of June 21, 2018.This information is current as

AsthmaMediated Allergic−C5a in Dendritic Cell

Distinct Roles of the Anaphylatoxins C3a and

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

http://www.jimmunol.org/content/193/11/5387doi: 10.4049/jimmunol.1400080October 2014;

2014; 193:5387-5401; Prepublished online 29J Immunol 

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0.DCSupplementalhttp://www.jimmunol.org/content/suppl/2014/10/29/jimmunol.140008

Referenceshttp://www.jimmunol.org/content/193/11/5387.full#ref-list-1

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The Journal of Immunology

Distinct Roles of the Anaphylatoxins C3a and C5a inDendritic Cell–Mediated Allergic Asthma

Carsten Engelke,*,1 Anna V. Wiese,*,1 Inken Schmudde,*,1 Fanny Ender,*

Heike A. Strover,* Tillmann Vollbrandt,† Peter Konig,‡ Yves Laumonnier,*,2 and

Jorg Kohl*,x,2

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. Mech-

anistically, 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.

Allergic asthma is a chronic pulmonary disease driven bya maladaptive Th2 immune response toward aero-allergens. In addition to Th2 effector cells, other Th cells

contribute to the development of allergic asthma, in particular Th17and 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

cells (DCs) are considered most important. DCs are professionalAPCs specialized in Ag/allergen uptake, processing, and presen-tation to naive CD4+ Th cells, promoting their differentiation intoTh1, Th2, and Th17 effector cells and Treg cells. Under steadystate conditions, different subtypes of DCs reside in the lung,lining the conducting airways. Resident DCs comprise plasma-cytoid DCs (pDCs), CD11b+CD1032, and CD11b2CD103+ con-ventional DCs (cDCs) (3). CD11b+ and CD103+ cDCs sample theenvironment for Ags. In addition to cDCs, CCR2+ monocyte-derived DCs are recruited to the conducting airways by CCL2or CCL7 chemokines produced by lung epithelial cells (ECs) inresponse to allergen stimulation (4), After allergen uptake, DCs ofboth origins undergo a switch from the Ag-sampling into an Ag-presenting 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, costimulatorymolecule interaction, and cytokines produced by allergen-loadedDCs then pave the way for maladaptive Th2/Th17 skewing ofnaive CD4+ T cells (7, 8).Several studies, in which complement factors or receptors have

been deleted, point toward an important role for the complementsystem as a critical regulator of asthma development through itsfunctions on DCs (9). Complement is an ancient member of theinnate immune system that becomes activated upon recognitionof exogenous and endogenous danger motifs by soluble sensingmolecules such as C1q, mannan-binding lectin, and differentficolin molecules (10, 11). Activation of the complement results inthe proteolytic cleavage of C3 and C5 generating the anaphyla-toxins (AT) C3a and C5a. In addition to the activation of thecomplement cascade, complex allergens such as house dust mite

*Institute for Systemic Inflammation Research, University of L€ubeck and AirwayResearch Center North, member of the German Center for Lung Research, 23538L€ubeck, Germany; †Cell Analysis Core, University of L€ubeck, 23538 L€ubeck, Ger-many; ‡Institute for Anatomy, University of L€ubeck and Airway Research CenterNorth, 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

1C.E., A.V.W., and I.S. contributed equally to this work.

2Y.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 For-schungsgemeinschaft) Collaborative Research Center/Transregio Projects A21 (toJ.K.), Z5 (to P.K.), and KO 1245 4/1 (to J.K.) and by International ResearchTraining 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.Jorg Kohl, Institute for Systemic Inflammation Research, University of L€ubeck,Ratzeburger Allee 160, 23538 L€ubeck, Germany. E-mail addresses: yves.laumonnier@uksh.de (Y.L.) and joerg.koehl@uksh.de (J.K.)

The online version of this article contains supplemental material.

Abbreviations used in this article: AHR, airway hyperresponsiveness; AT, anaphyla-toxin; BAL, bronchoalveolar lavage; BM, bone marrow; BMDC, BM-derived DC;cDC, conventional DC; DC, dendritic cell; EC, epithelial cell; HDM, house dustmite; i.t., intratracheal; mLN, mediastinal lymph node; PAS, periodic acid–Schiff;pDC, plasmacytoid DC; Treg, regulatory T; WT, wild-type.

Copyright� 2014 by The American Association of Immunologists, Inc. 0022-1767/14/$16.00

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(HDM) extract can cleave C3 and C5 and promote the generationof 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 inflamma-

tion in OVA-induced (15–17) and HDM-induced (18, 19) exper-

imental 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 fre-

quency 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 protec-

tive 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 ob-

servation 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 IL-

23 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 de-

velopment 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, C3aR2 /2, C5aR12 /2, and

C3aR2/2/C5aR12/2 cDCs to define the impact of ATR signaling

on Th1-, Th2-, and Th17-inducing cytokines, MHC-II, and co-

stimulatory 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 de-

velopment 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.

Materials and MethodsMice

The C3aR2/2 and C5aR12/2 mice (BALB/c background) have been de-scribed previously (15, 36). C3aR2/2/C5aR12/2 mice were generated bymating C3aR2/2 with C5aR12/2 mice. All mice were bred and maintainedtogether with WT BALB/c mice (Charles River) at the University ofL€ubeck specific pathogen-free facility and used at 8–12 wk of age. Animalcare was provided in accordance with German law. These studies werereviewed and approved by the Schleswig-Holstein state authorities(Nr. V312.72241.122-39).

BMDC preparation and induction of the allergic phenotypein vivo

BMDC preparation, allergen pulsing, and adoptive transfer into the airwaysof BALB/c WT mice were performed, as described (37). Briefly, BM cellswere isolated from naive BALB/c and the different knockout mice strainsby flushing femurs and tibias with RPMI 1640 medium. RBCs were lysedusing 155 mM NH4Cl, 10 mM NaHCO3, and 0.1 mM EDTA (all Sigma-Aldrich). BM cells were washed and cultured at 13 106 cells/ml incomplete 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 atmospherecontaining 5% CO2 for 10 d. On day 9, cells were pulsed overnight with60 mg/ml HDM extract (Greer; lot 125118) in vitro. To induce a pulmonaryallergic phenotype in vivo, HDM-pulsed BMDCs were harvested on day10 and washed, and 1 3 106 BMDCs were injected i.t. into the airways ofnaive BALB/c mice. Recipient mice were challenged once after 10 d with50 ml HDM (200 mg) i.t. Unpulsed BMDCs were injected into BALB/crecipients 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/mland 2%, respectively; Pfizer/Bayer). Muscle relaxation was induced by ad-ministration of 50 ml Esmeron (10 mg/ml; Organon). AHR was measured inanesthetized mice that were mechanically ventilated using a FlexiVentsystem(SciReq), as described (26). Aerosolized acetyl-b-methyl-choline (meth-acholine; 0, 1, 2.5, 5, 10, 25, 50 mg/ml; Sigma-Aldrich) was generated by anultrasonic 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 determinationof differential cell counts

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

Isolation of pulmonary cells and cytokine measurements

Collagenase/DNase I (both Sigma-Aldrich) digests of the lungs wereprepared to obtain single lung cell suspensions. Single cell suspensions(2.5 3 105) were restimulated ex vivo with 60 mg/ml HDM or with me-dium alone, and incubated at 37˚C for 72 h in RPMI 1640 culture mediumsupplemented with 10% FBS, 2 mM L-glutamine, 100 U/ml penicillin,100 mg/ml streptomycin (all from Life Technologies Invitrogen), 1 mM so-dium pyruvate (Cellgro Mediatech), and 50 mM 2-ME (Sigma-Aldrich). Theproduction of IL-4, IL-5, IL-10, IL-13, IL-17A, and IFN-g in culture super-natants was determined using DuoSet ELISA kits (R&D Systems), followingthe manufacturer’s protocol. The sensitivities were 16 pg/ml for IL-4 and IL-17A; 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 contentwere 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 withH&E and periodic acid-Schiff (PAS). For quantification of mucus productionin the airway epithelium, PAS-positive and PAS-negative airways werecounted by light microscopy for a total of four lung sections per animal. Thepercentages of PAS positive airways were calculated.

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In vitro stimulation of BMDCs

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 peni-cillin, 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, andTGF-b in culture supernatants was determined using DuoSet ELISA kits(R&D Systems), following the manufacturer’s protocol. The sensitivitieswere 16 pg/ml for IL-1b, IL-6, IL-23, IL-12p40, and TGF-b. Costimula-tory molecule expression was performed by flow cytometry using thefollowing Abs: CD11c allophycocyanin, CD11c PE, CD11b FITC, CD86PE, CD80 FITC, CD40 allophycocyanin, and MHC class II FITC (eBio-science), and analyzed on a BD LSRII (BD Biosciences). Raw data wereanalyzed using FCS Express 4.0 (De Novo software).

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

BMDCs were harvested on day 9, seeded at a density of 1 3 105 cells/200mL in a 48-well plate, and stimulated overnight with HDM (60 mg/ml) andOVA (Sigma-Aldrich; 10 mM). The next day, naive CD4+ T cells fromOVA-TCR transgenic DO11.10/RAG22/2 mice were isolated from spleenby magnetic selection using CD4 isolation kit II, according to the manu-facturer’s instructions (Miltenyi Biotec), and labeled with CFSE (1 mM;Molecular Probes). Labeled T cells (2.5 3 105) were cocultured withBMDCs from WT or C5aR12/2 mice. At day 4, cells were harvested,stained with an anti-CD4 allophycocyanin Ab (eBioscience), and assessedfor cell proliferation by flow cytometry on a BD LSRII (BD Biosciences).Furthermore, at day 9, levels of IL-13 were assessed by ELISA, cell sur-vival was evaluated by staining cells with CD4 allophycocyanin Ab and3 mM DAPI (Sigma-Aldrich), and RNA was isolated. The abundance ofpro (BimL/BimEL)- and anti-apoptotic (Bcl2) molecules as well as oftranscription factors T-bet, GATA3, Foxp3, and RORgT was evaluated byreal-time PCR, as described (37).

DC and T cell in vivo tracking

Unpulsed or HDM-pulsed BMDCs were labeled with CFSE (1 mM; Mo-lecular Probes). After washing, 23 106 cells were resuspended in PBS andadoptively transferred i.t. (Fig. 1B). After 48 or 120 h, lung and mLN werecollected and cells were isolated using mechanical disruption, followed byLiberase (Roche) digestion. After washing and blocking Fc receptors usinganti-CD16/CD32 Ab (eBioscience), cells were stained and analyzed byflow cytometry on a BD LSRII (BD Biosciences) using the gating strate-gies described in Supplemental Fig. 1. Raw data were analyzed using FCSExpress 4.0 (De Novo software). In lung and mLN isolations, T cells werefirst identified by anti-CD3 FITC and anti-CD4 PE Cy7 Abs. Then, effectorT cells were characterized as CD44+CD62L2 cells using anti-CD44BV421 and anti-CD62L PE Abs (all Abs from eBioscience), as shownin Supplemental Fig. 1A. Lung cells were stained with SiglecF-BV421 andanti-CD11c allophycocyanin Abs (eBioscience). BMDCs were identifiedas SiglecF2 CD11c+ CFSE+ cells. In mLN, cells were stained only forCD11c allophycocyanin, and BMDCs were identified as CD11c+ CFSE+

cells (Supplemental Fig. 1B).

RNA isolation from BMDCs and CD4+CD44+ T effector cellsand real-time PCR

RNA was isolated using TRIzol reagent, according to the manufacturer’sinstructions (Invitrogen). Reverse-transcription reaction was performedafter DNase I treatment of the RNA (Fermentas) using first-strand cDNAsynthesis kit (Revertaid Premium; Fermentas). Quantitative PCR wasdone using iQ Syber Green (Biorad) on a CFX96 real-time PCR system(Bio-Rad) using the following primers (Eurofin): actin, 59-GCACCA-CACCTTCTACAATGAG-39 (sense) and 59-AAATAGCACAGCCTGGA-TAGCAAC-39 (antisense); Bcl2, 59-ATGCCTTTGTGGAACTATATGGC-39 (sense) and 59-GGTATGCACCCAGAGTGATGC-39 (antisense); BimL,59-GACAGAACCGCAAGACAGGAG-39 (sense) and 59-GGACTTGGG-GTTTGTGTTGAC-39 (antisense); and BimEL, 59-GACAGAACCGCA-AGGTAATCC-39 (sense) and 59-ACTTGTCACAACTCATGGGTG-39(antisense). Differentiation of Th cells was evaluated by amplifying Th-specific transcription factors, as follows: TBX21 (Th1), 59-GGTGTCTG-GGAAGCTGAGAG-39 (sense) and 59-ATCCTGTAATGGCTTGTGGG-39(antisense); GATA3 (Th2), 59-GCCTGCGGACTCTACCATAA-39 (sense)and 59-AGGA TGTCCCTGCTCTCCTT-39 (antisense); RORgT (Th17), 59-CCGCTGAGAGGGCTTCAC-39 (sense) and 59-TGCAGGAGTAGGCCAC-ATTACA-39 (antisense); and Foxp3 (Treg), 59-CCCATCCCCAGGAGTCTTG-39 (sense) and 59-ACCATGACTAGGGGCACTGTA-39 (antisense).

Statistical analysis

Statistical analysis was performed using the SigmaSTAT 3.5 (SYSTATsoftware). 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.

ResultsC5aR1 but not C3aR signaling in BMDCs drivesHDM-induced allergic asthma

Following 9 d of differentiation in the presence of GM-CSF, BM-derived WT, C3aR2/2, C5aR12/2, and C3aR2/2/C5aR12/2 cDCswere pulsed overnight with 60 mg/ml HDM and transferred i.t.into WT BALB/c recipient mice. Ten days later, mice werechallenged i.t. with 200 mg HDM (Fig. 1A). Transfer of unpulsedBMDCs from either wt, C3aR2/2, or C3aR2/2/C5aR12/2 miceresulted in only minor increase in airway resistance from 2 to 6 cmH2O 3 s/ml in response to methacholine treatment, even afteradministration 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 thathad 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 unpulsedBMDCs at methacholine concentrations of 10, 25, and 50 mg/ml.In sharp contrast to WT and C3aR BMDCs, we found no signif-icant increase in airway resistance following adoptive transfer ofHDM-pulsed C5aR12/2 cells, even after a provocation with 50mg/ml methacholine. Similar to C5aR12/2 BMDCs, airway re-sistance in response to HDM-pulsed C3aR2/2/C5aR12/2 BMDCsremained at level of unpulsed controls following stimulation with10 mg/ml methacholine (Fig. 2B). After provocation with 25 or 50mg/ml methacholine, we observed a trend toward increased air-way resistance when we compared HDM-pulsed versus unpulsedC3aR2/2/C5aR12/2 BMDCs. These data suggest that C5aR1signaling in BMDCs is critical for the establishment of HDM-driven airway resistance. In contrast, C3aR does not seem toplay a major role in the development of AHR, but seems to in-terfere with C5aR1 signaling as C3aR2/2/C5aR12/2 show anintermediate phenotype and do not fully recapitulate the effect ofC5aR12/2 BMDCs.

C5aR1 but not C3aR signaling in BMDCs drives lungeosinophilia and neutrophilia

Next, we assessed the impact of C3aR and C5aR1 signaling inBMDCs on the development of airway inflammation. Upon transferof unpulsed BMDCs from WTor AT receptor–deficient strains, wefound a low number of total cells in BAL fluid. We found a minorbut significant increase in neutrophil numbers in responseto transfer of AT receptor–deficient BMDCs and increasedlymphocyte 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 significantincrease in the number of total cells as compared with unpulsedBMDCs from 4.256 0.58 to 46.596 5.39, 7.666 1.02 to 56.3264.86, 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 adoptivetransfer of C5aR12/2 BMDCs increased only from 7.3 6 0.8 to17.866 3.73 104/ml, which is significantly lower than the increasein 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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and C3aR2/2 BMDCs was mainly due to a strong increase ineosinophils to 13.23 6 1.19 (WT) or 12.53 6 1.26 (C3aR2/2) 3104/ml cells and in neutrophils to 18.07 6 2.13 (WT) and 24.16 62.79 (C3aR2/2) 3 104/ml. In sharp contrast, we observed onlya minor increase in eosinophil (0.59 6 0.14 3 104/ml) and neu-trophil (7.85 6 1.5 3 104/ml) numbers after adoptive transferof HDM-pulsed BMDCs from C5aR12/2 mice. The number oflymphocytes equally increased in response to transfer of HDM-pulsed from WT or AT receptor–deficient strains. In C3aR2/2/C5aR12/2 BMDC-transferred animals, we observed a minor in-crease in eosinophils (3.4 6 0.74 3 104/ml), but a marked increasein neutrophils (29.83 6 5.56 3 104/ml) (Fig. 3B). Histologicalexamination of the airways showed massive peribronchiolar accu-mulation of inflammatory cells following the transfer of HDM-pulsed wt, C3aR2/2, and C3aR2/2/C5aR12/2 BMDCs, whichwas 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 therecruitment of inflammatory cells by unpulsed BMDCs or BMDCsthat have been activated by allergen. Interestingly, C5a suppresses

the Ag-independent recruitment of neutrophils and lymphocytes byunpulsed BMDCs. In contrast, C5a drives the mixed eosinophilic/neutrophilic recruitment in the lung following BMDC-triggeredsensitization to HDM. Surprisingly, C3aR does not seem to beinvolved in inflammatory cell recruitment. However, C3aR sig-naling seems to suppress the inhibitory impact of C5aR1 signalingon BMDC-mediated neutrophil and eosinophil recruitment, asevidenced by the higher neutrophil and eosinophil numbers fol-lowing 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 Th2cytokine production

The increase in airway resistance and the development of airwayinflammation are mediated by broad range of cytokines producedby CD4+ Th cells differentiated into different T effector cells. Inaddition to Th1 and Th2 cells that produce IFN-g, IL-4, IL-5, IL-10, and IL-13, recent studies have shown the involvement of theTh17 subtype of T cells, producing IL-17A among other cytokines(19). To assess the impact of AT receptor signaling on BMDC-mediated induction of Th2, Th17, and Th1 cytokine production,we restimulated lung cells isolated from adoptively transferredanimals in vitro with HDM.The amounts of Th1, Th2, and Th17 cytokines produced by lung

cells following the adoptive transfer of unpulsed BMDCs from eitherstrain were low. As expected, Th2 cytokine levels significantly in-creased in lung cells isolated from mice transferred with WT HDM-pulsed BMDCs (Fig. 4A). The strongest increase occurred withregard to IL-4 production (70-fold from 42.6 6 11.32 to 2929 6593.6 pg/ml), followed by IL-10 (9.5-fold from 162.7 6 88.6 to1527 6 295.9 pg/ml), IL-13 (6.5-fold from 766.2 6 302.3 to4976 6 887.2 pg/ml), and IL-5 (4.9-fold from 1114 6 485.9to 5391 6 626.2 pg/ml). Furthermore, IL-17A and IFN-g produc-tion increased significantly from 534.9 6 145.6 to 1089 6 232.6pg/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 Th2cytokines. In fact, IL-4, IL-5, IL-10, and IL-13 production wassimilar to that induced by WT BMDCs. However, in contrast toWT BMDCs, we observed no increase in IL-17A production at all.Lung cells from mice treated with HDM-pulsed C3aR2/2 BMDCsproduced significantly lower concentrations of IL-17A than thosetreated with HDM-pulsed WT BMDCs (Fig. 4B). Similar to WTBMDCs, HDM-pulsed C3aR2/2 BMDCs promoted a significantincrease 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-10from 139.7 6 50.7 to 502.7 6 87.8 pg/ml.Restimulated lung cells isolated from lungs of animals trans-

ferred with C3aR2/2/C5aR12/2 BMDCs showed a significantincrease in IL-4 and IL-13 and also higher levels of IL-10, al-though the latter did not reach the level of statistical significance.IL-5 levels were only slightly elevated. When compared with lungcells from mice transferred with WT BMDCs, the Th2 cytokinelevels were either significantly lower (IL-10) or showed a cleartrend (IL-4, IL-5) toward lower levels. Importantly, IL-13 con-centrations were almost as high as in the WT group (Fig. 4A). Thehigh IL-13 may account for the high airway resistance and thesustained recruitment of inflammatory cells observed in thistransfer group (Figs. 1, 2). Similar to the WT group, we founda significantly increased IL-17A production, when compared withunpulsed controls. The significant relative increase in IL-17Aproduction may also add to the higher airway resistance and

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 23 106 cells were administered

i.t. into BALB/c recipients. After 48 or 120 h, recipient mice were sacri-

ficed. Subsequently, lung tissues and mLN were collected for further

analysis.

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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 pro-duction. Of note, the IFN-g levels in the C3aR2/2/C5aR12/2

group were even higher than those in the WT group, although theydid 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

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

fromWTor the different AT receptor–deficient strains. (B) Comparison of airway resistance in mice following adoptive transfer of either unpulsed or HDM-

pulsed 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.

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absence of C5aR1 on HDM-pulsed BMDCs. They further suggestthat C5aR1 signaling in adoptively transferred BMDCs has a majorimpact on the production of Th2 cytokines, but only a minor impacton IL-17A production, which is mainly under the control of C3a.The absence of both receptors shows again additional effects notobserved after the transfer of C3aR or C5aR1-deficient BMDCs, inparticular with regard to the production of IFN-g. In fact, both

receptors seem to control allergen-induced Th1 cell developmentin a codominant way.

C5aR1 signaling in BMDCs regulates the production of mucus

Another important hallmark of the asthmatic phenotype is theproduction of mucus by goblet cells of the bronchi. Histologicalexamination of mucus production in the airways showed an

FIGURE 3. C5aR1 signaling in

BMDCs drives eosinophilic and neu-

trophilic airway inflammation in asth-

matic lungs. Total and differential cell

counts in BAL fluid of animals trans-

ferred with (A) unpulsed or (B) HDM-

pulsed 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 treat-

ment groups.

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increased frequency of mucus-positive bronchi after transfer ofHDM-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-pulsedBMDCs resulted in a high frequency of mucus-producing bronchi(50.2 6 7.1%). This result is in agreement with the strong pro-duction of IL-13 observed in lung cells after transfer of BMDCs

from either WT or C3aR-deficient mice (Fig. 5A). It is well ap-preciated that IL-13 is an important driver of mucus production(38). Animals transferred with HDM-pulsed C5aR12/2 cDCsshowed only a minor increase in mucus-producing bronchi from6.65 6 1.8% to 16.4 6 1.6%, which was in the range of mucusproduction in response to transfer of unpulsed WT BMDCs(13.9 6 3.4%). This reduction in mucus production fits well with

FIGURE 4. C5aR1 signaling promotes pulmonary Th2

cytokine, whereas C3aR signaling controls pulmonary IL-

17A production. Cytokine profile of pulmonary cells har-

vested 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.

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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 from10.41 6 2.8% to 29.1 6 2.3%, but was somewhat lower than thatinduced by transfer of WT (40.9 6 8.8%) or C3aR2/2 (50.2 67.1%) BMDCs. The increase in mucus production following thetransfer of C3aR2/2/C5aR12/2 BMDCs is paralleled by an in-crease in airway resistance and IL-13 production from lung cells(Fig. 4A), which may explain the higher mucus production andairway resistance as compared with C5aR12/2 BMDCs.

C5aR1 and C3aR signaling in BMDCs are both necessary todrive IL-23 production in vitro

To delineate the mechanisms underlying the differences of Th1 andTh17 cytokine production that we observed in vivo, we determinedthe production of the Th1-promoting cytokine IL-12p70, the Th1/Th17-promoting cytokine IL-12p40, and the Th17-promotingcytokines IL-1b, IL-6, TGF-b, and IL-23 in vitro in response toHDM stimulation. Furthermore, we assessed IL-10 production.Surprisingly, we found no production of IL-12p70 in response toHDM stimulation of BMDCs from either strain (data not shown).These data suggest that the significant increase in IFN-g that wehad observed upon transfer of HDM-pulsed BMDCs does notresult from a direct impact on IL-12p70 production from trans-ferred DCs. With regard to Th17-promoting cytokines, we ob-served that unpulsed BMDCs from either strain produced minoramounts of IL-12p40, IL-1b, IL-6, or IL-23. In contrast, unpulsed

BMDCs from WT, C3aR2/2, and C3aR2/2/C5aR12/2 mice al-ready produced TGF-b levels in a range between 300 and 400 pg/ml (Fig. 6A). In contrast, unpulsed C5aR12/2 BMDCs secretedlower amounts of TGF-b than the BMDCs from WT or the twoother AT receptor–deficient mice. Although we observed a cleartrend, the data did not reach the level of statistical significance.Stimulation with HDM markedly increased the production ofIL-12p40, IL1-b, and IL-6 from WT BMDCs. Furthermore, itresulted in a modest but significant increase in IL-23, but had noeffect on TGF-b production (Fig. 6A). C3aR2/2 BMDCs showedthe same activation pattern except for IL-23, which remained atthe level of unpulsed C3aR2/2 BMDCs, suggesting that C3aR2/2

signaling is required for differentiation of Th17 cells with an in-flammatory phenotype (39). This finding may explain the lack ofIL-17A production observed in HDM-restimulated lung cells afteradoptive transfer of HDM-pulsed C3aR2/2 BMDCs. Similar toC3aR2/2 BMDCs, C5aR12/2 and C3aR2/2/C5aR12/2 BMDCsfailed to clearly increase the production of IL-23, which is in linewith the slightly lower IL-17 production in vivo (Fig. 4B). How-ever, HDM stimulation of AT receptor–deficient BMDCs resultedin a significant increase in IL-12p40, IL-1b, and IL-6 production,which was similar or even higher than that induced by WTBMDCs, for example, in case of IL-12p40 production in responseto C5aR12/2 BMDCs or IL-6 in response to C3aR2/2/C5aR12/2

BMDCs. HDM stimulation also induced IL-10 production. Al-though single deficiency of AT receptors did not affect IL-10production, C3aR2/2/C5aR12/2 BMDCs produced significantly

FIGURE 5. C5aR1 signaling in HDM-pulsed BMDCs drives mucus production. (A) Histological examination of mucus production in airways of re-

cipient mice transferred with unpulsed (unpls.) or HDM-pulsed BMDCs from WTor 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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higher amounts of this cytokine than BMDCs from all otherstrains. Altogether, our findings suggest that AT receptor single ofdouble deficiency alters the potency of BMDCs to produce IL-12family cytokines (IL-12p40, IL-23), IL-1b, IL-6, and IL-10, whichmay, at least in part, explain the reduced IL-17 production ob-served upon transfer of HDM-pulsed C3aR2/2 BMDCs. However,they do not explain the failure of HDM-pulsed C5aR12/2 BMDCsto drive an allergic phenotype.

C5a and C3a act in concert to control expression ofcostimulatory molecules at steady state and upon allergenstimulation

The expression of Ag-specific peptides via MHC-II and of co-stimulatory molecules is crucial for Th cell activation, subsetdifferentiation effector function, and survival (40). To determinea potential impact of AT receptor signaling on the expression ofsuch molecules, we determined the expression levels of MHC-II,CD80, CD86, and CD40, all of which have been implicated in thedevelopment of maladaptive Th2 and Th17 immune responses inallergic asthma (8).We found that naive WT, C3aR2/2, and C5aR12/2 BMDCs ex-

press low amounts of MHC-II, which were increased in BMDCsfrom C3aR2/2/C5aR12/2 mice (p = 0.069) (Fig. 6B). MHC-II ex-pression was not affected by 24-h HDM treatment. Similarly, CD86expression was low in unpulsed BMDCs from WT, C3aR2/2,and C5aR12/2 mice, but significantly higher in BMDCs fromC3aR2/2/C5aR12/2 mice. Again, we found no effect in response toHDM stimulation in BMDCs from either strain. CD80 and CD40expression was similar between BMDCs from all groups and slightlyincreased after HDM treatment, although this treatment did not reachthe level of statistical significance. Interestingly, CD40 expression inC3aR2/2/C5aR12/2 BMDCs was significantly higher than in WTDCs following HDM challenge (Fig. 6B). Taken together, HDMtreatment has only a minor impact on the expression of MHC-II andthe costimulatory molecules CD40, CD80, and CD86 in vitro in WT,C3aR2/2, and C5aR12/2 BMDCs and does not explain the failureof C5aR1 BMDCs to promote an asthmatic phenotype. Both ATreceptors together seem to control the expression of MHC-II andCD86 at steady state and of CD40 upon HDM challenge. Our find-ings of a similar expression pattern of MHC-II and costimulatorymolecule expression in WT and C5aR12/2 DCs suggest that the lackof HDM-pulsed C5aR12/2 BMDCs to drive allergic is not due to animpaired ability to regulate these molecules.

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

To evaluate the role of C5aR1 in T cell proliferation and differ-entiation, pulsed WT and C5aR12/2 BMDCs were cocultivatedwith naive CD4+ T cells, and their proliferation rate and differentsurvival parameters were assessed. In absence of a model allowingto test directly the proliferation of T cell clones in response toHDM Ag, we used a surrogate model (41), in which BMDCs werestimulated with a mixture of HDM and OVA in the presence ofCFSE-labeled CD4+ T cells from OVA-TCR transgenic DO11.10/Rag22/2 for 4–9 d. As expected, T cells strongly proliferated, asevidenced by the decreased CFSE signal 4 d after coculture.C5aR12/2 BMDCs were equally potent in driving CD4+ T cellproliferation (Fig. 7A). Recently, we observed in an OVA in vitromodel that C5aR12/2 BMDC-stimulated T cells suffer from ac-celerated cell death due to a decreased abundance of the anti-apoptotic molecule Bcl-2 associated with increased abundanceof proapoptotic molecules BimL/BimEL (37). Surprisingly, OVA/HDM-pulsed WT and C5aR12/2 BMDCs were equally potent tokeep T cells alive up to 9 d (Fig. 7B), which was associated with

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 6SEM; n = 3–6 per group, *p , 0.05.

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similar expression levels of anti- and proapoptotic molecules inCD4+ 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 byflow cytometry, and the abundance of transcription factors that arecritical for the differentiation into Th1 (T-bet), Th2 (GATA3),Th17 (RORgt), or Treg (Foxp3) cell subtypes was evaluated byreal-time PCR. We found that GATA3 and Foxp3 transcriptionfactors were most abundant, in cocultures from both WT andC5aR12/2 BMDCs (Fig. 7D). In line with the similar ability ofWT and C5aR12/2 BMDCs to drive Th2 differentiation of naiveT cells in vitro, we found comparable amounts of IL-13 insupernatants of WT and C5aR12/2 cocultures (Fig. 7E). Thesedata confirm that C5aR12/2 BMDCs have no general defect todrive a strong Th2 immune response in vitro, in accordance withour previous findings (37).

C5aR1 signaling in BMDCs regulates the homing of T effectorcells from the draining lymph nodes into the lung tissue

To determine a potential impact of the lung environment for thedevelopment of the allergen-specific Th cell response afteradoptive transfer of WT or C5aR12/2 BMDCs, we determinedthe numbers of CD44+CD62L2 effector T cells in lung and mLN120 h after BMDC transfer. First, we gated on CD3+CD4+

T cells. Within this gate, we identified T effector cells by theexpression of CD44 and the absence of CD62L (SupplementalFig. 1A). The number of CD44+CD62L2 effector T cells in mLNwas very low in mice that had received unpulsed BMDCs fromWT mice. Interestingly, this number was somewhat higher inmice transferred with HDM-pulsed BMDCs (Fig. 7F). As ex-pected, HMD-pulsed BMDCs from WT induced a significantincrease in T effector cells in mLN (from 0.02 6 0.01 3 106 to0.11 6 0.03 3 106; Fig. 7F). In contrast, although the absolutenumbers of T effector cells in mLN were similar after transfer ofHDM-pulsed BMDCs from C5aR12/2 mice, the relative in-crease 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 ef-fector cells than in the mLN after transfer of unpulsed BMDCsfrom either WT or C5aR12/2 mice (0.26 6 0.05 3 106, WTBMDCs; 0.27 6 0.06 3 106, C5aR12/2 BMDCs; Fig. 7G). Ofnote, we observed a modest increase in T effector cells followingthe adoptive transfer of HDM-pulsed WT BMDCs (to 0.43 60.06 3 106), which was almost completely lacking in response toC5aR12/2 BMDCs (0.33 6 0.04 3 106). The reduced T cellnumbers did not result from increased T cell death, as we foundcomparable expression of pro- and anti-apoptotic molecules inCD44+CD62L2 T cells following transfer of either HDM-pulsedWT 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 similarexpression of GATA3 and Foxp3 transcription factors suggestingsimilar potencies of WT and C5aR12/2 BMDCs to promoteearly 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 inresponse to WT BMDC transfer. This result is in accordance withprevious findings suggesting that C5aR1-deficient cDCs drivenaive T cells more toward Th17 differentiation (19, 28). Takentogether, our in vivo findings are in line with our in vitro data,suggesting that C5aR1 is not required for initial proliferationof effector T cells, but may contribute to their trafficking intothe lung.

C5aR1 signaling in BMDCs regulates the transmigration ofadoptively transferred BMDCs from the alveolar space into thelung

It is well appreciated that cDCs leave the lung compartment andmigrate to the mLN toward a CCL21 cytokine gradient (42). Toassess whether the missing asthma development upon transfer ofC5aR12/2 BMDCs results from a migration defect of such cellsfrom the alveolar space to the lung tissue and/or from the lung intothe 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 andmLN were collected 48 and 120 h after transfer, and CD11c+CFSE+

cells were tracked by flow cytometry. Forty-eight hours after trans-fer, a clear CFSE signal was detectable in the lung (SupplementalFig. 1B). The number of unpulsed CFSE+ BMDCs per lung fromeither WT or C5aR12/2 mice was similar (0.34 6 0.06 3 105, WTor 0.25 6 0.07 3 105 3 105, C5aR12/2 cells; Fig. 8A). Thenumbers increased significantly when WT HDM-pulsed BMDCswere transferred (1.02 6 0.03 3 105), suggesting that the activationof BMDCs by HDM increased their potency to cross the epithelialbarrier. The ability of BMDCs to penetrate the tissue in absence ofC5aR1 seems to be reduced, because we observed a lower number ofCD11c+CFSE+ cells in the lung tissues after the transfer of HDM-pulsed C5aR12/2 BMDCs (0.716 0.23 105; Fig. 8A). Of note, wefound no CD11c+CFSE+ cells in the mLN 48 h after transfer ofeither 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 thenumber of CFSE+ BMDCs 120 h after adoptive transfer. Indeed,we were able to detect low, but similar numbers of CFSE+ cells inmice transferred with unpulsed WT or C5aR12/2 BMDCs (203 6105, WT or 96 6 32, C5aR12/2; Fig. 8B). These numbers in-creased in recipients of WT or C5aR12/2 HDM-pulsed BMDCs(Fig. 8B). However, despite the clear migration of BMDCs to themLN, we found no impact of the C5aR1 on cell migration (cellnumbers: WT BMDCs, 546 6 175; C5aR12/2 BMDCs, 485 6196), suggesting that C5aR1 is dispensable for the migration ofBMDCs to the mLN.Finally, we also determined whether the C5a/C5aR1 axis has an

impact on BMDC migration into the lung 120 h after adoptivetransfer. First, we noticed that the number of CD11c+CFSE+

BMDCs in the lung tissue 120 h after transfer was markedlyhigher than after 48 h (Fig. 8B, Supplemental Fig. 2). This in-crease in pulmonary BMDC numbers was only partially depen-dent on allergen stimulation. Similar to our observation after 48 h,we found that the number of CFSE+ BMDCs in the lung was lowerin mice that had received HDM-pulsed C5aR12/2 BMDCs than inmice that had been treated with HDM-pulsed WT BMDCs (WT,3.016 0.93 105; C5aR12/2 1.326 0.43 105; Fig. 8B), indicatingthat the migration of HDM-pulsed BMDCs from the alveolarspace to the pulmonary tissue over time is C5aR1 dependent. Thereduced number of BMDCs in the lung after transfer of C5aR12/2

cells was not due to an accelerated cell death, because the expres-sion levels of the anti-apoptotic molecule Bcl2 were even higher insorted C5aR12/2 BMDCs as compared with WT cells. Further-more, the expression level of the proapoptotic molecule BimEL wassimilar in BMDCs from WT or C5aR12/2 mice (Fig. 8C). Insummary, our data suggest that the mechanisms underlying thefailure of C5aR12/2 BMDCs to induce an allergic phenotype in-clude a reduced capability to migrate into the lung tissue associatedwith a decreased potency to direct the homing of effector T cellsinto the lung tissue.

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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 fromWTand C5aR12/2mice were cocultivated with CFSE-labeled CD4+ OVA-TCR–specific T cells. After 4 d, cell proliferation was evaluated by flow cytometry. Thedashed 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; thedark 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 byDAPI incorporation. The DAPI signal was measured in CD4+-gated cells. Histograms are representative of five independent experiments; values shown are the mean6 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 mean6SEM of four independent experiments. (D) Evaluation of the relative abundance of mRNA encoding for the transcription factors T-bet, RORgT, GATA3, and Foxp3relative to actin. Values shown are the mean6 SEM of six independent experiments. (E) IL-13 production in coculture supernatants after 9 d. Values shown are themean6 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 themean 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 toactin in sorted CD44+CD62L2 effector T cells. Values shown are the mean 6 SEM from four independent experiments.

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DiscussionDCs are considered necessary and sufficient to drive maladaptiveT cell immune responses and to establish experimental allergicasthma (3). Because the low frequency of naive pulmonary DCsmakes it difficult to use them for in vitro priming and adoptivetransfer studies, in vitro GM-CSF–differentiated BMDCs havebeen widely used in adoptive transfer studies as they promote thedevelopment 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 fol-lowing i.t. adoptive transfer, when pulsed in vitro with HDM.Previous studies suggested that C3a and C5a both act on pul-

monary cDCs and pDCs (9), thereby regulating the development ofmaladaptive Th2 and Th17 development. We therefore expectedthat the adoptive transfer of in vitro differentiated DCs, deficientin either the C3aR, the C5aR1, or both AT receptors, will definethe contribution of AT receptor signaling in cDCs and help tobetter understand the observations made in former studies, inwhich C3aR or C5aR1 receptors were deleted (13, 15–20, 26) orpharmacologically targeted during the sensitization or effectorphases (13, 23, 24). Such studies clearly showed opposing rolesfor C3a and C5a during the sensitization phase, that is, C3a actingas a proasthmatic and C5a as an anti-asthmatic mediator. Fur-thermore, C5a was found to promote proinflammatory propertiesduring the effector phase of asthma (13, 23, 24). To our surprise,we found that C5aR1 signaling in BMDCs is crucial for asthmadevelopment. In fact, HDM-pulsed BMDCs lacking C5aR1 ex-pression failed to induce an allergic phenotype, including AHR,mucus production, recruitment of inflammatory eosinophils andneutrophils, 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 withadjuvant in C5aR12/2 mice or after C5aR1 blockade (13) resultedin an enhanced allergic phenotype. We found no differences inMHC-II and costimulatory molecule expression between WT,C5aR1-, and C3aR-deficient BMDCs in vitro following HDMstimulation, suggesting that BMDCs from C5aR12/2 mice do notdiffer in Ag-induced regulation of the MHC-II, B7 family, orCD40 axes. In sharp contrast to previous studies that describeda decreased allergic phenotype with low airway resistance, re-duced airway inflammation, and low Th2 cytokine production inC3aR-deficent mice (17–19), we observed a strong asthmaticphenotype following adoptive transfer of HDM-pulsed BMDCsfrom C3aR2/2 mice. Several effects may account for the opposingeffects of C5a and C3a that we observed upon adoptive transfer ofBMDCs as compared with in vivo priming (either i.t. or i.p. withadjuvant) models. First, although BMDCs have often been usedas surrogates for pulmonary cDCs, there is growing evidencethat BMDCs inappropriately reflect the two different subsets ofCD11b+CD1032 and CD11b2CD103+ pulmonary resident cDCs(3). In fact, GM-CSF–differentiated BMDCs express markers ofinflammatory DCs (SIRP-1a+, CD11c+, and CD11b+), suggestingthat they resemble monocyte-derived DCs (43, 44) that arerecruited to the lung upon allergen-mediated inflammation, inparticular following i.t. or intranasal HDM administration (6).Importantly, CD11b+ pulmonary DCs are the main DC populationthat migrates to the draining lymph node during allergen sensiti-zation to stimulate Th2 differentiation of naive Th cells (6). Thispopulation of resident CD11b+ cDCs does not contribute to theinduction of Th2 immunity mediated by adoptive transfer ofBMDCs, as the CD4+ T cell priming is largely dependent on thei.t. injected DCs (45). Thus, an inhibitory impact of C5a on

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 adop-

tively 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.

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resident pulmonary cDCs will not affect the development of Th2immunity following adoptive transfer of HDM-pulsed BMDCsfrom C5aR12/2 mice. Secondly, HDM contains the cysteinaseprotease allergens Der p1 and 9 as well as small amount of LPS andfungal products (4, 46, 47), which act in concert to activate TLR4and C-type lectin receptors not only on DCs, but also on bronchialECs (48). The emerging concept is that ECs instruct pulmonarycDCs to induce Th2 differentiation by the production of cytokines,including IL-1, GM-CSF, thymic stromal lymphopoietin, IL-25, andIL-33. The functional relevance of C5aR1 signaling in ECs has beenshown in a model of IgG-induced lung injury. C5aR1 targeting inECs improves the inflammatory response (49). Upon stimulationwith C5a alone or synergistically with TLR4, rat ECs secrete largeamounts of TNF-a and CCL2 and to a lower extent IL-1b (50),supporting the idea that C5a can cooperate with TLR signaling inECs similar to what has been observed in APCs (14). C3a inducesthe expression of Muc5A and promotes the recruitment of inflam-matory cells by ECs (51). Also, the C-type lectin receptor Dectin-1negatively regulates C5aR1 (52) and C3aR signaling (J. Kohl, un-published observation). Clearly, the cross-talk between ECs andcDCs and the potential role of C3a and C5a in the regulation of theEC/cDC axis are lacking in the adoptive transfer model. Thirdly,C5aR1 signaling regulates alveolar macrophage (50, 53) and pul-monary pDC function (13, 18), both of which have been shown tosuppress the development of Th2 immunity by the induction of Tregcells (54, 55). The potential regulation of such pathways by C5aR1signaling is also not affected in the adoptive transfer model usingC5aR1-deficient BMDCs. Finally, recent data provide evidence thatGM-CSF–differentiated BM cultures from C5aR1-deficient micecontain higher numbers of myeloid-derived suppressor cells thataccount, at least in part, for the decreased potency of C5aR12/2

BMDC preparations to promote Th2 immunity upon adoptivetransfer (37). In search for additional mechanisms that may accountfor 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 themajority of BMDCs express C5aR1 (J. Kohl, unpublished obser-vation). Taken together, these data support a model in which C5a,locally produced within the pulmonary tissue, may contribute to thetransmigration of BMDCs from the alveolar space into the lung.Furthermore, we found a decreased potency of C5aR12/2 BMDCsto direct the homing of effector T cells into the lung tissue. At thispoint, we do not know at which level C5aR1 signaling in BMDCsmay affect the complex pathways that regulate the migration ofeffector T cell from the efferent lymph to the lung (56). This will beevaluated 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 re-sponse with high IL-4, IL-5, and IL-13. In addition to the Th2 re-sponse, we discovered a modest increase in IL-17A and in IFN-gproduction upon transfer of HDM-pulsed WT BMDCs, but no suchincrease upon transfer of HDM-pulsed C3aR2/2 BMDCs. This lackof IL-17A production was associated with a lack or a reduced IL-23production by C3aR2/2 or C5aR12/2 BMDCs stimulated withHDM in vitro. The failure of HDM-pulsed C3aR2/2 BMDCs topromote an increase in IL-17A production is in line with previousfindings demonstrating that C3a drives the differentiation of Th17cells by an IL-23–mediated mechanism (19). Of note, a recent studyshowed an opposite effect of C3a in a model of Aspergillus-medi-ated pulmonary allergy, that is, a high percentage of Th17 cells inthe lungs and high IL-17 in BAL, which was associated with highpulmonary neutrophil numbers. These data suggest a complex roleof C3a in Th17 development depending on the nature of the al-lergen (20). The low IL-23 production from C5aR12/2 BMDCs in

response to HDM challenge confirms our recent finding of low IL-23 production from such cells in response to OVA stimulation (9).However, Lajoie et al. (19) reported a significant increase in IL-23production upon HDM challenge of BMDCs from C5aR1-deficientmice. Similarly, we previously observed increased IL-23 productionwhen we stimulated spleen-derived cDCs with the TLR2 ligandPam3Cys (28). Differences in microbiota composition and diversitymay account for the different cytokine responses. It is well appre-ciated that identical mouse strains can differ significantly in theirmicrobiome composition depending on their housing conditions andthat such differences have a strong impact on the emergence ofparticular CD4+ T cell subsets, in particular with regard to the de-velopment of Th17 cells (57). In support of this view, C5aR1 sig-naling has been shown to modulate the cutaneous microbiome,which was associated with alterations in innate and adaptive im-mune cells in the skin (58). Similarly, we observed an alteredcomposition and diversity of the intestinal microbiome of C5aR12/2

mice as compared with WT littermates (J. Kohl, unpublishedobservation).Given the dominant effects of C5aR1 DC signaling on all

asthma-related parameters, we expected that double deficiency ofC5aR1 and C3aR will copycat the effects of C5aR1 alone.Interestingly, C3aR2/2/C5aR12/2 BMDC-transferred mice showedan intermediate asthmatic phenotype with reduced AHR at lowmethacholine concentrations, decreased eosinophilia associatedwith low pulmonary IL-4, IL-5, and IL-10 levels. Interestingly,we observed C3aR2/2/C5aR12/2-specific effects that we did notsee upon transfer of either C3aR- or C5aR1-deficient BMDCs.In vivo, we found increased production of IFN-g from lung cellsrestimulated with HDM. In vitro, we observed an increasedrelease of IL-6 upon HDM challenge. Furthermore, C3aR2/2/C5aR12 /2 BMDCs showed a higher expression of CD86 atsteady state and of CD86 and CD40 upon HDM stimulation.The higher potency of C3aR2 /2C5aR12 /2 BMDCs to driveIFN-g production as compared with WT DCs is surprising. Re-cently, 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 cognate-interacting T cells in vivo (29, 59), and that C3aR2/2/C5aR12/2

BMDCs have an impaired potency to drive the differentiation ofIFN-g–producing effector cells in vitro (27). Our data provideevidence that HDM-pulsed C3aR2/2C5aR12/2 BMDCs are per-fectly suited to promote differentiation of IFN-g–producing Th1effector cells when administered i.t. into WT mice. These datasuggest that the absence of C3aR and C5aR1 signaling on DCsdoes not necessarily result in a default pathway that promotes thedifferentiation of inducible Treg cells, but that the nature of the Agand the local cellular and humoral environment in the lung alsoplay 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 serveas an appropriate surrogate to define the role of AT receptor sig-naling in resident pulmonary cDCs with regard to their potency topromote maladaptive Th2 and Th17 immunity in experimental al-lergic asthma. It is well appreciated that C3aR and C5aR1 areexpressed on several innate immune cells relevant for the initiationof the allergic response such as pulmonary ECs, macrophages, aswell as resident cDCs and pDCs. To understand the complex role ofC3a and C5a during allergen sensitization, it will be important infuture studies to delineate the interplay between AT receptor acti-vation on such lung-resident cells and their regulatory impact oninnate pattern recognition receptors, including TLR, C-type lectinreceptors, and other G protein–coupled receptors.

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AcknowledgmentsWe thank E. Strerath and G. Kohl for excellent technical support.

DisclosuresThe authors have no financial conflicts of interest.

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