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Retinoid-Related Orphan Receptor γ Controls Immunoglobulin Production and Th1/Th2 Cytokine Balance in the Adaptive Immune Response to Allergen This information is current as of September 27, 2021. Stephen L. Tilley, Maisa Jaradat, Cliona Stapleton, Darlene Dixon, Xiaoyang Hua, Christopher J. Erikson, Joshua G. McCaskill, Kelly D. Chason, Grace Liao, Leigh Jania, Beverly H. Koller and Anton M. Jetten

J Immunol 2007; 178:3208-3218; ; Downloaded from doi: 10.4049/jimmunol.178.5.3208 http://www.jimmunol.org/content/178/5/3208 http://www.jimmunol.org/ References This article cites 52 articles, 22 of which you can access for free at: http://www.jimmunol.org/content/178/5/3208.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2007 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Retinoid-Related Orphan Receptor ␥ Controls Immunoglobulin Production and Th1/Th2 Cytokine Balance in the Adaptive Immune Response to Allergen1

Stephen L. Tilley,2* Maisa Jaradat,‡ Cliona Stapleton,‡ Darlene Dixon,§ Xiaoyang Hua,* Christopher J. Erikson,* Joshua G. McCaskill,* Kelly D. Chason,* Grace Liao,‡ Leigh Jania,† Beverly H. Koller,† and Anton M. Jetten‡

The retinoid-related orphan receptors (ROR) comprise a distinct subfamily of nuclear receptors with the capacity to act as both repressors and activators of transcription. ROR␥, the most recently identified member of the ROR family, has been shown to be important for the development of normal lymphocyte compartments as well as organogenesis of some lymphoid organs. In this report, we examine the capacity of ROR␥-deficient mice to develop an adaptive immune response to Ag using OVA-induced Downloaded from inflammation in mice as a model for allergic airway disease. In sham-treated mice lacking ROR␥, low-grade pulmonary inflammation was observed and characterized by the perivascular accumulation of B and T lymphocytes, increased numbers of inflammatory cells in the lung lavage fluid, and polyclonal Ig activation. Following sensitization and challenge, the capacity of these animals to develop the allergic phenotype was severely impaired as evidenced by attenuated eosinophilic pulmonary inflammation, ␥-reduced numbers of CD4؉ lymphocytes, and lower Th2 cytokines/chemokine protein and mRNA expression in the lungs. IFN and IL-10 production was markedly greater in splenocytes from ROR␥-deficient mice following in vitro restimulation with OVA http://www.jimmunol.org/ compared with wild-type splenocytes, and a shift toward a Th1 immune response was observed in sensitized/challenged ROR␥- deficient animals in vivo. These data reveal a critical role for ROR␥ in the regulation of Ig production and Th1/Th2 balance in adaptive immunity. The Journal of Immunology, 2007, 178: 3208–3218.

uclear receptors constitute a superfamily of ligand-de- The ROR, ROR␣, ␤, and ␥, constitute a subfamily of nuclear pendent transcription factors that include receptors for orphan receptors whose roles in inflammation are less well under- N steroid hormones, retinoic acid, and thyroid hormone; stood. Similar to other nuclear receptors, ROR are believed to be and orphan receptors for which the ligands have yet to be identified activated by small lipophilic molecules that bind to the receptor, by guest on September 27, 2021 (1). Several studies have demonstrated a role for a number of nu- induce a conformational change resulting in the dissociation of clear receptors in the regulation of inflammation. These include the corepressor complexes, and facilitating translocation to the nucleus glucocorticoid receptor (GR),3 estrogen receptor, vitamin D recep- where gene expression is modulated. In addition to their capacity tor, retinoic acid receptors, peroxisome proliferator-activated re- to directly promote gene transcription, an anti-inflammatory role ceptors, and several orphan receptors, including members of the for ROR has been suggested based on findings that they can pos- retinoid-related orphan receptor (ROR) subfamily (2–11). The anti- itively regulate the expression of I␬B␣ (12). Thus, similar to GR, inflammatory action of glucocorticoids, ligands for the GR, is well ROR can act as both ligand-dependent transcription activators as established, and these agonists form the first line of treatment in well as ligand-dependent negative regulators of other transcription asthma. factors. Due in part to this complexity, the precise role of these receptors in inflammation has been difficult to predict. The newest member of the ROR subfamily, ROR␥, was first *Department of Medicine, Division of Pulmonary and Critical Care Medicine, and identified in skeletal muscle by its homology to retinoic acid re- †Department of Genetics University of North Carolina, Chapel Hill, NC 27599; and ‡ § ceptor but speculated to play a role in inflammation based upon its Laboratory of Respiratory Biology, Cell Biology Section, and Laboratory of Ex- ϩ ϩ perimental Pathology, Division of Intramural Research, National Institute of Envi- abundant expression in CD4 CD8 double-positive thymocytes ronmental Health Sciences, National Institutes of Health, Research Triangle Park, (13, 14). Forced expression of ROR␥ in T cell hybridomas resulted NC 27709 in inhibition of TCR-induced proliferation and cell death, suggest- Received for publication November 10, 2005. Accepted for publication December 12, 2006. ing an important role for this ROR subtype in T cell apoptosis (15, 16). Indeed, disruption of the ROR␥ gene in mice resulted in a The costs of publication of this article were defrayed in part by the payment of page ϩ ϩ charges. This article must therefore be hereby marked advertisement in accordance markedly increased rate of apoptosis in CD4 CD8 double-pos- with 18 U.S.C. Section 1734 solely to indicate this fact. itive thymocytes, confirming the physiological importance of 1 This work is supported by Intramural Research Program of the National Institute on ROR␥ in T cell biology in vivo (17, 18). Due to this accelerated Environmental Health Sciences, National Institutes of Health, and the extramural National Institutes of Health Grant HL071802. rate of apoptosis, the numbers of peripheral blood T lymphocytes 2 in ROR␥-deficient mice were decreased 6-fold, with a 10-fold re- Address correspondence and reprint requests to Stephen L. Tilley, 8033 Burnett- ϩ ϩ Womack, CB# 7219, University of North Carolina, Chapel Hill, NC 27599-7219. duction in CD4 cells and a 3-fold reduction in CD8 cells (17). E-mail address: [email protected] Despite a lower thymic output, T cells from ROR␥-deficient mice 3 Abbreviations used in this paper: GR, glucocorticoid receptor; EOS, eosinophil; appear to be exported normally to the periphery and have normal PAS, periodic acid-Schiff; PMN, polymorphonuclear neutrophil; Raw, airway resis- proliferative function (18). The numbers of CD4ϩ and CD8ϩ lym- tance; RL, dynamic resistance; ROR, retinoid-related orphan receptor; Th17, IL-17- producing Th. phocytes are normal to marginally increased in the spleens of www.jimmunol.org The Journal of Immunology 3209

FIGURE 1. Lung lavage ﬂuid cellularity in wt and ROR␥Ϫ/Ϫ mice. Total cells and differentials were evaluated in sensitized mice 24 h following the last of ﬁve daily aerosol challenges with saline (A)orOVA(B). Saline-exposed ROR␥Ϫ/Ϫ (Ⅺ, n ϭ 15), saline- exposed wt (f, n ϭ 13), OVA- exposed ROR␥Ϫ/Ϫ (Ⅺ, n ϭ 14), and OVA-exposed wt (f, n ϭ 16). Data represent mean cell number Ϯ SEM. -p Ͻ 0.05. C, Representative cy ,ء tospin from saline-exposed wt mouse showing macrophages and a few lymphocytes. D, Representative cytospin Ϫ/Ϫ from saline-exposed ROR␥ mouse Downloaded from showing granulocytes, lymphocytes, and macrophages. Slides were stained with Hema-3. http://www.jimmunol.org/

ROR␥-deficient mice, whereas the B cell compartment is substan- for 10 min at 4°C. The supernatants were aliquoted, 10% FBS was added tially expanded, ϳ3-fold the numbers found in wild-type (wt) con- to each aliquot, and samples subsequently frozen at Ϫ80°C for cytokine trols (19). In addition to these abnormalities in lymphocyte ho- analysis. Cell pellets were resuspended in 1 ml of HBSS and counted with ␥ a hemocytometer. Slides of lavage fluid cells were prepared using a Cy- meostasis, ROR -deficient mice fail to develop lymph nodes and tospin-3 centrifuge (Thermo Shandon) and stained with Mema-3 and Fast Peyer’s patches (17, 18). Based on these observations of abnor- Green (eosinophil stain) for determination of cellular differentials. malities in T cell homeostasis and lymphoid organogenesis, we Lung histopathology by guest on September 27, 2021 surmised that ROR␥-deficient mice might have an impaired capacity to generate an adaptive immune response to Ag. To test this Immediately following whole-lung lavage, the lungs were inflated with 10% hypothesis, we compared the induction of inflammatory responses neutral-buffered formalin under 20 cm of pressure, and the trachea was tied off. ␥ The lungs were then removed en bloc, immersed in 10% formalin, and dehy- between wt and ROR -deficient mice using OVA-induced inflam- drated with ethanol before paraffin embedding. Five- to 6-␮m serial sections mation as a model system for allergic asthma. were cut through the right lung lobes and stained with H&E for general mor- phology and periodic acid-Schiff (PAS)/Alcian blue for mucus. Materials and Methods Histopathology score Experimental animals The H&E-stained sections were semiquantitatively scored by a pathologist ROR␥-deficient mice (10, 17) were backcrossed four generations to the for the degree of inflammation. Seventy to 100 fields were evaluated and C57BL/6 background. Heterozygotes were intercrossed to produce scored in a blinded fashion from 0 to 4. A score of 0 indicated the absence Ϫ Ϫ Ϫ Ϫ ϩ ϩ ROR␥ / N4C57BL/6 (hereafter referred to as ROR␥ / ) and ROR␥ / of inflammation in the perivascular, peribronchial/bronchiolar, and intra- N4C57BL/6 wt, littermate controls. Mice were genotyped by PCR analysis alveolar regions of the lung. Two inflammatory scores were assigned. One of DNA from tail biopsy as described previously (17). All mice were used was based on perivascular infiltrates of lymphocytes only and the other on between 9 and 22 wk of age, and within experiments, mutant and wt mice peribronchial/bronchiolar infiltrates of mixtures of polymorphonuclear Ϫ Ϫ were matched for age and sex. For some experiments, ROR␥ / neutrophils (PMN), eosinophils (EOS), and lymphocytes. For the perivas- ϩ ϩ N4BALB/c and ROR␥ / N4BALB/c littermate controls were used. These cular lymphocytic infiltrates, the scores were assigned as follows: 1, min- animals were generated by intercrossing heterozygotes after four genera- imal: few scattered perivascular infiltrates of lymphocytes involving Ͻ15% tions of backcrossing to the BALB/c background. All experiments were of the blood vessels; no perivascular cuffing of the lymphocytes; 2, mild: conducted in accordance with institutional animal care and use guidelines infiltrates of lymphocytes with formation of perivascular cuffs (consisting of the University of North Carolina at Chapel Hill and the National Insti- of fewer than five layers of cells) involving 15–25% of the blood vessels, tute of Environmental Health Sciences. with few adjacent foci of associated lymphocytes; 3, moderate: infiltrates of lymphocytes with formation of perivascular cuffs (consisting of 5–10 Induction of allergic inflammation layers of cells) involving Ͼ25 but Ͻ75% of the blood vessels with some Mice were sensitized with an i.p. injection of 20 ␮g of chicken egg OVA adjacent foci of lymphocytes; and 4, severe: infiltrates of lymphocytes with ␮ formation of perivascular cuffs (consisting of Ͼ10 layers of cells) involv- (grade V; Sigma-Aldrich) emulsified in 200 l of aluminum hydroxide Ͼ (alum) adjuvant (Alhydrogel; Accurate Chemical & Scientific). Fourteen ing 75% of the blood vessels, with many adjacent foci of lymphocytes. days later, mice were challenged with either aerosolized 1% OVA or saline For the peribronchial/bronchiolar infiltrates of mixtures of PMN, EOS, and for 30 min on 5 consecutive days in a whole-body exposure chamber. lymphocytes, the scores were assigned as follows: 1, minimal: inflammatory infiltrates of primarily lymphocytic cells mixed with a few PMN/EOS Twenty-four hours following the last exposure, physiological, pathologi- Ͻ cal, and biochemical phenotyping was performed. within the tissue surrounding the bronchi and bronchioles, involving 15% of the airways; 2, mild: peribronchial/bronchiolar infiltrates of PMN and Whole-lung lavage EOS mixed with lymphocytes involving 15–25% of the airways; 3, moderate: peribronchiolar/bronchial infiltrates of predominantly EOS and PMN The chest was opened, and lungs were lavaged by instillation as described mixed with lymphocytes involving Ͼ25 but Ͻ75% of the airways; and 4, previously (11). Lavage fluid was kept on ice and centrifuged at 360 ϫ g severe: peribronchiolar infiltrates of predominantly PMN and EOS mixed 3210 DYSREGULATED ADAPTIVE IMMUNE RESPONSE IN ROR␥Ϫ/Ϫ MICE

FIGURE 2. OVA-induced lung in- ﬂammation in wt and ROR␥Ϫ/Ϫ mice. Twenty-four hours following the last of ﬁve daily aerosol challenges, lungs were evaluated in sensitized wt and ROR␥Ϫ/Ϫ mice. Saline-exposed H&E

(A and C), OVA-exposed H&E (B Downloaded from and D) mice, and oVa-exposed PAS/ Alcian blue for mucus (E and F)inwt (A, B, and E) and ROR␥Ϫ/Ϫ (C, D, and F) mice. Arrowheads denote perivascular lymphoid aggregates; arrows denote peribronchovascular inﬂammatory inﬁltrates. b, bronchus; http://www.jimmunol.org/ v, vessel. by guest on September 27, 2021

with lymphocytes involving Ͼ75% of the airways. In lung tissue sections that were scored moderate or severe, the mixed inflammatory infiltrates often also involved the perivascular and intra-alveolar regions. In cases of intra-alveolar infiltrates, there were alveolar macrophages present in addition to the mixed aggregates of EOS, PMN, and lymphocytes. Scores were subsequently grouped and averaged according to genotype and treatment. Immunohistochemistry To detect CD3, frozen mouse lungs sectioned on slides were thawed 5 min on ice, then dipped in ice cold acetone for 2 min. Slides were dipped in 0.3% hydrogen peroxide in methanol for 10 min, then blocked with the Avidin/Biotin Blocking kit (Vector Laboratories), according to the manufacturer’s instructions. CD3 Ab (hamster anti-mouse; Biolegend), 1/10 in Ab diluent (BD Pharmingen), was applied, and sections were incubated in a humid chamber overnight at room temperature (RT). Anti-hamster secondary Ab, 1/2000 in Ab diluent, was applied, and sections incubated for 1 h in a humid chamber at room temperature. Streptavidin-HRP (BD Pharm- ingen) was applied for 45 min in a humid chamber. The DAB Substrate kit FIGURE 3. Inflammatory scores for wt and ROR␥Ϫ/Ϫ mice. H&E- (BD Pharmingen) was used for detection following the manufacturer’s in- stained sections of lungs from sensitized wt and ROR␥Ϫ/Ϫ mice challenged structions. To detect CD45, paraffin-embedded lungs were sectioned on with either saline (Ϫ) or OVA (ϩ) were scored from 0 to 4 for the extent slides. Sections were deparaffinized in xylene for 5 min and rehydrated of inflammatory cell infiltration, as described in Materials and Methods. through an ethanol series. Slides were heated in 10 mM sodium citrate to Perivascular lymphocytes only (u) and mixed peribronchial/bronchiolar boiling in a microwave and allowed to stand in the hot solution for 30 min. infiltrates (f) were scored separately. Zero to 1 indicates no or little in- Slides were dipped in 0.3% hydrogen peroxide in PBS for 15 min and dipped in acetone for 30 s. Sections were otherwise treated the same as flammation, whereas 4 represents severe inflammation. Data represent -p Ͻ 0.05 compared with saline ,ء .frozen slides, except CD45 Ab (rat anti-mouse; BD Pharmingen), 1/25, was mean inflammatory score Ϯ SEM used as the primary Ab and anti-rat Ab, 1/2000, as the secondary Ab. PBS exposed group of the same genotype. ϩ, p Ͻ 0.05 compared with OVA- was used to wash the sections between each step. Control sections of each exposed wt mice. The Journal of Immunology 3211

FIGURE 4. Lymphocyte populations in the lungs of ROR␥Ϫ/Ϫ mice. Frozen and paraffin-embedded sections from saline-exposed ROR␥Ϫ/Ϫ mice were used for immunohistochemistry with CD3 and CD45 Abs, respectively. A and C, Controls with Downloaded from secondary Ab only. B, CD3 mAb. D, CD45/B220 mAb. E, Lung lavage fluid was collected from sensitized mice 24 h following the last of five daily aerosol challenges with saline (Ϫ) or OVA (ϩ). Lymphocytes were

sorted by ﬂow cytometry after Ab la- http://www.jimmunol.org/ beling of CD3 (f), B220 (Ⅺ), CD4 (o), and CD8 (u). Data represent mean cell number Ϯ SEM. n ϭ 6–8 p Ͻ 0.05 compared ,ء ;mice/group with respective saline-treated group. by guest on September 27, 2021

type were treated exactly as experimental sections, except for incubation (Tarc) and CCL-24 (eotaxin-2) by real-time quantitative RT-PCR. The with PBS rather than primary Ab in Ab diluent. Sections were counter- PCR were conducted in triplicate using a 7300 Real-Time PCR system stained with methylene green, dehydrated through alcohols, cleared in xy- using the TaqMan One-Step RT-PCR mix (Applied Biosystems). lene, and coverslipped using a permanent mounting medium. Splenocyte cultures Flow cytometric analysis Spleens were removed and homogenized with a pellet pestle (Kontes) in Lung lavage fluid lymphocytes expressing CD3, CD4, CD8, and B220 RPMI 1640 medium (Invitrogen Life Technologies) supplemented with 10 were stained as described previously (17). Briefly, 5 ϫ 105 cells were mM HEPES (Mediatech) and 10% FBS (Invitrogen Life Technologies). incubated with a combination of FITC- or PE-conjugated CD3, CD4, CD8, Cell suspension was collected and centrifuged 370 ϫ g at 4°C for 10 min. and B220 Abs (BD Pharmingen). Cell surface markers were sorted using The pellet was resuspended in 10 ml of ice-cold RBC lysis solution (0.14 a LSR flow cytometer (BD Biosciences). Data were analyzed using MNH4Cl and 0.017 M Tris (pH 7.2)), incubated on ice for 5 min, 5 ml of CellQuest software (BD Biosciences). RPMI-HEPES-FBS added, and centrifuged again. Lysis was repeated if RBC were apparent in the pellet. Once RBC lysis was complete, cells were Cytokine assays washed twice with RPMI 1640 medium and strained through a cell strainer (BD Biosciences). Cells were counted, pelleted, and resuspended in RPMI Lavage fluid supernatants were stored at Ϫ80°C until analysis. Samples 1640 medium with 10% FBS, 10 U/ml penicillin, and 0.1 mg/ml strepto- were thawed on ice, and levels of IL-13 were measured with a commer- mycin (Invitrogen Life Technologies) at 37°C at a density of 5 ϫ 106 cially available ELISA kit according to the manufacturer’s instructions cells/ml in medium alone or medium supplemented with OVA (100 ␮g/ (R&D Systems). Other cytokines were assayed with Bio-Plex multiplex ml). Culture supernatants were harvested after 72 h and stored at Ϫ80°C cytokine assay system (Bio-Rad) using the Luminex 100 instrument (Lu- until analysis. minex) according to the manufacturer’s instructions. Measurements of Ig isotypes Real-time quantitative RT-PCR analysis Blood was obtained from the aorta or heart while mice were deeply anes- RNA was isolated from the lungs of mice of each genotype and treatment thetized with pentobarbital. Blood was placed in an Eppendorf tube and group, reverse transcribed, and analyzed for the expression of CCL-17 allowed to coagulate. Samples were centrifuged at 6000 ϫ g for 2 min, and 3212 DYSREGULATED ADAPTIVE IMMUNE RESPONSE IN ROR␥Ϫ/Ϫ MICE

the serum was removed and stored at Ϫ80°C. The samples were thawed on ice, and the levels of several mouse Ig isotypes were determined by ELISA using biotinylated rat anti-mouse IgE (R35-92), IgG1, IgG2a and b, IgG3, IgA, and IgM according to the manufacturer’s instructions (BD Pharmin- gen). OVA-specific IgE was measured by the same ELISA with the following modification: the 96-well plate was coated with OVA (100 ␮lof20 mg/ml OVA in the coating buffer provided by the manufacturer). Samples, standard, and controls were incubated overnight at 4°C. For OVA-specific IgG1 and IgG2c, 96-well high-binding microplates (Fisher Scientific) were coated with 100 ␮l/well of 1 mg/ml OVA (Sigma-Aldrich) in PBS at 37°C for 2 h, followed by incubation with 200 ␮l/well blocking buffer (1% BSA in PBS) for1hatroom temperature. OVA-specific IgG2c measurements were made on serum samples diluted 1/300 and/or serially to 1/3000 in blocking buffer. For OVA-specific IgG1 measurements, samples were serially diluted to 1/5,000 and/or 1/500,000. A standard curve for each assay was prepared by diluting pooled sera that had been previously tested as highly concentrated in OVA-specific IgG1 or IgG2c. One hundred microliters of each standard or sample dilution was applied to the wells in triplicate and incubated overnight at 4°C. One hundred microliters of biotinylated anti-mouse IgG1 (BD Biosciences) (2 ␮g/ml in blocking buffer) and 100 ␮l of biotinylated anti-mouse IgG2c (Southern Biotechnology Asso- ciates) (1/5000 in blocking buffer) were added to each well and incubated

at room temperature for 30 min. One hundred microliters of streptavidin- Downloaded from HRP (BD Biosciences) (1/1000 in blocking buffer) was applied to each well and incubated for 30 min at room temperature, followed by the addition of HRP substrate (ABTS; Sigma-Aldrich). Color was developed for 30 min, and absorbances were read at 405 nm. Calculations were made using the serum dilution that fell closest to the middle of the linear part of the standard curve. Concentrations are expressed as arbitrary units. FIGURE 5. Th2 chemokine expression in the lungs of wt and ROR␥Ϫ/Ϫ Airway physiology mice. Eotaxin/CCL-24 (A) and TARC/CCL-17 (B) mRNA expression were http://www.jimmunol.org/ examined by quantitative real-time PCR in OVA-exposed animals and Airway mechanics were evaluated in anesthetized, paralyzed, mechanically ventilated mice using a computer-controlled mouse mechanical ventilator compared with expression in saline-exposed mice of the same genotype. .p Ͻ 0.01 ,ء with software specifically designed for evaluating respiratory mechanics (Scireq). Mice were anesthetized with pentobarbital (70–90 mg/kg), tra- cheostomized with a 19-gauge canula, and ventilated at a frequency of 300 ␮ breaths/min, 150 l of tidal volume, and 4 cm/H2O PEEP. After paralysis lavage fluid compared with saline-treated wt controls. While with pancuronium bromide (0.8 mg/kg), airway resistance (R ) was mea- Ϫ/Ϫ aw ROR␥ mice also developed increased lung lavage fluid cellu- sured using the forced oscillation technique, where an 8-s oscillatory flow larity in response to OVA, the numbers of EOS and lymphocytes waveform is delivered to the murine airway. Using this methodology, Raw is determined by fitting input impedance to the constant phase model. Fol- were significantly lower than OVA-exposed wt mice (Fig. 1B). by guest on September 27, 2021 lowing these measurements, dynamic resistance (RL) was determined be- Similar reductions in the proportion of EOS were also observed in fore and following aerosols of methacholine by measuring pressure ROR␥Ϫ/Ϫ N4BALB/c mice (data not shown). changes at the airway opening during the delivery of a 1-Hz sinusoidal ␥Ϫ/Ϫ breath of 150 ␮l and by fitting the data to the linear single-compartment Histologically, the lungs from saline-treated ROR mice model using multiple linear regression. After baseline measurements of showed perivascular accumulation of lymphocytes (Fig. 2C). Sa- resistance, aerosols of methacholine were delivered for 20 s each through line-treated wt mice did not show this lymphocytic accumulation a side-port in the ventilator circuit, and RL was measured every 10 s around blood vessels (Fig. 2A). Wt mice challenged with OVA postchallenge. displayed changes typical of allergen-induced inflammation: infil- Statistical analysis of data tration of many peribronchial/vascular EOS, PMN, and lymphocytes as well as moderate amounts of these cells in the alveolar Statistical analysis was conducted using one-way ANOVA, followed by Bon- ferroni method (multiple comparison) when comparing between groups and sacs (Fig. 2B). Sections of lungs were scored by a semiquantita- Student’s t test for comparison within the group using the Sigmastat 2.0 soft- tive, histopathological scoring system in a blinded fashion using ware (Jandel). All values are expressed as averages Ϯ SE. two distinctive criteria: 1) the extent of perivascular infiltration of lymphocytes; and 2) the extent of peribronchial/bronchiolar infil- Results tration of mixtures of PMN, EOS, and lymphocytes. The average ␥Ϫ/Ϫ Lung inflammation in ROR mice peribronchiolar inflammation score for OVA-exposed wt mice was To determine the role of ROR␥ in the development of allergic 3.2, indicating moderate inflammation (Fig. 3). Lungs from inflammation in the lung, we evaluated changes in lung lavage ROR␥Ϫ/Ϫ mice challenged with OVA revealed mild peribronchial/ fluid cellularity and lung histology in response to OVA. Interest- bronchiolar infiltration of EOS and PMN (Fig. 2D). The average ingly, while saline-treated wt mice showed predominantly macro- score for peribronchial/bronchiolar infiltration of inflammatory phages and a few lymphocytes in the lavage fluid, a significant cells for OVA-exposed ROR␥Ϫ/Ϫ was 1.4, indicating mild inflam- number of saline-treated ROR␥Ϫ/Ϫ mice had increased numbers of mation (Fig. 3). The perivascular infiltration of lymphocytes ob- inflammatory cells present (Fig. 1A). Cytospins of this fluid reserved in saline-treated ROR␥Ϫ/Ϫ mice was further enhanced fol- vealed that, while most of the cells were macrophages, lympho- lowing OVA treatment (Figs. 2D). In OVA-treated wt mice, a cytes and granulocytes were also present in significantly greater perivascular infiltration of a mixed population of inflammatory numbers than wt mice (Fig. 1, A, C, and D). Fast green stain (spe- cells was seen, consisting largely of EOS and PMN, similar to the cific for EOS granules) revealed that approximately half of these peribronchial/bronchiolar infiltrates. The primary lymphocytic granulocytes were EOS. perivascular infiltrate found in ROR␥Ϫ/Ϫ mice was not observed in As expected, OVA-exposed wt mice developed a robust inflam- the saline or OVA-treated wt mice. A similar marked reduction in matory cell influx into the lung with significant increases in num- pulmonary inflammation was also seen in OVA-exposed ROR␥Ϫ/Ϫ bers of EOS, lymphocytes, macrophages, and PMN in the lung N4BALB/c mice (data not shown). The Journal of Immunology 3213

FIGURE 6. Cytokines in lung lavage fluid of Downloaded from wt and ROR␥Ϫ/Ϫ mice. Lung lavage fluid su- pernatant was collected from sensitized mice 24 h following the last of five daily aerosol challenges with saline (u, Ϫ) or OVA (f, ϩ), and IL-4 (A), IL-2 (B), IL-5 (C), IL-12 (D), IL-13 (E), IFN-␥ (F), TNF-␣ (G), and IL-10 (H) pro- http://www.jimmunol.org/ tein levels were measured. Data represent -p Ͻ 0.05 com ,ء .mean cytokine level Ϯ SEM pared with saline-exposed group of the same genotype. #, p Ͻ 0.05 compared with similarly exposed wt mice. n ϭ 8 mice/group. by guest on September 27, 2021

Mucus metaplasia is another characteristic feature of OVA- Lymphocyte phenotyping induced inflammation. As shown in Fig. 2E, wt mice sensitized To further characterize the perivascular lymphocyte accumulation and challenged with OVA showed a large number of PAS/ observed in ROR␥Ϫ/Ϫ animals, immunohistochemistry was per- Alcian blue-positive staining goblet cells in the respiratory ep- formed using mAbs against CD3 and CD45/B220. As shown in ␥Ϫ/Ϫ ithelium. In contrast, similarly treated ROR mice showed Fig. 4, these perivascular lesions were composed of a mixed pop- significantly less PAS/Alcian blue-positive goblet cells (Fig. ulation of B and T lymphocytes. To further evaluate changes in Ϫ/Ϫ 2F). Both wt and ROR␥ saline-treated controls showed ab- lymphocyte populations following OVA challenge, flow cytom- sence of PAS/Alcian blue-positive goblet cells in the airway etry was performed on lavage fluid lymphocytes from saline and epithelium (data not shown). The attenuation of allergic inflam- OVA-exposed ROR␥Ϫ/Ϫ and wt animals. Significant increases in Ϫ Ϫ mation observed in OVA-treated ROR␥ / mice on two dif- CD3ϩ, B220ϩ, CD4ϩ, and CD8ϩ cells were observed in both wt ferent genetic backgrounds suggests that ROR␥ is critical for and ROR␥Ϫ/Ϫ mice compared with their respective saline-exposed the development of a normal adaptive immune response to controls (Fig. 4E). A trend toward lower numbers of CD4ϩ ( p ϭ allergen. 0.054) and CD8ϩ ( p ϭ 0.069) lymphocytes was observed in the 3214 DYSREGULATED ADAPTIVE IMMUNE RESPONSE IN ROR␥Ϫ/Ϫ MICE

FIGURE 7. Cytokine production by splenocytes from wt and ROR␥Ϫ/Ϫ mice. Splenocytes were cul- tured at 37°C at a density of 5 ϫ 106 cells/ml in RPMI 1640 medium alone (u, Ϫ) or medium supplemented with OVA (100 ␮g/ml) (f, ϩ). Culture supernatants were harvested after 72 h, and protein levels of IL-4 (A), IL-2 (B), IL-5 (C), IFN-␥ (D), and IL-10 (E) were Ϯ measured. Data represent mean cytokine level Downloaded from p Ͻ 0.05 compared with saline-exposed ,ء .SEM group of the same genotype. #, p Ͻ 0.05 compared with similarly exposed wt mice. n ϭ 6–8 mice/group. http://www.jimmunol.org/ by guest on September 27, 2021

OVA-treated ROR␥Ϫ/Ϫ group when compared with similarly ex- contribute to the reduced lung inflammation observed in these posed wt mice. Equally, a trend toward lower numbers of B cells animals. was also observed ( p ϭ 0.13). However, T/B cell ratios and To further examine the influence of ROR␥ deficiency on Ag- ϩ ϩ Ϫ Ϫ CD4 /CD8 ratios in ROR␥ / mice were similar to wt controls, induced cytokine production, OVA-sensitized splenocytes were ϩ and CD4 T cells constituted the majority of the T cell population examined following in vitro restimulation. As shown in Fig. 7, in OVA-treated animals of both genotypes. splenocytes from ROR␥Ϫ/Ϫ mice showed marked increases in all Ϫ Ϫ cytokines examined following stimulation with OVA, suggesting Reduced induction of Th2 chemokine mRNA in ROR␥ / mice that these animals are capable of generating a robust adaptive im- Eotaxin-2/CCL-24 and TARC/CCL-17 are high-affinity ligands for mune response to Ag. Significant increases in IFN-␥ levels were chemokine receptors (CCR3 and CCR4) selectively expressed on observed following OVA stimulation of ROR␥Ϫ/Ϫ splenocytes cells present in Th2-mediated inflammation (EOS, basophils, and ( p ϭ 0.03) at 6.8 times higher than levels produced by wt OVA- Th2 lymphocytes). To evaluate the expression of these chemo- stimulated cells ( p ϭ 0.06) (Fig. 7D). Production of the T regu- kines, RNA from lungs of OVA- and saline-challenged wt and Ϫ/Ϫ latory cytokine IL-10 was also examined following in vitro re- ROR␥ mice were examined using quantitative real-time RT- Ϫ Ϫ stimulation. OVA-stimulated splenocytes from ROR␥ / mice PCR. As shown in Fig. 5, the expression of both eotaxin-2/CCL-24 produced five times greater amounts of IL-10 than similarly treated and TARC/CCL-17 was markedly lower in the lungs of ROR␥Ϫ/Ϫ wt splenocytes ( p ϭ 0.04) (Fig. 7E). Taken together, these studies mice than in wt animals. suggest that ROR␥ can significantly influence the cytokine profile Aberrant cytokine production in ROR␥Ϫ/Ϫ mice induced by Ag. To further evaluate the immune response to OVA, Th1 and Th2 cytokines were examined in the lung lavage fluid from saline and Spontaneous polyclonal Ig production in ROR␥Ϫ/Ϫ mice OVA-exposed wt and ROR␥Ϫ/Ϫ mice. As shown in Fig. 6, A, C, ␥Ϫ/Ϫ and E, robust induction of IL-4, IL-5, and IL-13 was observed in Ig production by ROR mice was first examined by measuring wt animals. In contrast, modest induction of these Th2 cytokines total IgE in sera. As expected, total IgE levels increased signifi- was detected in ROR␥Ϫ/Ϫ mice. ROR␥Ϫ/Ϫ mice showed trends cantly in wt mice following OVA challenge (Fig. 8A). Total IgE Ϫ Ϫ toward paradoxical elevations in IL-2 and IFN-␥ after OVA chal- levels in saline-treated ROR␥ / mice were significantly greater lenge ( p ϭ 0.072, p ϭ 0.061), as well as significant elevations in than saline-treated wt mice and were as high as levels observed in TNF-␣ (Fig. 6, B, F, and G). These results show that ROR␥Ϫ/Ϫ OVA-treated wt animals. Total IgE did not change significantly in mice produce an aberrant cytokine response to OVA, which might ROR␥Ϫ/Ϫ mice after OVA treatment ( p ϭ 0.78). To determine The Journal of Immunology 3215

FIGURE 8. Ig levels in the serum of wt and ROR␥Ϫ/Ϫ mice. Serum was collected from sensitized mice 24 h following the last of ﬁve daily aerosol challenges with saline (u, Ϫ) or OVA (f, ϩ). Total IgE (A), IgA, IgM, and IgG (B), and OVA-spe-

ciﬁc IgE (C), IgG1 (D), and IgG2c (E) Downloaded from were measured by ELISA. Data are pre- sented as mean serum Ig level Ϯ SEM. p Ͻ 0.05 compared with saline-exposed ,ء group of the same genotype. ϩ, p Ͻ 0.05 compared with similarly exposed wt mice. n ϭ 8 mice/group. http://www.jimmunol.org/ by guest on September 27, 2021

␥Ϫ/Ϫ whether dysregulated Ig production by ROR mice was re- mice was no different from saline-treated controls. RL at baseline stricted to the IgE class, IgM, IgA, and IgG subclasses were mea- and following graded methacholine challenge was also no different sured. As shown in Fig. 8B, IgM and all IgG subclasses trended between OVA-exposed and saline-exposed ROR␥Ϫ/Ϫ mice (Fig. ␥Ϫ/Ϫ higher in ROR mice compared with wt controls. 9B). In contrast, RL in OVA-exposed wt mice was significantly greater than saline-exposed wt and OVA-exposed ROR␥Ϫ/Ϫ mice ␥Ϫ/Ϫ OVA-specific Ig production has Th1 bias in ROR mice at baseline and following most challenges with methacholine. Fail- In contrast to total IgE, OVA-specific IgE levels were low in sa- ure to detect characteristic physiological changes in the airways of Ϫ Ϫ line-treated ROR␥Ϫ/Ϫ mice but rose substantially following OVA OVA-exposed ROR␥ / mice is consistent with the markedly at- exposure (Fig. 8C). Next, IgG1 (Th2)- and IgG2c (Th1)-specific tenuated allergic inflammation observed in these animals. Igs were measured in sera from wt and ROR␥Ϫ/Ϫ animals. As shown in Fig. 8D, IgG1 levels increased substantially in wt ani- Discussion mals following OVA treatment. A much more modest induction of In this report, we examined the capacity of mice deficient in ROR␥ Ϫ Ϫ IgG1 was observed in ROR␥ / mice following OVA challenge, to develop allergic lung inflammation in response to OVA sensi- and levels were significantly lower than those from OVA-treated tization and challenge. Similar to the asthmatic airway, this murine Ϫ Ϫ wt controls. In contrast, many OVA-exposed ROR␥ / animals response is dominated by Th2 cells and a corresponding compli- showed marked elevations of IgG2c, whereas levels of this Th1 Ig ment of cytokines. We show that allergic lung inflammation pro- tended to be lower in wt mice following OVA exposure (109 Ϯ 51 duced by this model is severely blunted in ROR␥Ϫ/Ϫ mice and that vs 4.5 Ϯ 1.3 U, p ϭ 0.08) (Fig. 8E). cytokine profiles are altered in the absence of ROR␥. While a number of immunological lesions have been described in this ␥Ϫ/Ϫ Lack of airflow obstruction in OVA-exposed ROR mice mouse line, our findings here suggest additional roles for ROR␥ in To determine whether the attenuated allergic response in T cell homeostasis and response to allergen. ROR␥Ϫ/Ϫ mice impacted airway physiology, airway mechanics The most striking finding of the present study was the aberrant were evaluated in anesthetized, paralyzed, mechanically ventilated cytokine profile produced by ROR␥Ϫ/Ϫ mice. In contrast to wt animals. OVA exposure resulted in significant increases in Raw in mice, which developed a robust Th2 immune response character- ␥Ϫ/Ϫ wt mice (Fig. 9A). In contrast, Raw in OVA-exposed ROR ized by the induction of IL-4, IL-5, and IL-13, OVA-sensitized 3216 DYSREGULATED ADAPTIVE IMMUNE RESPONSE IN ROR␥Ϫ/Ϫ MICE

that IFN-␥ can inhibit several aspects of allergen-induced inflammation, including lung eosinophilia, goblet cell hyperplasia, and CD4ϩ T cell infiltration and proliferation (29–31). In our studies, we found IFN-␥ levels trended higher in the lung lavage fluid of OVA-treated ROR␥Ϫ/Ϫ mice compared with similarly treated wt controls. We also observed exaggerated IFN-␥ secretion by splenocytes stimulated with OVA in vitro. One possible mechanism for this aberrant immune response in ROR␥Ϫ/Ϫ mice could be that ROR␥ plays a critical role in the functional maturation of APC and their subsequent capacity to direct T cell differentiation. Another possibility is that ROR␥ expression by lymphocytes is critical for suppressing transcription of Th1 cytokines. Recently it has been suggested that IL-17-producing Th (Th17) cells may contribute to the pathogenesis of a number of inflammatory diseases, including allergic asthma (32). In established allergic inflammation, IL-17 has been shown to reduce inflammation by inhibiting the synthesis of Th2 chemokine and cytokine production (33). However, two independent investigations have

shown that IL-17 may play a critical role in the induction of al- Downloaded from lergic inflammation. Mice lacking IL-17 demonstrated attenuated eosinophilic lung inflammation, Th2 cytokine production, and airway hyperresponsive (33, 34). Recent studies showed that Th17 cells are absent in ROR␥Ϫ/Ϫ mice and indicated that ROR␥t (or FIGURE 9. Airway physiology in wt and ROR␥Ϫ/Ϫ mice. Twenty-four ROR␥2), a ROR␥ isoform, is responsible for orchestrating the dif- hours following the last of five daily exposures to saline (Ⅺ) or OVA (f),

ferentiation of Th17 cells both in vitro and in vivo (35). Thus, http://www.jimmunol.org/ baseline Raw (A) and resistance following challenge with aerosolized methacholine (B) was determined in anesthetized, paralyzed, and mechan- impaired IL-17 production during the induction phase of allergic ically ventilated mice. A,Raw was measured by oscillatory mechanics. B, inflammation could contribute to the reduced allergic inflammation F ␥Ϫ/Ϫ RL was measured during a single 1-hz breath. B, Baseline. represents wt that we observed in ROR mice. OVA-exposed mice (n ϭ 14), E represents OVA-exposed ROR␥Ϫ/Ϫ mice While aberrant cytokine production by ROR␥Ϫ/Ϫ mice in re- (n ϭ 10), f represents saline-exposed wt mice (n ϭ 15), and Ⅺ represents sponse to allergen is an attractive mechanism for the reductions in Ϫ/Ϫ saline-exposed ROR␥ mice (n ϭ 12). For both A and B, data represent allergic lung inflammation observed in these animals, several other Ͻ Ͻ Ϫ Ϫ ء Ϯ mean resistance SEM. , p 0.05 vs saline-exposed wt mice; #, p possibilities warrant discussion. ROR␥ / mice lack lymph nodes ␥Ϫ/Ϫ 0.05 vs OVA-exposed ROR mice. and Peyer’s patches, raising the possibility that lack of regional lymph nodes may be responsible for the attenuated response to by guest on September 27, 2021 aerosolized Ag (17, 18). Secondary lymphoid tissue provides an and -challenged ROR␥Ϫ/Ϫ animals showed unexpected increases important microenvironment for presentation of Ag and amplifi- in TNF-␣, IL-2, and IFN-␥ and reciprocal reductions in IL-4, IL-5, cation of T cell populations. Therefore, a lack of lymph nodes and IL-13. OVA-stimulated splenocytes from these animals also provides a plausible explanation for the attenuated response to showed excessive production of IFN-␥. Interestingly, ROR␥Ϫ/Ϫ OVA in ROR␥-deficient mice. However, this explanation is not mice showed a trend toward greater IL-10 levels in the lung lavage consistent with studies conducted with other mouse lines lacking fluid, and production of IL-10 by OVA-stimulated splenocytes was lymph nodes, where it has been shown that experimental allergic significantly greater than that produced by similarly treated spleno- inflammation can develop. Lymphotoxin-␣-deficient mice, similar cytes from wt animals. This enhanced IL-10 production by to ROR␥-deficient mice, are born without detectable lymph nodes ROR␥Ϫ/Ϫ mice is one potential mechanism for the suppression of or Peyer’s patches. Surprisingly, robust inflammation was ob- allergic lung inflammation observed in our experiments. IL-10 is a served in the lungs of sensitized animals following repeated airway major regulator of innate and adaptive immunity, and a number of challenges with OVA (36). Allergic inflammation was completely studies have shown that this cytokine can inhibit allergic inflam- abrogated in splenectomized lymphotoxin-␣-deficient mice, sug- mation by several different mechanisms. IL-10 has been shown to gesting that Ag presentation and allergen-specific T cell expansion reduce proinflammatory cytokine release by mast cells, down-reg- occur in the spleen of these mice. Thus, it is unlikely that the ulate EOS function, inhibit cytokine production and chemokine absence of lymph nodes alone can account for the drastic reduction receptor expression by immune cells, and inhibit T cell prolifera- in allergic lung disease seen in ROR␥Ϫ/Ϫ mice because their tion (20–24). In vivo, mice deficient in IL-10 demonstrate exag- spleens are available to subserve this function. gerated allergic responses (25–27). Conversely, IL-10 administra- Another possibility is an impairment of T cell migration to the tion to mice before allergen treatment induces Ag-specific T cell lung from secondary lymphoid organs. As discussed above, since unresponsiveness (28). ROR␥Ϫ/Ϫ mice lack regional lymph nodes, the spleen may serve Therefore, it is possible that the attenuated allergic inflammation as the major location of Ag presentation and T cell expansion. observed in ROR␥Ϫ/Ϫ mice is driven by excessive IL-10 produc- Previous reports have suggested that lymphocyte trafficking out of tion in response to Ag. the spleen is impaired in ROR␥-deficient mice. Zhang et al. (19) This shift in cytokine profiles, coupled with our findings of re- have shown that spleens from ROR␥-deficient mice contain of 2- duced IgG1 and elevated IgG2c levels in the sera of OVA-exposed to 3-fold more cells. This increase in cell number was predomi- ROR␥Ϫ/Ϫ mice, demonstrates a switch in the type of Ag-specific nantly due to increased numbers of B lymphocytes. The numbers immune response to a Th1 type. Thus, another possible mechanism of CD4ϩ and CD8ϩ T lymphocytes were only marginally in- for the attenuation of allergic inflammation is the unexpected increased; however, CD4 and CD8 cells in blood were significantly duction of Th1 cytokines, particularly IFN-␥. It is well-established reduced, suggesting that T and B cells accumulate in the spleens of The Journal of Immunology 3217

ROR␥-deficient mice. A defect in the splenic microenvironment been identified (50–52). Cholesterol and cholesterol derivatives was confirmed when it was shown by adoptive transfer experi- have been reported to bind as agonists to ROR, whereas certain ments that both B and T cells from C57BL/6 bone marrow donors retinoids may function as partial antagonists. These studies suggest accumulated in the spleens of ROR␥Ϫ/Ϫ recipients following bone that it might be possible to design synthetic ROR (ant)agonists that marrow transplantation but also that lymphocytes from ROR␥- influence the physiological functions of these orphan nuclear re- deficient mice did not accumulate in the spleens of C57BL/6 or ceptors, with potential applications to a number of human diseases. RAG-2 mice (19). Thus, if the spleen is functioning as the sec- Further studies of the role of ROR in immune cell function and ondary lymphoid organ for Ag presentation and trafficking out of host defense are needed to determine whether ROR ligands could the spleen is impeded, a reduced cellular response to Ag challenge provide useful therapeutic strategies for asthma and other inflam- may be observed, as was the case for our studies. Robust cytokine matory diseases. production by ROR␥Ϫ/Ϫ splenocytes stimulated in vitro with OVA, but reduced cell numbers and cytokine levels in the lung in Acknowledgments vivo, support the possibility of impaired migration of OVA-spe- We thank the Keck family for their generous support of the Keck Animal Ϫ/Ϫ cific T cells from the spleen to the lungs of ROR␥ mice. High Models Facility at the University of North Carolina. IFN-␥ levels produced by OVA-stimulated ROR␥Ϫ/Ϫ splenocytes supports this contention in light of the well-recognized suppressive Disclosures effects of IFN-␥ on CD4ϩ T cell migration (31). The authors have no financial conflict of interest. A reduction in the CD4ϩ lymphocyte pool is an additional mechanism that could contribute to the attenuated allergic lung Downloaded from inflammation observed ROR␥Ϫ/Ϫ mice. While we found similar References ϩ 1. Kumar, R., and E. B. Thompson. 1999. The structure of the nuclear hormone numbers of CD4 cells in the lung lavage fluid of saline-exposed receptors. Steroids 64: 310–319. Ϫ Ϫ ROR␥ / mice compared with wt controls, the number of periph- 2. Staples, J. E., T. A. Gasiewicz, N. C. Fiore, D. B. Lubahn, K. S. Korach, and eral blood CD4ϩ T cells is substantially reduced, a consequence of A. E. Silverstone. 1999. Estrogen receptor ␣ is necessary in thymic development ϩ ϩ and estradiol-induced thymic alterations. J. 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