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Immunity to Mucosal CandidiasisDependent−Role of Neutrophils in IL-17

Sarah L. GaffenHernández-Santos, Toni Darville, Partha S. Biswas and Anna R. Huppler, Heather R. Conti, Nydiaris

ol.1302265http://www.jimmunol.org/content/early/2014/01/17/jimmun

published online 17 January 2014J Immunol 

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is published twice each month byThe Journal of Immunology

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The Journal of Immunology

Role of Neutrophils in IL-17–Dependent Immunity toMucosal Candidiasis

Anna R. Huppler,*,† Heather R. Conti,† Nydiaris Hernandez-Santos,† Toni Darville,*

Partha S. Biswas,† and Sarah L. Gaffen†

Oropharyngeal candidiasis (OPC), caused by the commensal fungus Candida albicans, is an opportunistic infection associated with

infancy, AIDS, and IL-17–related primary immunodeficiencies. The Th17-associated cytokines IL-23 and IL-17 are crucial for

immunity to OPC, but the mechanisms by which they mediate immunity are poorly defined. IL-17RA–deficient humans and mice

are strongly susceptible to OPC, with reduced levels of CXC chemokines and concomitantly impaired neutrophil recruitment to

the oral mucosa. Paradoxically, humans with isolated neutropenia are typically not susceptible to candidiasis. To determine

whether immunity to OPC is mediated via neutrophil recruitment, mice lacking CXCR2 were subjected to OPC and were found

to be highly susceptible, although there was no dissemination of fungi to peripheral organs. To assess whether the entire neutrophil

response is IL-17 dependent, IL-17RA2/2 and IL-232/2 mice were administered neutrophil-depleting Abs and subjected to OPC.

These mice displayed increased oral fungal burdens compared with IL-17RA2/2 or IL-232/2 mice alone, indicating that addi-

tional IL-17–independent signals contribute to the neutrophil response. WT mice treated with anti–Gr-1 Abs exhibited a robust

infiltrate of CD11b+Ly-6GlowF4/802 cells to the oral mucosa but were nonetheless highly susceptible to OPC, indicating that this

monocytic influx is insufficient for host defense. Surprisingly, Ly-6G Ab treatment did not induce the same strong susceptibility to

OPC in WT mice. Thus, CXCR2+ and Gr-1+ neutrophils play a vital role in host defense against OPC. Moreover, defects in the IL-

23/17 axis cause a potent but incomplete deficiency in the neutrophil response to oral candidiasis. The Journal of Immunology,

2014, 192: 000–000.

Oropharyngeal candidiasis (OPC; thrush) is an opportu-nistic mucosal infection caused by the commensal fun-gus Candida albicans. C. albicans causes disease when

exposure is combined with host susceptibility through immuno-deficiency or a breach in normal barriers. A range of primary andsecondary immunodeficiency syndromes are associated with sus-ceptibility to OPC (1, 2), including the primary immunodeficiencyhyper-IgE syndrome and a family of genetic diseases leading tochronic mucocutaneous candidiasis (3). Secondary immunodefi-ciencies are now the most common predisposing factor for mu-cosal Candida disease. OPC is well described in patients with HIVand is considered an AIDS-defining illness. Cancer chemotherapy,immunosuppression for transplant recipients, renal failure, andhigh-dose corticosteroids increase the risk for candidiasis.

T cells, specifically the Th17 subset of CD4+ Th cells, play acentral role in resistance to OPC (4–6). Th17 cells secrete IL-17(IL-17A), a proinflammatory cytokine involved in defense againstextracellular pathogens (7, 8). Previous studies have shown thatmice deficient in the IL-17R (IL-17RA or IL-17RC) and IL-23(required for the expansion of the Th17 cell subset) exhibit dra-matic susceptibility to OPC (4, 9).The mechanisms of host defense induced by IL-17 are not fully

understood, but they appear to be mediated by the secretion of an-timicrobial peptides and recruitment of neutrophils through chemo-tactic chemokines and granulopoietic cytokines. A role for neutrophilsin OPC has been inferred based on the observation of neutrophilinflux into the tongue after oral Candida infection (4). Mice subjectedto OPC show an increased expression of genes associated withneutrophil regulation, including granulopoietic cytokines G-CSFand IL-6 and chemokines CXCL1 (KC, Groa), CXCL2 (MIP-2),and CXCL5 (LIX) (4, 10). Moreover, neutrophil recruitment to tis-sue is markedly attenuated in mice deficient in IL-17 signaling, thuslinking IL-17 to neutrophil function and susceptibility to OPC (4,9). Strikingly, recent reports of single gene defects in humansresulting in chronic mucocutaneous candidiasis cluster in the IL-17 pathway, including mutations in IL-17RA, IL-17F, and Act1,an adaptor molecule mediating IL-17 signaling (3, 11, 12).The requirement for neutrophils in host defense from dissemi-

nated candidiasis is well described (13, 14). However, the role ofneutrophils in host defense at the oral mucosa is less clear. Patientswith broad immunosuppression that includes neutropenia are typi-cally susceptible to OPC, as are those with hereditary myelo-peroxidase deficiency with defects in neutrophil and macrophageactivity. However, oral thrush is not frequently reported in patientswith isolated neutropenia (15–17), raising the question of whetherthe effect of IL-17 on OPC susceptibility is solely or primarily dueto its impact on neutrophils.

*Department of Pediatrics, Children’s Hospital of Pittsburgh of UPMC, Pittsburgh,PA 15224; and †Division of Rheumatology and Clinical Immunology, Department ofMedicine, University of Pittsburgh, Pittsburgh, PA 15261

Received for publication August 26, 2013. Accepted for publication December 10,2013.

This work was supported by the National Institutes of Health (Grant DE022550 toS.L.G.; Grant AI054624 to T.D.; Grant F32-DE023293 to H.R.C.), the Children’sHospital of Pittsburgh of UPMC Health System (to A.R.H.), a Pediatric InfectiousDisease Society Fellowship Award (funded by the Stanley A. Plotkin Sanofi PasteurFellowship Award; to A.R.H.), and the Division of Rheumatology and Clinical Im-munology, UPMC (to P.S.B.).

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. Sarah L. Gaffen, University ofPittsburgh, BST S702, 200 Lothrop Street, Pittsburgh, PA 15261. E-mail address:sig65@pitt.edu

Abbreviations used in this article: ANC, absolute neutrophil count; CMC, chronicmucocutaneous candidiasis; OPC, oropharyngeal candidiasis.

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

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There is no knockout mouse system to study profound neu-tropenia, but alternate tools are available to examine the role ofneutrophil recruitment and effector responses. Neutrophils, char-acterized by expression of Ly-6G (a component of the Gr-1 epi-tope), are regulated through the chemokine receptor CXCR2 (18–20). Neutrophils are the predominant CXCR2+ cell among bloodleukocytes, with lower expression on mast cells, monocytes, mac-rophages, endothelial cells, and epithelial cells (21–23). In addition,several Abs (anti–Ly-6G and anti–Gr-1) are available to depleteneutrophils peripherally, albeit with variable degrees of efficiency intissue and cell type specificity (24–27). To test the hypothesis thatneutrophils are essential in host defense from OPC, we evaluateddisease in CXCR22/2 mice and Ab-mediated neutrophil depletion.In addition, we assessed redundancy in host defense systems withneutrophil depletion in mice deficient in IL-23 or IL-17RA. Ourfindings indicate a central role for CXCR2 and Gr-1+ neutrophils inthe oral mucosa.

Materials and MethodsMice

IL-23p192/2 mice were provided by Genentech (South San Francisco,CA) and IL-17RA2/2 mice by Amgen (Seattle, WA) and bred in-house.All other mice were from The Jackson Laboratory (Bar Harbor, ME) onthe C57BL/6 background unless otherwise noted. Mice were 6–9 wk old,and age- and sex-matched for all experiments unless otherwise noted.Protocols were approved by the University of Pittsburgh InstitutionalAnimal Care and Use Committee and adhered to guidelines in the Guidefor the Care and Use of Laboratory Animals of the National Institutesof Health.

Ab-mediated neutrophil depletion

Mice were injected i.p. with mAb 24 h before and 2 d after inoculation withC. albicans (day 0). Anti–Ly-6G (clone 1A8; Bio X Cell, West Lebanon,NH) and isotype control (clone 2A3; Bio X Cell) were administered ata dose of 300 mg. Anti–Gr-1 (clone RB6-8C5; Bio X Cell) and isotypecontrol (clone LTF-2; Bio X Cell) were administered at a dose of 80 mg.All cohorts not receiving mAbs were treated with PBS. The degree ofneutrophil depletion was assessed in peripheral blood as follows: bloodwas collected by saphenous venous puncture or cardiac puncture and anti-coagulated in EDTA-containing tubes (Microvette Tubes, Sarstedt, Newton,NC, and Microtainer Tubes, BD, Franklin Lakes, NJ). Total WBC countswere determined by visual enumeration after trypan blue exclusion. Thepercentage of neutrophils was determined by enumeration of a blood smearstained with Giemsa (Fisher Scientific, Fair Lawn, NJ). One hundred to 200cells were counted per sample. The absolute neutrophil count (ANC)was determined by multiplying the percentage of neutrophils (matureand band forms) by total WBC.

Mouse model of OPC

OPC was performed as described previously (4, 28). In brief, mice wereinoculated under anesthesia by placing a 0.0025-g cotton ball saturated ina 23 107 CFU/ml C. albicans suspension (strain CAF2-1) sublingually for75 min. Sham-inoculated mice were inoculated with a cotton ball saturatedwith sterile PBS. The oral cavity was swabbed immediately before eachinfection and streaked on yeast peptone dextrose agar to verify the absenceof Candida. No antibiotics were administered. If indicated, control micewere immunosuppressed with cortisone acetate 225 mg/kg (Sigma-Aldrich)on days 21, 1, and 3. Mice were weighed daily, and weight was comparedwith baseline weight on day 21. Fungal burden in the indicated organs wasdetermined by dissociation on a GentleMACS Dissociator (Miltenyi Biotec,Auburn, CA) followed by plating serial dilutions on yeast peptone dextrosewith antibiotics (ampicillin, gentamicin). Plates were incubated for 48 h at30˚C followed by colony enumeration in triplicate.

Flow cytometry

Tongue tissue was processed with an enzyme mixture (EDTA, HEPES,collagenase-2 [Worthington Biochemical, Lakewood, NJ], Dispase [LifeTechnologies, Invitrogen], DNase I [Applied Biochemical], and definedtrypsin inhibitor [Life Technologies]). Tissue was mechanically homoge-nized and passed through a cell strainer to form a single-cell suspension.Abs were from BD Biosciences (San Jose, CA), eBioscience (San Diego,

CA) or Biolegend (San Diego, CA). One to 23 106 cells were stained withCD11b-allophycocyanin (clone M1/70), Gr-1–FITC or Alexa Fluor 700(AF700; clone RB6-8C5), CD45-AF700 (clone 30-F11), Ly-6G–PE (clone1A8), Ly-6C–PerCP–Cy5.5 (clone AL-21), CD115-AF488 (clone AFS98),CD11c-PerCP-Cy5.5 (clone HL3), and F4/80-PE-Cy7 (clone BM8). Iso-type control Abs and unstained samples were included in the analyses.Data were acquired on an LSRII (BD Biosciences) and analyzed withFlowJo (Tree Star).

RNA extraction and real-time reverse transcriptase PCR

Frozen tonguewas homogenized in RLT lysis buffer (RNAeasy Kit; Qiagen,Valencia, CA) with a GentleMACS Dissociator (M-tubes, RNA02 program;

FIGURE 1. CXCR2 deficiency induces persistent susceptibility to OPC.

(A) CXCR22/2 mice are susceptible to OPC. CXCR2+/2 and CXCR22/2

mice at 7–16 wk of age were subjected to OPC on day 0. Sham-infected

and WT mice treated with cortisone acetate were used as controls. Mice

were sacrificed at 4 d, and fungal burden in tongue was assessed by plating

and colony enumeration. Data are pooled from two independent experi-

ments and geometric means are shown. ***p , 0.001 versus CXCR2+/2.

(B) CXCR22/2 mice show steady weight loss after inoculation. Mice were

inoculated as in (A), and daily weight is shown as a percentage of day 21.

Means and SEM are shown. (C) CXCR22/2 mice show persistent

C. albicans infection. CXCR22/2 and WT littermates at 6–9 wk of age

were subjected to OPC on day 0, and tongues were assessed for fungal

burden on days 3–8. Geometric means are shown. *p , 0.05 comparing

samples at matched time points. (D) CXCR22/2 mice show persistent

weight loss. Mice were inoculated as in (C), and daily weight was com-

pared with day 21. Means and SEM are shown. (E) There is no dissem-

ination of C. albicans from gastrointestinal tract to peripheral organs in

CXCR22/2 mice. Four days after oral inoculation, CXCR22/2 or WT

mice 6 cortisone acetate were evaluated for fungal loads in the indicated

organs.

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Miltenyi). RNAwas extracted with RNAeasy Kit. In all, 0.732 mg total RNAwas used to synthesize cDNA with a SuperScript III First-Strand SynthesisSystem (Invitrogen). Relative quantification of indicated genes was deter-mined by real-time PCRwith SYBRGreen (Quanta BioSciences, Gaithersburg,MD) normalized to GAPDH. Primers were from SA Biosciences (Qiagen).Results were analyzed on a 7300 Real Time PCR System (Applied Bio-systems, Carlsbad, CA) in triplicate.

Statistics

Data were compared by Mann–Whitney U or unpaired Student t test usingGraphPad Prism (La Jolla, CA) or Microsoft Excel. The p values,0.05 wereconsidered significant. All experiments were performed a minimum of twice.

ResultsCXCR2 deficiency induces persistent susceptibility to OPC

To assess the contribution of neutrophils to host defense from OPC,we used a mouse deficient in neutrophil recruitment because of

knockout of the chemokine receptor CXCR2 (22, 29). CXCR22/2

mice exhibit abnormal neutrophil trafficking in response to thechemokines CXCL1, CXCL2, and CXCL5, all of which are up-regulated by IL-17 during oral C. albicans infection (22, 29–31).We subjected CXCR22/2 mice to OPC by sustained sublingualexposure (4, 28). In this model, WT mice are resistant to infection,but cortisone-treated or IL-17RA–deficient mice are susceptible(4, 28). Whereas heterozygous littermates were not susceptible toOPC, CXCR22/2 mice showed a mean oral fungal burden of 1.93103 CFU/g tongue tissue at 4 d after inoculation (Fig. 1A).CXCR22/2 mice also showed progressive weight loss after inoc-ulation, with a mean of 16% weight loss on day 4 compared withbaseline (Fig. 1B). To determine whether CXCR22/2 mice showedpersistent susceptibility or only a delayed clearance, we inoculatedCXCR22/2 mice and WT littermates with C. albicans on day 0, andoral fungal burden was assessed on days 3, 4, 6, and 8. CXCR22/2

FIGURE 2. Elevated oral fungal load with preserved CD11b+ tongue cellular infiltrate in IL-17RA2/2 mice after neutrophil depletion. (A) IL-17RA2/2

mice treated with anti–Ly-6G had sustained neutropenia. IL-17RA2/2 mice were treated with isotype or anti–Ly-6G Abs on days 21 and 2, and subjected

to OPC on day 0. Sham- and Candida-inoculated WT mice served as controls. Peripheral blood neutrophil counts were assessed by visual smear ex-

amination on days 0, 2, and 4. Geometric means are shown. Dashed line indicates threshold for clinically significant neutropenia. (B) IL-17RA2/2 mice

treated with anti–Ly-6G have elevated oral fungal burdens. Mice were treated with Abs and infected as in (A). Fungal burden in tongue was assessed 4 d

after inoculation. Geometric means are shown. ***p , 0.001. (C) IL-17RA2/2 mice treated with anti–Ly-6G display elevated weight loss. Mice were

treated with Ab and inoculated as in (A). Mice were weighed daily and weight loss was compared with baseline. Means and SEM are shown. (D) IL-17RA2/2

mice treated with anti–Gr-1 exhibit profound neutropenia. IL-17RA2/2 mice were treated with isotype or anti–Gr-1 Abs on days 21 and 2, and subjected to

OPC on day 0. Controls included: sham- or Candida-inoculated IL-17RA2/2 mice, IL-17RA2/2 mice treated with PBS, Candida-inoculated WT mice, and

cortisone-treated WT mice. Peripheral blood neutrophil counts were assessed as in (A). Geometric means are shown. Dashed line indicates threshold for

clinically significant neutropenia. (E) IL-17RA2/2 mice treated with anti–Gr-1 Abs show an elevated oral fungal burden. Mice were treated with Abs and

inoculated as in (D). Fungal burden in tongue tissue was assessed after 4 d. ***p , 0.001. Mice were evaluated in two independent experiments. (F) IL-

17RA2/2 mice treated with anti–Gr-1 exhibited profound weight loss. Mice were treated with Abs and inoculated as in (D). Mice were weighed daily and

weight loss was compared with baseline. (G) Candida-inoculated IL-17RA2/2 mice show a CD11b+ cellular infiltrate in tongue that is maintained with anti–Gr-1

treatment. Mice were treated with Abs and inoculated as in (D). Tongue was harvested on day 4 and tongue homogenates were analyzed by flow cytometry.

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mice had persistent oral fungal burden through day 8, in contrast withWT littermates that cleared the fungus fully by day 6 (Fig. 1C).Consistently, weight loss in CXCR22/2 mice was sustained throughday 8, whereas WT mice returned to baseline (Fig. 1D). AlthoughC. albicans was found in the mouth and gastrointestinal tract post-infection, CXCR22/2 mice did not show dissemination of yeast toperipheral organs such as kidney or brain, measured on day 4 afterinoculation (Fig. 1E). Thus, CXCR2 signaling is essential forimmunity to oral candidiasis, but not for barrier maintenanceand prevention of dissemination.

Neutrophil depletion augments OPC in IL-17/Th17–deficientmice

To investigate the degree to which IL-17 signaling is responsible forregulation of neutrophils in OPC, we administered Abs to depleteneutrophils during the course of infection in IL-17RA2/2mice. IL-17RA2/2 mice were treated with anti–Ly-6G (clone 1A8), whichrecognizes a highly expressed marker on neutrophils, 24 h beforeand 2 d postinfection. Anti–Ly-6G–treated mice were neutropenicthroughout the experimental period, with ANCs ,0.5 3 106

neutrophils/ml (Fig. 2A). As expected, IL-17RA2/2 mice treatedwith isotype Abs were susceptible to OPC, similar to untreated IL-17RA2/2 (4) (Fig. 2B). IL-17RA2/2 mice treated with anti–Ly-6Gshowed significantly higher oral fungal burdens than isotype-treatedmice (Fig. 2B). The anti–Ly-6G–treated mice also displayed moreweight loss than isotype-treated mice (Fig. 2C).To extend these findings, we treated IL-17RA2/2 mice with an

Ab directed against Gr-1 (clone RB6-8C5), which recognizes Ly-6G and, to a lesser extent, Ly-6C on mature granulocytes, mono-cytes (transiently during differentiation), and plasmacytoid dendriticcells (at low levels). Most mice treated with anti–Gr-1 demonstratedperipheral blood neutropenia (Fig. 2D). IL-17RA2/2 mice treatedwith anti–Gr-1 exhibited a significant increase in oral fungal burdencompared with isotype-treated mice 4 d after inoculation (Fig. 2E).Dissemination of C. albicans to the kidney was found in 10% of IL-17RA2/2 mice treated with anti–Gr-1, but not in mice treated withisotype Abs, PBS, or anti–Ly-6G (data not shown). Treatment withisotype did not alter the susceptibility of IL-17RA2/2 mice to OPCcompared with PBS. Treatment with anti–Gr-1 resulted in increased

weight loss in IL-17RA2/2 after OPC challenge, similar to theweight loss observed in WT mice treated with high-dose cortisone(Fig. 2F). Collectively, these data show that the susceptibility toOPC caused by defective IL-17 signaling can be augmented byneutrophil depletion.Although anti–Gr-1 and–Ly-6G can cause profound depletion

of peripheral blood neutrophils, this has been shown to correlatepoorly with neutrophil depletion in resident tissues (24). Accord-ingly, we assessed the effects of Ab treatment on neutrophil infil-tration into tongue by flow cytometry. At 4 d after inoculation, WTmice showed a small but measurable CD11b+Gr-1low population(Fig. 2G). Inoculated WT mice treated with cortisone showed atongue infiltrate of CD11b+Gr-1hi cells. Sham-treated IL-17RA2/2

mice showed minimal infiltrate of cells staining positive for CD11b,Ly-6G, or Gr-1. IL-17RA2/2 mice treated with isotype showeda robust infiltrate of CD11b+Ly-6Ghi and CD11b+Ly-6Glow cells. Ininoculated IL-17RA2/2 mice treated with anti–Gr-1, only thetongue infiltrate of CD11b+Ly-6Glow cells was observed, and thesecells were Gr-1low (Fig. 2G). These data indicate that OPC inducesa local CD11b+ cellular infiltrate that is preserved after treatmentwith anti–Gr-1 Abs, but the absence of CD11b+Ly-6Ghi neutrophilsresults in heightened susceptibility to OPC.IL-23 is critical for the maintenance of IL-17–producing cells

(32, 33), and deficiency in IL-23 results in susceptibility to OPC atlevels indistinguishable from IL-17RA2/2 mice (9). To determinewhether neutrophil depletion in IL-232/2 mice followed a similarprofile, we treated IL-232/2 mice with anti–Ly-6G 24 h beforeand 2 d after oral inoculation with C. albicans. Mice were neu-tropenic throughout the experimental period with peripheralANCs ,0.5 3 106 neutrophils/ml (Fig. 3A). Mice treated withanti–Ly-6G showed a significant increase in fungal burden on day4 compared with IL-232/2 mice treated with isotype Abs (Fig.3B). Similar to the IL-17RA2/2 mice, IL-232/2 mice treated withisotype Abs exhibited a robust infiltrate of CD11b+Gr-1hi cells intongue. IL-232/2 mice treated with anti–Ly-6G continued to havea CD11b+ population, but Gr-1 staining shifted to Gr-1low (Fig.3C). Low numbers of CD11b+Ly-6Glow cells were present insham-infected IL-232/2 and Candida-infected WT mice, whereasIL-232/2 mice treated with cortisone and inoculated with C. albicans

FIGURE 3. Neutrophil depletion in IL-232/2 mice

increases susceptibility to OPC. (A) IL-232/2 mice treated

with anti–Ly-6G show peripheral neutropenia. IL-232/2

mice were treated with isotype or anti–Ly-6G Abs on days

21 and 2, and subjected to OPC on day 0. Control mice

include Candida-inoculated WT, cortisone-treated WT, and

cortisone-treated IL-232/2 mice. Peripheral blood neutro-

phil counts were assessed as in Fig. 2A. Dashed line

indicates threshold for clinically significant neutropenia.

(B) IL-232/2 mice treated with anti–Ly-6G show elevated

oral fungal burdens. Mice were treated with Abs and in-

oculated as in (A), and fungal burden in tongue tissue was

assessed after 4 d. Mice were evaluated in two independent

experiments, and geometric means are shown. *p , 0.05.

(C) IL-232/2 mice exhibit a robust CD11b+ cell population

in tongue, which is Gr-1low in anti–Ly-6G–treated mice.

Mice were treated with Abs and inoculated as in (A).

Tongue was harvested on day 4.

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showed a robust infiltrate of CD11b+Ly-6Ghi and Gr-1hi cells (Fig.3C). Together, these results show that deficiency in Th17 cells orIL-17 signaling does not fully eliminate the neutrophilic responseto OPC.

Susceptibility to OPC is induced in normally resistant WT micewith myeloid cell depletion

Next, we assessed whether neutrophil depletion induced suscep-tibility in WTmice. WTmice were treated with isotype or anti–Ly-6G as described earlier. Peripheral ANCs demonstrated a transientneutropenia in anti–Ly-6G–treated mice, with recovery above 0.5 3106 cells/ml by days 4–5 (Fig. 4A). Neutrophil-depleted mice showeda bimodal response to inoculation, with 86% of mice completelyresistant to infection, whereas 14% were profoundly susceptible withhigh oral fungal burdens, sustained weight loss, and dissemina-tion to kidney at days 4–5 (Fig. 4B and data not shown). Thecohort treated with anti–Ly-6G experienced weight loss similar toisotype-treated mice (data not shown). There was no correlationbetween the degree of neutropenia and susceptibility to OPC.Isotype-treated mice had a large infiltrate of CD11b+Gr-1hi cells,

whereas anti–Ly-6G–treated mice showed an infiltrate of CD11b+

Gr-1low cells at day 2. Both populations were absent in sham-inoculated WT mice (Fig. 4C).Although anti–Ly-6G and anti–Gr-1 treatments cause similar

phenotypes in IL-17RA2/2 mice, their effects were different inWT mice. Anti–Gr-1 resulted in profound peripheral neutropeniaon day 0, with some count recovery by day 2 (Fig. 5A). Unlike theanti–Ly-6G–treated mice, these animals were profoundly ill, withdecreased activity and grooming seen as early as day 1. Mice weresacrificed on day 3 for humane reasons, at which time there wasa significant difference in the oral fungal burden between isotypeand anti–Gr-1–treated mice (Fig. 5B). At 2 d after inoculation,sham-treated WT mice treated with Gr-1 Abs showed minimalinfiltration of leukocytes staining positive for CD11b and Ly-6G(Fig. 5Ci). WT mice treated with isotype Abs and inoculated withCandida had a dominant population of CD11b+Ly-6Ghi cells anda smaller population of CD11b+Ly-6Glow cells. At day 3, bothpopulations were starting to wane as the infection cleared (datanot shown). In WT mice treated with anti–Gr-1 and infected withCandida, a much larger percentage of the population consisted ofCD11b+Ly-6Glow cells (Fig. 5Cii). The CD11b+ population wasfurther characterized by staining for macrophage/monocytemarkers including Ly-6C, F4/80, and CD115. Based on thesemarkers, the cell infiltrate in infected mice (isotype treated) wasconsistent with neutrophils (CD11b+Ly-6GhiLy-6Chi) and inflam-matory monocytes (CD11b+Ly-6GlowLy-6Chi; Fig. 5Cii). Theneutrophil population, but not the inflammatory monocyte pop-ulation, was depleted in anti–Gr-1–treated mice (Fig. 5Ciii). Theinflammatory monocytes that remained could not orchestrateCandida clearance, because these mice were highly susceptible toOPC (Fig. 5B). Markers for monocyte-derived phagocytes wereabsent from this population, because these cells did not stainstrongly for F4/80 or CD115 (Fig. 5C). We also looked for DCs bystaining for CD11c, but no positive cells were observed (data notshown).A Gr-1+ or CD11b+ cell capable of suppressing T cells has been

described in other settings, including disseminated candidiasis(34–36). To determine whether the cellular influx into the tongueafter oral Candida challenge suppressed the IL-17/Th17–mediatedhost defense, we examined expression of a panel of IL-17 signa-ture genes known to be induced in tongue after C. albicans ex-posure (4). There was no significant difference in expression ofil17a between mice treated with isotype or neutrophil-depletingAbs compared with untreated WT mice (Fig. 6A). There was alsono difference in expression of the neutrophil-associated genes100a9 (Fig. 6B). There was a small but significant decrease inexpression of the CXCR2 ligand cxcl5 in mice treated with theisotype for anti–Gr-1, but no other differences in treated comparedwith untreated WT mice were observed (Fig. 6C). As expected,there was no basal expression of il17a or IL-17 signature genes insham-inoculated mice, but there was an elevation in il17a ex-pression in CXCR22/2 mice (4, 22). These findings indicate thatthe tongue cellular influx does not appear to suppress IL-17 ex-pression or activity.

DiscussionIn this study, we demonstrate that Gr-1+ neutrophils are vital forthe elimination of C. albicans from the oral mucosa. Deficiency inthe CXCR2 chemokine receptor results in persistent oral fungalburdens, weight loss, and augmented IL-17 signature gene expres-sion in the oral tissue, although no dissemination to visceral organswas detectable. Ab-mediated neutrophil depletion also results insusceptibility to OPC in normally resistant WT mice. Susceptibilityis profound and consistent in mice broadly depleted of myeloid cells

FIGURE 4. Selective neutrophil depletion induces susceptibility to OPC

in a subset of WT mice. (A) WT mice treated with anti–Ly-6G exhibit

transient peripheral neutropenia. WT mice were treated with isotype

control or anti–Ly-6G on days 21 and 2, and subjected to OPC on day 0.

Controls were WT sham-inoculated mice, IL-232/2, and cortisone-treated

WT mice. Peripheral neutrophil counts were assessed as described in Fig.

2A. Dashed line indicates threshold for clinically significant neutropenia.

(B) WT mice treated with anti–Ly-6G show a bimodal response. Mice were

treated with Abs and inoculated as in (A). Fungal burden in tongue was

assessed after 4–5 d. Mice were evaluated in two independent experiments

and geometric means are shown. n.s., not significant. (C) WT mice show

a CD11b+ tongue infiltrate on day 2 after inoculation. Mice were treated

with Abs and inoculated as in (A). Tongue was harvested on day 2 and

analyzed by flow cytometry.

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with anti–Gr-1 mAb, and susceptibility is also induced with the morespecific anti–Ly-6G mAb. In addition, an increase in susceptibilityis induced in mice deficient in Th17 cells or IL-17 signaling uponAb-mediated neutrophil depletion, indicating that IL-17 does notaccount for all of the Ly-6G/Gr-1–dependent host protection in theoral cavity. All susceptible mice showed a CD11b+ tongue infiltrate,with alterations in Ly-6G and Gr-1 positivity depending on thespecific Ab treatment. It has been long assumed that neutrophils andCXC chemokines are the mediators of host defense at the oralmucosal surface, but the evidence supporting this assumption issurprisingly scant. This is, to our knowledge, the first study to di-rectly demonstrate a role for CXCR2 in OPC and the role of neu-trophils in the context of IL-23/IL-17 signaling deficiency.CXCR2, as the central chemokine receptor for neutrophil re-

cruitment, is considered a potential therapeutic target (37); thus, ourdata suggest a potential new infectious risk implication of blockingCXCR2. In the context of fungal infections, CXCR2 is linked to

fungal asthma and resistance to invasive pulmonary Aspergillusfumigatus (26, 38, 39). In the oral cavity, CXCR2 plays a host-protective role in a mouse model of periodontal disease, withCXCR2 deficiency resulting in a more severe phenotype of oralbone loss than IL-17 signaling deficiency alone (31). Depletion ofneutrophils, the primary CXCR2-expressing cell type, with anti-Ly6G Abs resulted in two distinct phenotypes. The majority ofmice were resistant to OPC, but a subset was profoundly ill withboth oral and disseminated candidiasis (Fig. 4B). The more severephenotype was observed in all anti–Gr-1–treated mice (Fig. 5B). Itremains controversial in the field whether Ly-6G Abs are simplyless efficient at depleting neutrophils than Gr-1 Abs, or whether analternative Ly-6G2 cell type exists that compensates for neu-trophils upon depletion. Our data are more consistent with theformer model, because our staining experiments did not reveal anyevidence for an alternative monocytic cell that might be function-ally compensatory. In disseminated candidiasis, however, there is

FIGURE 5. Neutrophil depletion with anti–Gr-1 induces

susceptibility to OPC in WT mice. (A) WT mice treated

with anti–Gr-1 Abs have peripheral neutropenia. Mice were

treated with isotype or anti–Gr-1 Abs on days 21 and 2,

and subjected to OPC on day 0. Controls include WT

sham-inoculated and IL-17RA2/2 mice. Peripheral neu-

trophil counts were determined as in Fig. 2A. Dashed line

indicates threshold for clinically significant neutropenia.

(B) WT mice treated with anti–Gr-1 are highly susceptible

to OPC. Mice were treated with Abs and inoculated as in

(A). Fungal burden was assessed after 3 d. Mice were

evaluated in two independent experiments, and geometric

means are shown. ***p , 0.001. (C) Anti–Gr-1–treated

WT mice lose neutrophils, but not inflammatory mono-

cytes, in tongue. WT mice were treated with anti–Gr-1 Abs

(i, iii) or isotype (ii) and inoculated with C. albicans (ii, iii)

as in (A). Tongue was harvested on day 2 and analyzed by

flow cytometry.

FIGURE 6. No evidence for suppression of IL-17 respon-

ses upon anti–Ly-6G or anti–Gr-1 treatment. WT mice were

treated with isotype control Abs (n = 3–5 mice), anti–Ly-6G

(n = 3), anti–Gr-1 (n = 5), or no Ab (n = 3) on day 21, and

subjected to OPC on day 0. Controls include sham-inoculated

WT mice (n = 3) or CXCR22/2 mice (n = 2). Tongue mRNA

was evaluated for il17a (A), s100a9 (B), or cxcl5 (C) by real-

time RT-PCR. Each sample was analyzed in triplicate, and

bars represent means with SEM. *p , 0.05 compared with

WT. n.d., Not detectable.

6 MECHANISMS OF IMMUNITY TO OROPHARYNGEAL CANDIDIASIS

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evidence that inflammatory monocytes can mediate Candida clear-ance from kidney (40), but immunity to mucosal candidiasis isknown to be considerably different from disseminated disease. Acaveat to mouse models of candidiasis is the potential differencein the human and mouse neutrophil response to C. albicans. A recentreport found a reduced ability to kill C. albicans and increasedphagocyte death after in murine versus human neutrophils (41).Therefore, it remains to be determined whether the compensatoryCD11b+ cell response we see in mice occurs in humans, where thedirect neutrophil response to C. albicans is more effective.It was unexpected to find that susceptibility to OPC in mice

lacking IL-23 or IL-17 signaling could be augmented by neutrophildepletion. Although signals for neutrophil recruitment are medi-ated by IL-17 in the context of OPC, alternative mechanisms ofneutrophil regulation and recruitment thus appear to be presentwhen IL-17 signaling is perturbed. Cellular influx of CD11b+ cellsinto the tongue was observed in mice experiencing active infection(Figs. 2G, 3C), which may be because of TLRs, C-type lectin re-ceptors, or other cytokines. In addition to mediating expression ofneutrophil-recruiting chemokines, IL-17 regulates expression ofantimicrobial peptides that exhibit antifungal activity, such asb-defensins and histatins (4, 42). IL-17 also mediates host defensethrough alterations in cytokine production and synergy with otherproinflammatory effectors (43). The entirety of IL-17–mediatedhost protection to candidiasis presumably comes from the com-bined effects of all these facets of the immune system.As noted, there are limited tools to study isolated states of

neutropenia in mice. Historically, many studies used cyclophos-phamide, whole-body irradiation or other chemotherapeutic agentsthat induce neutropenia, but these regimens also cause defects inother leukocytes and mucosal integrity. Selective depletion ofneutrophils with mAbs is a useful approach, but it has limitations.Commercially available Abs such as anti–Gr-1 and anti–Ly-6Gdeplete mature neutrophils but may also affect eosinophils, mono-cytes, and dendritic cells. In addition, these Abs deplete matureneutrophils, but not the immature band forms, which are none-theless fully functional. Therefore, in response to intact signals forneutrophil production in the bone marrow and/or neutrophil re-cruitment to the site of infection, an ongoing robust neutrophilicresponse may occur even in the face of continued Ab treatment.This is most evident in the affected tissue and is overlooked if onlyperipheral blood is evaluated to assess efficiency of depletion. Ina prior study attempting to address the issue of neutrophils inOPC, a swabbing method was used as a qualitative measure offungal infection (44). However, that approach does not accuratelyrecover hyphal forms of the yeast that have invaded the mucosaltissue. This study advances the field by providing a highly quan-titative evaluation of susceptibility to invasive mucosal Candidainfection, the hallmark of OPC, in states of neutrophil/myeloidcell impairment.The prevalence of immunodeficiency is on the rise, along with

an increasing awareness of the risk for opportunistic infection insusceptible patients. In clinical medicine, morbidity and mor-tality caused by fungal infections are observed in patients with neu-tropenia. Early reports of primary immunodeficiency with isolatedneutropenia focused on invasive rather than mucosal candidiasis, butmore recent case reports of congenital neutropenia highlight sus-ceptibility to oral candidiasis (45). Neutropenia can also besecondary to immunosuppressive medications, medication sideeffects, or autoimmunity. Notably, neutropenia is rarely an isolatedimmunodeficiency. Combination defects in branches of the im-mune system are common and are likely to become more so withthe increasing use of selective immunomodulation with biologicagents. This study is directly relevant to the use of drugs currently

in development that target IL-17 and the Th17 pathway (46–48).Along these lines, a recent analysis of IBD patients showed thatanti–TNF-a therapy is linked to oral candidiasis (49). Our findingssuggest that the addition of IL-17 blockade to neutropenia-inducingtherapies is likely to lead to a substantially increased risk for mu-cosal candidiasis.

AcknowledgmentsIL-232/2 mice were provided by Genentech and IL-17RA2/2 mice by

Amgen. We thank M. Simpson-Abelson, M. McGeachy, M. Michaels,

M.H. Nguyen, P.L. Lin, and B. Mehrad for helpful suggestions and A.J.

Mamo for technical assistance.

DisclosuresS.L.G. has consulted for and received a research grant from Novartis (Basel,

Switzerland).

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Corrections

Huppler, A. R., H. R. Conti, N. Hernandez-Santos, T. Darville, P. S. Biswas, and S. L. Gaffen. 2014. Role of neutrophils in IL-17–dependent immunity to mucosal candidiasis. J. Immunol. 192: 1745–1752.

There was an inadvertent error in the calculation of the absolute neutrophil count due to incorrect total WBC counts. The error does notchange the conclusions of the paper.

In the Results section, in the second paragraph under the subheading Susceptibility to OPC is induced in normally resistant WT micewith myeloid cell depletion, the sentence “Anti–Gr-1 resulted in profound peripheral neutropenia on day 0, with some count recovery byday 2 (Fig. 5A)” should read “Anti–Gr-1 resulted in profound peripheral neutropenia on day 0, persisting through day 2 (Fig. 5A).”

A corrected panel for Fig. 5A is shown below. The new Fig. 5A shows a more profound neutropenia in mice treated with anti–Gr-1,which supports the conclusion that anti–Gr-1 treatment induces susceptibility to oropharyngeal candidiasis in wild-type mice due toneutrophil depletion. The new Fig. 5A also shows that there are normal peripheral neutrophil numbers in IL-17RA2/2 mice.

The legend for Fig. 5A was correct as published and is shown below for reference.

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1490053

FIGURE 5. Neutrophil depletion with anti–Gr-1 induces susceptibility to OPC in WT mice. (A) WT mice treated with anti–Gr-1 Abs have peripheral

neutropenia. Mice were treated with isotype or anti–Gr-1 Abs on days21 and 2, and subjected to OPC on day 0. Controls include WT sham-inoculated and

IL-17RA2/2 mice. Peripheral neutrophil counts were determined as in Fig. 2A. Dashed line indicates threshold for clinically significant neutropenia.

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