bs_bs_banner Clinical and Experimental Immunology ORIGINAL ARTICLE doi:10.1111/cei.12376 Interleukin (IL)-17A, F and AF in inflammation: a study in collagen-induced arthritis and rheumatoid arthritis S. Sarkar,* S. Justa,* M. Brucks,* Summary J. Endres,† D. A. Fox,† X. Zhou,* Interleukin (IL)-17 plays a critical role in inflammation. Most studies to date F. Alnaimat,* B. Whitaker,‡ have elucidated the inflammatory role of IL-17A, often referred to as IL-17. J. C. Wheeler,‡ B. H. Jones§ and IL-17F is a member of the IL-17 family bearing 50% homology to IL-17A S. R. Bommireddy* and can also be present as heterodimer IL-17AF. This study elucidates the *Section of Rheumatology, Department of Medicine, and the Arizona Arthritis Center, distribution and contribution of IL-17A, F and AF in inflammatory arthritis. University of Arizona, Tucson, AZ, †Divison of Neutralizing antibody to IL-17A alone or IL-17F alone or in combination Rheumatology, Department of Internal Medicine, was utilized in the mouse collagen-induced arthritis (CIA) model to eluci- University of Michigan, Ann Arbor, MI, and date the contribution of each subtype in mediating inflammation. IL-17A, F ‡Biologics Research and §Immunology Discovery and AF were all increased during inflammatory arthritis. Neutralization of Research, Janssen Research and Development, IL-17A reduced the severity of arthritis, neutralization of IL-17A+IL-17F had Spring House, PA, USA the same effect as neutralizing IL-17A, while neutralization of IL-17F had no effect. Moreover, significantly higher levels of IL-17A and IL-17F were detected in peripheral blood mononuclear cells (PBMC) from patients with rheumatoid arthritis (RA) in comparison to patients with osteoarthritis (OA). IL-17A and AF were detected in synovial fluid mononuclear cells (SFMC) in RA and OA, with IL-17A being significantly higher in RA patients. Enriched CD3+ T cells from RA PBMCs produced singnificantly high levels of IL-17A and IL-17AF in comparison to OA peripheral blood + Accepted for publication 7 May 2014 CD3 T cells. IL-17A, F and AF were undetectable in T cells from SFMCs Correspondence: S. Sarkar, Section of from RA and OA. While IL-17A, F, and AF were all induced during CIA, Rheumatology, Department of Medicine, and IL-17A played a dominant role. Furthermore, production of IL-17A, and not the Arizona Arthritis Center, University of IL-17F or IL-17AF, was elevated in PBMCs, SFMCs and enriched peripheral Arizona, 1501 N Campbell Avenue, AHSC Rm blood CD3+ TinRA. 6309, Tucson, AZ 85724, USA. E-mail: [email protected] Keywords: arthritis, cytokine, inflammation and neutralization of IL-17B reduced the severity of Introduction CIA [5]. The vast majority of studies on rheumatoid Interleukin (IL)-17 plays an undisputed role in mediating arthritis (RA) reporting increased levels of IL-17 are inflammation. The IL-17 family consists of six subtypes, focused on IL-17A, frequently referred to as IL-17 [6–10]. IL-17A, IL-17B, IL-17C, IL-17D, IL-17E and IL-17F, with a There is limited information regarding the expression of 16–50% homology of B–F to IL-17A. IL-17F has about 50% other subtypes. IL-15-mediated induction of IL-17C, E homology to IL-17A. IL-17A and F can be secreted as and F has been reported in mononuclear cells from RA homodimers as well as the heterodimer IL-17AF [1,2]. [11]. IL-17A, IL-17F and IL-17C were increased signifi- The pathogenic role of IL-17A in inflammatory arthritis cantly in psoriatic skin [12]. Moreover, IL-17A and, to a has been reported in studies involving neutralization lesser extent, IL-17F was shown to induce a similar set of of IL-17A and in IL-17A-deficient mice [3,4]. The inflammatory genes in RA synovial fibroblasts [13,14]. pathogenic potential of other IL-17 subtypes has been These reports suggest that inflammatory potential is not reported in studies where T cells transduced with IL-17A, restricted to IL-17A alone, but also extends to other sub- IL-17F, IL-17B or IL-17C all induced joint inflammation types of IL-17 in preclinical models as well as in human in the mouse model of collagen-induced arthritis (CIA) diseases. 652 © 2014 The Authors. Clinical & Experimental Immunology published by John Wiley & Sons Ltd on behalf of British Society for Immunology, Clinical and Experimental Immunology, 177: 652–661 This is an open access article under the terms of the Creative Commons Attribution-NonCommercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes. IL-17 subtypes in inflammatory arthritis The studies in this report are focused on elucidating the incubating overnight at 30°C [17]. The panning process was contribution of IL-17A, F and AF responses in CIA and RA. repeated for a total of three rounds. Following the third We have also performed head-to-head studies of in-vivo round of panning, cells were harvested for plasmid DNA neutralization of IL-17A, IL-17F, and IL-17A+IL-17F in CIA miniprep. DNA was cut with NheI and SpeI enzymes to to establish the contribution each of these cytokines in remove the pIX gene and the vector DNA was gel isolated, mediating joint inflammation. ligated and transformed to obtain single colonies for screening. Single colonies were picked and expressed in deep 96-well plates by induction with IPTG. Cultures were Materials and methods centrifuged and supernatants were used for soluble Fab screening against mouse IL-17F coated on a Maxisorp plate Collagen-induced arthritis (1 μg/ml) and for Fab expression by binding to sheep anti- Experiments involving animals were carried out in accord- human FD (The Binding Site, Birmingham, UK) coated on ance with institutional guidelines under protocol approved a Maxisorp plate. Fabs were detected in both enzyme-linked by the Animal Care and Use Committee of the University of immunosorbent assays (ELISAs) using anti-kappa horse- Arizona. All efforts were made to minimize suffering. Eight radish peroxidase (HRP) (Southern Biotech, Birmingham, to 10-week-old male DBA1 mice were purchased from AL, USA) and detection with electrochemical luminescence Taconic Farms (Hudson, NY, USA). Arthritis was induced substrate (Roche, Basel, Switzerland). Clones that demon- following immunization with collagen and complete strated inhibition of IL-17F binding to mouse IL-17RC Freund’s adjuvant (CFA). CFA was prepared by mixing were subjected to affinity maturation. Polymerase chain heat-inactivated mycobacterial strain H37Ra in incomplete reaction (PCR)-amplified heavy chains were cloned into LC Freund’s adjuvant (4 mg/ml) and was mixed with libraries as described [16]. Panning was executed as lympholized chicken collagen (Chondrex, Redmond, WA, described above, but with lower concentrations of bt-mIL- USA) dissolved overnight in acetic acid (4 mg/ml) at 1:1 to 17F, 1 nM for all three rounds. Clones were screened as form an emulsion which was injected intradermally at the detailed above. Unique clones were converted to immuno- base of the tail. Clinical scoring of arthritis, assay of anti- globulin (Ig)G and further characterized for affinity and collagen antibodies and tissue histology of paws were inhibition of IL-17F activity. Neutralization activity of undertaken as described previously [15]. I17M502 was demonstrated in vitro via inhibition of IL-17F-induced epithelial cell-derived chemokine produc- tion from CMT93 cells (data not shown). Generation of mouse IL-17F antibody mAb (I17M502) Solution-based panning of the Centocor pIX de-novo Fab Administration of neutralizing antibodies libraries was performed based on previously used Centocor de-novo library panning techniques [16]. Specifically, Fab Mouse anti-IL-17A and isotype control antibodies were a library phage libraries were mixed and separated by heavy kind gift from Dr Merle Elloso (Janssen Research and chain gene, then blocked for 1 h with 3% milk in 1 × phos- Development, L.L.C., Spring House, PA, USA). Mouse anti- phate-buffered saline Tween 20 (PBST). Microcentrifuge IL-17F was generated as above. For experiments involving tubes were blocked with 3% milk in 1 × PBST for 1 h; 50 μl administration of neutralizing antibodies, each mouse μ + μ of Dynabeads (Dynal/Life Technologies, Paisley, Scotland, received 100 g of specific antibody 100 g of respective μ UK) per library were washed with 1 × PBS and blocked with isotype control antibody or 100 g each of both isotype 3% milk in PBST for 1 h at room temperature (RT) rotat- control antibodies, daily via the intraperitoneal (i.p.) route. ing. Bt-mIL-17F at 100 nM was also blocked for 1 h at RT rotating with 0·3% milk in PBST, then mixed with the Mouse tissue harvest and culture set-up blocked library phage for 1 h. Blocked streptavidin beads Splenocytes or draining inguinal lymph nodes were har- were incubated with the phage/antigen solution for 30 min. vested from mice 2 weeks following immunization with col- Beads were captured magnetically and washed 10 times lagen (initiation phase) and from arthritic mice. Single-cell with PBST. Cells were plated out evenly on 3–150 mm suspensions (5 × 106/ml) of splenocytes were stimulated Luria-Bertani LB broth with carbenicillin (LB/Carb)/1% with collagen (100 μg/ml; Chondrex, Redmond, WA, USA), glucose plates (Teknova, Hollister, CA, USA) and incubated in the presence or absence of IL-23 (100 ng/ml; Peprotech, overnight at 37°C. Cells were scraped from the agar plates Rocky Hill, NJ, USA) for 6 days. Culture supernatants were × μ into 2 yeast extract and tryptone (YT) media and 50 l collected and frozen in −80°C for further analyses.