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 http://www.jimmunol.org/content/early/2017/01/20/jimmun Downloaded from ol.1601359 Supplementary http://www.jimmunol.org/content/suppl/2017/01/20/jimmunol.160135 Material 9.DCSupplemental http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! 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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