Humans Arthritic Bone Destruction in Mice and T Cells, Drive Δγ + Th17

Th17 Cells, Not IL-17+ δγ T Cells, Drive Arthritic Bone Destruction in Mice and Humans

This information is current as Bernadette Pöllinger, Tobias Junt, Barbara Metzler, Ulrich of September 29, 2021. A. Walker, Alan Tyndall, Cyril Allard, Serkan Bay, Roland Keller, Friedrich Raulf, Franco Di Padova, Terrence O'Reilly, Nicole J. Horwood, Dhavalkumar D. Patel and Amanda Littlewood-Evans

J Immunol 2011; 186:2602-2612; Prepublished online 7 Downloaded from January 2011; doi: 10.4049/jimmunol.1003370 http://www.jimmunol.org/content/186/4/2602 http://www.jimmunol.org/ Supplementary http://www.jimmunol.org/content/suppl/2011/01/07/jimmunol.100337 Material 0.DC1 References This article cites 53 articles, 20 of which you can access for free at: http://www.jimmunol.org/content/186/4/2602.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 © 2011 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Th17 Cells, Not IL-17+ gd T Cells, Drive Arthritic Bone Destruction in Mice and Humans

Bernadette Po¨llinger,* Tobias Junt,* Barbara Metzler,* Ulrich A. Walker,† Alan Tyndall,† Cyril Allard,* Serkan Bay,* Roland Keller,* Friedrich Raulf,* Franco Di Padova,* Terrence O’Reilly,‡,1 Nicole J. Horwood,x Dhavalkumar D. Patel,* and Amanda Littlewood-Evans*

The mechanism whereby IL-17 drives rheumatoid arthritis remains incompletely understood. We demonstrate that anti–IL-17 therapy in collagen-induced arthritis ameliorates bone damage by reducing the number of osteoclasts in joints. We found equal numbers of CD4+ Th17 and IL-17 producing gd T cells in the joints of arthritic mice, and in vitro, both populations similarly

induced osteoclastogenesis. However, individual depletion and adoptive transfer studies revealed that in vivo, Th17 cells domi- Downloaded from nated with regard to bone destruction. Unlike gd T cells, Th17 cells were found in apposition to tartrate-resistant acid phosphatase positive osteoclasts in subchondral areas of inflamed joints, a pattern reproduced in patient biopsies. This localization was caused by Ag-specific retention, because OVA-primed Th17 cells showed a gd T cell-like diffuse distribution. Because IL-23, as produced by osteoclasts, enhanced T cell-mediated osteoclastogenesis, we propose that Ag-specific juxtaposition is key to foster the molecular cross talk of Th17 cells and osteoclasts, thus driving arthritic bone destruction. The Journal of Immunology, 2011, 186:

2602–2612. http://www.jimmunol.org/

heumatoid arthritis (RA) is a chronic autoimmune disease In collagen-induced arthritis (CIA), disease was markedly sup- characterized by immune cell invasion into the joint space, pressed in IL-17–deficient mice (13). Major sources of IL-17 in R cytokine imbalance, inflammation of the synovial mem- this model are CD4+ T cells (Th17) and IL-17–secreting gd branes, and progressive synovial fibroblast- and osteoclast-mediated T cells. Although both cell types have the potential to exacerbate joint destruction (1). There is mounting evidence that osteoclasts, CIA (14, 15), the relative significance of IL-17+ gd T cells and synoviocytes, and immune cells communicate via cytokines and other Th17 cells in arthritis remains to be shown (16–18). signaling molecules. Factors derived from activated T cells are known A strong link between the proinflammatory activity of IL-23 and to affect the differentiation and activation of pathogenic osteoclasts cells of the Th17 lineage has been established. IL-23 stabilizes the by guest on September 29, 2021 (2, 3). The role of IL-17, a T cell-derived inflammatory cytokine has Th17 lineage, thereby generating large numbers of IL-17–producing been of particular interest recently because it was found that it effector cells (19). Consequently, IL-23–deficient mice are protected exacerbates autoimmune diseases such as RA (4). The relevance of from autoimmune diseases such as CIA (20) and resistance corre- this cytokine in RA is highlighted by the current success of clinical lated with an absence of IL-17–producing CD4+ T cells. In addition, trials with neutralizing Abs to IL-17 (5, 6). This cytokine has been there is evidence that IL-23 affects cells of the innate immune sys- described to orchestrate diverse immune functions such as elicitation tem. For example, IL-23 is required to induce IL-17A production of proinflammatory mediators from joint-resident cells, chemo- from NKT cells, gd T cells, and Thy1+ SCA1+ innate immune cells attraction of neutrophils, stimulation of receptor activator for NF-kB (21–25). The IL-23/IL-17 axis has been described to play a pivotal ligand (RANKL) expression, a factor that is crucial for osteoclasto- role in the autoimmune inflammation in joints. Indeed, in RA, IL- genesis, as well as mediate direct pro-osteoclastogenic effects (7–12). 23p19 levels correlate with those of IL-17 in the synovial fluid and with IL-17, TNF-a, and IL-1b levels in the serum. Furthermore, synovial IL-23p19 levels were found to be greater in patients with *Department of Autoimmunity, Transplantation and Inflammation, Novartis Insti- tutes for BioMedical Research, 4002 Basel, Switzerland; †Department of Rheuma- bone erosions (26). Cross talk between IL-23 and IL-17 is particu- tology, Felix Platter Spital, 4012 Basel, Switzerland; ‡Department of Oncology, larly associated with the destruction phase of RA characterized by x Novartis Institutes for BioMedical Research, 4002 Basel, Switzerland; and Kennedy the T cell-mediated activation of osteoclastogenesis (27). Institute of Rheumatology, Imperial College of Science, Technology and Medicine, London, W6 8LH United Kingdom In this study, we identified the relative pro-osteoclastogenic + 1Retired. contributions of IL-17–secreting gd and CD4 T cells in the CIA Received for publication October 12, 2010. Accepted for publication December 8, model and relate these findings to RA. 2010. Address correspondence and reprint requests to Dr. Amanda Littlewood-Evans, De- partment of Autoimmunity, Transplantation and Inflammation, Novartis Institutes for Materials and Methods BioMedical Research, Forum 1 Novartis Campus, Basel 4002, Switzerland. E-mail Mice address: [email protected] Eight- to 10-wk-old DBA/1 male mice (Janvier) were used in accordance The online version of this article contains supplemental material. with Swiss Federal and Cantonal Authorities. Abbreviations used in this article: CIA, collagen-induced arthritis; IHC, immunohistochemistry; qRT-PCR, quantitative real-time PCR; RA, rheumatoid arthritis; Human samples RANKL, receptor activator for NF-kB ligand; ROR, retinoic acid-related orphan receptor; TRAP, tartrate-resistant acid phosphatase. Synovial fluid from three patients (60–68 y old) with RA based on American College of Rheumatology criteria were obtained from the Felix- Copyright Ó 2011 by The American Association of Immunologists, Inc. 0022-1767/11/$16.00 Platter and University Hospital Basel. Duration of RA ranged from 16 to www.jimmunol.org/cgi/doi/10.4049/jimmunol.1003370 The Journal of Immunology 2603

20 y. Patients were undergoing cortisone treatment and one received ionomycin (Sigma-Aldrich). After activation, cells were stained for in- abatacept. Informed consent from patients was obtained, and studies were tracellular cytokines using an intracellular cytokine staining kit (BD Phar- conducted under guidance/agreement from the Basel Ethical Commission. mingen). Abs were purchased from BD Pharmingen or eBioscience. Before Paraffin-embedded RA synovial tissue from a patient with chronic RA (53 staining, Fc-blockade (anti-CD16/32) was performed. For FACS, T cell y) treated with prednisolone, diclofenac, and leflunomide was purchased populations were first gated on CD45 and subsequently on CD3+CD4+ and from Asterand. Activities of Asterand and their collaborators were con- CD3+gdTCR+CD42, respectively. For mouse, we employed the following ducted in accordance with applicable laws, regulations, and ordinances. Abs: FITC-IFN-g, PE–IL-17, FITC- or PerCP-CD45, allophycocyanin-CD3, Human metacarpal joints were obtained by informed consent from five RA biotinylated gdTCR, Alexa 488- or PerCP-CD4, PE-CD3, CD11b-PerCP, patients (four women and one man) undergoing joint replacement surgery FITC–Ki-67, allophycocyanin-Gr1, streptavidin-PerCP, and streptavidin- under the terms of the ethical approval granted by the combined office of allophycocyanin. For human, we used the following Abs: FITC-IFN-g, PE- research ethics committee (COREC No. 07/Q0411/30). Patients were aged gdTCR, PE-CD4, PerCP-CD3, and AlexaFluor647-IL-17A. Flow cytometry 44–65 y of age at the time of surgery. data were acquired on a FACSCalibur (BD Bioscience) and analyzed with FlowJo 6.42 software (Tree Star). Cell sorting was performed on a FACSAria CIA and histopathology II (BD Bioscience). The CIA model and histopathology were performed as described previously Quantitative real-time PCR analysis of FACS-sorted T cells by others (28). In brief, 100 mg bovine collagen type II (Chondrex, Red- mond, WA) emulsified in complete Freund’s adjuvant was administered FACS-sorted spleen,lymphnodes,or jointcellswere placedin RNeasyMicro intradermally in the base of the tail of anesthetized DBA/1 mice. Twenty- RNA Isolation Kit lysis buffer (Qiagen). cDNAwas generated using the High one days later, a boost of collagen (100 mg) in PBS was given i.p. Mice Capacity cDNA RT Kit (Applied Biosystems). Quantitative real-time PCR generally developed arthritis shortly after boost. Histopathology was (qRT-PCR) analyses were performed using a TaqMan Low Density Array scored for bone erosion by two independent researchers with blinded (Applied Biosystems) with the following assays (assay ID): IL-17A (Mm0- 0439618_m1), IL-17B (Mm00444686_m1), IL-17C (Mm00521397_m1), samples: 0 = no bone erosion; 1 = mild (only surface erosion, not overall); Downloaded from 2 = moderate (moderate surface erosion, overall, and/or 1–2 breakthrough IL-17D (Mm01313472_m1), IL-17F(Mm00521423_m1), IL-23A (p19) of inflammatory cells into the bone marrow); 3 = strong (strong surface (Mm00518984_m1), IL-23R (Mm00519943_m1), RANKL (Mm004419- erosion, overall, with deep lacunas, and $3 breakthroughs of inflammatory 08_m1), IL-1b(Mm01336189_m1), TNF-a (Mm00443258_m1), IL-6 (Mm- cells in the bone marrow). Tartrate-resistant acid phosphatase (TRAP)- 99999064_m1), IL-21 (Mm00517640_m1), IL-22 (Mm00444241_m1), positive stained osteoclasts were assessed in tissue sections covering ta- retinoic acid-related orphan receptor a (RORa; Mm00443103_m1), RORg lus, tibia, calcaneus, and metatarsal bones. Infiltration of cells was scored (Mm01264022_m1), CCR6 (Mm99999114_s1), and 18S rRNA (Hs999- on a scale of 0–3, depending on the amount of inflammatory cells in the 99901_s1) for normalization. joint cavity. Cartilage proteoglycan loss as assessed by Safranin O staining http://www.jimmunol.org/ was: 0 = fully stained cartilage; 1 = 10–20%; 2 = 20–60%; 3 = 60–100% Adoptive transfer proteoglycan loss visible. A total of 1 3 105 FACS-purified gd and CD4+ T cells from spleen and + Depletion of IL-17A in CIA lymph nodes of CIA mice or CD4 T cells from spleen and lymph nodes of collagen type II or OVA-primed mice (day 10) were labeled with Cell- CIA mice were treated i.p. three times per week from days 19–40 with 200 Tracker CM-DIL (Invitrogen) and injected i.p. into CIA recipient mice. mg/mouse of anti-mouse IL-17A (mAb 3-784-9; Novartis), anti-mouse IL- Forty-eight hours later, animals were sacrificed, paws snap-frozen, and 20- 17A (mAb 421; R&D Systems), and mouse IgG1 and rat IgG2a control mm sections generated on a cryostat (Microm). Abs. In vitro induction of osteoclasts + Depletion of gd and CD4 T cells in CIA by guest on September 29, 2021 FACS-sorted gd or CD4+ T cells (5 3 103) isolated from organs of arthritic CIA mice were injected on day 28 with 500 mg/mouse anti-mouse CD4 mice were cocultured with bone marrow cells (2 3 106) for 6–10 d. In one (clone GK1.5), anti-mouse gd TCR Abs (clone UC7-13D5; BioLegend), or experiment, FACS-purified gd or CD4+ T cells were preincubated with 10 isotype control Ab (anti-chicken lysozyme IgG1; Novartis), followed by ng/ml recombinant mouse IL-23 for 24 h before coculture. Positive control 200 mg/mouse every third day until day 40. Depletion efficacy was tested was bone marrow cells incubated with M-CSF (10 mg/ml), RANKL (10 by FACS analysis of PBLs. mg/ml), and IL-1a (5 mg/ml; R&D Systems). Tartrate-resistant acid phosphatase-positive cells were counted. Immunohistochemistry Statistical analysis For mouse, serial frozen unfixed sections were stained with H&E and TRAP according to standard techniques. Another serial section was mounted for Unless otherwise noted, data are presented as means with the error bars analysis of fluorescently labeled T cells. For human, deparaffinized sec- representing SEM. Data were analyzed either by two sample tests (t test for tions were subjected to Ag retrieval in 10 mM citrate buffer, pH 6.0 at 98˚C normally distributed data, and rank sum test for data that was either not for 20 min. Immunohistochemistry (IHC) was performed using standard normally distributed or the groups had unequal variance), or by ANOVA techniques. Rabbit anti-human IL-17 Ab (Santa Cruz Biotechnology) with (one way for normally distributed data of equal variance or ANOVA on biotinylated anti-rabbit IgG Ab (Vector Laboratories) was used for both ranks when data were not normally distributed or of unequal variance). In human and mouse. Mouse anti-human CD4 (RPA-T4) purified rat anti- addition, two-way ANOVA was used as appropriate (see figure legends). mouse CD4 (RM4-5), mouse anti-human gdTCR (B1), and hamster anti- Post hoc tests for one-way ANOVA used Tukey’s test and for ANOVA on mouse gdTCR (GL3) were all from BD Pharmingen. ranks used Dunn’s test, the latter was used because of unequal group sizes. Normality was detected by Shapiro–Wilk tests, and equal variance among Microcomputerized tomography analysis groups was determined by an F-test. x2 test was used to determine the ratio of human T cells in proximity to bone. Global p a value was set at 0.05. Paws were scanned on a VIVA MicroCT40 machine (Scando Medical) with All tests were done using SigmaPlot 11 (Systat Software). calibration settings of 70 kV, 200 mg HA, 4096 scaling, and diameter of 21 mm. Optical slice thickness was 10 mm. High-quality three-dimensional images of the calcaneus bone were produced. Results Isolation of cells from joints, spleen, and lymph nodes Anti–IL-17A therapy protects bone destruction in CIA Paws were digested in 0.125% Dispase II (Roche), 0.2% collagenase II, and IL-17 is known to be involved in the pathogenesis of arthritis. To 0.2% collagenase IV (Sigma-Aldrich) in HBSS for 75 min at 37˚C and understand the role of this cytokine with regard to bone destruction, passed through a cell strainer. Spleen and draining lymph nodes were di- we treated arthritic mice with two different neutralizing anti–IL- rectly minced through a cell strainer. RBCs were lysed by incubating the 17A Abs (mAb 3-784-9 and mAb 421). Anti–IL-17A treatment preparations in 0.83% NH4Cl for 3 min on ice. before onset of clinical signs of disease potently inhibited disease Intracellular cytokine and surface staining severity (Fig. 1A). This was accompanied by a lower histopatho- For intracellular cytokine staining, 1 3 106 cells/ml were cultured at 37˚C for logical disease score with respect to inflammatory cell infiltrate, 4 h in medium containing 5 mg/ml brefeldin A, 50 ng/ml PMA, and 0.5 mg/ml cartilage damage, and bone loss (Fig. 1B,1C, Supplemental Fig. 2604 Th17 NOT IL-17+ gd T CELLS DRIVE BONE DESTRUCTION

FIGURE 1. Anti–IL-17 therapy in CIA. A, Clinical score of CIA mice treated with isotype control (mouse IgG1, rat IgG2a), anti–IL-17 (mAb [mAB] 3-784-9), and anti–IL-17 (mAB 421) Abs. Treatment started at day 19, 2 d before boost and before onset of clinical signs of disease (n = 9 mice/group). Abs were applied with adosageof200mg/animal three times a week. Data are average + SEM. Statistical analysis by Student t test is shown for the last day only. Mouse IgG1 was compared with mAB 3-784-9, and rat IgG2a was compared with mAB 421. B, Histopathological analysis of inflammation, bone erosion, and proteoglycan loss in sections of hind paws of animals treated with the various Abs. n = 7–9 animals/group. Bars 6 SEM. C, Histopathology (Giemsa stain) of hind paws of CIA mice treated withmouse IgG1 control (top panel) and anti–IL-17 (mAB 3-784-9) Ab (bottom panel). Asterisk indicates influx of inflammatory cells; arrows highlight bone de- Downloaded from struction. Scale bars, 500 mm. D, Number of TRAP+ osteoclasts in histopathological hind- paw sections (n = 5/group). Representative TRAP stain sections showing osteoclast activity in mouse IgG1 control (top right panel) and anti–IL-17 (mAB 3-784-9) Ab (bottom right http://www.jimmunol.org/ panel). Scale bars, 50 mm. A–D, One representative of two experiments is shown. B–D, The p values were calculated by a RANK sum test comparing mouse IgG1 with mAB 3-784- 9 or rat IgG2a compared with mAB 421.

1). Because osteoclast-mediated bone destruction is an integral To gain insight into the effector mechanisms used by these two component of CIA, TRAP+ cells were quantified in paw sections. T cell types in CIA, we performed qRT-PCR analysis for ex- As expected, osteoclast numbers were significantly reduced in pression patterns of Th17 lineage markers on FACS-sorted gd and by guest on September 29, 2021 joints from anti–IL-17A–treated animals (Fig. 1D). This con- CD4+ T cells from the spleen, lymph nodes, and joints of CIA firmed the relevance of IL-17A for the induction of osteoclast- mice (Supplemental Fig. 2). gd T cells in arthritic mice always mediated bone destruction in arthritis. displayed features of Th17 cells, most notably in inflamed joints.

+ Both T cell populations when isolated from this tissue showed an Arthritic joint-derived gd and CD4 T cells take on Th17 upregulation of IL-17A, IL-17F, and IL-22, and transcription characteristics, express pro-osteoclastogenic cytokines, and + factors RORa and RORg when compared with peripherally de- induce TRAP osteoclast formation rived counterparts (Fig. 3A). Of note, both gd and CD4+ T cells Next, we defined the source of IL-17 in arthritic joints. We observed from arthritic joints expressed high and similar levels of IL-23R that in CIA, IL-17 is produced solely by gd and CD4+ T cells (data and the Th17-associated CCR6 (Fig. 3A). not shown). To define the net contribution of both T cell pop- Th17 cells display a pro-osteoclastogenic potential and en- ulations, we characterized these cells on isolation from different hance osteoclast activity via the induction of RANKL in locations during CIA. This enabled us to compare peripherally osteoclastogenesis-supporting cells, thus linking T cell activation primed cells (in spleen and draining lymph nodes) with patho- and bone destruction (29). However, the participation of IL-17– genic effector T cells after invasion into arthritic joints. To eval- producing gd T cells in this paradigm is not described. To com- uate any potential cross-contamination caused by preparative pro- pare gd and CD4+ T cells for their potential to contribute to bone cedures when analyzing joint-derived lymphocytes, we deter- damage, they were isolated from the spleen, draining lymph mined the frequency of gd and CD4+ T cells in purified bone mar- nodes, or arthritic joints of CIA mice, and analyzed for the ex- row preparations. We found that the frequencies were low (∼0.4% pression of cytokines known to be involved in osteoclast activa- gd and ∼7.4% CD4+ T cells), and of these low percentages, ∼7.6 tion. In addition to IL-17, gd and CD4+ T cells from the arthritic and ∼1.0%, respectively, were IL-17+ (data not shown). joints displayed a comparable expression pattern of other pro- Within the gd T cell population, IL-17–producing cells were 3- osteoclastogenic factors such as RANKL, TNF-a, IL-1b, and fold enriched in arthritic joints as compared with the periphery. IL-6 (Fig. 3B). To address the functional significance of these This enrichment was more pronounced for the CD4+ T cell frac- results, we assessed the bone pathogenic potential of gd and CD4+ tion where a 14-fold greater accumulation of the percentage of T cells. These cells were cocultured with bone marrow cells using IL-17+ cells was observed in the joint (Fig. 2A). TRAP staining to indicate de novo osteoclast formation. Because Although there were about 4-fold more CD4+ T cells than gd there is as yet no means to enrich for the IL-17+ subpopulations, T cells in the arthritic joints (Fig. 2B, left column), the number of we performed our studies with whole preparations of gd and CD4+ IL-17–producing CD4+ and gd T cells was almost equal (Fig. 2B, T cells. Identical numbers of FACS-sorted gd and CD4+ T cells right column,2C) because the latter population contained a greater isolated from the peripheral organs could induce some TRAP+ proportion of IL-17+ cells compared with the CD4+ counterpart. cells. However, in line with the previous molecular findings, both The Journal of Immunology 2605 Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 2. Arthritic joint-derived gd and CD4+ T cells take on Th17 characteristics. A, Intracellular costaining for IL-17 and IFN-g in gd and CD4+ T cells from spleen, lymph node, and joints of naive and acute CIA mice. The percentages + SEM of IL-17+ and IFN-g+ cells are indicated. gd T cells (top panels) and CD4+ T cells (bottom panels). Fold increase of IL-17+ cells in the joint versus periphery is shown above for each T cell type. Data combined from a total of n = 8–10 animals analyzed per time point from 6 independent experiments. B, Absolute number of gd T cells (top panels) and CD4+ T cells (bottom panels) in the joints of naive and acute CIA mice (left panels), and IL-17+ gd T cells and CD4+ T cells in the same tissue (right panels). Data are average + SEM. One representative of six experiments with at least n = 4 animals per time point is shown. A and B, Rank sum tests were performed for statistical analysis. C, Representative FACS blots showing intracellular costaining for IL-17 and IFN-g in gd and CD4+ T cells from joints of naive (top panels) and CIA mice (bottom panels). Percentages of gated cell populations are indicated. gd and CD4+ T cells exerted a higher potential to induce oste- Depletion of CD4+ T cells protects bone damage in CIA oclasts when derived from arthritic joints (Fig. 3C). We saw an + The in vitro data could not entirely exclude pro-osteoclastogenic equipotent ability of gd and CD4 T cells from arthritis joints to effects from contaminating cells. Thus, to unequivocally define induce osteoclast formation despite the knowledge that a greater the individual relevance of the pathogenic capability of gd and percentage of gd T cells produce IL-17 in this organ, highlighting CD4+ T cells in vivo, cell type-specific depletion studies were the fact that IL-17 may not be the only driver of osteoclasto- performed on early onset of disease in arthritic mice. These genesis. experiments revealed a strikingly distinct role for these T cell Because of experimental procedures, we cannot exclude that populations. Whereas ablation of gd T cells delayed disease, albeit a low percentage of contaminating non-T cells such as macrophages nonsignificantly, depletion of CD4+ T cells suppressed arthritis and fibroblasts were contributing to the pro-osteoclastogenic sig- progression (Fig. 4A, Supplemental Fig. 3A,3B). This was nature of joint-derived T cell populations (Supplemental Fig. 2). reflected in the histopathological and microcomputerized tomog- Nevertheless, these experiments demonstrate that gd T cells raphy analysis where anti-CD4+ T cell treatment led to a strong analogous to CD4+ T cells exhibit an IL-17–biased and pro- protection of bone integrity (Fig. 4B–D). These dichotomous osteoclastogenic signature especially on isolation from arthritic in vivo results were unexpected because our previous experiments joints and have the potential to compromise bone integrity. revealed comparable numbers of IL-17–producing CD4+ and gd 2606 Th17 NOT IL-17+ gd T CELLS DRIVE BONE DESTRUCTION Downloaded from http://www.jimmunol.org/

FIGURE 3. Arthritic joint-derived gd and CD4+ T cells take on Th17 characteristics, express pro-osteoclastogenic cytokines, and induce TRAP+ os- by guest on September 29, 2021 teoclast formation. qRT-PCR analysis for the expression of (A) IL-17 signature genes and (B) genes involved in osteoclastogenesis on FACS-sorted gd T cells and CD4+ T cells from spleen, lymph nodes, and joints of acute CIA mice. A and B, Mean + SEM of two independent experiments. C, In vitro coculture of mouse bone marrow cells either left unstimulated (negative control [2]); stimulated with RANKL, IL-1a, and M-CSF (positive control [+]); or cultured in the presence of FACS-sorted gd or CD4+ T cells from organs of acute CIA mice. TRAP+ multinucleated cells (MNC) were counted after 6–10 d. Data are average + SEM of three independent experiments. Statistical analysis used ANOVA on ranks (overall p = 0.027) with Dunn’s test for comparisons versus control (cultures minus growth factor).

T cells in the arthritic joints (Fig. 2B) with in vitro capacities to of the gd T cells could be identified in this area (Fig. 5B,5C, affect bone integrity (Fig. 3C). Supplemental Fig. 4C). Histopathological analysis of serial sec- In histopathological joint sections, TRAP+ osteoclasts were tions revealed that the labeled gd T cells were predominantly prominent within areas of high bone turnover in control and anti- positioned near soft tissue structures such as tendons where gd T cell-treated animals. However, ablation of CD4+ T cells led osteoclasts and other bone-associated cells were absent. Thus, to a drastic reduction in osteoclast number (Fig. 4E). This indi- although gd T cells were found in areas of IL-17 staining, they cates that presence of CD4+ but not gd T cells is an essential were not associated with TRAP+ osteoclasts (Fig. 5C, left column, component for osteoclast activity in murine arthritis. Supplemental Fig. 4C). In contrast, many fluorescently labeled + + CD4 T cells (confirmed by IHC analysis in Supplemental Fig. Differential localization of gd and CD4 T cells within the 4D) could be found adjacent to TRAP+ osteoclasts in areas of IL- arthritic joint 17 positivity (Fig. 5C, middle and right columns). However, we To define the mechanism behind these in vivo findings, we in- assume that not all of the CD4+ T cells were positive for this vestigated whether there may be a differential localization of gd cytokine. and CD4+ T cells within the inflamed tissue. Labeled gd or CD4+ One major difference between gd and CD4+ T cell subsets is T cells from collagen-primed animals were adoptively transferred their responsiveness to the priming Ag collagen type II, with only into arthritic recipients to assess their migration patterns. Al- the latter mounting an Ag-specific response (30, 31). Proliferation though both cell types possessed comparable capacities to migrate (thymidine incorporation) assays with CD4+ T cells were per- to the spleen (Supplemental Fig. 4A,4B), there were markedly less formed to monitor the collagen-specific recall response using total gd T cells infiltrating the joint (Fig. 5A), in line with our splenocytes and lymphocytes from collagen-immunized DBA/1 findings in Fig. 2B. These adoptive transfer studies revealed major animals. In both organs, an Ag-specific response with a stimula- differences in the localization of gd or CD4+ T cells within the tion index between 2 and 3 was routinely measured on incubation inflamed joint. Although about half of the CD4+ T cell population with 50 mg/ml collagen II over baseline (data not shown). To was found in close proximity (within 100 mm) to bone, only 10% dissect whether Ag-specific retention of CD4+ T cells is an The Journal of Immunology 2607

FIGURE 4. Prevalent role of CD4+ T cells causing bone damage in CIA. A, Clinical score of CIA mice treated with control (anti-lysozyme IgG1), anti-gd, and anti-CD4+ T cell depleting Abs on early clinical signs of disease (n = 5 mice/group). Abs were applied from days 26–40 starting with a loading dose of 500 mg/animal and subsequent dosages of 200 mg/ animal every third day. Data are average + SEM. Statistical analysis was a two-way ANOVA using treatment and days as factors, starting from day 30. Analysis showed a significant effect of treatment and time (both p , 0.001), and these factors interacted (p = 0.006). Multiple-comparison procedure used Tukey’s test; significant differences observed for anti- CD4+ T cell versus control from day 30, whereas anti-gd T cell therapy showed no such effects when compared with control. The p values on the graph are from day 40. B,Scoring of inflammation, bone damage, and pro- Downloaded from teoglycan loss from histopathological sections of paws. Data are average + SEM. The p values are calculated by a rank sum test comparing anti-CD4 T cell-treated with control animals (for inflammation and bone damage). Student t test was performed for statistical analysis of http://www.jimmunol.org/ proteoglycan loss. C, Representative Giemsa- stained histopathological sections of paws from treated animals. Scale bars, 250 mm. D, Microcomputerized tomography analysis of calcaneus bone from hind paws of two representative animals per group. Scale bar, 500 mm. E,NumberofTRAP+ osteoclasts in histopathological paw sections of treated animals. Data are average + SEM. The p value on the graph is ANOVA on ranks using Dunn’s tests by guest on September 29, 2021 for pairwise comparisons. Control versus anti- gd Tcells,p . 0.05. underlying cause for the observed phenotype, we performed T cells far outnumbered those producing IL-17 (Supplemental Fig. adoptive transfer studies using CD4+ T cells from donors primed 5). To study the localization of gd and CD4+ T cell populations, with collagen II or OVA in adjuvant. Although both Ag-primed we performed IHC on biopsy sections of RA synovial tissue CD4+ T cells had an equal capacity to migrate into the arthritic showing bone destruction, vessel-rich regions, and inflammatory joints (data not shown), only the collagen type II-specific T cells cell infiltrates. Similar to the CIA mouse model, there were less associated with bone. In contrast, OVA-primed CD4+ T cells ex- total numbers of infiltrating gd versus CD4+ T cells in the hibited a similar localization pattern as the innate gd T cells inflamed tissue (Figs. 2B,6A). Many CD4+ T cells were localized (Fig. 5D). in perivascular cuffs in blood vessel-rich areas, whereas the gd Because we have previously shown that IL-17–producing cells T cells were more disperse within similar regions (Fig. 6A). In were enriched in joint versus peripheral CD4+ T cell fractions addition, and in striking analogy to the mouse system, we found (Fig. 2), we also wanted to determine how many of the adoptively ∼55% of CD4+ T cells in close proximity to bone (Fig. 6B), often transferred collagen II- and OVA-primed peripheral CD4+ T cells in clusters surrounding multinucleated osteoclasts (Fig. 6C). gd became IL-17+ on invasion into arthritic joints. Although we T cells were more diffusely distributed with only ∼35% of the managed to recover the transferredCFSE-labeledcells,their cells close to bone surfaces. Similar to the CIA model, IL-17 numbers were so low that a solid determination of IL-17 positivity immunoreactivity was detected in proximity of the bone (Fig. was not possible. 6D), highlighting the potential for bone-resorbing osteoclasts to These data suggest that gd and CD4+ T cells have different respond to this secreted proinflammatory cytokine. These findings functions in the pathogenesis of arthritis. Collagen-specific CD4+ again point toward a differential contribution of gd and CD4+ T cells have, by nature of their localization, a greater capacity to T cells to bone pathology in RA patients in analogy to the CIA interact with osteoclasts in vivo and thus accentuate joint pa- model. thology. CD4+ T cells in close apposition to osteoclasts in human RA IL-23 produced by osteoclasts enhances the osteoclastogenic To assess whether our findings translate to human RA, we analyzed profile of T cells gd and CD4+ T cells in synovium and synovial fluid of RA Increasing evidence points toward a bidirectional cytokine com- patients. Analogous to studies by Ito et al. (18), we found that in munication between cells of the immune system and osteoclasts. the synovial fluid, the percentage of IFN-g–positive gd or CD4+ In human RA, the relevance of the IL-23/IL-17 axis has been 2608 Th17 NOT IL-17+ gd T CELLS DRIVE BONE DESTRUCTION Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 5. Differential localization of gd and Ag-specific CD4+ T cells within the arthritic joint. A, Quantification of total infiltrating CM-DIL+ gd and CD4+ T cells in CIA joints after adoptive transfer. Statistical analysis was a rank sum test. B, Percentage of bone-associated (within 100 mm) gd and CD4+ T cells in CIA joints after adoptive transfer. A and B, Data are average + SEM. Blinded scoring of three midsections per inflamed paw with n = 3–6 animals per adoptive transfer group. Pooled data from two separate experiments. C, Histopathological analysis of joints from CIA recipient mice after adoptive transfer of gd T cells (left column) or CD4+ T cells (middle and right columns). Photomicrograph showing H&E-stained frozen sections. Boxes in first row indicate areas displayed in rows 3–5. Influx of fluorescently labeled CM-DIL gd T cells or CD4+ T cells. Dashed lines show outline of bone/tendon. Asterisk shows autofluorescence in the lower power CM-DIL images. IHC for IL-17 (faint staining in gd T cell section [arrows]). Asterisk shows nonspecific staining of tendon with anti–IL-17 Ab. TRAP staining for presence of active osteoclasts. Scale bars, 50 mm. D, Percentage of bone-associated (within 100 mm) collagen II- or OVA-primed CD4+ T cells in CIA joints after adoptive transfer. Data are average + SEM. Blinded scoring of a midsection per inflamed paw with n = 6–7 paws per group. Statistical analysis for B and D was a two-way ANOVA using Tukey’s test for pairwise comparisons. B, bone; T, tendon. The Journal of Immunology 2609 Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 6. CD4+ T cells in close apposition to osteoclasts in human RA. A, IHC showing infiltrating gd and CD4+ T cells in blood vessel-rich areas of rheumatoid synovium (low-power views in color (scale bar, 250 mm) and higher power views in black and white: numbers of infiltrating cells per serial section are indicated at the top right. Scale bars, 25 mm. Arrows point to the infiltrating cells. B, Percentage of bone-associated (within 100 mm) gd and CD4+ T cells in bone-rich RA synovial tissue sections. Data are average + SEM. Scoring of a midsection from six individual RA patients. Student t test was performed for statistical analysis. C, Photomicrographs of serial sections showing the proximity of CD4+ T cells (asterisk) to osteoclasts (arrows) in areas of high bone turnover. Scale bars, 25 mm. D, IHC for IL-17 positivity and negative control in bone-rich areas in synovial tissue. Scale bars, 50 mm. described to be associated with the T cell-mediated bone de- It is known that IL-23 is essential to generate pathogenic IL-17+ struction phase of the disease (26, 27). Studies with IL-23–de- effector T cells (19). Indeed, conditioning peripheral CD4+ T cells ficient mice further support the requirement for this cytokine with recombinant mouse IL-23 overnight led to a significant in- toward development of arthritis (20). We found that IL-23p19 crease in IL-17 production (data not shown) and an enhancement mRNA was highly expressed in the joints of arthritic mice, of TRAP+ osteoclasts on coculture with bone marrow cells (Fig. whereas it was only marginally present in the periphery of arthritic 7D). We conclude that there is a cross talk between IL-17+ Ag- animals and joints of naive mice (Fig. 7A). By RT-PCR, we found specific CD4+ T cells and IL-23–producing osteoclasts that leads expression of IL-23p19 mRNA on FACS-sorted cells from ar- to aggravated bone erosion (Fig. 8). thritic joints. Monocytic cells (Gr1med CD11bhigh) and neutrophils (Gr1high CD11bhigh) were positive for this cytokine (Fig. 7B). Discussion Because osteoclasts are derived from the monocytic lineage, we We demonstrate that in arthritic joints, collagen II-specific Th17 were interested to investigate whether IL-23 is also produced by cells rather than IL-17+ gd T cells are the main effectors that the mature bone-resorbing cells. A mixture of IL-1b, RANKL, trigger osteoclast-mediated joint destruction in an IL-23–depen- and M-CSF induced de novo generation of TRAP+ osteoclasts, dent manner. which was accompanied by a significant upregulation of IL-23p19 Pathogenic bone erosion in RA is mediated by the mobilization mRNA as the cells matured in culture (Fig. 7C). and subsequent differentiation of osteoclast precursors within the 2610 Th17 NOT IL-17+ gd T CELLS DRIVE BONE DESTRUCTION

FIGURE 7. Osteoclast-derived IL-23 enhances the osteoclastogenic profile of T cells. A, qRT-PCR analysis for IL-23p19 mRNA from total cell preparations of arthritic spleen, lymph nodes, joints, and naive joints. Data show mean + SEM from n = 2–4 mice. B, qRT-PCR for IL-23p19 mRNA on FACS-sorted inflammatory cells from arthritic joints and periphery of CIA mice. Typical FACS plot showing gates for neutrophils and monocytes. One representative experiment with mean + SEM of three independent experiments is shown. C,qRT- PCR for IL-23mRNA on de novo formed osteoclasts from mouse bone marrow cultures 6 RANKL, IL-1a, and M-CSF. Statistical analysis is by Student t test. Representative TRAP stained sections showing cultures 6 growth factors. Scale bars, 10 mm. Insert in bottom right panel shows the multinuclei Downloaded from from TRAP+ multinucleated cells. Scale bar, 5 mm. D, Bone marrow cells either left unstimulated (negative control [2]); stimulated with RANKL, IL-1a,andM-CSF (positive control [+]); or cocultured with isolated peripheral CD4+ T cells from arthritic mice that had been incubated with or http://www.jimmunol.org/ without recombinant IL-23 (10 ng/ml) for 24 h before the initiation of coculture. TRAP+ multinucleated cells were counted. Data from one experiment with three to four replicates. Statistical analysis is by ANOVA with Tukey. inflamed synovial tissue. Active osteoclasts localize at the counterparts (11, 26, 27, 33, 34), and are predictive for bone de- boundary between synovium and articular bone, leading to erosion struction (35). by guest on September 29, 2021 (32). These cells require complex cytokine signals from sur- We confirmed that blockade of endogenous IL-17A significantly rounding stroma and immune cells, to generate and maintain reduced bone erosion in arthritic mice (28, 36, 37), and that gd and osteoclast activity (2). In RA patients, pro-osteoclastogenic cyto- CD4+ T cells are responsible for production of this cytokine. kines such as RANKL, IL-23, and IL-17 are increased in synovial Furthermore, both T cell populations, when derived from the ar- fluid and synovium compared with osteoarthritic and normal thritic joint, displayed a remarkably similar in vitro profile with respect to Th17 gene signatures; however, because our study inhibited only IL-17A, we cannot exclude an additive effect by blocking other Th17-associated cytokines. Both joint-derived CD4+ and gd T cells similarly upregulated pro-osteoclastogenic genes including RANKL and induced osteoclastogenesis in in vitro cocultures, revealing a comparable potential to destroy bone. IL-17 can exert its osteoclastogenic effects via RANKL (11), but the cellular source thereof is under debate (3). Sato et al. (29) used in vitro polarized Th17 cells and showed that these induced osteoclastogenesis only in the presence of osteoblasts. In our studies, we used cell populations isolated from arthritic animals to show ex vivo osteoclastogenic capa- bilities of CD4+ and gd T cells, which may highlight a direct function for T cells in the disease situation, in line with a role of IL-17 in osteoclastogenesis (12). Interestingly, the mRNA of IL- 1b, although rarely recognized as a product of T cells, was present in low and high levels in the spleen and joint-derived T cell FIGURE 8. Self-perpetuating feedback loop in CIA. 1, Peripheral CD4+ + populations, respectively. However, studies in other disease set- and IL-23R gd T cells release low levels of cytokines. 2, On formation of tings have indicated that IL-1 may be produced by T cells (38–40). the immunological synapse with Ag-presenting osteoclasts, IL-17+ colla- Therefore, we cannot eliminate the possibility that Ag-specific gen-specific CD4+ T cells stay at bone surfaces. 3, IL-17+ CD4+ T cells differentiate/activate osteoclasts via osteoclastogenic cytokine release. 4, T cells are producing this cytokine in the CIA model. IL-23+ osteoclasts, in turn, maintain/enhance the pathogenic capacity of Despite the many aforementioned similarities of the IL-17– + IL-17+ CD4+ T cells generating a positive feedback loop of joint de- producing CD4 and gd T cells, our studies highlighted the par- + struction. 5, IL-17+ gd T cells recruit IL-23–producing neutrophils to the ticular importance of CD4 T cells over gd T cells for bone pa- joint cavity. thology. Ag-specific retention appeared to account for the The Journal of Immunology 2611 accumulation of CD4+ T cells to the collagenous surfaces of bone iela Benz for provision of human samples. We also thank Dr. Jose´ Carbal- and cartilage in the CIA mouse because CD4+ T cells primed for lido for fruitful scientific discussions. an irrelevant epitope were less frequently found near bone. Indeed, osteoclasts may serve as APCs: they express MHC class I and II, Disclosures CD80, CD86, and CD40, and have the capacity to present soluble B.P., T.J., B.M., C.A., S.B., R.K., F.R., F.D.P., T.O.R., D.D.P., and A.L.-E. Ags and activate T cells (41). We speculate that in CIA, osteo- are or were employees of Novartis Institutes for BioMedical Research and/ clasts, among other APCs, present peptides to CD4+ T cells or own Novartis shares. leading to their subsequent activation and local retention on formation of a stable immunological synapse. This preferential re- References tention of Ag-specific Th17 cells in the arthritic joint may explain 1. Emery, P., I. B. McInnes, R. van Vollenhoven, and M. C. Kraan. 2008. Clinical + + the enrichment of IL-17 CD4 T cells in arthritic joints and in identification and treatment of a rapidly progressing disease state in patients with proximity to the bone. rheumatoid arthritis. Rheumatology (Oxford) 47: 392–398. + 2. Takayanagi, H. 2009. Osteoimmunology and the effects of the immune system In human RA, the close proximity of CD4 T cells and osteo- on bone. Nat. Rev. Rheumatol. 5: 667–676. clasts in areas of IL-17 production within the rheumatoid syno- 3. McInnes, I. B., and G. Schett. 2007. Cytokines in the pathogenesis of rheumatoid vium closely resembled our observations in the CIA model. This arthritis. Nat. Rev. Immunol. 7: 429–442. + 4. Tesmer, L. A., S. K. Lundy, S. Sarkar, and D. A. Fox. 2008. 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Iwakura. 2003. Suppression of immune induction of collagen-induced arthritis in IL-17-deficient mice. J. Immunol. 171: Yago et al., who demonstrated that IL-23 stimulates human os- 6173–6177. teoclast differentiation indirectly via Th17 cell activation (53). We 14. Goldschmidt, T. J., L. Jansson, and R. Holmdahl. 1990. In vivo elimination of demonstrated that the osteoclastogenic potential of T cells from T cells expressing specific T-cell receptor V b chains in mice susceptible to collagen-induced arthritis. Immunology 69: 508–514. the periphery was enhanced when cells were either precultured 15. Peterman, G. M., C. Spencer, A. I. Sperling, and J. A. Bluestone. 1993. Role of with IL-23 or derived from IL-23–rich arthritic joints. gamma delta T cells in murine collagen-induced arthritis. J. Immunol. 151: 6546–6558. Besides monocytes and neutrophils, we identified osteoclasts as 16. Corthay, A., A. Johansson, M. Vestberg, and R. Holmdahl. 1999. Collagen- the main expressors of IL-23 in CIA joints. This suggested the ex- induced arthritis development requires abT cells but not gdT cells: studies istence of a self-perpetuating feedback loop between these cells with T cell-deficient (TCR mutant) mice. Int. Immunol. 11: 1065–1073. 17. Roark, C. L., J. D. French, M. A. Taylor, A. M. Bendele, W. K. Born, and and IL-17–producing T cells. We propose that the joint-destructive R. L. O’Brien. 2007. Exacerbation of collagen-induced arthritis by oligoclonal, + + aspect of IL-17 mostly stems from the IL-17 CD4 T cells that IL-17-producing gdT cells. J. Immunol. 179: 5576–5583. localize to bone and activate/differentiate osteoclasts. IL-23–ex- 18. Ito, Y., T. Usui, S. Kobayashi, M. Iguchi-Hashimoto, H. Ito, H. Yoshitomi, T. Nakamura, M. Shimizu, D. Kawabata, N. Yukawa, et al. 2009. 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