EXTENDED REPORT Type 3 innate lymphoid cells producing IL-17 and IL-22 are expanded in the gut, in the peripheral blood, synovial fluid and bone marrow of patients with ankylosing spondylitis Francesco Ciccia, 1 Giuliana Guggino, 1 Aroldo Rizzo, 2 Laura Saieva, 3 Sergio Peralta, 4 AnnaRita Giardina, 1 Alessandra Cannizzaro, 2 Guido Sireci, 3 Giacomo De Leo, 3 Riccardo Alessandro, 3 Giovanni Triolo 1 Handling editor Tore K Kvien ▸ Additional material is published online only. To view please visit the journal online (http://dx.doi.org/10.1136/ annrheumdis-2014-206323). 1 Dipartimento Biomedico di Medicina Interna e Specialistica, Sezione di Reumatologia, Università degli Studi di Palermo, Palermo, Italy 2 Unità Operativa di Anatomia Patologica, Azienda Ospedaliera Ospedali Riuniti “Villa Sofia-Cervello”, Palermo, Italy 3 Dipartimento di Biopatologia e Biotecnologie Mediche e Forensi, Università di Palermo, Palermo, Italy 4 Dipartimento Biomedico di Medicina Interna e Specialistica, Sezione di Gastroenterologia, Università degli Studi di Palermo, Palermo, Italy Correspondence to Professor Giovanni Triolo, Department of Internal Medicine, Division of Rheumatology, Piazza delle Cliniche 2, Palermo 90127, Italy; giovanni.triolo@unipa.it Received 21 July 2014 Revised 13 March 2015 Accepted 5 April 2015 Published Online First 22 April 2015 ▸ http://dx.doi.org/10.1136/ annrheumdis-2015-207735 To cite: Ciccia F, Guggino G, Rizzo A, et al. Ann Rheum Dis 2015;74:1739–1747. ABSTRACT Background The aim of the study was to better characterise the immunological origin and the behaviour of interleukin (IL)-23-responsive innate lymphoid cells (ILCs) in the gut, synovial fluid (SF) and bone marrow (BM) of patients with ankylosing spondylitis (AS). Methods ILC1, ILC2 and ILC3 cells were determined and characterised by confocal microscopy and flow cytometry in ileal and BM biopsies, in peripheral blood (PB) and SF mononuclear cells obtained from patients with AS and controls. Mucosal vascular addressin cell adhesion molecule 1 (MADCAM-1), IL-7, IL-15 and aggregates of lymphoid tissue inducer cells (LTi) were evaluated by immunohistochemistry. The in vitro ability of epithelial cells in driving the differentiation of ILC3 and the effect of tumour necrosis factor inhibitors (TNFi) on the frequency of ILC3 and the expression of MADCAM1 were also assessed. Results ILC3 characterised as Lyn - RORc - Tbet + NKp44 + cells were significantly expanded in the gut, SF and BM of patients with AS compared with controls, produced high levels of IL-17 and IL-22 and expressed α4β7. MADcAM1 was overexpressed in BM and ileal high endothelial venules. IL-7 was significantly increased in AS gut, especially in the context of Paneth cells, and accompanied by the presence of aggregates of c-kit/IL- 7R + cells (LTi). In in vitro experiments, epithelial cells from patients with AS actively induced differentiation of ILC3 from LTi. TNFi efficacy was accompanied by a significant decrease in the percentage of intestinal and circulating ILC3 and in the expression of MADCAM1. Conclusions Gut-derived IL-17 + and IL-22 + ILC3 are expanded in the peripheral blood, SF and inflamed BM of patients with AS, suggesting the presence of an active homing axis between the gut and the inflamed sacroiliac joints. INTRODUCTION Interleukin (IL)-23 is considered to be a central cytokine in ankylosing spondylitis (AS). 1 2 The intestine appears to be the main site of IL-23 pro- duction, 23 and there is emerging clinical evidence that blocking IL-23 in AS may be effective. 4 IL-23-sensitive entheseal resident T cells (IL-23R + RAR-related orphan receptor γt (ROR-γt) ( + )CD3( + )CD4( - )CD8( - ), stem cell antigen 1 (Sca1)( + ) cells) have been observed in a murine model of AS. 5 These cells have been found to release IL-17 and IL-22, cytokines considered important actors in driving spinal inflammation and osteoproliferation. 5 Although well charac- terised from a phenotypic point of view, these cells have not been demonstrated in humans, and the exact nature of these cells and their developmental origin in patients with AS remain elusive. Interestingly, these cells share several immuno- logical similarities with specific subsets of innate lymphoid cells (ILCs). ILCs populations are specialised cells involved in the regulation of innate immunity and inflamma- tion through the secretion of polarised cytokines and chemokines. 6 Based on the cytokine properties, ILCs are classified into three groups (ILC1, ILC2 and ILC3): ILC1 express the transcription factor T-bet, produce interferon-γ (IFN-γ) and mediate immunity against intracellular pathogens and tumors 7 ; ILC2 mainly produce IL-5 and IL-13; ILC3s are an important source of ‘type 17’ cyto- kines, IL-22 and IL-17, in response to IL-23, express the retinoic acid-ROR-γ and are required for mediating immunity to extracellular bacterial infections, 8 also providing help to marginal zone B cells, 9 and may also play a proinflammatory role. 10 ILC3 seem to be essentially mucosal-restricted cells, developmentally related to lymphoid tissue-induced cells (LTi) and dependent on IL-7 for their differen- tiation. 11 12 These ROR-γ + cells are essentially involved in protective response by negatively regu- lating Th17 cells through the modulation of intes- tinal microflora. 13 14 Specifically in the gut of patients with AS, ILC3 expressing the natural cyto- toxicity receptor NKp44 are significantly expanded, produce IL-22 and induce mucins production and goblet cells hyperplasia. 15 Although the ligand for NKp44 in normal tissues has not yet been identi- fied, it seems to bind to an unusual isoform of the mixed lineage leukaemia-5 protein. 16 The contin- ued engagement of NKp44 and cytokine receptors, however, seems to induce a potent proinflamma- tory programme in NKp44 + ILC3 cells. 10 Since the prevalent expression of IL-23 in the gut of patients with AS 2 and the strong expansion of NKp44 + ILC3s we previously demonstrated, 15 in this study we aimed to better characterise ILC3 and to study ILC1 and ILC2 in the gut of AS. In Editor’s choice Scan to access more free content Ciccia F, et al. Ann Rheum Dis 2015;74:1739–1747. doi:10.1136/annrheumdis-2014-206323 1739 Basic and translational research group.bmj.com on October 22, 2015 - Published by http://ard.bmj.com/ Downloaded from