CXCR3/CXCL10 Axis Regulates Neutrophil–NK Cell Cross-Talk Determining the Severity of Experimental Osteoarthritis

CXCR3/CXCL10 Axis Regulates Neutrophil− NK Cell Cross-Talk Determining the Severity of Experimental Osteoarthritis

This information is current as Giorgia Benigni, Petya Dimitrova, Fabrizio Antonangeli, of September 26, 2021. Emilio Sanseviero, Viktoriya Milanova, Arjen Blom, Peter van Lent, Stefania Morrone, Angela Santoni and Giovanni Bernardini J Immunol published online 20 January 2017

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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 © 2017 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published January 20, 2017, doi:10.4049/jimmunol.1601359 The Journal of Immunology

CXCR3/CXCL10 Axis Regulates Neutrophil–NK Cell Cross-Talk Determining the Severity of Experimental Osteoarthritis

Giorgia Benigni,*,1 Petya Dimitrova,† Fabrizio Antonangeli,* Emilio Sanseviero,* Viktoriya Milanova,† Arjen Blom,‡ Peter van Lent,‡ Stefania Morrone,x Angela Santoni,{,‖ and Giovanni Bernardini*,‖

Several immune cell populations are involved in cartilage damage, bone erosion, and resorption processes during osteoarthritis. The purpose of this study was to investigate the role of NK cells in the pathogenesis of experimental osteoarthritis and whether and how neutrophils can regulate their synovial localization in the disease. Experimental osteoarthritis was elicited by intra- articular injection of collagenase in wild type and Cxcr32/2 8-wk old mice. To follow osteoarthritis progression, cartilage Downloaded from damage, synovial thickening, and osteophyte formation were measured histologically. To characterize the inflammatory cells involved in osteoarthritis, synovial fluid was collected early after disease induction, and the cellular and cytokine content were quantified by flow cytometry and ELISA, respectively. We found that NK cells and neutrophils are among the first cells that accumulate in the synovium during osteoarthritis, both exerting a pathogenic role. Moreover, we uncovered a crucial role of the CXCL10/CXCR3 axis, with CXCL10 increasing in synovial fluids after injury and Cxcr32/2 mice being protected from disease development. Finally, in vivo depletion experiments showed that neutrophils are involved in an NK cell increase in the http://www.jimmunol.org/ synovium, possibly by expressing CXCL10 in inflamed joints. Thus, neutrophils and NK cells act as important disease- promoting immune cells in experimental osteoarthritis and their functional interaction is promoted by the CXCL10/CXCR3 axis. The Journal of Immunology, 2017, 198: 000–000.

atural killer cells are innate cytotoxic lymphocytes able to replacement, and in other arthropathies (6–8). However, their role kill virus-infected, aberrant, or transformed cells (1, 2). in joint diseases is poorly understood. N Moreover, NK cells can exert immunoregulatory func- Osteoarthritis is the most common joint disease worldwide, tions and promote inflammation thanks to their capacity to rapidly mainly affecting the elderly population. Secondary disease can by guest on September 26, 2021 release cytokines and growth factors (3–5). NK cells have been develop in younger individuals as a consequence of an irregular found as a prominent population in the leukocyte infiltrate of the recovery process after joint/bone trauma. Progressive loss of articular synovial tissue of osteoarthritis patients undergoing total joint cartilage, resulting from the altered balance between degradation and synthesis, leads to abnormal bone remodeling also involving sub- *Department of Molecular Medicine, Sapienza University of Rome, 00161 Rome, chondral bone outgrowths at the joint edge that generates osteo- Italy; †Department of Immunology, Institute of Microbiology, Bulgarian Academy of phytes (9, 10). This unbalance is the result of several pathogenic Sciences, 1113 Sofia, Bulgaria; ‡Department of Rheumatology, Radboud University Medical Center, Nijmegen 86525, the Netherlands; xDepartment of Experimental mechanisms, including enzymatic degradation of the extracellular Medicine, Sapienza University of Rome, 00161 Rome, Italy; {Laboratory of Immu- matrix, defects in new matrix formation, chondrocyte death, and nology and Molecular Immunopathology Institute Pasteur Italy–Cenci Bolognetti abnormal activation and hypertrophic differentiation of cartilage Foundation, Department of Molecular Medicine, Sapienza University of Rome, 00161 Rome, Italy; and ‖Istituto di Ricovero e Cura a Carattere Scientifico, Neu- cells. romed, Pozzilli, 86077 Isernia, Italy Increasing evidence supports a pivotal role of synovitis in os- 1Current address: Innate Immunity Unit, Pasteur Institute, Paris, France. teoarthritis. Synovitis is often found in osteoarthritis patients with ORCIDs: 0000-0003-0920-3712 (P.D.); 0000-0002-6369-2249 (F.A.); 0000-0002- active disease at early as well as advanced stages, indicating that a 2643-0573 (E.S.); 0000-0002-3578-4744 (V.M.); 0000-0003-2441-9749 (S.M.); plethora of ongoing immune processes perpetuates local tissue 0000-0003-1206-7731 (A.S.); 0000-0002-3705-2598 (G.B.). damage and leads to chronic joint inflammation (11–13). Signif- Received for publication August 4, 2016. Accepted for publication December 21, icant amounts of inflammatory mediators, including the cytokines 2016. TNF, IL-1b, and IL-6, and the chemokines CXCL8 and CCL2 are This work was supported by Inter-Pasteurien Concerted Actions Grant A05_11, France, and grants from the Ministero dell’Istruzione, dell’Universita` e della produced by the synovial cells during osteoarthritis (14–18). Al- Ricerca–Fondo per gli Investimenti della Ricerca di Base (Futuro in Ricerca program though several groups characterized the immune cells infiltrating and Grant MIUR-L.297 FAR), Istituto Italiano di Tecnologia, and the Sapienza University of Rome. the synovial tissue of end-stage osteoarthritis patients, the cellular immune players of the inflammatory processes underlying the Address correspondence and reprint requests to: Prof. Angela Santoni, Department of Molecular Medicine, Sapienza University of Rome, Viale Regina Elena 291, Rome disease are still unclear (7, 19, 20). NK cells and neutrophils can 00161, Italy. E-mail address: [email protected] be a source of proinflammatory mediators, degradation enzymes The online version of this article contains supplemental material. and growth factors, triggering not only cartilage damage but also Abbreviations used in this article: BM, bone marrow; CIOA, collagenase-induced altering bone metabolism and repair. Although their function has osteoarthritis; i.a., intra-articular; TRAP, tartrate-resistant acid phosphatase; WT, been linked to several inflammatory diseases, including arthropa- wild type. thies, it is still unclear how and at which stage a particular pop- Copyright Ó 2017 by The American Association of Immunologists, Inc. 0022-1767/17/$30.00 ulation migrates to the synovial fluid/synovium and contributes

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1601359 2 NEUTROPHIL–NK CELL CROSS-TALK BY CXCL10 IN OSTEOARTHRITIS to cartilage/bone damage or remodeling. In addition, several in vitro Immunofluorescence and flow cytometry and in vivo studies have demonstrated the ability of neutrophils to Spleen, blood, and BM were collected and processed as previously de- modulate NK cell biology (21–24). scribed (31). Synovial fluid cells were obtained by washing the knee cavity In the current study, we investigated the role of chemokines, with 25 ml PBS/1 mM EDTA twice, followed by extensive washing after small structurally related cytokines that exert fundamental roles in careful removal of patella and frontal ligaments. Cells were washed and 2+/ 2+ leukocyte migration, in the orchestration of reciprocal neutrophil– resuspended in staining buffer (PBS without Ca Mg , 0.5% BSA, 2 mM EDTA, and 0.05% NaN3) and incubated for 10 min at 4˚C with Fc-blocking NK cell interaction during experimental osteoarthritis. To evaluate the (24G2) mAb. Then, cells were stained with the indicated fluorochrome- relevance of these cell populations in disease progression, the dy- conjugated mAbs for 25 min at 4˚C. Flow cytometric analysis was per- namics of their recruitment into joints was analyzed in the early formed using FACSCanto II (Becton Dickinson) and data were elaborated phases of collagenase-induced osteoarthritis (CIOA) (25), in parallel using FlowJo Version 8.5.2 software. For macrophage and neutrophil isolation, cells were harvested from pooled with the chemokine pattern in the synovial fluid, and CIOA was 2/2 synovial fluids (n = 7 mice), spleens (n =2),orBM(n =2)ofmice15hafter followed in neutrophil- or NK cell–depleted and Cxcr3 mice. PBS or collagenase injection. Synovial fluid (entire population), spleen, and BM cells (5 3 106/ml) were labeled with fluorochrome-conjugated Ly6G, F4/80 and CD11b mAbs and Ly6G+CD11b+ (neutrophils), and F4/80+CD11b+ Materials and Methods (macrophages) cells were sorted using FACSAria (Becton Dickinson). Purity Animal and mouse models of osteoarthritis of the isolated cell populations was always $98%. + + Female wild type (WT) Ly5.1 (CD45.1 ) and Ly5.2 (CD45.2 ) C57BL/6 mRNA expression analysis by real time quantitative PCR mice (Charles River, Calco, Italy) and Ly5.2 Cxcr32/2 (B6.129P2- Cxcr3tm1Dgen/J) mice (Jackson Laboratory, Bar Harbor, ME) were RNA was isolated from sorted populations using a total RNA minikit housed in filter-top cages in the animal facility of the histology unit (Geneaid, Taiwan) and reverse transcribed to cDNA. Real time quantita- Downloaded from (Sapienza University of Rome) under standard conditions (temperature, tive PCR was performed with the following primers from Applied Bio- diet, and water ad libitum). All animal studies were designed according to system (Foster City, CA): mouse CXCL10 (Mm00445235_m1) conjugated Animal Research: Reporting of In Vivo Experiments, approved by the with fluorochrome FAM and b-actin (Mm01205647_g1) or hypoxanthine Italian Ministry of Health according to art. 7 D.lgs. 116/92 and conducted phosphoribosyltransferase 1 (Mm00446968_m1) conjugated with fluoro- in accordance with National Guidelines for Animal Care and Use (D.lgs. chrome VIC. cDNAs were amplified in duplicate using specific TaqMan 116/92). CIOA was elicited in anesthetized mice (8- to 12-wk old) by a Gene Expression Assays (Applied Biosystems).

single intra-articular (i.a.) injection of 10 U/10 ml of bacterial collagenase http://www.jimmunol.org/ in endotoxin-free PBS with Ca2+/Mg2+ (Sigma-Aldrich, St. Louis, MO), as Immunofluorescence of intracellular CXCL10 expression previously described (26). Control mice were i.a. injected with vehicle. Sorted neutrophils and macrophages were plated on poly-L-lysine–coated 3 4 Abs multichamber glass plates (2 10 cells per chamber). Cells were then fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton-X-100 for 5 min, mAbs directly conjugated to FITC, PE, PerCP 5.5, allophycocyanin, PE- and stained with the goat anti-CXCL10 polyclonal Ab (R&D, Minneapolis, cyanine (cy)7, allophycocyanin-eFluor 780, allophycocyanin-cy7, or bio- MI), then FITC-conjugated rabbit anti-goat IgG (Sigma-Aldrich) diluted in tin, and specific for the following Ags (clone name in parentheses) were PBS and Hoechst Stain solution (Sigma-Aldrich). After extensive washing, used in this study: NK1.1 (PK136), CD3ε (145-2C11), CD11b (M1/70), cover slips were mounted using SlowFade Gold reagent (Life Technologies) Ly6G (1A8), F4/80 (CI:A3-1), CD69 ([1H].2F3), CD45.2 (104), CD45.1 and acquired using an ApoTome Observer Z.1 microscope with 340/0.75 NA (A20). Abs and PE-cy7, allophycocyanin-cy7 conjugated streptavidin were Plan-Neofluar objective and an Axiocam MR equipped with Axio-Vision by guest on September 26, 2021 purchased from Pharmingen (Becton Dickinson, San Diego, CA), eBioscience Version 4.6.3 software for image acquisition (all from Carl Zeiss). Images (San Diego, CA), and BioLegend (San Diego, CA). F4/80 was from AbD were processed with Photoshop Version 7 software (Adobe Systems). Serotec (Kidlington, U.K.). Generation of mixed BM chimera In vivo cell depletion C57BL/6 mice CD45.2 were irradiated with 900 rads (two 450 rad doses with an To deplete neutrophils in vivo, mice were injected i.p. with 100 mg of anti- interval of 3 h). After 1 d, irradiated mice were reconstituted by i.v. injection of Ly6G mAb (clone 1A8; BioXCell, West Lebanon) in 200 ml of endotoxin- 1 3 107 BM cells from CD45.1 WT and CD45.2 CXCR3 KO mice at 1:1 free PBS at day 21, +1 and +3 of CIOA induction. NK cells were depleted ratio. Seven weeks later, reconstituted mice were injected i.a. with collagenase by i.p injection of 100 mg of anti-NK1.1 mAb (clone PK136; BioXCell) in at both knees and sacrificed at day 1 and 3. Cells from synovial fluid, blood, 200 ml of endotoxin-free PBS at day 22, 0 and +2 of CIOA induction. BM, and spleen were stained with anti-NK1.1, -CD3, -CD19, or anti-F4/80, -Ly6G, -CD11b mAbs coupled with CD45.1 and -CD45.2 specific mAbs and Histological procedures analyzed by flow cytometry. To analyze differences between CXCR32/2 and WT NK cell numbers, we determined the CD45.2:CD45.1 ratio of NK1.1+ At week 1 and 4 after collagenase injection, total knee joints were dissected and CD32 cells and their respective numbers within tissues. The obtained ratio and processed as previously described (27), stained by Safranin O and Toluidine numbers were normalized to that of B cells, which do not express CXCR3, blue methods and CIOA development was defined in a blinded protocol by the used as reference population as described (31). Grade-Stage scoring system recommended by Osteoarthritis Research Society International (28). A tartrate-resistant acid phosphatase (TRAP) kit (Sigma- Statistics Aldrich) was used to identify activated osteoclasts. Van Gieson staining was performed to evaluate collagen elastic fibers in cartilage and subchondral bone Unpaired Student t test was used to compare data from experimental groups (27). The classical Jenner-Giemsa method was used to distinguish osteoblasts after FACS and ELISA. Histological analysis was performed in a blinded (basophilic cytoplasm, blue) from osteoclasts (eosinophilic cytoplasm, purple) manner with an interclass correlation $0.8 and Kendall’s coefficient of in subchondral bone marrow (BM) (29). Photos were captured at different concordance r $0.5 showing the degree of agreement between the ob- magnifications and image analyses to quantify the intensity of collagen staining servers. Histological scores were compared by Mann–Whitney U test. Data and to count osteoblasts/osteoclasts in the gated region were accomplished by were analyzed using Graphpad Prism Software (GraphPad Software, La Image J 1.42 software (Research Services Branch, National Institutes of Jolla) version 6.01. A p value , 0.05 was considered statistically significant. Health, Bethesda, MD) (30). Chemokine measurement Results Neutrophils, macrophages, and NK cells are recruited to the Synovial fluids were obtained by injecting 25 ml PBS/1 mM EDTA twice in inflamed joint at the early phase of CIOA development the intact knee cavity and flushing, centrifugation, and collection of the supernatant. The levels of the following chemokines were analyzed in To identify and characterize the innate immune cells involved in synovial fluids, by Milliplex MAP (Millipore, Darmstadt, Germany) and CIOA development, synovial fluid cells of PBS and collagenase Mouse Chemokine 9plex (eBioscience) kits: KC, CXCL1/GROa, CCL2/ injected mice were analyzed and quantified by flow cytometry. MCP-1, CCL4/MIP-1b, CCL3/MIP-1a, CXCL10/IP-10, CCL5/RANTES, + and CCL7/MCP-3. Chemokine expression is shown as total picograms in Synovial fluid cellularity and CD45 cell numbers increased at 6 h, two knee joints. day 1, and day 3 following osteoarthritis induction, and returned to The Journal of Immunology 3 basal levels at day 7 (Supplemental Fig. 1A). As shown in Fig. 1A, according to the protocols shown in Supplemental Fig. 2A or were neutrophils (Ly6G+CD11b+), macrophages (F4/80+CD11b+), and NK left untreated. Histological analysis at week 1 showed that disease cells (NK1.1+CD32) represented a large proportion of CD45+ cells in ameliorated in the absence of either cell type (histological score, the synovial fluid, where they were recruited with different kinetics. Fig. 2A). Details are presented in Fig. 2B, which shows more Neutrophils accumulated at very early time points, reaching a peak at intact joint architecture with reduced cell infiltration (indicative of 6 h and progressively decreasing thereafter; macrophage numbers reduced synovitis), chondrocyte apoptosis and glycosaminoglycan peaked at day 1 and decreased at day 3, whereas NK cells were loss in cartilage of neutrophil-depleted CIOA mice (Safranin O detectable at 6 h and progressively increased, reaching their maximum and Toluidine blue staining). Neutrophil removal also resulted in a at day 3. Higher levels of CD11b on neutrophils and of CD69 on NK diminished osteoclast number and activation in subchondral bone cells from CIOA mouse synovial fluid, as compared respectively to (TRAP staining in Fig. 2B), which was confirmed by measuring control synovial fluid and to the CIOA blood counterpart, indicated an TRAP activity in joint bone tissue homogenates (Supplemental activated phenotype (Fig. 1B). The marked reduction of synovial fluid Fig. 2B). In regards to NK cell-depleted CIOA mice, the de- cells at day 7 corresponded to a deeper infiltration in the joint tissues, creased histological score at day 7 (Fig. 2A) was due to increased consistent with histological identification of cell infiltrates in synovial glycosaminoglycans and extensive chondrocyte proliferation in membrane, proteoglycan/collagen loss in cartilage and osteoclast ac- cartilage, and osteophyte formation at joint edges (Safranin O, tivation (Supplemental Fig. 1B). In the remodeling phase occurring at Toluidine blue staining, Fig. 2B). Furthermore, depletion of NK week 4, focal cellular nodules were identified together with increased cells in CIOA mice abrogated osteoclast activation in subchondral proteoglycan/collagen fibers in cartilage, decreased activation of os- bone (TRAP staining, Fig. 2B) and increased osteoblast-related teoclasts and osteophyte formation, characteristic features of abnor- ALP activity in the synovium (ALP enzymatic activity assay of Downloaded from mal bone repair (Supplemental Fig. 1B, 1C) joint bone tissue homogenates, Supplemental Fig. 2B). Altogether our results show that NK cell and neutrophil accumulation in the Neutrophils and NK cells promote CIOA development synovial fluid was detrimental for tissue repair and joint integrity To analyze the role of neutrophils and NK cells in CIOA onset as its inhibition can restore the balance between destruction and and progression, mice were depleted of Ly6G+ or NK1.1+ cells repair processes in osteoarthritis. http://www.jimmunol.org/

FIGURE 1. Neutrophils, macrophages, by guest on September 26, 2021 and NK cells are recruited to collagenase- injected joints with different kinetics. Synovial cells from knee joints of PBS- and collagenase-injected mice were collected and analyzed by flow cytometry, using anti-CD45.2, -NK1.1, -CD3, -Ly6G, -F4/80, -CD11b, and -CD69 specific mAbs. (A) Number and frequency of neutrophils (Ly6G+CD11b+), macrophages (F4/80+CD11b+) and NK cells (NK1.1+CD32) among CD45+ cells are shown in the representative dot plots after gating and in the graphs (column represents mean value 6 SEM, n $ 8micepergroupin three independent experiments). (B) CD11b expression level (MFI, median fluorescence intensity) on mature neutrophils and percentage of CD69+ NK cells. Graphs on the left represent the mean value 6 SEM of three independent experiments (n $ 8 mice per group) whereas the histogram plots on the right show one representative sample (IC, isotype control). Student t test, *p , 0.05, **p , 0.01, ***p , 0.001, ****p , 0.0001. 4 NEUTROPHIL–NK CELL CROSS-TALK BY CXCL10 IN OSTEOARTHRITIS

FIGURE 2. Neutrophils and NK cells have a disease-promoting role in CIOA. (A) Graph represents mean value of histological score 6 SEM in non-depleted versus NK cell and neutrophil depleted CIOA mice (n $ 6 mice per group in two independent experiments). Mann–Whitney U test, **p , 0.01, ***p , 0.001, ****p , 0.0001. (B) Paraffin-embedded knee sections of mice non-depleted (left, control PBS, or CIOA) and depleted of Ly6G+ cells (middle) or of NK1.1+ cells (right) were stained by Safranin O, Toluidine blue, TRAP, or Jenner-Giemsa methods. Rep- resentative photomicrographs of Safranin O stained knee joint sections made at frontal plane direction show lateral side of Downloaded from femur (F) and tibia (T). Arrows indicate cell infiltration (1); inflammation of synovial membrane (2); glycosaminoglycan loss (3); chondrocyte apoptosis (4); lack of inflammation in synovial membrane (6); normal staining intensity for http://www.jimmunol.org/ glycosaminoglycans (7); lack of apoptotic chondrocytes (8); increased glycosaminoglycan density (10); and chondrocyte proliferation (12); osteophyte formation at joint edges (13). In subchondral bone (SB), arrows show activated osteoclasts (Os) near to BM trabeculae (5); lack of activated osteoclasts (9); decreased number of activated osteoclasts (14). by guest on September 26, 2021

Immune cells are mobilized from BM to blood circulation at increase in the synovial fluid. The pattern of chemokine expression early CIOA phases in synovial fluid from control and CIOA mice was thus analyzed to To clarify the tissue origin of the joint-infiltrating cells, we asked identify the crucial molecules involved in cell recruitment to the whether the cell increase in CIOA synovial fluid was associated with destabilized CIOA joint (Fig. 4A): KC and CXCL1 (ligands for changes in the cellular pools from BM, spleen, and circulation. To this CXCR2) mainly increased at 6 h, in accordance with neutrophil aim, we compared neutrophil and NK cell tissue distribution at 6 h, day infiltration. Similarly, the macrophage chemotactic molecules 1, 3, and 7 following collagenase versus PBS injection. When ana- CCL2 and CCL7 (shared ligands for CCR2) were found elevated lyzing BM, we found reduced frequency and numbers of neutrophil at 6 h and gradually declined at later times. CCL4 and CCL5 (6 h and day 1) and NK cells (day 1 and 3), which coincided with their (shared ligands for CCR1 and CCR5) were detectable only in the respective increase in the synovial fluid (Fig. 3A, Supplemental Fig. CIOA group at 6 h and day 1. Among other chemokines acting on 3); both cell populations remained constant in the spleen, whereas the NK cells, CXCL10 (a CXCR3 ligand) levels increased at 6 h numbers of peripheral blood neutrophils increased and of NK cells reaching a peak at day 1, which coincided with the accumulation decreased at day 1. These results are indicative of a rapid mobilization of neutrophils and macrophages in the synovial fluid. Further- of BM neutrophils, followed by a delayed mobilization of BM NK more, CXCL10 mRNA could be found in the synovium up to cells into the bloodstream after CIOA induction, likely in charge of day 7, suggesting that this chemokine is still produced although replacing and enriching the circulating immune cell pools entering in undetectable in synovial fluids, and may contribute to the early the synovium. In regard to residual cells in BM, neutrophils showed synovial tissue immune cell infiltration (data not shown). reduced expression levels of the activation marker CD11b, whereas The cellular sources and the role of CXCL10 expression in NK NK cells displayed progressively increased CD69 expression from 6 h cell accumulation in synovial fluids and in disease progression were to day 3 (Fig. 3B). This suggests that neutrophils exit from BM in an further investigated as it was the only chemokine active on NK cells activated state, whereas NK cells exit in a resting CD692 state and are whose expression levels persisted after day 1. Because neutrophils activated after migration to the synovium. and macrophages are documented to produce CXCR3 ligands (32– 35), we collected synovial cells 15 h post collagenase injection in Cell migration is associated with elevated chemokine levels in the knee, when neutrophils and macrophages were equally rep- CIOA synovial fluids resented (data not shown). Afterwards, CXCL10 mRNA expres- The overall changes of immune cell tissue distribution indicated the sion was analyzed in highly purified synovial Ly6G+CD11b+ involvement of chemotactic mechanisms in innate immune cell mature neutrophils and F4/80+CD11b+macrophages. Interestingly, The Journal of Immunology 5

FIGURE 3. Joint inflammation influ- ences cell distribution in BM and peripheral blood. Blood, BM, and spleen were collected at 6 h, day 1, 3 and 7 from control (PBS) or CIOA mice and cell suspensions were stained with anti-Ly6G, anti-NK1.1, anti-CD3, anti-CD11b, and anti-CD69 specific Abs. (A)Graphsshow the number of NK cells (NK1.1+CD32) and neutrophils (Ly6G+CD11b+). (B) CD11b expression level was analyzed in BM neutrophils following collagenase injections. NK cell activation state was

assessed by analysis of CD69 expression Downloaded from in BM. Histograms show mean 6 SEM of CD11b (MFI, median ﬂuorescence intensity) expression in BM neutrophils and frequency of CD69+ NK cells (n $ 4mice per group), whereas the right panels show histogram plots from representative sam- ples at 6 h, 1 and 3 d (IC, isotype control). http://www.jimmunol.org/ Student t test, *p , 0.05, **p , 0.01.

CXCL10 mRNA expression was markedly increased in synovial (Supplemental Fig. 2C), indicating that neutrophil-produced or in- by guest on September 26, 2021 neutrophils in comparison with BM neutrophils (Fig. 4B). CXCL10 duced chemotactic factors in synovial fluids are indeed important mRNA was also up-regulated in CIOA synovial macrophages, but for NK cell localization. less than in neutrophils. Intracellular CXCL10 protein was detected To investigate how neutrophils could affect NK cell recruitment by immunofluorescence, confirming the ability of neutrophils to to the joint, we analyzed the chemokine microenvironment in the produce this chemokine (Fig. 4C). synovial fluid of neutrophil-depleted mice. Although the chemokine Collectively, these results describe a process in which neutro- directing neutrophil recruitment, KC, was increased in neutrophil- phils, recruited as a consequence of tissue damage, are activated in depleted CIOA mice, we found an impaired increase of several the CIOA synovium and produce CXCL10. chemokine levels, including CCL2, CCL7, and CXCL10, at day 1 that persisted at day 3 (Fig. 5B). Neutrophil depletion diminishes CXCL10 levels and NK cell numbers in CIOA synovial fluids CXCR3 promotes NK cell and macrophage accumulation in the Given that neutrophils and NK cells were sequentially recruited in joint and is crucial for disease development the synovial fluid during experimental osteoarthritis, and that To understand if CXCL10/CXCR3 axis was involved in NK cell neutrophil-produced CXCL10 could promote NK cell migration, recruitment in the inflamed synovium, we analyzed cell accumu- we asked if neutrophils were involved in the accumulation of NK lation in CXCR32/2 mice joints at the peak of NK cell increase, cells in CIOA joints by analyzing neutrophil-depleted CIOA mice. i.e., at day 3 after CIOA induction. Both macrophage and NK cell Despite neutrophil removal, synovial total cellularity did not numbers and frequency were reduced by more than 50% in change at day 1, and only decreased at day 3 (Fig. 5A). Macro- CXCR3-deficient mice, despite constant CD45+ cell and neutrophil phages were the most abundant population infiltrating the joints of numbers, suggesting that CXCR3 is important for recruitment/ neutrophil-depleted CIOA mice with no change in recruitment accumulation of specific cell populations in the CIOA joints kinetics, suggesting that the depletion of neutrophils does not (Fig. 6A). affect macrophage recruitment. On the contrary, neutrophil de- To determine if the role of CXCR3 on NK cell recruitment in the pletion reduced NK cell accumulation at day 3, but not day 1 synovial fluids of CIOA mice was direct, we generated mixed (Fig. 5A). Previous evidence indicates that NK cell function is CD45.1+ WT and CD45.2+Cxcr32/2 BM chimeras. We found that impaired in mice lacking neutrophils and thus NK cell defective Cxcr32/2 NK cells had a significant advantage in BM engraftment migration to the synovium could be attributable to a direct or in agreement with our previous observations (Supplemental Fig. indirect effect (24). Nevertheless, our preliminary evidence dem- 4A, right panel) (36), whereas no differences could be observed onstrates that NK cells purified from healthy mice have a reduced for neutrophils and monocytes (data not shown). Osteoarthritis in vitro chemotaxis toward synovial fluids of neutrophil-depleted induction similarly promoted Cxcr32/2 and WT macrophage in- CIOA mice as compared with the undepleted counterpart crease in synovial fluids at all time points analyzed. Cxcr32/2 6 NEUTROPHIL–NK CELL CROSS-TALK BY CXCL10 IN OSTEOARTHRITIS

FIGURE 4. Cell accumulation in synovial fluid is associated to inflammatory chemokine expression during CIOA. (A) Synovial fluids Downloaded from from control and CIOA mice were collected by washing the synovial cavity with PBS, as described in the Materials and Methods section. Analysis of chemokine content was performed by multiplex assays for the chemokines indicated. Histograms show mean values of che- 6 mokine total amount (picogram) in two knees http://www.jimmunol.org/ SEM (n $ 4 mice per group in three independent experiments). (B) Neutrophils and macrophages from synovial cells, splenocytes, and BM were stained with anti-Ly6G, -F4/80, and -CD11b specific Abs and purified by FACS sorting for RNA extraction. Histograms represent the fold increase in CXCL10 mRNA levels in CIOA synovium-derived neutrophils and macrophages, compared with corresponding cells in BM and spleen (SP), respectively of PBS- and by guest on September 26, 2021 collagen-treated mice. Data were obtained from two independent experiments. (C)Immunofluo- rescence and microscopic analysis of sorted cells for intracellular CXCL10 expression. Original magnification 3400. Student t test, *p , 0.05, **p , 0.01.

synovial fluid NK cell number was significantly higher at day 3 n = 9). On the other hand, activation of CXCR3-deficient NK cells but not at day 1 as compared with WT NK cells (Fig. 6B), but this at day 1 was markedly reduced in the synovium, as shown by an increase was due to higher tissue accumulation of NK cells in impaired increase of CD69 expression (Supplemental Fig. 4B). Cxcr32/2 mice (i.e., in spleen during CIOA, Supplemental Fig. As the CXCL10/CXCR3 axis is involved in the NK cell in- 4A), as demonstrated by normalizing the number of NK cells in crease in synovial fluids, and given the pathogenic role of NK cells synovial fluid with those in spleen (ratio synovial fluid/spleen: in CIOA, we analyzed if CXCR3 absence could affect disease 8.7 6 1.6 3 1024 in WT versus 8.7 6 1.7 3 1024 in Cxcr32/2; development. To this aim, we analyzed disease progression in The Journal of Immunology 7

FIGURE 5. Neutrophil depletion leads to reduced NK cell numbers and CXCL10/ IP-10 protein levels in the synovium. (A) Synovial cells from control (PBS), CIOA, and neutrophil-depleted CIOA (anti-Ly6G) mice were collected at the time points indicated after disease induction and analyzed by ﬂow cytometry after staining for anti-NK1.1, anti-F4/80, and anti-CD11b speciﬁc Abs. Graphs show total synovial cellularity (3104, left), macrophages (F4/80+CD11b+ cells, 3104, middle) and NK cells (NK1.1+CD32, 3103,right). Each circle represents a single mouse, whereas the horizontal bar represents the mean value. Data were obtained by at Downloaded from least three independent experiments. (B) Chemokines in the synovium of neutrophil-depleted mice injected with collagenase were compared with those found in non-depleted CIOA mice by multiplex analysis. Histograms show mean 6 SEM. http://www.jimmunol.org/ At least eight mice per group, in three independent experiments were used. Stu- dent t test, *p , 0.05, **p , 0.01.

Cxcr32/2 mice by histological assessment of collagenase-injected week 4) of extensive bone remodeling. We observed that cell in- by guest on September 26, 2021 joints 1 and 4 wk following the treatment. Interestingly, the his- filtration and inflammation coexist with both destruction and tological score of 1-wk old CIOA Cxcr32/2 mice was comparable remodeling processes. Neutrophils were the first cells recruited in to that of PBS-injected WT and Cxcr32/2 mice (Fig. 6C). Safranin the joint (6 h), followed by macrophages (day 1). NK cells were O staining showed intact cartilage with lack of proteoglycan loss also found in synovial fluids where they accumulated with delayed and absence of activated osteoclasts in CIOA Cxcr32/2 mice kinetics (day 3). Thus, the alterations induced rapidly in CIOA (Fig. 6C). Histological evaluation demonstrated the accumulation caused the increase, likely due to fast recruitment, of damage- of activated osteoclasts in subchondral bone (TRAP staining) of sensing cells in synovial fluids in a sequential process likely WT but not in Cxcr32/2 CIOA mice, whereas activated osteo- triggered by extracellular matrix breakdown. Both neutrophils and blasts were detected in Cxcr32/2 CIOA mice. No signs of ongoing NK cells decreased in BM, coincidently to their increase in the or resolved disease were evident at 1 wk. Cartilage abnormalities synovial fluid, suggesting that BM is a major source of immune were reduced at 4 wk in Cxcr32/2 mice, and were accompanied cells recruited to the inflamed joint (Fig. 3A), as previously by some changes in bone metabolism, as demonstrated by positive documented in other pathologies (39, 40). Thus, understanding the Alizarin S red staining (Fig. 6C, Supplemental Fig. 4C). Overall, mechanisms regulating BM immune cell mobilization in osteo- these results support a crucial role of CXCR3 for disease devel- arthritis may enable to better control the amount and type of cells opment (Fig. 7). directed to the joint. Our data also indicate that neutrophils and NK cells are dele- Discussion terious for joint integrity as Ab-mediated depletion of either cell In this study we identified a crucial role of neutrophils and NK cells type ameliorates disease. The neutrophil pathogenic role is likely in the promotion of osteoarthritis using a mouse model charac- mediated by their ability to promote inflammation, and bone de- terized by high synovial activation (CIOA), which was shown to struction due to production of factors directly regulating osteoclast resemble the disease in patients displaying high serum levels function and bone resorption (41). NK cells were shown to induce of inflammatory mediators (37, 38). In addition, we found that osteoclast differentiation by receptor activator of NF k-B ligand neutrophils produce CXCL10 in inflamed synovial fluids, thus and M-CSF upon activation and to promote osteoblast apoptosis contributing to NK cell localization/activation in the joints, and to when activated by IL-15, so interfering with bone metabolism disease establishment. We found that abrogated immune cell in- toward promotion of bone degradation (8, 42). filtration in CIOA after NK cell depletion or in Cxcr32/2 mice In regard to the mechanisms of NK cell and neutrophil re- directly impacted on disease severity. cruitment in the joint, accumulating reports have provided evidence Experimental osteoarthritis induced by enzymatic damage of of the presence of several chemokines in synovial fluids from knee joint develops in two phases: an early phase (until week 1) of osteoarthritis patients that were associated with metalloprotease cartilage destruction/bone resorption, and a late phase (observed at production and cartilage abnormalities (43, 44). However, no studies 8 NEUTROPHIL–NK CELL CROSS-TALK BY CXCL10 IN OSTEOARTHRITIS Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 6. Cxcr3 deficiency dampens NK cell and macrophage accumulation in synovial fluid and protects mice from experimental osteoarthritis. (A) Synovial fluid was collected at day 3 from WT and CXCR32/2 CIOA mice. Synovial cells were collected and stained with mAbs against CD45.2, NK1.1, CD3, Ly6G, F4/80, and CD11b and analyzed by flow cytometry. Graphs represent mean value 6 SEM of the number of CD45+ cells, neutrophils (Ly6G+ CD11b+), macrophages (F4/80+CD11b+) and NK cells (NK1.1+CD32)(n = 8 knees per group). Student t test: *p , 0.05, **p , 0.01. (B) Seven weeks after the generation of mixed CD45.1+ (WT) and CD45.2+ (Cxcr32/2) BM chimeras, mice were injected with collagenase or PBS and sacrificed at day 1 and 3 to analyze BM, spleen, and synovial fluid. Graphs show the number of WT and Cxcr32/2 macrophages (F4/80+CD11b+) and NK (NK1.1+CD32) cells in synovial fluid (n $ 5 mice per group in two independent experiments). Student t test: *p , 0.05. (C) WT and Cxcr32/2 mice were injected i.a. with collagenase and knee joints were dissected after 1 and 4 wk and assessed by histology. Representative photomicrographs show Safranin O, TRAP and Jenner-Giemsa stained sagittal knee joint sections from WT and Cxcr32/2 mice at 1 wk after injection with PBS or collagenase (CIOA). The lack of cartilage (C) destruction in femur (F) or tibia (T) and of osteoclast (Os) activation are demonstrated along with presence of activated osteoblasts (Ob) in subchondral bone (SB). Arrows point proteoglycan (PG) loss in WT mice and intact cartilage (1) in Cxcr32/2 mice (Safranin O staining), activated osteoclasts (Os) and osteoblasts (Ob). Graph represents mean value of the histological score 6 SEM of PBS (n = 4) and CIOA (n = 4) groups. Mann–Whitney U test, **p , 0.01, ***p , 0.001. have correlated the levels of chemokines in synovial fluid with Among the NK cell chemotactic molecules we found, CXCL10 recruitment/activation of immune cells in experimental osteo- was also present in the joints of osteoarthritis patients at the time of arthritis. We found increased levels of neutrophil, macrophage, diagnosis or in those who were undergoing total joint replacement and NK cell chemotactic molecules in CIOA joints, with their (7, 45). Its expression was linked to osteoclastogenic processes expression paralleling infiltration of the corresponding cell mediated by receptor activator of NF k-B ligand in other ar- types. thropathies (46, 47). In addition, this chemokine is crucial for NK The Journal of Immunology 9

FIGURE 7. Joint injury induces recruitment of neutrophils in the synovium. In addition to directly promoting tissue damage, they can produce CXCL10, which is a crucial mediator in the OA process. This chemokine plays its detrimental role by different mechanisms: it induces recruitment of macrophages (directly) and NK cells (in- directly) and it directly activates NK cells, both cells promoting OA progression. In particular, NK cells are able to interfere with tissue repair mechanisms, delaying the restoration of tissue integrity and contributing to disease severity.

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