Distinct Roles of the Anaphylatoxins C3a and C5a in Dendritic Cell −Mediated Allergic Asthma This information is current as of August 11, 2017.

Carsten Engelke, Anna V. Wiese, Inken Schmudde, Fanny Ender, Heike A. Ströver, Tillmann Vollbrandt, Peter König, Yves Laumonnier and Jörg Köhl

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http://www.jimmunol.org/content/suppl/2014/10/29/jimmunol.140008 0.DCSupplemental This article cites 56 articles, 17 of which you can access for free at: http://www.jimmunol.org/content/193/11/5387.full#ref-list-1 Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts

The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2014 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606.

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J Immunol 2014; 193:5387-5401; Prepublished online 29 October 2014; doi: 10.4049/jimmunol.1400080 http://www.jimmunol.org/content/193/11/5387

The Journal of Immunology

Distinct Roles of the Anaphylatoxins C3a and C5a in Dendritic Cell–Mediated Allergic Asthma Carsten Engelke,*,1 Anna V. Wiese,*,1 Inken Schmudde,*,1 Fanny Ender,* Heike A. Stro¨ver,* Tillmann Vollbrandt,† Peter Ko¨nig,‡ Yves Laumonnier,*,2 and Jo¨rg Ko¨hl*,x,2

A

llergic asthma is a chronic pulmonary disease driven by a maladaptive Th2 immune response toward aeroallergens. In addition to Th2 effector cells, other Th cells contribute to the development of allergic asthma, in particular Th17 and regulatory T (Treg) cells (1, 2). Among the different cell types of the innate immune system that may play a role in the development of allergic asthma, dendritic *Institute for Systemic Inflammation Research, University of L€ubeck and Airway Research Center North, member of the German Center for Lung Research, 23538 L€ubeck, Germany; †Cell Analysis Core, University of L€ubeck, 23538 L€ubeck, Germany; ‡Institute for Anatomy, University of L€ubeck and Airway Research Center North, member of the German Center for Lung Research, 23538 L€ubeck, Germany; and xDivision of Immunobiology, Cincinnati Children’s Hospital Medical Center, University of Cincinnati, Cincinnati, OH 45229 1

C.E., A.V.W., and I.S. contributed equally to this work.

2

Y.L. and J.K. shared supervision of this work.

Received for publication January 14, 2014. Accepted for publication September 29, 2014. This work was supported by the German Research Foundation (Deutsche Forschungsgemeinschaft) Collaborative Research Center/Transregio Projects A21 (to J.K.), Z5 (to P.K.), and KO 1245 4/1 (to J.K.) and by International Research Training Group 1911 Projects A1 (to Y.L. and J.K.) and A2 (to P.K.). Address correspondence and reprint requests to Dr. Yves Laumonnier and Prof. Jo¨ rg Ko¨ hl, Institute for Systemic Inflammation Research, University of L€ubeck, Ratzeburger Allee 160, 23538 L€ubeck, Germany. E-mail addresses: yves. [email protected] (Y.L.) and [email protected] (J.K.) The online version of this article contains supplemental material. Abbreviations used in this article: AHR, airway hyperresponsiveness; AT, anaphylatoxin; BAL, bronchoalveolar lavage; BM, bone marrow; BMDC, BM-derived DC; cDC, conventional DC; DC, dendritic cell; EC, epithelial cell; HDM, house dust mite; i.t., intratracheal; mLN, mediastinal lymph node; PAS, periodic acid–Schiff; pDC, plasmacytoid DC; Treg, regulatory T; WT, wild-type. Copyright Ó 2014 by The American Association of Immunologists, Inc. 0022-1767/14/$16.00 www.jimmunol.org/cgi/doi/10.4049/jimmunol.1400080

cells (DCs) are considered most important. DCs are professional APCs specialized in Ag/allergen uptake, processing, and presentation to naive CD4+ Th cells, promoting their differentiation into Th1, Th2, and Th17 effector cells and Treg cells. Under steady state conditions, different subtypes of DCs reside in the lung, lining the conducting airways. Resident DCs comprise plasmacytoid DCs (pDCs), CD11b+CD1032, and CD11b2CD103+ conventional DCs (cDCs) (3). CD11b+ and CD103+ cDCs sample the environment for Ags. In addition to cDCs, CCR2+ monocytederived DCs are recruited to the conducting airways by CCL2 or CCL7 chemokines produced by lung epithelial cells (ECs) in response to allergen stimulation (4), After allergen uptake, DCs of both origins undergo a switch from the Ag-sampling into an Agpresenting mode associated with the upregulation of adhesion, MHC-II, and costimulatory molecules, including CD40, CD80, and CD86 (5), and migrate to the mediastinal lymph nodes (mLN) (5, 6). The three signals provided by MHC-II/TCR, costimulatory molecule interaction, and cytokines produced by allergen-loaded DCs then pave the way for maladaptive Th2/Th17 skewing of naive CD4+ T cells (7, 8). Several studies, in which complement factors or receptors have been deleted, point toward an important role for the complement system as a critical regulator of asthma development through its functions on DCs (9). Complement is an ancient member of the innate immune system that becomes activated upon recognition of exogenous and endogenous danger motifs by soluble sensing molecules such as C1q, mannan-binding lectin, and different ficolin molecules (10, 11). Activation of the complement results in the proteolytic cleavage of C3 and C5 generating the anaphylatoxins (AT) C3a and C5a. In addition to the activation of the complement cascade, complex allergens such as house dust mite

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Conventional dendritic cells (cDC) are necessary and sufficient to drive mixed maladaptive Th2/Th17 immune responses toward aeroallergens in experimental allergy models. Previous studies suggest that the anaphylatoxin C3a promotes, whereas C5a protects from the development of maladaptive immunity during allergen sensitization. However, only limited evidence exists that such effects are directly mediated through anaphylatoxin-receptor signaling in cDCs. In this study, we assessed the impact of C3a and C5a on cDC-mediated induction pulmonary allergy by adoptively transferring house dust mite (HDM)–pulsed bone marrow–derived DCs (BMDC) from wild-type (WT) C3aR2/2, C5aR12/2, or C3aR2/2/C5aR12/2 into WT mice. Transfer of HDM-pulsed WT BMDCs promoted a strong asthmatic phenotype characterized by marked airway resistance, strong Th2 cytokine, and mucus production, as well as mixed eosinophilic and neurophilic airway inflammation. Surprisingly, C3aR2/2 cDCs induced a strong allergic phenotype, but no IL-17A production, whereas HDM-pulsed C5aR12/2 cDCs failed to drive pulmonary allergy. Transfer of C3aR2/2/C5aR12/2 cDCs resulted in a slightly reduced allergic phenotype associated with increased IFN-g production. Mechanistically, C3aR and C5aR1 signaling is required for IL-23 production from HDM-pulsed BMDCs in vitro. Furthermore, C3aR2/2 BMDCs produced less IL-1b. The mechanisms underlying the failure of C5aR12/2 BMDCs to induce experimental allergy include a reduced capability to migrate into the lung tissue and a decreased potency to direct pulmonary homing of effector T cells. Thus, we uncovered a crucial role for C5a, but only a minor role for C3a in BMDC-mediated pulmonary allergy, suggesting that BMDCs inappropriately reflect the impact of complement on lung cDC-mediated allergic asthma development. The Journal of Immunology, 2014, 193: 5387–5401.

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C3a AND C5a IN DENDRITIC CELL–MEDIATED ALLERGIC ASTHMA

Materials and Methods Mice The C3aR2/2 and C5aR12/2 mice (BALB/c background) have been described previously (15, 36). C3aR2/2/C5aR12/2 mice were generated by mating C3aR2/2 with C5aR12/2 mice. All mice were bred and maintained together with WT BALB/c mice (Charles River) at the University of L€ubeck specific pathogen-free facility and used at 8–12 wk of age. Animal care was provided in accordance with German law. These studies were reviewed and approved by the Schleswig-Holstein state authorities (Nr. V312.72241.122-39).

BMDC preparation and induction of the allergic phenotype in vivo BMDC preparation, allergen pulsing, and adoptive transfer into the airways of BALB/c WT mice were performed, as described (37). Briefly, BM cells were isolated from naive BALB/c and the different knockout mice strains by flushing femurs and tibias with RPMI 1640 medium. RBCs were lysed using 155 mM NH4Cl, 10 mM NaHCO3, and 0.1 mM EDTA (all SigmaAldrich). BM cells were washed and cultured at 13 106 cells/ml in complete RPMI 1640 culture medium (PAA) supplemented with 10% FBS (PAA), 2 mM L-glutamine, 100 U/ml penicillin, 100 mg/ml streptomycin (all from Life Technologies Invitrogen), and 20 ng/ml murine rGM-CSF (PeproTech). Cultures were incubated at 37˚C in a humidified atmosphere containing 5% CO2 for 10 d. On day 9, cells were pulsed overnight with 60 mg/ml HDM extract (Greer; lot 125118) in vitro. To induce a pulmonary allergic phenotype in vivo, HDM-pulsed BMDCs were harvested on day 10 and washed, and 1 3 106 BMDCs were injected i.t. into the airways of naive BALB/c mice. Recipient mice were challenged once after 10 d with 50 ml HDM (200 mg) i.t. Unpulsed BMDCs were injected into BALB/c recipients following the same protocol (Fig. 1A) as controls. After 72 h, AHR was determined and lung tissue was harvested for further analysis.

Allergen-induced AHR Mice were anesthetized by i.p. injection of 50 ml Ketavet/Rompun (50 mg/ml and 2%, respectively; Pfizer/Bayer). Muscle relaxation was induced by administration of 50 ml Esmeron (10 mg/ml; Organon). AHR was measured in anesthetized mice that were mechanically ventilated using a FlexiVentsystem (SciReq), as described (26). Aerosolized acetyl-b-methyl-choline (methacholine; 0, 1, 2.5, 5, 10, 25, 50 mg/ml; Sigma-Aldrich) was generated by an ultrasonic nebulizer and delivered in-line through the inhalation port for 10 s. Airway resistance was measured 2 min later.

Collection of bronchoalveolar lavage cells and determination of differential cell counts Bronchoalveolar lavage (BAL) samples were obtained by cannulating the trachea, injecting 1.0 ml ice-cold PBS, and subsequently aspirating the BAL fluid. BAL cells were washed once in PBS and counted using a hemocytometer (Paul Marienfeld). Differential cell counts were obtained from BAL cells spun down onto slides and treated with May–Gr€unwald–Giemsa stain (Sigma-Aldrich). A minimum of 200 cells was morphologically differentiated by light microscopy, as described (13).

Isolation of pulmonary cells and cytokine measurements Collagenase/DNase I (both Sigma-Aldrich) digests of the lungs were prepared to obtain single lung cell suspensions. Single cell suspensions (2.5 3 105) were restimulated ex vivo with 60 mg/ml HDM or with medium alone, and incubated at 37˚C for 72 h in RPMI 1640 culture medium supplemented with 10% FBS, 2 mM L-glutamine, 100 U/ml penicillin, 100 mg/ml streptomycin (all from Life Technologies Invitrogen), 1 mM sodium pyruvate (Cellgro Mediatech), and 50 mM 2-ME (Sigma-Aldrich). The production of IL-4, IL-5, IL-10, IL-13, IL-17A, and IFN-g in culture supernatants was determined using DuoSet ELISA kits (R&D Systems), following the manufacturer’s protocol. The sensitivities were 16 pg/ml for IL-4 and IL17A; 31 pg/ml for IL-5, IL-10, and IFN-g; and 62.5 pg/ml for IL-13.

Lung histology Lung histological staining, detection, and quantification of mucus cell content were done, as described (38). Briefly, lungs were excised and fixed in 3.7% formalin. Fixed tissues were then washed with 70% ethanol, dehydrated, embedded in paraffin, and cut into 5-mm sections. Slides were stained with H&E and periodic acid-Schiff (PAS). For quantification of mucus production in the airway epithelium, PAS-positive and PAS-negative airways were counted by light microscopy for a total of four lung sections per animal. The percentages of PAS positive airways were calculated.

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(HDM) extract can cleave C3 and C5 and promote the generation of the ATs, in vitro (12) and during allergen sensitization (13). Both ATs bind their cognate AT receptors, that is, C3a binds to the C3aR, whereas C5a can bind to C5aR1 and C5aR2 (C5L2) (14). Studies that aimed at delineating the function of the AT receptors in allergic asthma development have shown that genetic deficiency of the C3aR decreases the allergic phenotype, including airway hyperresponsiveness (AHR), bronchoconstriction, and inflammation in OVA-induced (15–17) and HDM-induced (18, 19) experimental allergic asthma models. The reduced allergic phenotype in C3aR-deficient mice is associated with decreased Th2 (17) and Th17 (19) cytokine production. In contrast, an increased frequency of Th17 cells and increased airway neutrophilia have been described in C3aR2/2 mice following pulmonary exposure to a mixture of OVA and Aspergillus melleus proteinase (20). C5a plays a dual role during the sensitization and the effector phases. During allergen sensitization, genetic deletion or pharmacologic targeting of C5 (21, 22) or the C5aR1 (13, 18, 19) results in an increased asthma phenotype, suggesting that C5aR1 has a protective role during asthma development. In contrast, blockade of the C5a signal during the effector phase decreased the asthmatic phenotype (13, 23, 24), indicating that C5a is proallergic in established allergic asthma. Interestingly, genetic targeting of the second C5a receptor, C5aR2, also leads to a decreased asthmatic phenotype (25, 26). AT receptor signaling in cDCs and pDCs serves as an important regulator of CD4+ Th cell differentiation toward either effector (13, 19, 27, 28) or Treg cells (28–30). Stimulation of C5aR12/2 splenic DCs with OVA together with TLR2 results in minor secretion of IL-12 and IL-6, but strong production of TGF-b and IL-23, resulting in the development of Th17 cells (28). This observation was recapitulated by in vitro stimulation of bone marrow (BM)–derived DCs (BMDCs) with HDM (19). In contrast, the C3a/C3aR signaling axis supports the development of Th17 cells upon HDM stimulation by driving the production of IL-6 and IL23 and the suppression of IL-10 (19). Up to now, the direct impact of C5aR1 and/or C3aR signaling in HDM-pulsed cDCs and its impact for the development of allergic asthma have not been determined. In this study, we used in vitro differentiated BMDCs of wild-type (WT), C3aR2/2, C5aR12/2, or C3aR2/2/C5aR12/2 origin as a surrogate for pulmonary cDCs to evaluate the role of AT receptor signaling in cDCs for the development of HDM-induced allergic asthma. BMDCs have been frequently used in the past as a surrogate for pulmonary APCs to study the role of cDCs in asthma development (31–35). This study has been designed to provide new insights into the role of C3aR and C5aR1 signaling in cDCs on the development of AHR, airway inflammation, mucus production, as well as Th1, Th2, and Th17 cytokine production. Additionally, we have performed in vitro studies with BMDCs from WT, C3aR 2/2 , C5aR1 2/2 , and C3aR2/2/C5aR12/2 cDCs to define the impact of ATR signaling on Th1-, Th2-, and Th17-inducing cytokines, MHC-II, and costimulatory molecule expression. To our surprise, we found that, in contrast to the intratracheal (i.t.) HDM sensitization model (13, 19), C5aR1 signaling in cDCs is a critical driver of allergic asthma development in the adoptive transfer model. It promotes the development of Th2-driven allergic immune response to HDM and is also involved in Th17 induction. Interestingly, C3aR-deficient BMDCs induce a strong Th2-dependent asthmatic phenotype, but failed to induce IL-17A production, whereas C3aR2/2/C5aR12/2 BMDCs exhibit effects beyond what we observed in the absence of either C3aR or C5aR1, suggesting cross-talk between the different AT receptors in allergic asthma.

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In vitro stimulation of BMDCs

Statistical analysis

WT, C3aR2/2, C5aR12/2, or C3aR2/2/C5aR12/2 BMDCs (1 3 106/well) were stimulated with HDM (60 mg/ml), in RPMI 1640 culture medium (PAA) supplemented with 10% FBS, 2 mM L-glutamine, 100 U/ml penicillin, and 100 mg/ml streptomycin (all from Life Technologies Invitrogen) at 37˚C for 24 h. Secretion of IL-1b, IL-6, IL-10, IL-12p40, IL-12p70, and TGF-b in culture supernatants was determined using DuoSet ELISA kits (R&D Systems), following the manufacturer’s protocol. The sensitivities were 16 pg/ml for IL-1b, IL-6, IL-23, IL-12p40, and TGF-b. Costimulatory molecule expression was performed by flow cytometry using the following Abs: CD11c allophycocyanin, CD11c PE, CD11b FITC, CD86 PE, CD80 FITC, CD40 allophycocyanin, and MHC class II FITC (eBioscience), and analyzed on a BD LSRII (BD Biosciences). Raw data were analyzed using FCS Express 4.0 (De Novo software).

Statistical analysis was performed using the SigmaSTAT 3.5 (SYSTAT software). Statistical difference of data was assessed by either Newman– Keuls ANOVA or unpaired t test. A p value , 0.05 was considered as * or † significant, p , 0.01 as ** or †† significant, and p , 0.001 as *** or ††† significant.

Coculture of BMDCs with OVA-TCR transgenic T cells and T cell proliferation assay

DC and T cell in vivo tracking Unpulsed or HDM-pulsed BMDCs were labeled with CFSE (1 mM; Molecular Probes). After washing, 2 3 106 cells were resuspended in PBS and adoptively transferred i.t. (Fig. 1B). After 48 or 120 h, lung and mLN were collected and cells were isolated using mechanical disruption, followed by Liberase (Roche) digestion. After washing and blocking Fc receptors using anti-CD16/CD32 Ab (eBioscience), cells were stained and analyzed by flow cytometry on a BD LSRII (BD Biosciences) using the gating strategies described in Supplemental Fig. 1. Raw data were analyzed using FCS Express 4.0 (De Novo software). In lung and mLN isolations, T cells were first identified by anti-CD3 FITC and anti-CD4 PE Cy7 Abs. Then, effector T cells were characterized as CD44+CD62L2 cells using anti-CD44 BV421 and anti-CD62L PE Abs (all Abs from eBioscience), as shown in Supplemental Fig. 1A. Lung cells were stained with SiglecF-BV421 and anti-CD11c allophycocyanin Abs (eBioscience). BMDCs were identified as SiglecF2 CD11c+ CFSE+ cells. In mLN, cells were stained only for CD11c allophycocyanin, and BMDCs were identified as CD11c+ CFSE+ cells (Supplemental Fig. 1B). +

+

RNA isolation from BMDCs and CD4 CD44 T effector cells and real-time PCR RNA was isolated using TRIzol reagent, according to the manufacturer’s instructions (Invitrogen). Reverse-transcription reaction was performed after DNase I treatment of the RNA (Fermentas) using first-strand cDNA synthesis kit (Revertaid Premium; Fermentas). Quantitative PCR was done using iQ Syber Green (Biorad) on a CFX96 real-time PCR system (Bio-Rad) using the following primers (Eurofin): actin, 59-GCACCACACCTTCTACAATGAG-39 (sense) and 59-AAATAGCACAGCCTGGATAGCAAC-39 (antisense); Bcl2, 59-ATGCCTTTGTGGAACTATATGGC39 (sense) and 59-GGTATGCACCCAGAGTGATGC-39 (antisense); BimL, 59-GACAGAACCGCAAGACAGGAG-39 (sense) and 59-GGACTTGGGGTTTGTGTTGAC-39 (antisense); and BimEL, 59-GACAGAACCGCAAGGTAATCC-39 (sense) and 59-ACTTGTCACAACTCATGGGTG-39 (antisense). Differentiation of Th cells was evaluated by amplifying Thspecific transcription factors, as follows: TBX21 (Th1), 59-GGTGTCTGGGAAGCTGAGAG-39 (sense) and 59-ATCCTGTAATGGCTTGTGGG-39 (antisense); GATA3 (Th2), 59-GCCTGCGGACTCTACCATAA-39 (sense) and 59-AGGA TGTCCCTGCTCTCCTT-39 (antisense); RORgT (Th17), 59CCGCTGAGAGGGCTTCAC-39 (sense) and 59-TGCAGGAGTAGGCCACATTACA-39 (antisense); and Foxp3 (Treg), 59-CCCATCCCCAGGAGTCTTG39 (sense) and 59-ACCATGACTAGGGGCACTGTA-39 (antisense).

C5aR1 but not C3aR signaling in BMDCs drives HDM-induced allergic asthma Following 9 d of differentiation in the presence of GM-CSF, BMderived WT, C3aR2/2, C5aR12/2, and C3aR2/2/C5aR12/2 cDCs were pulsed overnight with 60 mg/ml HDM and transferred i.t. into WT BALB/c recipient mice. Ten days later, mice were challenged i.t. with 200 mg HDM (Fig. 1A). Transfer of unpulsed BMDCs from either wt, C3aR2/2, or C3aR2/2/C5aR12/2 mice resulted in only minor increase in airway resistance from 2 to 6 cm H2O 3 s/ml in response to methacholine treatment, even after administration of high methacholine concentrations (50 mg/ml). Surprisingly, airway resistance in response to 10, 20, and 50 mg/ ml methacholine stimulation was significantly lower in mice that had received unpulsed BMDCs from C5aR12/2 mice (Fig. 2). Adoptive transfers of HDM-pulsed cDCs from WT or C3aR2/2 origin triggered strong airway resistance (Fig. 2, lower panel), which was significantly higher than that induced with unpulsed BMDCs at methacholine concentrations of 10, 25, and 50 mg/ml. In sharp contrast to WT and C3aR BMDCs, we found no significant increase in airway resistance following adoptive transfer of HDM-pulsed C5aR12/2 cells, even after a provocation with 50 mg/ml methacholine. Similar to C5aR12/2 BMDCs, airway resistance in response to HDM-pulsed C3aR2/2/C5aR12/2 BMDCs remained at level of unpulsed controls following stimulation with 10 mg/ml methacholine (Fig. 2B). After provocation with 25 or 50 mg/ml methacholine, we observed a trend toward increased airway resistance when we compared HDM-pulsed versus unpulsed C3aR2/2/C5aR12/2 BMDCs. These data suggest that C5aR1 signaling in BMDCs is critical for the establishment of HDMdriven airway resistance. In contrast, C3aR does not seem to play a major role in the development of AHR, but seems to interfere with C5aR1 signaling as C3aR2/2/C5aR12/2 show an intermediate phenotype and do not fully recapitulate the effect of C5aR12/2 BMDCs. C5aR1 but not C3aR signaling in BMDCs drives lung eosinophilia and neutrophilia Next, we assessed the impact of C3aR and C5aR1 signaling in BMDCs on the development of airway inflammation. Upon transfer of unpulsed BMDCs from WT or AT receptor–deficient strains, we found a low number of total cells in BAL fluid. We found a minor but significant increase in neutrophil numbers in response to transfer of AT receptor–deficient BMDCs and increased lymphocyte numbers in response to transfer of C5aR12/2 and C3aR2/2/C5aR12/2 BMDCs (Fig. 3A). After transfer of HDM-pulsed BMDCs from WT, C3aR2/2, and C3aR2/2/C5aR12/2 mice, we observed a marked and significant increase in the number of total cells as compared with unpulsed BMDCs from 4.25 6 0.58 to 46.59 6 5.39, 7.66 6 1.02 to 56.32 6 4.86, and 8.29 6 1.79 to 52.27 6 9.32 3 104/ml, respectively (Fig. 3B). In contrast, the total cell number in response to adoptive transfer of C5aR12/2 BMDCs increased only from 7.3 6 0.8 to 17.86 6 3.7 3 104/ml, which is significantly lower than the increase in response to WT, C3aR2/2, or C3aR2/2/C5aR12/2 BMDCs (Fig. 3B). The increase in the total cell number upon transfer of WT

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BMDCs were harvested on day 9, seeded at a density of 1 3 105 cells/200 mL in a 48-well plate, and stimulated overnight with HDM (60 mg/ml) and OVA (Sigma-Aldrich; 10 mM). The next day, naive CD4+ T cells from OVA-TCR transgenic DO11.10/RAG22/2 mice were isolated from spleen by magnetic selection using CD4 isolation kit II, according to the manufacturer’s instructions (Miltenyi Biotec), and labeled with CFSE (1 mM; Molecular Probes). Labeled T cells (2.5 3 105) were cocultured with BMDCs from WT or C5aR12/2 mice. At day 4, cells were harvested, stained with an anti-CD4 allophycocyanin Ab (eBioscience), and assessed for cell proliferation by flow cytometry on a BD LSRII (BD Biosciences). Furthermore, at day 9, levels of IL-13 were assessed by ELISA, cell survival was evaluated by staining cells with CD4 allophycocyanin Ab and 3 mM DAPI (Sigma-Aldrich), and RNA was isolated. The abundance of pro (BimL/BimEL)- and anti-apoptotic (Bcl2) molecules as well as of transcription factors T-bet, GATA3, Foxp3, and RORgT was evaluated by real-time PCR, as described (37).

Results

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C3a AND C5a IN DENDRITIC CELL–MEDIATED ALLERGIC ASTHMA the Ag-independent recruitment of neutrophils and lymphocytes by unpulsed BMDCs. In contrast, C5a drives the mixed eosinophilic/ neutrophilic recruitment in the lung following BMDC-triggered sensitization to HDM. Surprisingly, C3aR does not seem to be involved in inflammatory cell recruitment. However, C3aR signaling seems to suppress the inhibitory impact of C5aR1 signaling on BMDC-mediated neutrophil and eosinophil recruitment, as evidenced by the higher neutrophil and eosinophil numbers following adoptive transfer of HDM-pulsed C3aR2/2/C5aR12/2 BMDCs as compared with transfer of C5aR12/2 BMDCs. C5aR1 but not C3aR signaling in BMDCs is driving Th2 cytokine production

and C3aR2/2 BMDCs was mainly due to a strong increase in eosinophils to 13.23 6 1.19 (WT) or 12.53 6 1.26 (C3aR2/2) 3 104/ml cells and in neutrophils to 18.07 6 2.13 (WT) and 24.16 6 2.79 (C3aR2/2) 3 104/ml. In sharp contrast, we observed only a minor increase in eosinophil (0.59 6 0.14 3 104/ml) and neutrophil (7.85 6 1.5 3 104/ml) numbers after adoptive transfer of HDM-pulsed BMDCs from C5aR12/2 mice. The number of lymphocytes equally increased in response to transfer of HDMpulsed from WT or AT receptor–deficient strains. In C3aR2/2/ C5aR12/2 BMDC-transferred animals, we observed a minor increase in eosinophils (3.4 6 0.74 3 104/ml), but a marked increase in neutrophils (29.83 6 5.56 3 104/ml) (Fig. 3B). Histological examination of the airways showed massive peribronchiolar accumulation of inflammatory cells following the transfer of HDMpulsed wt, C3aR2/2, and C3aR2/2/C5aR12/2 BMDCs, which was minor after C5aR12/2 BMDC transfer (Fig. 3C). Taken together, these findings suggest that activation of the C5a/ C5aR1 axis plays an important role in the coordination of the recruitment of inflammatory cells by unpulsed BMDCs or BMDCs that have been activated by allergen. Interestingly, C5a suppresses

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FIGURE 1. Experimental design of the adoptive transfer model using HDM-pulsed BMDCs. (A) BM cells were isolated from WT BALB/c, C3aR2/2, C5aR12/2, or C3aR2/2/C5aR12/2 mice and differentiated for 10 d in the presence of 20 ng/ml murine rGM-CSF. On day 9, BMDCs were pulsed overnight with 60 mg/ml HDM. The next day, 1 3 106 unpulsed or HDM-pulsed BMDCs were administered i.t. into BALB/c recipients. After 10 d, recipient mice were challenged i.t. with 200 mg HDM. Seventy-two hours after the injection, airway responsiveness was determined. Subsequently, BAL fluid, lung cells, and tissues were collected for further analysis. BALB/c mice receiving unpulsed BMDCs served as controls. (B) as in (A), except that unpulsed or HDM-pulsed BMDCs were labeled with 1 mM CFSE and washed, and 2 3 106 cells were administered i.t. into BALB/c recipients. After 48 or 120 h, recipient mice were sacrificed. Subsequently, lung tissues and mLN were collected for further analysis.

The increase in airway resistance and the development of airway inflammation are mediated by broad range of cytokines produced by CD4+ Th cells differentiated into different T effector cells. In addition to Th1 and Th2 cells that produce IFN-g, IL-4, IL-5, IL10, and IL-13, recent studies have shown the involvement of the Th17 subtype of T cells, producing IL-17A among other cytokines (19). To assess the impact of AT receptor signaling on BMDCmediated induction of Th2, Th17, and Th1 cytokine production, we restimulated lung cells isolated from adoptively transferred animals in vitro with HDM. The amounts of Th1, Th2, and Th17 cytokines produced by lung cells following the adoptive transfer of unpulsed BMDCs from either strain were low. As expected, Th2 cytokine levels significantly increased in lung cells isolated from mice transferred with WT HDMpulsed BMDCs (Fig. 4A). The strongest increase occurred with regard to IL-4 production (70-fold from 42.6 6 11.32 to 2929 6 593.6 pg/ml), followed by IL-10 (9.5-fold from 162.7 6 88.6 to 1527 6 295.9 pg/ml), IL-13 (6.5-fold from 766.2 6 302.3 to 4976 6 887.2 pg/ml), and IL-5 (4.9-fold from 1114 6 485.9 to 5391 6 626.2 pg/ml). Furthermore, IL-17A and IFN-g production increased significantly from 534.9 6 145.6 to 1089 6 232.6 pg/ml and from 70.7 6 23.5 to 418.6 6 102.4 pg/ml (Fig. 4B). Surprisingly, lung cells isolated from animals transferred with HDM-pulsed C3aR2/2 BMDCs produced high amounts of Th2 cytokines. In fact, IL-4, IL-5, IL-10, and IL-13 production was similar to that induced by WT BMDCs. However, in contrast to WT BMDCs, we observed no increase in IL-17A production at all. Lung cells from mice treated with HDM-pulsed C3aR2/2 BMDCs produced significantly lower concentrations of IL-17A than those treated with HDM-pulsed WT BMDCs (Fig. 4B). Similar to WT BMDCs, HDM-pulsed C3aR2/2 BMDCs promoted a significant increase in IFN-g production. The lung cells from C5aR12/2 BMDC-treated mice almost completely failed to induce Th1, Th2, or Th17 cytokine responses. Of note, we observed a clear trend toward an increase in IL-10 from 139.7 6 50.7 to 502.7 6 87.8 pg/ml. Restimulated lung cells isolated from lungs of animals transferred with C3aR2/2/C5aR12/2 BMDCs showed a significant increase in IL-4 and IL-13 and also higher levels of IL-10, although the latter did not reach the level of statistical significance. IL-5 levels were only slightly elevated. When compared with lung cells from mice transferred with WT BMDCs, the Th2 cytokine levels were either significantly lower (IL-10) or showed a clear trend (IL-4, IL-5) toward lower levels. Importantly, IL-13 concentrations were almost as high as in the WT group (Fig. 4A). The high IL-13 may account for the high airway resistance and the sustained recruitment of inflammatory cells observed in this transfer group (Figs. 1, 2). Similar to the WT group, we found a significantly increased IL-17A production, when compared with unpulsed controls. The significant relative increase in IL-17A production may also add to the higher airway resistance and

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FIGURE 2. C5aR1 signaling in BMDCs is critical for airway constriction at steady state and in response to HDM challenge. (A) AHR in response to i.t. administration of methacholine measured as airway resistance. Shown are dose-response curves after transfer of either unpulsed or HDM-pulsed BMDCs from WT or the different AT receptor–deficient strains. (B) Comparison of airway resistance in mice following adoptive transfer of either unpulsed or HDMpulsed WT or AT receptor–deficient BMDCs using 10, 25, or 50 mg/ml methacholine. Values shown are the mean 6 SEM; n = 8–10 per group, * or †p , 0.05, ** or ††p , 0.01, *** or †††p , 0.001. †, ††, or ††† is used to indicate significant differences between unpulsed and pulsed treatment groups.

may explain the increased neutrophil numbers observed in BAL (Fig. 2B). Similar to the WT group, lung cells from the C3aR2/2/ C5aR12/2 group showed a significant increase in IFN-g production. Of note, the IFN-g levels in the C3aR2/2/C5aR12/2

group were even higher than those in the WT group, although they did not reach the level of statistical significance. In summary, these findings are consistent with the decreased airway resistance and the low airway inflammation observed in the

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FIGURE 3. C5aR1 signaling in BMDCs drives eosinophilic and neutrophilic airway inflammation in asthmatic lungs. Total and differential cell counts in BAL fluid of animals transferred with (A) unpulsed or (B) HDMpulsed WT or AT receptor–deficient BMDCs. (C) Histological examination of airway inflammation. Sections were stained with H&E (original magnification 3200). Values shown are the mean 6 SEM; n = 8–10 per group, *p , 0.05, ** or ††p , 0.01, *** or †††p , 0.001. †† or ††† is used to indicate significant differences between unpulsed and pulsed treatment groups.

absence of C5aR1 on HDM-pulsed BMDCs. They further suggest that C5aR1 signaling in adoptively transferred BMDCs has a major impact on the production of Th2 cytokines, but only a minor impact on IL-17A production, which is mainly under the control of C3a. The absence of both receptors shows again additional effects not observed after the transfer of C3aR or C5aR1-deficient BMDCs, in particular with regard to the production of IFN-g. In fact, both

receptors seem to control allergen-induced Th1 cell development in a codominant way. C5aR1 signaling in BMDCs regulates the production of mucus Another important hallmark of the asthmatic phenotype is the production of mucus by goblet cells of the bronchi. Histological examination of mucus production in the airways showed an

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FIGURE 4. C5aR1 signaling promotes pulmonary Th2 cytokine, whereas C3aR signaling controls pulmonary IL17A production. Cytokine profile of pulmonary cells harvested from lungs of mice 72 h after adoptive transfer of unpulsed or HDM-pulsed BMDCs from WT or AT receptor– deficient animals. Supernatants were collected after in vitro restimulation with 60 mg/ml HDM (72 h). (A) Th2 cytokine (IL-4, IL-5, IL-13, IL-10) profile of pulmonary cells. (B) Th1 (IFN-g) and Th17 (IL-17A) cytokine profiles of pulmonary cells. Values shown are the mean 6 SEM; n = 8–10 per group, * or †p , 0.05, ** or ††p , 0.01, *** or †††p , 0.001. † †† , , or ††† is used to indicate significant differences between unpulsed and pulsed treatment groups.

increased frequency of mucus-positive bronchi after transfer of HDM-pulsed WT BMDCs from 13.9 6 3.4% to 40.9 6 8.8% (p = 0.004) as compared with unpulsed WT BMDCs (Fig. 5). Similar to WT BMDCs, adoptive transfer of C3aR2/2-pulsed BMDCs resulted in a high frequency of mucus-producing bronchi (50.2 6 7.1%). This result is in agreement with the strong production of IL-13 observed in lung cells after transfer of BMDCs

from either WT or C3aR-deficient mice (Fig. 5A). It is well appreciated that IL-13 is an important driver of mucus production (38). Animals transferred with HDM-pulsed C5aR12/2 cDCs showed only a minor increase in mucus-producing bronchi from 6.65 6 1.8% to 16.4 6 1.6%, which was in the range of mucus production in response to transfer of unpulsed WT BMDCs (13.9 6 3.4%). This reduction in mucus production fits well with

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the low IL-13 levels (Fig. 4) and the low airway resistance (Fig. 2). In mice transferred with C3aR2/2/C5aR12/2 BMDCs, the frequency of PAS-positive bronchi significantly increased from 10.41 6 2.8% to 29.1 6 2.3%, but was somewhat lower than that induced by transfer of WT (40.9 6 8.8%) or C3aR2/2 (50.2 6 7.1%) BMDCs. The increase in mucus production following the transfer of C3aR2/2/C5aR12/2 BMDCs is paralleled by an increase in airway resistance and IL-13 production from lung cells (Fig. 4A), which may explain the higher mucus production and airway resistance as compared with C5aR12/2 BMDCs. C5aR1 and C3aR signaling in BMDCs are both necessary to drive IL-23 production in vitro

FIGURE 5. C5aR1 signaling in HDM-pulsed BMDCs drives mucus production. (A) Histological examination of mucus production in airways of recipient mice transferred with unpulsed (unpls.) or HDM-pulsed BMDCs from WT or AT receptor–deficient mice. Sections were stained with PAS for mucus production (original magnification 3200). (B) Evaluation of the frequency of PAS-positive bronchi in recipient mice. Mucus-producing airways are plotted relative to all analyzed airways. Values shown are the mean 6 SEM; n = 5 per group, * or †p , 0.05, ** or ††p , 0.01, †††p , 0.001. †, ††, or ††† is used to indicate significant differences between unpulsed and pulsed treatment groups.

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To delineate the mechanisms underlying the differences of Th1 and Th17 cytokine production that we observed in vivo, we determined the production of the Th1-promoting cytokine IL-12p70, the Th1/ Th17-promoting cytokine IL-12p40, and the Th17-promoting cytokines IL-1b, IL-6, TGF-b, and IL-23 in vitro in response to HDM stimulation. Furthermore, we assessed IL-10 production. Surprisingly, we found no production of IL-12p70 in response to HDM stimulation of BMDCs from either strain (data not shown). These data suggest that the significant increase in IFN-g that we had observed upon transfer of HDM-pulsed BMDCs does not result from a direct impact on IL-12p70 production from transferred DCs. With regard to Th17-promoting cytokines, we observed that unpulsed BMDCs from either strain produced minor amounts of IL-12p40, IL-1b, IL-6, or IL-23. In contrast, unpulsed

BMDCs from WT, C3aR2/2, and C3aR2/2/C5aR12/2 mice already produced TGF-b levels in a range between 300 and 400 pg/ ml (Fig. 6A). In contrast, unpulsed C5aR12/2 BMDCs secreted lower amounts of TGF-b than the BMDCs from WT or the two other AT receptor–deficient mice. Although we observed a clear trend, the data did not reach the level of statistical significance. Stimulation with HDM markedly increased the production of IL-12p40, IL1-b, and IL-6 from WT BMDCs. Furthermore, it resulted in a modest but significant increase in IL-23, but had no effect on TGF-b production (Fig. 6A). C3aR2/2 BMDCs showed the same activation pattern except for IL-23, which remained at the level of unpulsed C3aR2/2 BMDCs, suggesting that C3aR2/2 signaling is required for differentiation of Th17 cells with an inflammatory phenotype (39). This finding may explain the lack of IL-17A production observed in HDM-restimulated lung cells after adoptive transfer of HDM-pulsed C3aR2/2 BMDCs. Similar to C3aR2/2 BMDCs, C5aR12/2 and C3aR2/2/C5aR12/2 BMDCs failed to clearly increase the production of IL-23, which is in line with the slightly lower IL-17 production in vivo (Fig. 4B). However, HDM stimulation of AT receptor–deficient BMDCs resulted in a significant increase in IL-12p40, IL-1b, and IL-6 production, which was similar or even higher than that induced by WT BMDCs, for example, in case of IL-12p40 production in response to C5aR12/2 BMDCs or IL-6 in response to C3aR2/2/C5aR12/2 BMDCs. HDM stimulation also induced IL-10 production. Although single deficiency of AT receptors did not affect IL-10 production, C3aR2/2/C5aR12/2 BMDCs produced significantly

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In vitro proliferation and differentiation of Th cells are independent of C5aR1 expression in BMDCs

FIGURE 6. Impact of C5aR1 and C3aR signaling in BMDCs on the HDM-driven production of cytokines and the expression of MHC-II and costimulatory molecules. (A) Cytokine profiles from BMDCs of WT or AT receptor–deficient mice before or after stimulation with 60 mg/ml HDM extract for 24 h. Values shown are the mean 6 SEM; n = 3–6 per group, *p , 0.05, ** or ††p , 0.01, *** or †††p , 0.001. ††, or ††† is used to indicate significant differences between unpulsed and pulsed treatment groups. (B) BMDCs from WT or AT receptor–deficient mice were analyzed for the expression of MHC-II, CD80, CD86, or CD40 at steady state (unpulsed) or 24 h after stimulation with 60 mg/ml HDM. Values shown are the mean 6 SEM; n = 3–6 per group, *p , 0.05.

To evaluate the role of C5aR1 in T cell proliferation and differentiation, pulsed WT and C5aR12/2 BMDCs were cocultivated with naive CD4+ T cells, and their proliferation rate and different survival parameters were assessed. In absence of a model allowing to test directly the proliferation of T cell clones in response to HDM Ag, we used a surrogate model (41), in which BMDCs were stimulated with a mixture of HDM and OVA in the presence of CFSE-labeled CD4+ T cells from OVA-TCR transgenic DO11.10/ Rag22/2 for 4–9 d. As expected, T cells strongly proliferated, as evidenced by the decreased CFSE signal 4 d after coculture. C5aR12/2 BMDCs were equally potent in driving CD4+ T cell proliferation (Fig. 7A). Recently, we observed in an OVA in vitro model that C5aR12/2 BMDC-stimulated T cells suffer from accelerated cell death due to a decreased abundance of the antiapoptotic molecule Bcl-2 associated with increased abundance of proapoptotic molecules BimL/BimEL (37). Surprisingly, OVA/ HDM-pulsed WT and C5aR12/2 BMDCs were equally potent to keep T cells alive up to 9 d (Fig. 7B), which was associated with

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The expression of Ag-specific peptides via MHC-II and of costimulatory molecules is crucial for Th cell activation, subset differentiation effector function, and survival (40). To determine a potential impact of AT receptor signaling on the expression of such molecules, we determined the expression levels of MHC-II, CD80, CD86, and CD40, all of which have been implicated in the development of maladaptive Th2 and Th17 immune responses in allergic asthma (8). We found that naive WT, C3aR2/2, and C5aR12/2 BMDCs express low amounts of MHC-II, which were increased in BMDCs from C3aR2/2/C5aR12/2 mice (p = 0.069) (Fig. 6B). MHC-II expression was not affected by 24-h HDM treatment. Similarly, CD86 expression was low in unpulsed BMDCs from WT, C3aR2/2, and C5aR12/2 mice, but significantly higher in BMDCs from C3aR2/2/C5aR12/2 mice. Again, we found no effect in response to HDM stimulation in BMDCs from either strain. CD80 and CD40 expression was similar between BMDCs from all groups and slightly increased after HDM treatment, although this treatment did not reach the level of statistical significance. Interestingly, CD40 expression in C3aR2/2/C5aR12/2 BMDCs was significantly higher than in WT DCs following HDM challenge (Fig. 6B). Taken together, HDM treatment has only a minor impact on the expression of MHC-II and the costimulatory molecules CD40, CD80, and CD86 in vitro in WT, C3aR2/2, and C5aR12/2 BMDCs and does not explain the failure of C5aR1 BMDCs to promote an asthmatic phenotype. Both AT receptors together seem to control the expression of MHC-II and CD86 at steady state and of CD40 upon HDM challenge. Our findings of a similar expression pattern of MHC-II and costimulatory molecule expression in WT and C5aR12/2 DCs suggest that the lack of HDM-pulsed C5aR12/2 BMDCs to drive allergic is not due to an impaired ability to regulate these molecules.

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similar expression levels of anti- and proapoptotic molecules in CD4+ T cells (Fig. 7C). To test the ability of WT and C5aR12/2 BMDCs to trigger Th2 differentiation in vitro, day 9 CD4+ DAPI2 T cells were sorted by flow cytometry, and the abundance of transcription factors that are critical for the differentiation into Th1 (T-bet), Th2 (GATA3), Th17 (RORgt), or Treg (Foxp3) cell subtypes was evaluated by real-time PCR. We found that GATA3 and Foxp3 transcription factors were most abundant, in cocultures from both WT and C5aR12/2 BMDCs (Fig. 7D). In line with the similar ability of WT and C5aR12/2 BMDCs to drive Th2 differentiation of naive T cells in vitro, we found comparable amounts of IL-13 in supernatants of WT and C5aR12/2 cocultures (Fig. 7E). These data confirm that C5aR12/2 BMDCs have no general defect to drive a strong Th2 immune response in vitro, in accordance with our previous findings (37). C5aR1 signaling in BMDCs regulates the homing of T effector cells from the draining lymph nodes into the lung tissue

It is well appreciated that cDCs leave the lung compartment and migrate to the mLN toward a CCL21 cytokine gradient (42). To assess whether the missing asthma development upon transfer of C5aR12/2 BMDCs results from a migration defect of such cells from the alveolar space to the lung tissue and/or from the lung into the mLN, we set up cell-tracking experiments. WT or C5aR12/2 BMDCs were stimulated overnight with HDM, labeled with CSFE, and adoptively transferred into WT recipient mice. Lung tissues and mLN were collected 48 and 120 h after transfer, and CD11c+CFSE+ cells were tracked by flow cytometry. Forty-eight hours after transfer, a clear CFSE signal was detectable in the lung (Supplemental Fig. 1B). The number of unpulsed CFSE+ BMDCs per lung from either WT or C5aR12/2 mice was similar (0.34 6 0.06 3 105, WT or 0.25 6 0.07 3 105 3 105, C5aR12/2 cells; Fig. 8A). The numbers increased significantly when WT HDM-pulsed BMDCs were transferred (1.02 6 0.03 3 105), suggesting that the activation of BMDCs by HDM increased their potency to cross the epithelial barrier. The ability of BMDCs to penetrate the tissue in absence of C5aR1 seems to be reduced, because we observed a lower number of CD11c+CFSE+ cells in the lung tissues after the transfer of HDMpulsed C5aR12/2 BMDCs (0.71 6 0.2 3 105; Fig. 8A). Of note, we found no CD11c+CFSE+ cells in the mLN 48 h after transfer of either WT or C5aR12/2-pulsed BMDCs (data not shown). We reasoned that the migration of activated BMDCs from the lung to the mLN may occur at a low pace. Thus, we determined the number of CFSE+ BMDCs 120 h after adoptive transfer. Indeed, we were able to detect low, but similar numbers of CFSE+ cells in mice transferred with unpulsed WT or C5aR12/2 BMDCs (203 6 105, WT or 96 6 32, C5aR12/2; Fig. 8B). These numbers increased in recipients of WT or C5aR12/2 HDM-pulsed BMDCs (Fig. 8B). However, despite the clear migration of BMDCs to the mLN, we found no impact of the C5aR1 on cell migration (cell numbers: WT BMDCs, 546 6 175; C5aR12/2 BMDCs, 485 6 196), suggesting that C5aR1 is dispensable for the migration of BMDCs to the mLN. Finally, we also determined whether the C5a/C5aR1 axis has an impact on BMDC migration into the lung 120 h after adoptive transfer. First, we noticed that the number of CD11c+CFSE+ BMDCs in the lung tissue 120 h after transfer was markedly higher than after 48 h (Fig. 8B, Supplemental Fig. 2). This increase in pulmonary BMDC numbers was only partially dependent on allergen stimulation. Similar to our observation after 48 h, we found that the number of CFSE+ BMDCs in the lung was lower in mice that had received HDM-pulsed C5aR12/2 BMDCs than in mice that had been treated with HDM-pulsed WT BMDCs (WT, 3.01 6 0.9 3 105; C5aR12/2 1.32 6 0.4 3 105; Fig. 8B), indicating that the migration of HDM-pulsed BMDCs from the alveolar space to the pulmonary tissue over time is C5aR1 dependent. The reduced number of BMDCs in the lung after transfer of C5aR12/2 cells was not due to an accelerated cell death, because the expression levels of the anti-apoptotic molecule Bcl2 were even higher in sorted C5aR12/2 BMDCs as compared with WT cells. Furthermore, the expression level of the proapoptotic molecule BimEL was similar in BMDCs from WT or C5aR12/2 mice (Fig. 8C). In summary, our data suggest that the mechanisms underlying the failure of C5aR12/2 BMDCs to induce an allergic phenotype include a reduced capability to migrate into the lung tissue associated with a decreased potency to direct the homing of effector T cells into the lung tissue.

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To determine a potential impact of the lung environment for the development of the allergen-specific Th cell response after adoptive transfer of WT or C5aR12/2 BMDCs, we determined the numbers of CD44+CD62L2 effector T cells in lung and mLN 120 h after BMDC transfer. First, we gated on CD3+CD4+ T cells. Within this gate, we identified T effector cells by the expression of CD44 and the absence of CD62L (Supplemental Fig. 1A). The number of CD44+CD62L2 effector T cells in mLN was very low in mice that had received unpulsed BMDCs from WT mice. Interestingly, this number was somewhat higher in mice transferred with HDM-pulsed BMDCs (Fig. 7F). As expected, HMD-pulsed BMDCs from WT induced a significant increase in T effector cells in mLN (from 0.02 6 0.01 3 106 to 0.11 6 0.03 3 106; Fig. 7F). In contrast, although the absolute numbers of T effector cells in mLN were similar after transfer of HDM-pulsed BMDCs from C5aR12/2 mice, the relative increase was lower (from 0.04 6 0.01 3 106 to 0.11 6 0.02 3 106). In the lung, we found a 6- to 10-fold higher number of T effector cells than in the mLN after transfer of unpulsed BMDCs from either WT or C5aR12/2 mice (0.26 6 0.05 3 106, WT BMDCs; 0.27 6 0.06 3 106, C5aR12/2 BMDCs; Fig. 7G). Of note, we observed a modest increase in T effector cells following the adoptive transfer of HDM-pulsed WT BMDCs (to 0.43 6 0.06 3 106), which was almost completely lacking in response to C5aR12/2 BMDCs (0.33 6 0.04 3 106). The reduced T cell numbers did not result from increased T cell death, as we found comparable expression of pro- and anti-apoptotic molecules in CD44+CD62L2 T cells following transfer of either HDM-pulsed WT or C5aR12/2 BMDCs (data not shown). Next, we sought to determine the Th cell differentiation profile of the T effector cells in the lung. We found strong and similar expression of GATA3 and Foxp3 transcription factors suggesting similar potencies of WT and C5aR12/2 BMDCs to promote early Th2 and Treg differentiation in response to HDM (Fig. 7H). Interestingly, we also found expression of RORgt in C5aR12/2 BMDC-treated mice that was almost completely absent in response to WT BMDC transfer. This result is in accordance with previous findings suggesting that C5aR1-deficient cDCs drive naive T cells more toward Th17 differentiation (19, 28). Taken together, our in vivo findings are in line with our in vitro data, suggesting that C5aR1 is not required for initial proliferation of effector T cells, but may contribute to their trafficking into the lung.

C5aR1 signaling in BMDCs regulates the transmigration of adoptively transferred BMDCs from the alveolar space into the lung

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FIGURE 7. Impact of C5aR1 signaling in BMDCs on T cell proliferation, apoptosis, and differentiation in vitro and in vivo. (A) In vitro generated BMDCs from WT and C5aR12/2 mice were cocultivated with CFSE-labeled CD4+ OVA-TCR–specific T cells. After 4 d, cell proliferation was evaluated by flow cytometry. The dashed line represents the CFSE signal in naive CD4+ T cells; the gray histogram represents the CFSE signal in coculture of WT BMDCs and CD4+ T cells; the dark line represents the CFSE signal in coculture of C5aR12/2 BMDCs and CD4+ T cells. (B) After 9 d of coculture, survival of CD4+ T cells was evaluated by DAPI incorporation. The DAPI signal was measured in CD4+-gated cells. Histograms are representative of five independent experiments; values shown are the mean 6 SEM (n = 5/group). (C) Relative abundance of mRNA encoding for Bcl-2, BimL, and BimEL relative to actin in sorted BMDCs. Values shown are the mean 6 SEM of four independent experiments. (D) Evaluation of the relative abundance of mRNA encoding for the transcription factors T-bet, RORgT, GATA3, and Foxp3 relative to actin. Values shown are the mean 6 SEM of six independent experiments. (E) IL-13 production in coculture supernatants after 9 d. Values shown are the mean 6 SEM (n = 6/group, *p , 0.05). Number of CD44+CD62L2 T cells isolated from (F) mLN or (G) lung 120 h after adoptive transfer. Values shown are the mean 6 SEM (n = 5–6/group, *p , 0.05). (H) Relative abundance of mRNA encoding for the transcription factors T-bet, RORgT, GATA3, or Foxp3 relative to actin in sorted CD44+CD62L2 effector T cells. Values shown are the mean 6 SEM from four independent experiments.

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C3a AND C5a IN DENDRITIC CELL–MEDIATED ALLERGIC ASTHMA

Discussion DCs are considered necessary and sufficient to drive maladaptive T cell immune responses and to establish experimental allergic asthma (3). Because the low frequency of naive pulmonary DCs makes it difficult to use them for in vitro priming and adoptive transfer studies, in vitro GM-CSF–differentiated BMDCs have been widely used in adoptive transfer studies as they promote the development of maladaptive Th2 and Th17 responses in the lung (31–35). In agreement with such studies, we found that GM-CSF– differentiated BMDCs trigger a strong asthmatic phenotype following i.t. adoptive transfer, when pulsed in vitro with HDM. Previous studies suggested that C3a and C5a both act on pulmonary cDCs and pDCs (9), thereby regulating the development of maladaptive Th2 and Th17 development. We therefore expected that the adoptive transfer of in vitro differentiated DCs, deficient in either the C3aR, the C5aR1, or both AT receptors, will define the contribution of AT receptor signaling in cDCs and help to better understand the observations made in former studies, in which C3aR or C5aR1 receptors were deleted (13, 15–20, 26) or pharmacologically targeted during the sensitization or effector phases (13, 23, 24). Such studies clearly showed opposing roles for C3a and C5a during the sensitization phase, that is, C3a acting as a proasthmatic and C5a as an anti-asthmatic mediator. Furthermore, C5a was found to promote proinflammatory properties during the effector phase of asthma (13, 23, 24). To our surprise, we found that C5aR1 signaling in BMDCs is crucial for asthma development. In fact, HDM-pulsed BMDCs lacking C5aR1 expression failed to induce an allergic phenotype, including AHR, mucus production, recruitment of inflammatory eosinophils and neutrophils, as well as production of Th2 and Th17 cytokines. Interestingly, OVA-pulsed C5aR1-deficient BMDCs also failed to

drive an allergic phenotype (37), whereas i.p. OVA priming with adjuvant in C5aR12/2 mice or after C5aR1 blockade (13) resulted in an enhanced allergic phenotype. We found no differences in MHC-II and costimulatory molecule expression between WT, C5aR1-, and C3aR-deficient BMDCs in vitro following HDM stimulation, suggesting that BMDCs from C5aR12/2 mice do not differ in Ag-induced regulation of the MHC-II, B7 family, or CD40 axes. In sharp contrast to previous studies that described a decreased allergic phenotype with low airway resistance, reduced airway inflammation, and low Th2 cytokine production in C3aR-deficent mice (17–19), we observed a strong asthmatic phenotype following adoptive transfer of HDM-pulsed BMDCs from C3aR2/2 mice. Several effects may account for the opposing effects of C5a and C3a that we observed upon adoptive transfer of BMDCs as compared with in vivo priming (either i.t. or i.p. with adjuvant) models. First, although BMDCs have often been used as surrogates for pulmonary cDCs, there is growing evidence that BMDCs inappropriately reflect the two different subsets of CD11b+CD1032 and CD11b2CD103+ pulmonary resident cDCs (3). In fact, GM-CSF–differentiated BMDCs express markers of inflammatory DCs (SIRP-1a+, CD11c+, and CD11b+), suggesting that they resemble monocyte-derived DCs (43, 44) that are recruited to the lung upon allergen-mediated inflammation, in particular following i.t. or intranasal HDM administration (6). Importantly, CD11b+ pulmonary DCs are the main DC population that migrates to the draining lymph node during allergen sensitization to stimulate Th2 differentiation of naive Th cells (6). This population of resident CD11b+ cDCs does not contribute to the induction of Th2 immunity mediated by adoptive transfer of BMDCs, as the CD4+ T cell priming is largely dependent on the i.t. injected DCs (45). Thus, an inhibitory impact of C5a on

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FIGURE 8. Impact of C5aR1 signaling in BMDCs on their migration from the alveolar space into the lung and from the lung to mLN. In vitro generated BMDCs from WT and C5aR12/2 mice were pulsed with HDM and subsequently labeled with CFSE. A total of 2 3 106 BMDCs was adoptively transferred to WT recipient mice. (A) Number of CD11c+CFSE+ BMDCs in the lung 48 h after transfer. (B) Number of CD11c+CFSE+ BMDCs in the lung (left panel) or the mLN (right panel) 120 h after transfer. Values shown are the mean 6 SEM; n = 3–6 per group, *p , 0.05. (C) Relative abundance of mRNA encoding for Bcl-2 and BimEL relative to actin in sorted BMDCs from lung tissue. Values shown are the mean 6 SEM of four independent experiments. **p , 0.01.

The Journal of Immunology

response to HDM challenge confirms our recent finding of low IL23 production from such cells in response to OVA stimulation (9). However, Lajoie et al. (19) reported a significant increase in IL-23 production upon HDM challenge of BMDCs from C5aR1-deficient mice. Similarly, we previously observed increased IL-23 production when we stimulated spleen-derived cDCs with the TLR2 ligand Pam3Cys (28). Differences in microbiota composition and diversity may account for the different cytokine responses. It is well appreciated that identical mouse strains can differ significantly in their microbiome composition depending on their housing conditions and that such differences have a strong impact on the emergence of particular CD4+ T cell subsets, in particular with regard to the development of Th17 cells (57). In support of this view, C5aR1 signaling has been shown to modulate the cutaneous microbiome, which was associated with alterations in innate and adaptive immune cells in the skin (58). Similarly, we observed an altered composition and diversity of the intestinal microbiome of C5aR12/2 mice as compared with WT littermates (J. Ko¨hl, unpublished observation). Given the dominant effects of C5aR1 DC signaling on all asthma-related parameters, we expected that double deficiency of C5aR1 and C3aR will copycat the effects of C5aR1 alone. Interestingly, C3aR2/2/C5aR12/2 BMDC-transferred mice showed an intermediate asthmatic phenotype with reduced AHR at low methacholine concentrations, decreased eosinophilia associated with low pulmonary IL-4, IL-5, and IL-10 levels. Interestingly, we observed C3aR2/2/C5aR12/2-specific effects that we did not see upon transfer of either C3aR- or C5aR1-deficient BMDCs. In vivo, we found increased production of IFN-g from lung cells restimulated with HDM. In vitro, we observed an increased release of IL-6 upon HDM challenge. Furthermore, C3aR2/2/ C5aR1 2/2 BMDCs showed a higher expression of CD86 at steady state and of CD86 and CD40 upon HDM stimulation. The higher potency of C3aR2/2 C5aR1 2/2 BMDCs to drive IFN-g production as compared with WT DCs is surprising. Recently, it has been reported that, in the absence of C3aR2/2/ C5aR12/2, DCs enter into an autocrine TGF-b1–producing state, which amplifies the induction of Treg cells in the cognateinteracting T cells in vivo (29, 59), and that C3aR2/2/C5aR12/2 BMDCs have an impaired potency to drive the differentiation of IFN-g–producing effector cells in vitro (27). Our data provide evidence that HDM-pulsed C3aR2/2C5aR12/2 BMDCs are perfectly suited to promote differentiation of IFN-g–producing Th1 effector cells when administered i.t. into WT mice. These data suggest that the absence of C3aR and C5aR1 signaling on DCs does not necessarily result in a default pathway that promotes the differentiation of inducible Treg cells, but that the nature of the Ag and the local cellular and humoral environment in the lung also play an important role for the lineage decision of naive CD4+ T cells. Taken together, data presented in this and in previous studies (26, 37) challenge the view that GM-CSF–differentiated BMDCs serve as an appropriate surrogate to define the role of AT receptor signaling in resident pulmonary cDCs with regard to their potency to promote maladaptive Th2 and Th17 immunity in experimental allergic asthma. It is well appreciated that C3aR and C5aR1 are expressed on several innate immune cells relevant for the initiation of the allergic response such as pulmonary ECs, macrophages, as well as resident cDCs and pDCs. To understand the complex role of C3a and C5a during allergen sensitization, it will be important in future studies to delineate the interplay between AT receptor activation on such lung-resident cells and their regulatory impact on innate pattern recognition receptors, including TLR, C-type lectin receptors, and other G protein–coupled receptors.

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resident pulmonary cDCs will not affect the development of Th2 immunity following adoptive transfer of HDM-pulsed BMDCs from C5aR12/2 mice. Secondly, HDM contains the cysteinase protease allergens Der p1 and 9 as well as small amount of LPS and fungal products (4, 46, 47), which act in concert to activate TLR4 and C-type lectin receptors not only on DCs, but also on bronchial ECs (48). The emerging concept is that ECs instruct pulmonary cDCs to induce Th2 differentiation by the production of cytokines, including IL-1, GM-CSF, thymic stromal lymphopoietin, IL-25, and IL-33. The functional relevance of C5aR1 signaling in ECs has been shown in a model of IgG-induced lung injury. C5aR1 targeting in ECs improves the inflammatory response (49). Upon stimulation with C5a alone or synergistically with TLR4, rat ECs secrete large amounts of TNF-a and CCL2 and to a lower extent IL-1b (50), supporting the idea that C5a can cooperate with TLR signaling in ECs similar to what has been observed in APCs (14). C3a induces the expression of Muc5A and promotes the recruitment of inflammatory cells by ECs (51). Also, the C-type lectin receptor Dectin-1 negatively regulates C5aR1 (52) and C3aR signaling (J. Ko¨hl, unpublished observation). Clearly, the cross-talk between ECs and cDCs and the potential role of C3a and C5a in the regulation of the EC/cDC axis are lacking in the adoptive transfer model. Thirdly, C5aR1 signaling regulates alveolar macrophage (50, 53) and pulmonary pDC function (13, 18), both of which have been shown to suppress the development of Th2 immunity by the induction of Treg cells (54, 55). The potential regulation of such pathways by C5aR1 signaling is also not affected in the adoptive transfer model using C5aR1-deficient BMDCs. Finally, recent data provide evidence that GM-CSF–differentiated BM cultures from C5aR1-deficient mice contain higher numbers of myeloid-derived suppressor cells that account, at least in part, for the decreased potency of C5aR12/2 BMDC preparations to promote Th2 immunity upon adoptive transfer (37). In search for additional mechanisms that may account for the failure of C5aR12/2 BMDCs to drive an allergic phenotype, we observed a reduced capability to migrate into the lung tissue. Using a novel floxed GFP-C5aR1 reporter mouse, we found that the majority of BMDCs express C5aR1 (J. Ko¨hl, unpublished observation). Taken together, these data support a model in which C5a, locally produced within the pulmonary tissue, may contribute to the transmigration of BMDCs from the alveolar space into the lung. Furthermore, we found a decreased potency of C5aR12/2 BMDCs to direct the homing of effector T cells into the lung tissue. At this point, we do not know at which level C5aR1 signaling in BMDCs may affect the complex pathways that regulate the migration of effector T cell from the efferent lymph to the lung (56). This will be evaluated in further studies. We found that the allergic phenotype in response to HDM-pulsed BMDCs is mainly driven by the development of a strong Th2 response with high IL-4, IL-5, and IL-13. In addition to the Th2 response, we discovered a modest increase in IL-17A and in IFN-g production upon transfer of HDM-pulsed WT BMDCs, but no such increase upon transfer of HDM-pulsed C3aR2/2 BMDCs. This lack of IL-17A production was associated with a lack or a reduced IL-23 production by C3aR2/2 or C5aR12/2 BMDCs stimulated with HDM in vitro. The failure of HDM-pulsed C3aR2/2 BMDCs to promote an increase in IL-17A production is in line with previous findings demonstrating that C3a drives the differentiation of Th17 cells by an IL-23–mediated mechanism (19). Of note, a recent study showed an opposite effect of C3a in a model of Aspergillus-mediated pulmonary allergy, that is, a high percentage of Th17 cells in the lungs and high IL-17 in BAL, which was associated with high pulmonary neutrophil numbers. These data suggest a complex role of C3a in Th17 development depending on the nature of the allergen (20). The low IL-23 production from C5aR12/2 BMDCs in

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C3a AND C5a IN DENDRITIC CELL–MEDIATED ALLERGIC ASTHMA

Acknowledgments We thank E. Strerath and G. Ko¨hl for excellent technical support.

Disclosures The authors have no financial conflicts of interest.

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