Kapp, F. Heidenreich, Old dogs—new tricks: immunoregulatory properties of C3 and C5 cleavage O. Go¨tze, and J. Zwirner. 2000. Activated human T lymphocytes express a fragments. Immunol. Rev. 274: 112–126. functional C3a receptor. J. Immunol. 165: 6599–6605. 4. Coulthard, L. G., and T. M. Woodruff. 2015. Is the complement activation 28. Cravedi, P., J. Leventhal, P. Lakhani, S. C. Ward, M. J. Donovan, and product C3a a proinflammatory molecule? Re-evaluating the evidence and the P. S. Heeger. 2013. Immune cell-derived C3a and C5a costimulate human T cell myth. J. Immunol. 194: 3542–3548. alloimmunity. Am. J. Transplant. 13: 2530–2539. 5. Ames, R. S., Y. Li, H. M. Sarau, P. Nuthulaganti, J. J. Foley, C. Ellis, Z. Zeng, 29. Kwan, W. H., W. van der Touw, E. Paz-Artal, M. O. Li, and P. S. Heeger. 2013. K. Su, A. J. Jurewicz, R. P. Hertzberg, et al. 1996. Molecular cloning and Signaling through C5a receptor and C3a receptor diminishes function of murine characterization of the human anaphylatoxin C3a receptor. J. Biol. Chem. 271: natural regulatory T cells. J. Exp. Med. 210: 257–268. 20231–20234. 30. Liszewski, M. K., M. Kolev, G. Le Friec, M. Leung, P. G. Bertram, A. F. Fara, 6. Crass, T., U. Raffetseder, U. Martin, M. Grove, A. Klos, J. Ko¨hl, and M. Subias, M. C. Pickering, C. Drouet, S. Meri, et al. 2013. Intracellular W. Bautsch. 1996. Expression cloning of the human C3a anaphylatoxin re- complement activation sustains T cell homeostasis and mediates effector dif- ceptor (C3aR) from differentiated U-937 cells. Eur. J. Immunol. 26: 1944–1950. ferentiation. Immunity 39: 1143–1157. 7. Settmacher, B., C. Rheinheimer, H. Hamacher, R. S. Ames, A. Wise, 31. Strainic, M. G., J. Liu, D. Huang, F. An, P. N. Lalli, N. Muqim, V. S. Shapiro, L. Jenkinson, D. Bock, M. Schaefer, J. Ko¨hl, and A. Klos. 2003. Structure- G. R. Dubyak, P. S. Heeger, and M. E. Medof. 2008. Locally produced com- function studies of the C3a-receptor: C-terminal serine and threonine residues plement fragments C5a and C3a provide both costimulatory and survival sig- which influence receptor internalization and signaling. Eur. J. Immunol. 33: nals to naive CD4+ T cells. Immunity 28: 425–435. 920–927. 32. Braun, M. C., R. Y. Reins, T. B. Li, T. J. Hollmann, R. Dutta, W. A. Rick, 8. Settmacher, U., H. D. Volk, S. Jahn, K. Neuhaus, F. Kuhn, and R. von Baehr. B. B. Teng, and B. Ke. 2004. Renal expression of the C3a receptor and func- 1991. Characterization of human lymphocytes separated from fetal liver and tional responses of primary human proximal tubular epithelial cells. J. Immunol. spleen at different stages of ontogeny. Immunobiology 182: 256–265. 173: 4190–4196. 9. Klos, A., E. Wende, K. J. Wareham, and P. N. Monk. 2013. International union 33. Kiafard, Z., T. Tschernig, S. Schweyer, A. Bley, D. Neumann, and J. Zwirner. of basic and clinical pharmacology. [corrected]. LXXXVII. Complement pep- 2007. Use of monoclonal antibodies to assess expression of anaphylatoxin tide C5a, C4a, and C3a receptors. Pharmacol. Rev. 65: 500–543. receptors in tubular epithelial cells of human, murine and rat kidneys. Immu- 10. Hsu, M. H., J. A. Ember, M. Wang, E. R. Prossnitz, T. E. Hugli, and R. D. Ye. nobiology 212: 129–139. 1997. Cloning and functional characterization of the mouse C3a anaphylatoxin 34. Tschernig, T., Z. Kiafard, C. Dibbert, D. Neumann, and J. Zwirner. 2007. Use receptor gene. Immunogenetics 47: 64–72. of monoclonal antibodies to assess expression of anaphylatoxin receptors in rat 11. Tornetta, M. A., J. J. Foley, H. M. Sarau, and R. S. Ames. 1997. The mouse and murine models of lung inflammation. Exp. Toxicol. Pathol. 58: 419–425. anaphylatoxin C3a receptor: molecular cloning, genomic organization, and 35. Engelke, C., A. V. Wiese, I. Schmudde, F. Ender, H. A. Stro¨ver, T. Vollbrandt, functional expression. J. Immunol. 158: 5277–5282. P. Ko¨nig, Y. Laumonnier, and J. Ko¨hl. 2014. Distinct roles of the anaphyla- 12. Dillard, P., R. A. Wetsel, and S. M. Drouin. 2007. Complement C3a regulates toxins C3a and C5a in dendritic cell–mediated allergic asthma. J. Immunol. Muc5ac expression by airway Clara cells independently of Th2 responses. Am. 193: 5387–5401. J. Respir. Crit. Care Med. 175: 1250–1258. 36. Zhang, X., I. Schmudde, Y. Laumonnier, M. K. Pandey, J. R. Clark, P. Ko¨nig, 13. Drouin, S. M., J. Kildsgaard, J. Haviland, J. Zabner, H. P. Jia, P. B. McCray, Jr., N. P. Gerard, C. Gerard, M. Wills-Karp, and J. Ko¨hl. 2010. A critical role for B. F. Tack, and R. A. Wetsel. 2001. Expression of the complement anaphyla- C5L2 in the pathogenesis of experimental allergic asthma. J. Immunol. 185: toxin C3a and C5a receptors on bronchial epithelial and smooth muscle cells in 6741–6752. models of sepsis and asthma. J. Immunol. 166: 2025–2032. 37. de Felipe, P., G. A. Luke, L. E. Hughes, D. Gani, C. Halpin, and M. D. Ryan. 14. Drouin, S. M., D. B. Corry, T. J. Hollman, J. Kildsgaard, and R. A. Wetsel. 2006. E unum pluribus: multiple proteins from a self-processing polyprotein. 2002. Absence of the complement anaphylatoxin C3a receptor suppresses Th2 Trends Biotechnol. 24: 68–75. effector functions in a murine model of pulmonary allergy. J. Immunol. 169: 38. Kim, J. H., S. R. Lee, L. H. Li, H. J. Park, J. H. Park, K. Y. Lee, M. K. Kim, 5926–5933. B. A. Shin, and S. Y. Choi. 2011. High cleavage efficiency of a 2A peptide 15. Gerardy-Schahn, R., D. Ambrosius, D. Saunders, M. Casaretto, C. Mittler, derived from porcine teschovirus-1 in human cell lines, zebrafish and mice. G. Karwarth, S. Go¨rgen, and D. Bitter-Suermann. 1989. Characterization of PLoS One 6: e18556. C3a receptor-proteins on guinea pig platelets and human polymorphonuclear 39. Schmudde, I., H. A. Stro¨ver, T. Vollbrandt, P. Ko¨nig, C. M. Karsten, leukocytes. Eur. J. Immunol. 19: 1095–1102. Y. Laumonnier, and J. Ko¨hl. 2013. C5a receptor signalling in dendritic cells 16. Martin, U., D. Bock, L. Arseniev, M. A. Tornetta, R. S. Ames, W. Bautsch, controls the development of maladaptive Th2 and Th17 immunity in experi- J. Ko¨hl, A. Ganser, and A. Klos. 1997. The human C3a receptor is expressed on mental allergic asthma. Mucosal Immunol. 6: 807–825. neutrophils and monocytes, but not on B or T lymphocytes. J. Exp. Med. 186: 40. Weischenfeldt, J., and B. Porse. 2008. Bone marrow-derived macrophages 199–207. (BMM): isolation and applications. CSH Protoc. 2008: pdb.prot5080. 17. Asgari, E., G. Le Friec, H. Yamamoto, E. Perucha, S. S. Sacks, J. Ko¨hl, 41. Helft, J., J. Bo¨ttcher, P. Chakravarty, S. Zelenay, J. Huotari, B. U. Schraml, H. T. Cook, and C. Kemper. 2013. C3a modulates IL-1b secretion in human D. Goubau, and C. Reis e Sousa. 2015. GM-CSF mouse bone marrow cultures monocytes by regulating ATP efflux and subsequent NLRP3 inflammasome comprise a heterogeneous population of CD11c+MHCII+ macrophages and activation. Blood 122: 3473–3481. dendritic cells. Immunity 42: 1197–1211. 18. Daffern, P. J., P. H. Pfeifer, J. A. Ember, and T. E. Hugli. 1995. C3a is a 42. Karsten, C. M., Y. Laumonnier, B. Eurich, F. Ender, K. Bro¨ker, S. Roy, chemotaxin for human eosinophils but not for neutrophils. I. C3a stimulation of A. Czabanska, T. Vollbrandt, J. Figge, and J. Ko¨hl. 2015. Monitoring and cell- neutrophils is secondary to eosinophil activation. J. Exp. Med. 181: 2119–2127. specific deletion of C5aR1 using a novel floxed GFP-C5aR1 reporter knock-in 19. Elsner, J., M. Oppermann, W. Czech, G. Dobos, E. Scho¨pf, J. Norgauer, and mouse. J. Immunol. 194: 1841–1855. A. Kapp. 1994. C3a activates reactive oxygen radical species production and in- 43. Karsten, C. M., Y. Laumonnier, and J. Ko¨hl. 2014. Functional analysis of C5a tracellular calcium transients in human eosinophils. Eur. J. Immunol. 24: 518–522. effector responses in vitro and in vivo. Methods Mol. Biol. 1100: 291–304. 20. Bischoff, S. C., A. L. de Weck, and C. A. Dahinden. 1990. Interleukin 3 and 44. Glovsky, M. M., T. E. Hugli, T. Ishizaka, L. M. Lichtenstein, and granulocyte/macrophage-colony-stimulating factor render human basophils B. W. Erickson. 1979. Anaphylatoxin-induced histamine release with human responsive to low concentrations of complement component C3a. Proc. Natl. leukocytes: studies of C3a leukocyte binding and histamine release. J. Clin. Acad. Sci. USA 87: 6813–6817. Invest. 64: 804–811. 21. Kretzschmar, T., A. Jeromin, C. Gietz, W. Bautsch, A. Klos, J. Ko¨hl, 45. Daley, J. M., A. A. Thomay, M. D. Connolly, J. S. Reichner, and J. E. Albina. G. Rechkemmer, and D. Bitter-Suermann. 1993. Chronic myelogenous 2008. Use of Ly6G-specific monoclonal antibody to deplete neutrophils in leukemia-derived basophilic granulocytes express a functional active receptor mice. J. Leukoc. Biol. 83: 64–70. for the anaphylatoxin C3a. Eur. J. Immunol. 23: 558–561. 46. Guglietta, S., A. Chiavelli, E. Zagato, C. Krieg, S. Gandini, P. S. Ravenda, 22. Legler, D. F., M. Loetscher, S. A. Jones, C. A. Dahinden, M. Arock, and B. Bazolli, B. Lu, G. Penna, and M. Rescigno. 2016. Coagulation induced by B. Moser. 1996. Expression of high- and low-affinity receptors for C3a on the C3aR-dependent NETosis drives protumorigenic neutrophils during small in- human mast cell line, HMC-1. Eur. J. Immunol. 26: 753–758. testinal tumorigenesis. Nat. Commun. 7: 11037. The Journal of Immunology 705

47. Bautsch, W., H. G. Hoymann, Q. Zhang, I. Meier-Wiedenbach, U. Raschke, 73. Zhang, X., I. P. Lewkowich, G. Ko¨hl, J. R. Clark, M. Wills-Karp, and J. Ko¨hl. R. S. Ames, B. Sohns, N. Flemme, A. Meyer zu Vilsendorf, M. Grove, et al. 2009. A protective role for C5a in the development of allergic asthma associ- 2000. Cutting edge: guinea pigs with a natural C3a-receptor defect exhibit ated with altered levels of B7-H1 and B7-DC on plasmacytoid dendritic cells. decreased bronchoconstriction in allergic airway disease: evidence for an in- J. Immunol. 182: 5123–5130. volvement of the C3a anaphylatoxin in the pathogenesis of asthma. J. Immunol. 74. Hollmann, T. J., D. L. Haviland, J. Kildsgaard, K. Watts, and R. A. Wetsel. 1998. 165: 5401–5405. Cloning, expression, sequence determination, and chromosome localization of the 48. Humbles, A. A., B. Lu, C. A. Nilsson, C. Lilly, E. Israel, Y. Fujiwara, mouse complement C3a anaphylatoxin receptor gene. Mol. Immunol. 35: 137–148. N. P. Gerard, and C. Gerard. 2000. A role for the C3a anaphylatoxin receptor in 75. Kildsgaard, J., T. J. Hollmann, K. W. Matthews, K. Bian, F. Murad, and the effector phase of asthma. Nature 406: 998–1001. R. A. Wetsel. 2000. Cutting edge: targeted disruption of the C3a receptor gene 49. Morris, L., C. F. Graham, and S. Gordon. 1991. Macrophages in haemopoietic demonstrates a novel protective anti-inflammatory role for C3a in endotoxin- and other tissues of the developing mouse detected by the monoclonal antibody shock. J. Immunol. 165: 5406–5409. F4/80. Development 112: 517–526. 76. Mamane, Y., C. Chung Chan, G. Lavallee, N. Morin, L. J. Xu, J. Huang, 50. Plantinga, M., M. Guilliams, M. Vanheerswynghels, K. Deswarte, F. Branco- R. Gordon, W. Thomas, J. Lamb, E. E. Schadt, et al. 2009. The C3a anaphy- Madeira, W. Toussaint, L. Vanhoutte, K. Neyt, N. Killeen, B. Malissen, et al. latoxin receptor is a key mediator of insulin resistance and functions by 2013. Conventional and monocyte-derived CD11b+ dendritic cells initiate and modulating adipose tissue macrophage infiltration and activation. Diabetes 58: maintain T helper 2 cell-mediated immunity to house dust mite allergen. Im- 2006–2017. munity 38: 322–335. 77. Davoust, N., J. Jones, P. F. Stahel, R. S. Ames, and S. R. Barnum. 1999. Re- 51. Rothenberg, M. E., and S. P. Hogan. 2006. The eosinophil. Annu. Rev. Immunol. ceptor for the C3a anaphylatoxin is expressed by neurons and glial cells. Glia 24: 147–174. 26: 201–211. 52. Wu, D., A. B. Molofsky, H. E. Liang, R. R. Ricardo-Gonzalez, H. A. Jouihan, 78. Mo¨ller, T., C. Nolte, R. Burger, A. Verkhratsky, and H. Kettenmann. 1997. 2+ J. K. Bando, A. Chawla, and R. M. Locksley. 2011. Eosinophils sustain adipose Mechanisms of C5a and C3a complement fragment-induced [Ca ]i signaling alternatively activated macrophages associated with glucose homeostasis. Sci- in mouse microglia. J. Neurosci. 17: 615–624. ence 332: 243–247. 79. Kiwamoto, T., T. Katoh, C. M. Evans, W. J. Janssen, M. E. Brummet, 53. Guilliams, M., F. Ginhoux, C. Jakubzick, S. H. Naik, N. Onai, B. U. Schraml, S. A. Hudson, Z. Zhu, M. Tiemeyer, and B. S. Bochner. 2015. Endogenous E. Segura, R. Tussiwand, and S. Yona. 2014. Dendritic cells, monocytes and airway mucins carry glycans that bind Siglec-F and induce eosinophil apo- macrophages: a unified nomenclature based on ontogeny. Nat. Rev. Immunol. ptosis. J. Allergy Clin. Immunol. 135: 1329–1340.e1321–1329. 14: 571–578. 80. Westphalen, K., G. A. Gusarova, M. N. Islam, M. Subramanian, T. S. Cohen, 54. Mayer, C. T., P. Ghorbani, A. Nandan, M. Dudek, C. Arnold-Schrauf, C. Hesse, A. S. Prince, and J. Bhattacharya. 2014. Sessile alveolar macrophages commu- L. Berod, P. Stuve,€ F. Puttur, M. Merad, and T. Sparwasser. 2014. Selective and nicate with alveolar epithelium to modulate immunity. Nature 506: 503–506. efficient generation of functional Batf3-dependent CD103+ dendritic cells from 81. Petering, H., J. Ko¨hl, A. Weyergraf, Y. Dulkys, D. Kimmig, R. Smolarski, mouse bone marrow. Blood 124: 3081–3091. A. Kapp, and J. Elsner. 2000. Characterization of synthetic C3a analog peptides 55. Hoffmann, F., F. Ender, I. Schmudde, I. P. Lewkowich, J. Ko¨hl, P. Ko¨nig, and on human eosinophils in comparison to the native complement component C3a. Y. Laumonnier. 2016. Origin, localization, and immunoregulatory properties of J. Immunol. 164: 3783–3789. pulmonary phagocytes in allergic asthma. Front. Immunol. 7: 107. 82. DiScipio, R. G., P. J. Daffern, M. A. Jagels, D. H. Broide, and P. Sriramarao. 56. Varol, C., A. Vallon-Eberhard, E. Elinav, T. Aychek, Y. Shapira, H. Luche, 1999. A comparison of C3a and C5a-mediated stable adhesion of rolling eo- H. J. Fehling, W. D. Hardt, G. Shakhar, and S. Jung. 2009. Intestinal lamina propria sinophils in postcapillary venules and transendothelial migration in vitro and dendritic cell subsets have different origin and functions. Immunity 31: 502–512. in vivo. J. Immunol. 162: 1127–1136. 57. Gilliet, M., W. Cao, and Y. J. Liu. 2008. Plasmacytoid dendritic cells: sensing 83. Soruri, A., J. Grigat, Z. Kiafard, and J. Zwirner. 2008. Mast cell activation is nucleic acids in viral infection and autoimmune diseases. Nat. Rev. Immunol. 8: characterized by upregulation of a functional anaphylatoxin C5a receptor. BMC 594–606. Immunol. 9: 29. 58. Nakano, H., M. Yanagita, and M. D. Gunn. 2001. CD11c+B220+Gr-1+ cells in 84. Li, E., E. A. Tako, and S. M. Singer. 2016. Complement activation by Giardia mouse lymph nodes and spleen display characteristics of plasmacytoid den- duodenalis parasites through the lectin pathway contributes to mast cell re- dritic cells. J. Exp. Med. 194: 1171–1178. sponses and parasite control. Infect. Immun. 84: 1092–1099. 59. Ko¨hl, J., R. Baelder, I. P. Lewkowich, M. K. Pandey, H. Hawlisch, L. Wang, 85. Scha¨fer, B., A. M. Piliponsky, T. Oka, C. H. Song, N. P. Gerard, C. Gerard, J. Best, N. S. Herman, A. A. Sproles, J. Zwirner, et al. 2006. A regulatory role M. Tsai, J. Kalesnikoff, and S. J. Galli. 2013. Mast cell anaphylatoxin receptor for the C5a anaphylatoxin in type 2 immunity in asthma. J. Clin. Invest. 116: expression can enhance IgE-dependent skin inflammation in mice. J. Allergy 783–796. Clin. Immunol. 131: 541–548.e541–549. 60. Goubier, A., B. Dubois, H. Gheit, G. Joubert, F. Villard-Truc, C. Asselin- 86. Hartmann, K., B. M. Henz, S. Kruger-Krasagakes,€ J. Ko¨hl, R. Burger, S. Guhl, Paturel, G. Trinchieri, and D. Kaiserlian. 2008. Plasmacytoid dendritic cells I. Haase, U. Lippert, and T. Zuberbier. 1997. C3a and C5a stimulate chemotaxis mediate oral tolerance. Immunity 29: 464–475. of human mast cells. Blood 89: 2863–2870. 61. Ghosh, A. R., R. Bhattacharya, S. Bhattacharya, T. Nargis, O. Rahaman, 87. Venkatesha, R. T., E. Berla Thangam, A. K. Zaidi, and H. Ali. 2005. Distinct P. Duttagupta, D. Raychaudhuri, C. S. Liu, S. Roy, P. Ghosh, et al. 2016. regulation of C3a-induced MCP-1/CCL2 and RANTES/CCL5 production in Adipose recruitment and activation of plasmacytoid dendritic cells fuel meta- human mast cells by extracellular signal regulated kinase and PI3 kinase. Mol. flammation. Diabetes 65: 3440–3452. Immunol. 42: 581–587. 62. Bjo¨rck, P. 2001. Isolation and characterization of plasmacytoid dendritic cells 88. Ahamed, J., R. T. Venkatesha, E. B. Thangam, and H. Ali. 2004. C3a enhances from Flt3 ligand and granulocyte-macrophage colony-stimulating factor-treated nerve growth factor-induced NFAT activation and chemokine production in a mice. Blood 98: 3520–3526. human mast cell line, HMC-1. J. Immunol. 172: 6961–6968. 63. Dunkelberger, J., L. Zhou, T. Miwa, and W. C. Song. 2012. C5aR expression in 89. Kirshenbaum, A. S., C. Akin, Y. Wu, M. Rottem, J. P. Goff, M. A. Beaven, a novel GFP reporter gene knockin mouse: implications for the mechanism of V. K. Rao, and D. D. Metcalfe. 2003. Characterization of novel stem cell factor action of C5aR signaling in T cell immunity. J. Immunol. 188: 4032–4042. responsive human mast cell lines LAD 1 and 2 established from a patient with 64. Kantor, A. B., A. M. Stall, S. Adams, L. A. Herzenberg, and L. A. Herzenberg. mast cell sarcoma/leukemia; activation following aggregation of FcεRI or 1992. Differential development of progenitor activity for three B-cell lineages. FcgRI. Leuk. Res. 27: 677–682. Proc. Natl. Acad. Sci. USA 89: 3320–3324. 90. Erdei, A., and I. Pecht. 1996. Complement peptides and mast cell triggering. 65. Walker, J. A., J. L. Barlow, and A. N. McKenzie. 2013. Innate lymphoid cells— Immunol. Lett. 54: 109–112. how did we miss them? Nat. Rev. Immunol. 13: 75–87. 91. Gutzmer, R., M. Lisewski, J. Zwirner, S. Mommert, C. Diesel, M. Wittmann, 66. Qiu, J., X. Guo, Z. M. Chen, L. He, G. F. Sonnenberg, D. Artis, Y. X. Fu, and A. Kapp, and T. Werfel. 2004. Human monocyte-derived dendritic cells are L. Zhou. 2013. Group 3 innate lymphoid cells inhibit T-cell-mediated intestinal chemoattracted to C3a after up-regulation of the C3a receptor with interferons. inflammation through aryl hydrocarbon receptor signaling and regulation of Immunology 111: 435–443. microflora. Immunity 39: 386–399. 92. Li, K., H. Fazekasova, N. Wang, Q. Peng, S. H. Sacks, G. Lombardi, and 67. Wang, H., Y. Liu, and Z. Liu. 2013. Clara cell 10-kD protein in inflammatory W. Zhou. 2012. Functional modulation of human monocytes derived DCs by upper airway diseases. Curr. Opin. Allergy Clin. Immunol. 13: 25–30. anaphylatoxins C3a and C5a. Immunobiology 217: 65–73. 68. Corbie`re, V., V. Dirix, S. Norrenberg, M. Cappello, M. Remmelink, and 93. Coombes, J. L., K. R. Siddiqui, C. V. Arancibia-Ca´rcamo, J. Hall, C. M. Sun, F. Mascart. 2011. Phenotypic characteristics of human type II alveolar epithelial Y. Belkaid, and F. Powrie. 2007. A functionally specialized population of cells suitable for antigen presentation to T lymphocytes. Respir. Res. 12: 15. mucosal CD103+ DCs induces Foxp3+ regulatory T cells via a TGF-b and 69. Settmacher, B., D. Bock, H. Saad, S. Ga¨rtner, C. Rheinheimer, J. Ko¨hl, retinoic acid-dependent mechanism. J. Exp. Med. 204: 1757–1764. W. Bautsch, and A. Klos. 1999. Modulation of C3a activity: internalization of 94. Gross, M., T. M. Salame, and S. Jung. 2015. Guardians of the gut—murine the human C3a receptor and its inhibition by C5a. J. Immunol. 162: 7409–7416. intestinal macrophages and dendritic cells. Front. Immunol. 6: 254. 70. Chao, T. H., J. A. Ember, M. Wang, Y. Bayon, T. E. Hugli, and R. D. Ye. 1999. 95. Laudisi, F., R. Spreafico, M. Evrard, T. R. Hughes, B. Mandriani, Role of the second extracellular loop of human C3a receptor in agonist binding M. Kandasamy, B. P. Morgan, B. Sivasankar, and A. Mortellaro. 2013. Cutting and receptor function. J. Biol. Chem. 274: 9721–9728. edge: the NLRP3 inflammasome links complement-mediated inflammation and 71. Gupta, A., R. Rezvani, M. Lapointe, P. Poursharifi, P. Marceau, S. Tiwari, IL-1b release. J. Immunol. 191: 1006–1010. A. Tchernof, and K. Cianflone. 2014. Downregulation of complement C3 and 96. Kobayashi, T., K. Iijima, J. L. Checkel, and H. Kita. 2013. IL-1 family cyto- C3aR expression in subcutaneous adipose tissue in obese women. PLoS One kines drive Th2 and Th17 cells to innocuous airborne antigens. Am. J. Respir. 9: e95478. Cell Mol. Biol. 49: 989–998. 72. Norgauer, J., G. Dobos, E. Kownatzki, C. Dahinden, R. Burger, R. Kupper, and 97. Esnault, S., E. A. Kelly, L. M. Nettenstrom, E. B. Cook, C. M. Seroogy, and P. Gierschik. 1993. Complement fragment C3a stimulates Ca2+ influx in neutro- N. N. Jarjour. 2012. Human eosinophils release IL-1b and increase expression phils via a pertussis-toxin-sensitive G protein. Eur. J. Biochem. 217: 289–294. of IL-17A in activated CD4+ T lymphocytes. Clin. Exp. Allergy 42: 1756–1764. 706 C3aR EXPRESSION IN A tdTomato-C3aR KNOCK-IN MOUSE

98. Melendi, G. A., S. J. Hoffman, R. A. Karron, P. M. Irusta, F. R. Laham, 104. Ducruet, A. F., B. G. Hassid, W. J. Mack, S. A. Sosunov, M. L. Otten, A. Humbles, B. Schofield, C. H. Pan, R. Rabold, B. Thumar, et al. 2007. C5 D. J. Fusco, Z. L. Hickman, G. H. Kim, R. J. Komotar, J. Mocco, and modulates airway hyperreactivity and pulmonary eosinophilia during enhanced E. S. Connolly. 2008. C3a receptor modulation of granulocyte infiltration after respiratory syncytial virus disease by decreasing C3a receptor expression. J. murine focal cerebral ischemia is reperfusion dependent. J. Cereb. Blood Flow Virol. 81: 991–999. Metab. 28: 1048–1058. 99. Thangam, E. B., R. T. Venkatesha, A. K. Zaidi, K. L. Jordan-Sciutto, 105. Lian, H., A. Litvinchuk, A. C. Chiang, N. Aithmitti, J. L. Jankowsky, and D. A. Goncharov, V. P. Krymskaya, Y. Amrani, R. A. Panettieri, Jr., and H. Ali. H. Zheng. 2016. Astrocyte-microglia cross talk through complement activation 2005. Airway smooth muscle cells enhance C3a-induced mast cell degranula- modulates amyloid pathology in mouse models of Alzheimer’s disease. J. tion following cell-cell contact. FASEB J. 19: 798–800. Neurosci. 36: 577–589. 100. Ghannam, A., J. L. Fauquert, C. Thomas, C. Kemper, and C. Drouet. 2014. 106. Wenderfer, S. E., H. Wang, B. Ke, R. A. Wetsel, and M. C. Braun. 2009. C3a Human complement C3 deficiency: Th1 induction requires T cell-derived receptor deficiency accelerates the onset of renal injury in the MRL/lpr mouse. complement C3a and CD46 activation. Mol. Immunol. 58: 98–107. Mol. Immunol. 46: 1397–1404. 101. Strainic, M. G., E. M. Shevach, F. An, F. Lin, and M. E. Medof. 2013. Absence of 107. Vasek, M. J., C. Garber, D. Dorsey, D. M. Durrant, B. Bollman, A. Soung, signaling into CD4+ cells via C3aR and C5aR enables autoinductive TGF-b1 J. Yu, C. Perez-Torres, A. Frouin, D. K. Wilton, et al. 2016. A complement- signaling and induction of Foxp3+ regulatory T cells. Nat. Immunol. 14: 162–171. microglial axis drives synapse loss during virus-induced memory impairment. 102. Melo-Gonzalez, F., and M. R. Hepworth. 2017. Functional and phenotypic Nature 534: 538–543. heterogeneity of group 3 innate lymphoid cells. Immunology 150: 265–275. 108. Hannedouche, S., V. Beck, J. Leighton-Davies, M. Beibel, G. Roma, 103. Yuan, X., M. Shan, R. You, M. V. Frazier, M. J. Hong, R. A. Wetsel, S. Drouin, E. J. Oakeley, V. Lannoy, J. Bernard, J. Hamon, S. Barbieri, et al. 2013. A. Seryshev, L. Z. Song, L. Cornwell, et al. 2015. Activation of C3a receptor is Identification of the C3a receptor (C3AR1) as the target of the VGF-derived required in cigarette smoke-mediated emphysema. Mucosal Immunol. 8: 874–885. peptide TLQP-21 in rodent cells. J. Biol. Chem. 288: 27434–27443.