of June 27, 2016.This information is current as

Regulated by the Gut MicrobiomePulmonary Th17 Antifungal Immunity Is

K. KollsDean Sheppard, Wenjun Ouyang, Lora V. Hooper and JayVikram, Kyle Bibby, Yoshinori Umesaki, Amariliz Rivera, Armentrout, Derek A. Pociask, Aaron Hein, Amy Yu, AmitPawan Kumar, David M. Ricks, Matthew Binnie, Rachel A. Jeremy P. McAleer, Nikki L. H. Nguyen, Kong Chen,

http://www.jimmunol.org/content/197/1/97doi: 10.4049/jimmunol.15025662016;

2016; 197:97-107; Prepublished online 23 MayJ Immunol 

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6.DCSupplemental.htmlhttp://www.jimmunol.org/content/suppl/2016/05/20/jimmunol.150256

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, 24 of which you can access for free at: cites 61 articlesThis article

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is published twice each month byThe Journal of Immunology

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The Journal of Immunology

Pulmonary Th17 Antifungal Immunity Is Regulated by theGut Microbiome

Jeremy P. McAleer,*,1 Nikki L. H. Nguyen,*,† Kong Chen,* Pawan Kumar,*

David M. Ricks,*,† Matthew Binnie,‡ Rachel A. Armentrout,* Derek A. Pociask,*

Aaron Hein,* Amy Yu,† Amit Vikram,x Kyle Bibby,x,{ Yoshinori Umesaki,‖

Amariliz Rivera,# Dean Sheppard,** Wenjun Ouyang,†† Lora V. Hooper,‡‡ and

Jay K. Kolls*

Commensal microbiota are critical for the development of local immune responses. In this article, we show that gut microbiota can

regulate CD4 T cell polarization during pulmonary fungal infections. Vancomycin drinking water significantly decreased lung Th17

cell numbers during acute infection, demonstrating that Gram-positive commensals contribute to systemic inflammation. We next

tested a role for RegIIIg, an IL-22–inducible antimicrobial protein with specificity for Gram-positive bacteria. Following infection,

increased accumulation of Th17 cells in the lungs of RegIIIg2/2 and Il222/2 mice was associated with intestinal segmented

filamentous bacteria (SFB) colonization. Although gastrointestinal delivery of rRegIIIg decreased lung inflammatory gene ex-

pression and protected Il222/2 mice from weight loss during infection, it had no direct effect on SFB colonization, fungal

clearance, or lung Th17 immunity. We further show that vancomycin only decreased lung IL-17 production in mice colonized

with SFB. To determine the link between gut microbiota and lung immunity, serum-transfer experiments revealed that IL-1R

ligands increase the accumulation of lung Th17 cells. These data suggest that intestinal microbiota, including SFB, can regulate

pulmonary adaptive immune responses. The Journal of Immunology, 2016, 197: 97–107.

CD4+ Th cells orchestrate pulmonary host defense by dif-ferentiating into specialized subsets (1). IL-12 induces thegeneration of IFN-g–secreting Th1 cells, whereas the

combination of IL-6 plus TGF-b induces IL-17–producing Th17cells. Although the cytokines that program Th differentiation arefairly well defined, less is known regarding the regulation of Thimmunity in vivo. Studies on opportunistic fungal infections illus-trated complementary functions for effector Th subsets. Themechanisms through which CD4 T cells control pulmonary infec-tions include stimulating antimicrobial protein release from epi-thelium, macrophage activation, and neutrophil recruitment (1).Aspergillus fumigatus is a ubiquitous spore-forming soil fungus

that people inhale every day (2). Once conidia reach the alveoli, theyare usually destroyed by macrophages and neutrophils within 24–48 h.Immunocompromised patients are susceptible to invasive aspergillo-

sis, during which conidia germinate into hyphae and disseminate toother tissues, such as the brain, kidneys, or heart (2). Th1 cells areprotective against aspergillosis (3), whereas Th17 cells are criticalfor vaccine-induced antifungal immunity (4). These powerful inflam-matory responses to fungi can also cause pathology. For instance,chronic exposure to conidia may lead to allergic bronchopulmonaryaspergillosis in cystic fibrosis or asthma patients, corresponding tostrong Th2 responses (1, 2). A. fumigatus is the most prevalent causeof severe pulmonary allergic disease (5). In chronic granulomatousdisease, defective superoxide generation in neutrophils leads toincreased IL-17 production, which exacerbates acute lung injuryduring A. fumigatus infection (6). Regulatory T cells (Tregs) are in-duced by the tryptophan metabolite kynurenine, reducing lung damageduring aspergillosis but also potentially increasing fungal persistence(6). Understanding the mechanisms that regulate inflammatory

*Richard King Mellon Foundation Institute for Pediatric Research, Children’s Hos-pital of Pittsburgh of University of Pittsburgh Medical Center, Pittsburgh, PA 15224;†Department of Genetics, Louisiana State University Health Sciences Center, NewOrleans, LA 70112; ‡Division of Respirology, Department of Medicine, Universityof Toronto, Ontario M5B 1W8, Canada; xDepartment of Civil and EnvironmentalEngineering, University of Pittsburgh, Pittsburgh, PA 15261; {Department of Com-putational and Systems Biology, University of Pittsburgh, Pittsburgh, PA 15260;‖Yakult Central Institute for Microbiological Research, Kunitachi-shi, Tokyo 186-8650, Japan; #Department of Pediatrics, Center for Immunity and Inflammation,New Jersey Medical School, Newark, NJ 07101; **Lung Biology Center, Universityof California, San Francisco, San Francisco, CA 94143; ††Department of Immunology,Genentech, South San Francisco, CA 94080; and ‡‡Department of Immunology,Howard Hughes Medical Institute, The University of Texas Southwestern MedicalCenter, Dallas, TX 75390

1Current address: Department of Pharmaceutical Science and Research, MarshallUniversity School of Pharmacy, Huntington, WV.

ORCIDs: 0000-0003-1522-4122 (P.K.); 0000-0002-6266-7105 (D.M.R.); 0000-0001-8665-1389 (M.B.); 0000-0002-1256-6410 (D.A.P.); 0000-0002-8867-6122 (A.H.);0000-0002-1144-3371 (A.Y.); 0000-0003-3142-6090 (K.B.); 0000-0003-2422-9187(Y.U.); 0000-0002-6277-2036 (D.S.).

Received for publication December 9, 2015. Accepted for publication April 25, 2016.

This work was supported by National Institute of Allergy and Infectious Dis-eases, National Institutes of Health Award R37-HL079142 (to J.K.K.) andGrant R01 DK070855 (to L.V.H.); J.P.M. was supported in part by FellowshipF32AI096772. L.V.H. also receives support from the Howard Hughes MedicalInstitute.

The sequences presented in this article have been submitted to the MetagenomicsRAST server under accession number 4541676.3.

The content is solely the responsibility of the authors and does not necessarilyrepresent the official views of the National Institutes of Health.

Address correspondence and reprint requests to Dr. Jay K. Kolls, Children’s Hospitalof Pittsburgh of University of Pittsburgh Medical Center, Rangos Research Building,4401 Penn Avenue, Pittsburgh, PA 15224. E-mail address: jay.kolls@chp.edu

The online version of this article contains supplemental material.

Abbreviations used in this article: B6, C57BL/6; BSS, balanced salt solution; DC,dendritic cell; GF, germ-free; IL-1RA, IL-1R antagonist; o.p., oropharyngeal; OTU,operational taxonomic unit; SFB, segmented filamentous bacteria; siLP, small intes-tinal lamina propria; Treg, regulatory T cell; WT, wild-type.

Copyright� 2016 by TheAmerican Association of Immunologists, Inc. 0022-1767/16/$30.00

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T cell cytokine production toward A. fumigatus has substantialtherapeutic value in invasive disease, as well as fungal allergy.Commensal bacteria residing in the intestinal lumen have con-

siderable effects on host defense. Studies in germ-free (GF) micerevealed that commensals protect against pathogenic infectionsthrough competitive growth inhibition or stimulation of innate im-mune receptors (7). Colonization of the gut is critical for the mat-uration of lymphoid tissues, such as Peyer’s patches, facilitatinginteractions between B cells and T cells (8). Although helpful forcontrolling infections, activated T cells in the gut also have thepotential to drive colitis (9, 10). These studies demonstrated thatmicrobial products, as well as T cell–intrinsic properties, influenceintestinal inflammation. Microbiota also affect immune responses inperipheral tissues (11) and contribute to systemic antiviral immunity(12–14). Loss of microbial diversity was also implicated in allergicinflammation (15). Altogether, this suggests that host factors thatmodulate microbial composition in the gut may also regulate pe-ripheral T cell activation during infectious challenges.The intestinal mucosa provides a physical barrier against

microbes, maintained by the generation of a mucus layer, anti-microbial proteins, and IgA (16). Paneth cells shape the compo-sition of intestinal microbiota by producing antimicrobial peptides.For instance, a-defensin 5 increases the ratio of Bacteroidetes/Firmicutes in the distal small intestine (17). RegIIIg is producedby Paneth cells in response to IL-22 and has bactericidal activityagainst Gram-positive organisms (18, 19). RegIIIg contributes tomaintaining the spatial separation between microbiota and theintestinal cell wall (20). In the absence of RegIIIg, the microbiotais enriched for segmented filamentous bacteria (SFB), which aresufficient to induce Th17 cells (21, 22). Although RegIIIg protectsagainst Gram-positive pathogens in the gut (19, 23), its impact onpulmonary immunity to fungal pathogens is unknown.In this study, we investigated a role for commensal bacteria

in lung immunity to A. fumigatus. We found that vancomycin-sensitive bacteria amplify lung IL-17 production early followingfungal infection. Endogenous expression of RegIIIg or Il22 waslinked to decreased intestinal SFB colonization, leading to de-creased IL-17 production in the lungs and small intestine. Thephenotype of expanded CD4+ IL-17+ cells in Il222/2 mice wastransferrable with intestinal microbiota or serum to wild-type(WT) mice. Preincubation of serum with an IL-1R antagonist (IL-1RA) blunted the IL-17 response, demonstrating that commensalsinduce serum IL-1R ligands that contribute to lung IL-17 pro-duction. Finally, reconstituting Il222/2 mice with local RegIIIgprotected them from weight loss during A. fumigatus infection andreduced inflammatory cytokines in lung tissue. Thus, factors thatshape the balance of intestinal commensal species regulate lunginflammatory responses, including Th17 cell priming.

Materials and MethodsMice

C57BL/6 mice (B6) from The Jackson Laboratory (Bar Harbor, ME;Figs. 2, 3, 5D) or Taconic Farms (Germantown, NY or Cambridge City,IN; Figs. 1, 4, 5A–C, 5E, 6) were used. OT-II mice were purchased fromThe Jackson Laboratory. Il222/2 mice (24) were bred at Taconic Farms(Cambridge City, IN). Mice with a targeted deletion for all six exons ofRegIIIg were generated as described below and backcrossed to B6 mice for10 generations. Mice were housed at the Louisiana State University HealthSciences Center and the University of Pittsburgh under specific pathogen–free conditions and were handled in accordance with National Institutes ofHealth federal guidelines.

Generation of RegIIIg2/2 mice

Regions from the mouse RegIIIg locus were amplified from genomic DNAby PCR and cloned into the pDelboy 3X targeting vector. The fragments

included a 3.5-kb 59 flanking region ending at the RegIIIg transcriptionstart site, a 2.5-kb fragment cloned between two loxP sites and comprisingall six exons of the RegIIIg gene, and a 3.4-kb 39 flanking region. Thetargeting vector also contains a neomycin resistance gene, a thymidylatekinase gene, and a b-gal gene. Cre-mediated recombination between theloxP sites results in deletion of all coding portions of the RegIIIg gene andbrings the b-gal gene immediately downstream of the RegIIIg 59 promoter.

The vector was linearized and electroporated into embryonic stem cells,and clones underwent positive and negative selection with G418 andganciclovir. Successfully targeted clones were analyzed by PCR andSouthern blot, and three successfully targeted clones were identified. Oneclone was microinjected into blastocysts using standard techniques, and theresulting chimeric mice were bred to generate heterozygous mutant off-spring. Male RegIIIgfl/+ heterozygotes were crossed with Tie2 Cre femalesto generate RegIIIg+/2 offspring. These were interbred to generate RegIIIg2/2

offspring and were also backcrossed onto the B6 background for 10generations.

By quantitative PCR, RegIIIg2/2 mice showed a mean 240-fold lowerexpression of RegIIIg mRNA in the trachea compared with control mice.Because these mice have no detectable RegIIIg coding region, we believethat the residual RegIIIg signal is from transcripts of other highly relatedReg family genes, including RegIIIb.

Infections and treatments

Mice were infected with A. fumigatus via oropharyngeal (o.p.) aspiration(107 organisms). For some experiments (Figs. 1, 4B–E, 5B–E, 7), neutrophilswere depleted by i.p. injection of Ab 1A8 (0.6 mg; Bio X Cell, West Lebanon,NH) 1 d prior to infection. Serum was given i.v. (0.25 ml) in the presence ofanakinra (Kineret; Swedish Orphan Biovitrum; Stockholm, Sweden) or ve-hicle 7 h following infection. Vancomycin was administered in drinking waterat 0.5 g/l for$ 4 wk and then mice were placed on normal water 1 d prior toinfection. Il222/2 mice were gavaged with rRegIIIg protein (40 mg) (25) orvehicle every other day, beginning on day 29 prior to infection, for a total ofsix treatments. For fecal-transplant experiments, stool pellets were homoge-nized in water and poured over a 100-mm cell strainer, and recipient micewere gavaged with 0.4 ml fecal matter.

To examine pulmonary Th17 cell priming, TCR-transgenic OT-II T cellswere negatively selected using a CD4 T Cell Isolation Kit (Miltenyi Biotec,Bergisch Gladbach, Germany), and 0.5 million cells were adoptivelytransferred into mice via retro-orbital injection. The next day, mice wereinoculated via o.p. aspiration with OVA (100 mg; Sigma-Aldrich, St. Louis,MO) plus cholera toxin (1 mg; List Biological Laboratories, Campbell, CA).Lungs were collected on day 7. To examine intestinal Th17 cell priming,mice were injected i.p. with OVA (1 mg) at time 0 and Escherichia coli–derived LPS (50–60 mg; Sigma-Aldrich) 18 h later. Small intestinal laminapropria (siLP) lymphocytes were isolated 3 wk later.

Tissue processing

To analyze gene expression, lungs and distal small intestine were placed inTRIzol reagent (Life Technologies, Carlsbad, CA), homogenized, and pro-cessed according to the manufacturer’s protocol. One microgram of RNAwasused to synthesize cDNA (iScript; Bio-Rad, Hercules, CA). Real-time PCRprimers were purchased from Applied Biosystems (Foster City, CA) andused with TaqMan Universal PCR Master Mix (Applied Biosystems) orSsoFast Probes Supermix (Bio-Rad). PCRs were run on a Bio-Rad CFX96Real-Time System.

For ex vivo cytokine production, tissues were placed in cell lysis buffer(Cell Signaling Technology, Danvers, MA) with protease inhibitor mixturetablets (Roche Applied Science, Penzberg, Germany), homogenized, andcentrifuged at 10,000 3 g. Cytokine levels in supernatants were measuredby ELISA (eBioscience, San Diego, CA; BioLegend, San Diego, CA) orMilliplex Mouse Cytokine/Chemokine Immunoassay (EMD Millipore,Billerica, MA) and normalized to total protein levels (Pierce BCA ProteinAssay Kit; Thermo Fisher Scientific, Waltham, MA).

For flow cytometry, lungs were digested with collagenase D (3 mg/ml;Roche Applied Science) in DMEM for 1 h at 37˚C, crushed through 100-mmcell strainers (BioExpress, Kaysville, UT), and treated with ammoniumchloride to lyse RBCs. siLP lymphocytes were isolated as previously de-scribed (26), with some modifications. Briefly, small intestinal tissue wasflushed, sliced open longitudinally, cut into ∼1-cm pieces, and washed withCa/Mg-free balanced salt solution (BSS). Tissue was stirred at 37˚C in Ca/Mg-free BSS containing 5 mM EDTA and 0.15 mg/ml dithioerythritol, withthe supernatant removed. Then, tissue was incubated at 37˚C in BSS con-taining 1 mM CaCl2, 1 mM MgCl2, and 1 mg/ml collagenase D (RocheApplied Science). Cells were fractionated on a 44% and 67% Percoll gra-dient (Amersham Biosciences, Piscataway, NJ), with lymphocytes parti-tioning at the interface. Single-cell suspensions were incubated in IMDM

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with 10% FBS, PMA (50 ng/ml; Sigma-Aldrich), ionomycin (750 ng/ml;Sigma-Aldrich), and GolgiPlug (Becton Dickinson Pharmingen, FranklinLakes, NJ) for 4 h at 37˚C. Cells were stained with Abs directed againstCD3ε, CD4, CD8, TCR Va2, TCR gd, IL-17A, IL-22, or IFN-g (eBiosci-ence and BD Pharmingen).

For in vitro cytokine production, splenic dendritic cells (DCs) werepurified using CD11c MicroBeads (Miltenyi Biotec). Cells were stimulatedovernight with heat-killed swollen A. fumigatus (10:1 organisms/DC), LPS(50 ng/ml), or zymosan (100 mg/ml; InvivoGen). Cytokine levels weredetermined by an Milliplex Mouse Cytokine/Chemokine Immunoassay(EMD Millipore).

Microbiota analysis

Real-time PCR. DNA from small intestinal stool or cecum was isolatedusing a QIAamp DNA Stool Mini Kit (QIAGEN, Venlo, the Netherlands),and 90–180 ng was used as a template for PCR. In some experiments,cDNA from distal small intestine was used as a template. Primers forEubacteria, SFB (27), Bacteroides, Clostridium leptum (28), and Rumino-coccaceae were purchased from Integrated DNA Technologies (Coralville,IA). Samples were diluted with RNase/DNase-free water and mixed withiQ SYBR Green Supermix (Bio-Rad). PCRs were performed on a CFX96Real-Time System (Bio-Rad). Relative gene expression for bacteria species(SFB, Bacteroides, C. leptum) was normalized to Eubacteria.

16S next-generation sequencing.DNA from cecal contents was prepared forsequencing as previously described (29). Briefly, DNA was amplified byPCR using 515F and 806R bacterial 16S PCR primers containing sample-specific barcodes and sequence adaptors. PCR products were pooled inequimolar amounts and sequenced on an Illumina MiSeq using a 300 cycleV2 sequencing kit. Sequences were processed in the QIIME pipeline (30).Sequences were split and trimmed to a minimum quality score of 20, alignedto the reference greengenes database (31) using uclust (32) and a referenceoperational taxonomic unit (OTU) threshold of 97%, and analyzed fortaxonomic and a diversity. Data are available on the MetagenomicsRAST server under accession number 4541676.3 (http://metagenomics.anl.gov/metagenomics.cgi?page=MetagenomeOverview&metagenome=4541676.3).

Histopathology

B6-Taconic mice on normal water or vancomycin water and Il222/2 micewere injected i.p. with a neutrophil-depleting Ab (1A8; 0.6 mg) 1 d prior too.p. infection with A. fumigatus. Two days later, lungs were inflated withformalin and stained with H&E. The severity of disease was scored by apathologist (D.A.P.) blinded to the groups. Individual fields were scored at3200 magnification, with a minimum of 20 fields/slide. Scoring in eachfield was based on the percentage of inflammation: 0 = no inflammation,1 = 1–25% inflammation, 2 = 26–50% inflammation, 3 = 51–75% inflam-mation, and 4 = 76–100% inflammation.

DC/T cell cocultures

Naive CD4 T cells (CD4+ CD62L+) and CD11c+ cells were isolated fromspleens and lungs, respectively, using magnetic bead selection (MiltenyiBiotec). T cells and DCs were mixed at a 2.4:1 ratio (Supplemental Fig. 3A)or a 3:1 ratio (Supplemental Fig. 3B) in IMDM plus 10% FBS for 3 d.Cultures were treated with heat-killed swollen A. fumigatus, anti-CD3 (1 mg/ml),or A. fumigatus hyphae or were left unstimulated.

Statistical analysis

A two-tailed Student t test was used when variance between the groups wassimilar, as determined by an F test. For comparing groups with an unequalvariance, a two-tailed Mann–Whitney U test was performed.

ResultsCommensal bacteria amplify IL-17 production in lungs duringfungal infection

To study the role of commensal bacteria in pulmonary antifungalimmunity, B6 mice (Taconic Farms) known to be colonized withSFB (22) were placed on drinking water containing vancomycin(0.5 g/l) for $4 wk and then infected with A. fumigatus (107 organ-isms) via o.p. aspiration. A. fumigatus is an opportunistic pathogenthat colonizes immunocompromised patients. To mimic this situation,mice were injected with the neutrophil-depleting Ab 1A8 1 d prior toinfection. Although vancomycin treatment did not affect A. fumigatusburden in the lungs at early time points, there was a substantial

reduction in lung IL-17 and IL-22 levels during this acute infection(Fig. 1). In contrast, IFN-g was not affected by vancomycin,whereas IL-4 was significantly increased. This suggested thatcommensal bacteria in the intestine preferentially increase theproduction of cytokines associated with type 17 immunity in thelung and decrease type 2 responses. Overall, vancomycin treat-ment did not affect disease severity determined by H&E-stainedlung sections at the 48-h time point (Supplemental Fig. 1A),possibly as a result of neutrophil depletion. Intestinal colonizationinduces production of antimicrobial proteins, including the RegIIIfamily (18). Accordingly, vancomycin diminished the expression ofRegIIIb and RegIIIg in small intestinal tissue but not in the lungs(Fig. 1B). Therefore, vancomycin-sensitive commensals (i.e., Gram-positive bacteria) influence the expression of local antimicrobialfactors in the gut, as well as type 17 cytokines in the lung.

Endogenous RegIIIg suppresses lung IL-17 production duringfungal infection

The bactericidal activity of RegIIIg is attributed to its binding ofcell wall peptidoglycan on Gram-positive bacteria (18). Becauseoral vancomycin treatment potently decreased IL-17 and IL-22production in lung tissue (Fig. 1A), and RegIIIg2/2 mice werereported to have increased colonization with SFB (20), we hy-pothesized that the endogenous antimicrobial factor RegIIIg maysimilarly regulate pulmonary Th17 cytokines. Of note, our line ofRegIIIg2/2 mice was colonized with SFB, as previously reportedfor an independent line (20). To test our hypothesis, SFB+

RegIIIg2/2 and B6 Jackson mice that lacked SFB were infectedwith A. fumigatus via o.p. aspiration. Lungs were harvested 2 dlater and analyzed for gene expression by real-time PCR. Pul-monary fungal burden was similar between WT and RegIIIg2/2

mice, suggesting that RegIIIg was dispensable for antifungal hostdefense (Fig. 2A, A.f.). However, expression of the Th17-relatedgenes Il17a, Il17f, and Il22 was elevated in the lungs of RegIIIg2/2

mice, indicating more activated T cells. To determine which T cellsubsets contribute to IL-17 production, lymphocytes were isolatedfrom lungs 1 mo following infection, restimulated in vitro withPMA plus ionomycin, and analyzed by flow cytometry. Approx-imately 1% of total CD3+ T cells in WT mice produced IL-17A(Fig. 2B). In RegIIIg2/2mice, ∼2% of total T cells produced IL-17,indicating that SFB colonization in RegIIIg2/2 mice was linkedto increased pulmonary Th17 cell priming and/or maintenance(Fig. 2B). This trend was consistent when analyzing the individualCD4+, CD8+, and TCR gd+ T cell subsets. Thus, lung-residentT cells from infected RegIIIg2/2 mice had a 2-fold increase inIL-17 potential compared with T cells isolated from WT mice.

Microbiota in RegIIIg-deficient mice regulate pulmonary Th17cell priming

To test whether intestinal Gram-positive bacteria drive pulmonaryTh17 cell priming in RegIIIg2/2 mice, they were placed on van-comycin drinking water for $4 wk. In one of two experiments,vancomycin was combined with ampicillin, metronidazole, andneomycin, whereas control mice were maintained on normalwater. Following antibiotic treatment, mice were adoptivelytransferred with TCR-transgenic OT-II CD4 T cells. One day later,mice were immunized with OVA plus the mucosal adjuvantcholera toxin (33) via o.p. aspiration. On day 7, lung single-cellsuspensions were stimulated with PMA plus ionomycin and ana-lyzed for IL-17 production. The total number of OT-II CD4+

T cells recovered from the lungs of RegIIIg2/2 mice on normalwater was 1.8-fold higher compared with WT Jackson mice(Fig. 2C). Vancomycin drinking water significantly reduced lungT cell accumulation in RegIIIg2/2 mice to similar numbers as WT

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mice, demonstrating that the gut microbiota in RegIIIg2/2 micecontributes to pulmonary Th17 cell accumulation. In contrast,vancomycin did not significantly affect Th17 cells in WT micefrom The Jackson Laboratory that were not colonized with SFB,suggesting that their microbiota did not influence Th17 cell ac-cumulation in the lungs (Fig. 2D). These data demonstrate thatgut microbiota in RegIIIg2/2 mice inhibits pulmonary Th17 cellpriming in a T cell–extrinsic manner.To further test the link between SFB colonization and pulmonary

cytokines, we purchased SFB2 RegIIIg2/2 mice from The JacksonLaboratory. Following neutrophil depletion and infection, lung IL-17A, IL-22, IFN-g, and IL-10 were comparable to B6 Jacksonmice (Supplemental Fig. 2, top panel). Fungal burdens inRegIIIg2/2 Jackson mice were elevated slightly compared withB6 Jackson mice, but the difference was not statistically different(Supplemental Fig. 2, middle panel). Thus, elevated IL-17 pro-

duction in RegIIIg2/2 mice (Fig. 2) was linked to SFB coloni-zation.

RegIIIg suppresses intestinal Th17 cell priming

Because RegIIIg is highly expressed by intestinal epithelium (18),we next determined its effect on intestinal Th cell priming to themodel Ag OVA. Mice were immunized i.p. with OVA plus LPS.Three weeks later, lymphocytes from siLP were restimulatedin vitro with PMA plus ionomycin. The total numbers of CD4+

T cells isolated from siLP were 8-fold higher in RegIIIg2/2 micecompared with WT Jackson mice (Fig. 3B), suggesting that endog-enous RegIIIg suppresses T cell priming in the intestinal mucosa.This was accompanied by increased total numbers of CD4+ T cellsproducing the effector cytokine IL-17, IL-22, or IFN-g. Notably,immunized RegIIIg2/2 mice had 32-fold more Th17 cells than WTmice. As noted above, RegIIIg limits colonization with SFB by

FIGURE 1. Commensal bacteria

amplify lung IL-17 production

during fungal infection. B6 mice on

vancomycin drinking water for 1

mo or maintained on normal water

were injected i.p. with the neutro-

phil-depleting Ab 1A8, followed by

o.p. infection with A. fumigatus the

next day. Tissues were collected on

day 2. (A) Cytokine levels in lung

homogenates normalized to total

protein. (B) Gene expression in distal

small intestine (left panel) or lung

tissue (right panel) normalized to

Hprt. Data are combined from two

experiments with n = 10. *p , 0.05,

**p , 0.01, ***p , 0.001.

FIGURE 2. RegIIIg inhibits pulmonary type 17 im-

munity by modulating intestinal microbiota. B6 Jackson

mice (black bars) or RegIIIg2/2 mice (white bars) were

infected with A. fumigatus via o.p. aspiration. (A) Lung

gene expression on day 2 normalized to Hprt (n = 5). (B)

The percentage of IL-17–producing cells within indi-

vidual T cell populations 1 mo after infection (n = 3–4).

(C and D) Mice on vancomycin drinking water for 1 mo

or normal water were adoptively transferred with OT-II

cells, followed by o.p. immunization with OVA plus

cholera toxin. One week later, lung cells were restimu-

lated and stained for intracellular IL-17. (C) Total

numbers of lung-resident OT-II T cells (CD4+ TCR

Va2+). (D) Total numbers of IL-17–producing OT-II

CD4 T cells. Data in (C) and (D) are combined from two

experiments with n = 6–8. In one experiment, vanco-

mycin was combined with neomycin, metronidazole,

and ampicillin. *p , 0.05, **p , 0.01, ****p , 0.0001.

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maintaining the spatial separation between commensal microbes andmucosal epithelia (20). Our RegIIIg2/2 mice were also colonizedwith SFB prior to (data not shown) and after (Fig. 3C) immunization,correlating with increased percentages of CD4+ T cells producingIL-17 (Fig. 3A). Unimmunized RegIIIg2/2 mice contained 2.5- and3-fold more Th17 cells in the siLP and spleen, respectively, com-pared with unimmunized WT mice (data not shown). Altogether,these data demonstrate an association between SFB colonization inRegIIIg2/2 mice and preferential induction of the Th17 pathway inmucosal tissues.

Endogenous IL-22 expression suppresses lung IL-17 levelsduring fungal infection

RegIIIb and RegIIIg expression in intestinal epithelium is regu-lated by IL-22 during gastrointestinal infections (19). In thesteady-state, we observed that some naive Il222/2 mice expressedlower levels of RegIII family members in the distal small intestinecompared with WT Taconic controls, although this was not truefor all mice examined. However, on a consistent basis, we foundthat Il222/2 mice had increased levels of cecal SFB colonizationcompared with WT Taconic mice (Fig. 4A). If SFB influencespulmonary IL-17 responses, we reasoned that SFB-colonizedIl222/2 mice should have a similar phenotype as the RegIIIg2/2

strain (Figs. 2, 3). To test this, WT-Taconic and Il222/2 mice wereinfected with A. fumigatus via o.p. aspiration. One day prior toinfection, neutrophils were depleted by i.p. injection of Ab 1A8.To determine whether any potential differences between themouse strains were influenced by gut microbiota, some mice wereplaced on vancomycin-drinking water for 1 mo prior to the ex-periment. On day 2, Il222/2 mice had a 1.5-fold increase in lungIL-17 compared with WT Taconic mice (Fig. 4B), consistent withthe results observed in RegIIIg2/2 mice colonized with SFB(Fig. 2). Vancomycin drinking water decreased IL-17 levels by25% in Il222/2 mice, suggesting that IL-22–mediated alterationsin intestinal flora regulate pulmonary IL-17 production. In support,vancomycin water significantly decreased SFB colonization inIl222/2 mice (Fig. 4E). In B6 control mice, vancomycin decreased

lung IL-17 levels in only one of two experiments (Fig. 4B). Furtheranalysis revealed that B6 Taconic mice were SFB2 in one of theexperiments. Thus, vancomycin only decreased lung IL-17 levels inSFB-colonized mice. Levels of C. leptum and Ruminococcaceaewere similar between WT and Il222/2 mice and were decreased byvancomycin. This is consistent with a study demonstrating thatseveral Clostridium spp., including Ruminococcus, are sensitive tovancomycin (34). Therefore, lung IL-17 levels correlated with in-testinal SFB colonization. When analyzing cytokines in the smallintestine, Il222/2 mice on normal water had a dramatic 3000-foldincrease in Il17a expression compared with WT controls, whichwas reduced by 99.8% upon pretreatment with vancomycin drink-ing water (Fig. 4C). There was also a 3.7-fold increase in Ifngexpression in the small intestine of Il222/2 mice, which was de-creased by vancomycin. Expression of Il4 and Foxp3 was similarbetween WT Taconic and Il222/2 mice in the intestine and lung(Fig. 4C, 4D), suggesting that endogenous IL-22 did not affect Th2or Treg differentiation. Lung Il17a expression correlated with Il1ain the lungs and small intestine of Il222/2 mice (Fig. 4C, 4D).Therefore, the absence of IL-22 creates a permissive environmentfor SFB growth, augmenting mucosal IL-17 production in the gutand lung.To examine the pulmonary T cell subsets affected by microbiota,

B6 Taconic mice on vancomycin water and normal water wereinfected with A. fumigatus via o.p. aspiration. Two days later, lungsingle-cell suspensions were stimulated with PMA plus ionomycinand stained for intracellular IL-17. Vancomycin significantly de-creased the number of lung CD4+ cells producing IL-17 but didnot change the number of gd T cells making IL-17 (Fig. 5A).Next, the impact of IL-22–conditioned microbiota was assessed byreturning vancomycin-treated mice to normal water, followed by afecal transplant from B6 Taconic mice (low SFB) or Il222/2 donormice (high SFB). Fifteen days following the microbiota transfer,mice were depleted of neutrophils and infected with A. fumigatusthe next day. Recipients of microbiota from SFB-high colonizedIl222/2 mice had significantly greater CD4+ IL-17+ cells, whereasthe numbers of gd+ IL-17+ cells were not affected (Fig. 5B).

FIGURE 3. RegIIIg suppresses intestinal Th17 cell

priming. Mice were injected i.p. with OVA plus LPS.

Three weeks later, siLP lymphocytes were stimulated

with PMA plus ionomycin. (A) Flow cytometry plots of

IL-17 versus IFN-g staining, gated on CD4 T cells. (B)

Total numbers of siLP CD4 T cells producing IL-17, IL-

22, or IFN-g. Data in (A) and (B) are combined from two

experiments with n = 6. (C) SFB colonization deter-

mined by PCR from small intestinal cDNA, normalized

to Eubacteria. Data are combined from four experiments

with n = 8–9. **p , 0.01.

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Notably, the number of lung CD4+ IL-17+ cells directly correlatedwith SFB colonization in the small intestine (Fig. 5C) and thececum (data not shown). A previous study (35) found thatmicrobiota-induced IL-1b increases intestinal Th17 cell numbers.We found that SFB-high microbiota from Il222/2 donors in-creased Il1a/b gene expression in the lungs following fungal in-fection (Fig. 5E). Our data demonstrate that vancomycin-sensitivebacteria in the gut augment IL-17 production by CD4+ T cellsduring pulmonary fungal infection and that increased Th17 re-sponses observed in Il222/2 mice are transferrable with SFB-containing microbiota.

IL-22 suppresses pulmonary Th17 cell accumulation throughserum IL-1R ligands

We next addressed a potential role for SFB-containing microbiotain the regulation of lung DC function. In vitro, lung CD11c+ cells

from B6 Taconic mice on normal water or vancomycin werenearly equivalent at inducing IL-17 from polyclonal naive CD4T cells in the presence of heat-killed A. fumigatus (SupplementalFig. 1B). This suggested that lung DCs from vancomycin-treatedmice can mount a normal response to A. fumigatus in vitro. Insupport, overnight stimulation of lung CD11c+ cells from B6Taconic mice on normal water or vancomycin resulted in similarsecretion of IL-1a, IL-1b, and IL-6 (Supplemental Fig. 1D). Thiscontrasted with what we observed for splenic DCs; vancomycin-sensitive bacteria were required for optimal cytokine production inresponse to heat-killed A. fumigatus, zymosan, or LPS (Fig. 6).In vivo, we found that infection increased the number of lungCD11c+ CD11b+ cells similarly in B6 Taconic, B6 plus vanco-mycin, and Il222/2 mice (Supplemental Fig. 1E). Further, SFBcolonization did not impact IL-1a or IL-1b production by lungAPCs, as measured by intracellular cytokine staining. If SFB

FIGURE 4. IL-22 suppresses lung IL-17 levels during fungal infection. (A) SFB and C. leptum expression from cecum stool DNA of untreated WT-

Taconic and Il222/2 mice normalized to Eubacteria. Data (mean 6 SEM) are combined from four experiments with n = 17. (B–E) WT and Il222/2 mice

were placed on vancomycin drinking water for 1 mo (gray bars) or maintained on normal water. Then, all mice were placed on normal water and injected

i.p. with 1A8 Ab. The next day, mice were infected with A. fumigatus. (B) IL-17 levels in lung homogenate on day 2, normalized to total protein. Real-time

PCR for the indicated genes in distal small intestine (C) or lung (D) on day 2, normalized to Hprt. (E) Real-time PCR on small intestine cDNA for SFB,

C. leptum, or Ruminococcaceae 16S rRNA, normalized to Eubacteria. Data in (B)–(E) are combined from two experiments with n = 7–9. *p , 0.05, **p ,0.01, ***p , 0.001, ****p , 0.0001.

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colonization activates lung DCs, then Il222/2 DCs would be morepotent than B6 at inducing Th17 differentiation. Coculture of lungCD11c+ cells with A. fumigatus–specific naive T cells revealedthat IL-17 secretion in vitro did not correlate with SFB colonizationin DC donor mice (Supplemental Fig. 1C). Overall, these datasuggest that lung DCs were not directly influenced by gastroin-testinal microbiota, and additional factors contribute to IL-17production following pulmonary fungal infection.To determine the role of serum factors in pulmonary Th17 cell

accumulation, SFB2 B6 Jackson mice were infected withA. fumigatus and adoptively transferred with serum from naive B6Taconic or Il222/2 SFB-colonized mice 7 h later. Some groups ofmice received serum premixed with IL-1RA (anakinra), becauseIl1a expression was upregulated in the gut of Il222/2 mice in-fected with A. fumigatus (Fig. 4C). Two days following infection,lung single-cell suspensions were restimulated with PMA plusionomycin and stained for intracellular IL-17A. Mice receivingsera from SFB-colonized Il222/2 mice had increased numbers ofCD4+ IL-17+ T cells (Fig. 5D). This was dependent on IL-1Rsignaling, because preincubation of Il222/2 serum with IL-1RAsignificantly decreased Th17 cell numbers, as well as TCR gd+ IL-17+

cells. Notably, donor serum from Il222/2 mice contained elevatedlevels of IL-1a protein (Il222/2 serum: 9586 198 pg/ml; B6 Taconicserum: 5016 35 pg/ml), whereas IL-1b protein levels were similar toB6 Taconic (Il222/2 serum: 996 4 pg/ml; B6 serum: 1076 3 pg/ml).In contrast to the role of IL-1R signaling, systemic IL-23 adminis-

tration did not significantly affect IL-17 levels in the lung on day 2(data not shown). Overall, these data demonstrate that IL-22modulates serum IL-1R ligands that augment lung Th17 cell ac-cumulation during fungal infection.

Intestinal RegIIIg delivery is anti-inflammatory duringpulmonary fungal infection

Next, we tested whether rRegIIIg can rescue the phenotype inIl222/2 mice, because RegIIIg is an IL-22–inducible gene. Todirectly deliver RegIIIg to the gut, mice were gavaged with therecombinant protein at 48-h intervals from 1 to 9 d prior to in-fection. A total of five treatments were given, and tissues werecollected 2 d following infection. RegIIIg gavage protected micefrom weight loss, losing only 4% of body mass on day 1, whereasthe group receiving PBS vehicle lost 9% (Fig. 7A). A. fumigatusburden was not significantly different between the two groups(Fig. 7B, A.f.), suggesting that, rather than having a direct anti-fungal effect, RegIIIg was anti-inflammatory in this model. Sup-porting this, expression of Il1a, Il1b, Il6, and Ifng was significantlyreduced in lungs of mice receiving rRegIIIg (Fig. 7B), and thecytokines IL-1a, IL-1b, and IL-6 were also decreased (Fig. 7C).Surprisingly, IL-17 protein levels were comparable betweenRegIIIg-treated and control mice. Analysis of the small intestineand cecum revealed that RegIIIg had no effect on SFB colonization(data not shown). FACS was used to sort RegIIIg+ and RegIIIg2

fractions. This was followed by PCR, which revealed that

FIGURE 5. IL-22 suppresses pulmonary Th17 cell accumulation through serum IL-1R ligands. (A) Vancomycin-treated B6 Taconic mice and normal

water controls were infected with A. fumigatus via o.p. aspiration. Two days following infection, lung cells were stimulated with PMA plus ionomycin and

stained for intracellular IL-17. Shown are total numbers of IL-17+ cells, CD4+ IL-17+ cells, and TCR gd+ IL-17+ cells. Data are combined from two

experiments with n = 5. (B, C, and E) Vancomycin-treated B6 mice were placed on normal water and given a fecal transplant from B6 Taconic or Il222/2

donors. Fifteen days following gavage, mice were depleted of neutrophils and infected with A. fumigatus. (B) Total numbers of lung IL-17–producing cells

2 d following infection. (C) Distal small intestine cDNA was analyzed for bacterial species–specific 16S rRNA primers, normalized to Eubacteria. (E)

Expression of Il1a and Il1b in lung homogenates on day 2. Data in (B), (C), and (E) are combined from two experiments with n = 6–8. (D) B6 Jackson mice

were depleted of neutrophils and infected. Seven hours later, serum from naive B6 Taconic or Il222/2 mice was transferred i.v. Additional groups received

serum premixed with IL-1RA (anakinra). Shown are the total numbers of IL-17+ cells in lungs on day 2. Data are combined from two or three experiments

with n = 8–12. *p , 0.05, ***p , 0.001.

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endogenous RegIIIg does not preferentially bind to SFB-enrichedmicrobiota (P. Kumar, unpublished observations). Overall, thesefindings complement our data demonstrating that SFB coloniza-tion correlates with lung Th17 responses. To characterize bacteriaspecies that may contribute to weight loss during fungal infection,16S rDNA sequencing was performed on cecal contents. Exoge-nous RegIIIg preferentially decreased species in the Clostridialesorder but had no discernable effect on Bacteroidetes (Fig. 8).Many of the species affected were non-Lachnospiraceae, includ-ing Ruminococcaceae. Our data demonstrate that RegIIIg deliveryto the gut reduces the expression of lung inflammatory genesduring fungal infection.

SFB colonization augments Th17 immunity during pulmonaryfungal infection

Our data suggest that colonization with SFB, rather than hostgenotype for RegIIIg or Il22, determines the strength of pulmo-nary Th17 cell priming. To address this, we compared B6 micethat were SFB-high versus SFB-low. Routine screening revealed acohort of Taconic mice that was SFB2 upon arrival, in contrast

with previous experiments using this vendor. This cohort wasgavaged with SFB-high fecal suspensions from other Taconicdonors in-house and rested for 2 wk on normal or vancomycinwater. For comparison, B6 mice from The Jackson Laboratory(SFB-low) were also used. One day prior to infection, mice weredepleted of neutrophils. Results demonstrated that vancomycinwater significantly decreased lung Th17 cell numbers in SFB-highTaconic mice but not in SFB-low Jackson mice (SupplementalFig. 3). Accordingly, vancomycin significantly decreased SFBcolonization in Taconic mice. Although C. leptum was also de-creased by vancomycin, there were no differences in C. leptumcolonization between Taconic and Jackson mice (SupplementalFig. 3). This provides further evidence that intestinal SFB colo-nization promotes lung Th17 immunity.To confirm that SFB-enriched microbiota augment lung Th17

immunity, vancomycin-treated mice were gavaged with fecalsuspensions from Taconic donors (SFB-high) or Jackson donors(SFB-low). Three weeks following microbiota transfer, mice weredepleted of neutrophils and infected as described in Materials andMethods. Transfer of SFB-enriched microbiota into Jackson re-cipients significantly increased total numbers of IL-17+ and CD4+

IL-17+ cells in the lungs (Supplemental Fig. 4A). In contrast,transferring SFB-low microbiota from Jackson donors intoTaconic recipients did not significantly impact Th17 cell num-bers (Supplemental Fig. 4B). Altogether, our data demonstratea link between intestinal SFB colonization and pulmonaryTh17 immunity.

DiscussionCommensal microbiota support the development of immune re-sponses by stimulating pattern-recognition receptors. By targetinggrowth pathways that are shared among classes of bacteria, anti-biotics decrease intestinal species diversity, which modulates theimmune tone of the gut (36). For instance, Clostridium spp. havebeen shown to induce the development of colonic Tregs and aredepleted by metronidazole (28, 37). In contrast, vancomycin de-pletes Gram-positive bacteria and decreases the population ofsmall intestinal Th17 cells (38). In this article, we show that oralvancomycin has a significant effect on lung IL-17 productionduring an acute fungal infection (Fig. 1). Antibiotics also wereshown to inhibit Th17-mediated asthma (39). These data dem-onstrate that antibiotics, although required for treating intestinalinfections, have systemic anti-inflammatory effects that can in-hibit responses to secondary infections. In support, oral vanco-mycin renders mice susceptible to vancomycin-resistant enterococcithrough the downregulation of antimicrobial proteins, includingRegIIIg (40). We demonstrate that another consequence of RegIIIgdepletion may lead to increased accumulation of Th17 cells fol-lowing pulmonary challenges (Fig. 2). This was attributed tocommensal bacteria, including SFB, because the phenotype ob-served in RegIIIg2/2 mice was restored with vancomycin drinkingwater. Thus, our data show that modulating the balance of intes-tinal bacteria species significantly affects T cell polarization atnonlocal sites during infections.Beneficial effects of commensal bacteria include stimulating the

development of intestinal lymphoid tissue, antimicrobial proteinsecretion, and lymphocyte activation. It is not surprising that GF orcommensal-depleted animals are susceptible to intestinal infec-tions; however, they also succumb to systemic, skin, and respiratorypathogens (12–14, 41). Two mechanisms were proposed to explainthe role of commensals in systemic inflammation (42). First, in-nate immune agonists, such as bacterial cell wall products, maydirectly provide an adjuvant effect. Second, chronic low-level

FIGURE 6. Microbiota condition splenic DCs for antifungal cytokine

responses. Splenic CD11c+ cells were purified and stimulated overnight

with heat-killed swollen A. fumigatus (A.f.), LPS, or zymosan, and cyto-

kine levels were measured in supernatants. Concentrations of IL-1b (A),

IL-6 (B), and TNF (C) in culture supernatants are shown. Data (mean 6SEM) are from one representative experiment of two performed (four or

five wells). *p , 0.05, **p , 0.01, ***p , 0.001, ****p , 0.0001.

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stimulation of APCs by commensal products, termed “tonic sig-naling,” epigenetically programs them to respond robustly to afuture infection (12, 13). We observed that vancomycin-sensitivecommensals condition splenic DCs to produce increased levels ofinflammatory cytokines in response to fungal products (Fig. 6),providing evidence for epigenetic programming. Further, Quintinet al. (43) demonstrated that a primary fungal infection trainsmonocytes for increased cytokine production during a secondaryinfection. In our model, lung DCs from vancomycin-treated micewere not hyporesponsive to A. fumigatus (Supplemental Fig. 1D),and it remains possible that commensals provide an acute adjuvanteffect. Future studies will be aimed at deciphering the mechanismof how Gram-positive commensals amplify lung IL-17 production.Secretion of RegIIIg into the small intestine promotes the

physical separation of luminal bacteria from epithelium (20).Therefore, RegIIIg may block DC stimulation with commensalproducts or microbial translocation across the gastrointestinal

tract. SFB adhere directly to intestinal villi and provide an adju-vant effect that restores small intestinal Th17 cells in GF mice (21,22, 44). Because RegIIIg was found to inhibit mucosa-associatedSFB colonization (20), this may explain why small intestinal CD4T cells, including Th17 and Th1 cells, are significantly increasedin RegIIIg2/2 mice following i.p. immunization (Fig. 3). It is notknown whether RegIIIg has direct bactericidal activity againstSFB or inhibits colonization indirectly. Nevertheless, several in-flammatory diseases are associated with a compromised intestinalepithelial barrier. Microbial translocation is increased during in-flammatory bowel disease, chronic HIV infection, hepatitis B or Cinfection, and pancreatitis, contributing to systemic immune ac-tivation (45). IL-22 is critical for epithelial repair following injury(19, 46), and production of IL-22 by innate lymphoid cells preventsmicrobial translocation in the steady-state (47). This suggests thatintestinal IL-22 activity may have systemic anti-inflammatory ef-fects. We found that Il222/2 mice have increased IL-17 in the lungs

FIGURE 7. Intestinal RegIIIg delivery is anti-inflammatory during pulmonary fungal infection. (A) Il222/2 mice were gavaged with recombinant mouse

RegIIIg (40 mg; N) or vehicle control (n), as described in Materials and Methods. Mice were depleted of neutrophils and infected. Percentage body weight

relative to day 0 (mean 6 SEM). (B) Lung gene expression on day 2, normalized to Hprt. (C) Lung cytokines measured by ELISA on day 2, normalized to

total protein. Data are combined from two experiments with n = 8–10. *p , 0.05, **p , 0.01, ***p , 0.001, ****p , 0.0001.

FIGURE 8. Selective depletion of

Clostridiales Ruminococcaceae by RegIIIg

treatment. DNA from cecal contents was

isolated from Il222/2 mice used in Fig. 7

and used as a template for bacterial 16S

rDNA PCR analysis, as described previ-

ously (29). Amplicons were sequenced

using Illumina MiSeq and analyzed for

OTUs at the phylum, class, order, and

family levels. (A) Total number of OTUs

defined as species in PBS versus RegIIIg

treatment groups (upper panel) and the

distribution of major phyla (lower panel).

(B) Total number of individual species

that were changed by $2-fold in re-

sponse to RegIIIg treatment, as catego-

rized by phyla or order (upper panel).

Total number of individual species within

Clostridiales that were changed by RegIIIg

treatment, categorized by family (lower

panel). Data are combined from two ex-

periments with n = 8–10.

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and small intestine during acute fungal infection compared withWT mice (Fig. 4). This phenotype was vancomycin sensitive, cor-related with SFB colonization, and was transferrable with intestinalmicrobiota (Fig. 5B), demonstrating that IL-22 was modulatingmicrobial composition in the gut. Overall, cumulative evidencepoints to a model suggesting that IL-22 helps to prevent systemicimmune activation by inhibiting SFB colonization, blocking mi-crobial translocation, and decreasing pulmonary Th17 cell polari-zation toward fungal Ags.The cellular sources of IL-17 during infections may influence its

physiological effects. Deletion of integrin avb8 on CD11c+ cellsinhibits Ag-specific Th17 responses and protects against airwayhyperresponsiveness independently of airway inflammation (48).IL-17 produced by CD4 T cells, not gd T cells, enhances airwaysmooth muscle contraction. In our study, SFB colonization led topreferential accumulation of Th17 cells in lung tissue and had noeffect on the number of gd T cells making IL-17 (Fig. 5A, 5B).Further studies are necessary to define the role of SFB coloniza-tion on airway hyperresponsiveness.The regulation and function of RegIIIg in vivo are compart-

mentalized. In Paneth cells, IL-22 upregulates RegIIIg mRNA (19,47, 49), whereas stimulation of the MyD88 pathway inducesRegIIIg release from secretory granules in an inactive proform(50, 51). Following its secretion into the intestine, trypsin cleaves11 aa from the N terminus of RegIIIg, resulting in a 15-kDaisoform with potent bactericidal activity (51). In lung epithelium,IL-6 upregulates RegIIIg expression during Staphylococcusaureus infection (52). We showed that RegIIIg neutralizationdelays S. aureus clearance, whereas rRegIIIg delivery to the lungsaccelerates clearance. Although the cleaved 15-kDa isoform isdetectable in bronchoalveolar lavage fluid, the enzyme responsiblefor RegIIIg cleavage in lungs is not known. Thus, IL-22 and IL-6control RegIIIg expression in the gut and lungs, respectively.Because both of these cytokines activate the STAT3 transcriptionfactor, the compartmentalized regulation of RegIIIg expressionmay be determined by the local release of STAT3-activating cy-tokines and/or epithelial receptor expression. The regulation ofRegIIIg at multiple checkpoints is one way that the innate immunesystem controls bacteria colonization on mucosal surfaces.The role of RegIIIg in host defense is pathogen specific. Al-

though RegIIIg binds to the surface of A. fumigatus, it does notsignificantly affect fungal viability at pH 7 (data not shown). Thisis similar to what was reported for other fungi (18). Followinginfection, RegIIIg did not significantly affect pulmonary fungalclearance (Figs. 2A, 7B, Supplemental Fig. 2). Our finding thatvancomycin drinking water decreases lung Th17 cell numbers inRegIIIg2/2 mice demonstrates that RegIIIg expression is notsufficient to regulate Th17 immunity (Fig. 2D). The increasedTh17 cell numbers in RegIIIg2/2 mice compared with B6 controlswas due to microbiota differences (Fig. 2). Thus, when using SFB-negative strains, fungal clearance and IL-17 levels were compa-rable in B6 and RegIIIg2/2 mice (Supplemental Fig. 2). Our datasuggest that RegIIIg does not play a direct role in pulmonaryantifungal immunity.In this article we demonstrated that differences in microbiota

between strains of mice can impact pulmonary immunity. Certainbacteria, including SFB, may stimulate the systemic release of IL-1Rligands, augmenting lung Th17 cell accumulation during fungalinfections. The source of IL-1R ligands remains an interestingquestion. We observed that SFB-enriched microbiota increased theexpression of Il1b in lung tissue (Fig. 5E) but did not affect thenumber of lung CD11c+ CD11b+ cells producing IL-1a or IL-1b(Supplemental Fig. 1E). This suggests that non-DCs may provide asource of IL-1R ligands during infection. It also remains possible

that T cell responsiveness to IL-1R ligands might be regulated bymicrobiota. Our work complements a study showing that increasedactivity of the NLRP3 inflammasome in response to a high-fat dietleads to IL-17–dependent airway hyperresponsiveness (53). Theinvestigators found that IL-1b produced by macrophages drives theaccumulation of group 3 innate lymphoid cells that mediate diseasethrough IL-17. Our findings suggest that microbiota can influencesusceptibility to autoimmune disease. Animal studies identifiedcommensals that provoke or prevent autoimmunity by modulatingthe balance of Th17 cells and Tregs (54, 55). Intestinal Tregs rec-ognize microbial Ags (56, 57) and increase the ratio of Firmicutesto Bacteroidetes species (58), suggesting that T cells can, in turn,influence microbial composition. Thus, commensal organisms haveintegral roles in the development of local and systemic immuneresponses.Intestinal SFB colonization may regulate pulmonary host de-

fense through various mechanisms, including antimicrobial proteinsecretion, immune cell activation, and recruitment. A recent studyfound that SFB protects against acute S. aureus pneumonia (59).SFB-colonized mice had significantly increased levels of IL-6 andIL-22, as well as neutrophil cell numbers, in bronchoalveolar la-vage fluid. In that study, IL-22 action in the lung was critical forhost defense against S. aureus. Our data show that IL-22 action inthe gut may also regulate lung immunity by modulating themicrobiota. This is consistent with other studies measuring theimpact of IL-22 on gut microbiota (60, 61). Although we provideevidence that systemic IL-1R ligands contribute to pulmonaryTh17 immunity (Fig. 5D), more studies are necessary to fullydefine the link between SFB colonization and host defense.

DisclosuresW.O. is an employee of Genentech; the other authors have no financial

conflicts of interest.

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