Background/Purpose: Innate lymphoid cells (ILCs) are a group of lymphocytes that lack antigen-specific receptors and have important roles in mediating immune responses and in regulating tissue homeostasis.　ILCs are classified into three subsets, ILCs 1, 2, and 3, based on their patterns of cytokine production and transcription factors (TFs) expression. ILC3s are dependent on Retinoic-acid-receptor-relates orphan receptorγt (RORγt) and produce cytokines such as IL-17, IL-22, and Tumor necrosis factor -α (TNF-α), which play critical roles in inflammatory arthritis. ILC3s are further subdivided into several populations based on the expression of surface markers. LTi cells, which express chemokine receptor 6 (CCR6), are important for development and maintenance of lymph nodes and secondary lymphoid tissues. Another subgroup is natural cytotoxicity receptor (NCR) ⁺ ILC3s, which are essential for the epithelial barrier and host protection in the intestine. In rheumatic diseases, the increase of the number of NCR⁺ ILC3s was reported in patients with psoriasis, psoriatic arthritis (PsA) and ankylosing spondylitis (AS). It has also been shown that CCR6⁺ILC3s have increased in synovium fluid (SF) of patients with PsA. In this study, we tried to evaluate the ILCs 1,2, and 3 both in quantity and quality in various tissues isolated from collagen induced arthritis (CIA) model mice in order to clarify the role of ILCs in the development of rheumatoid arthritis (RA).

Methods: We isolated lymphocytes from bone marrow (BM), spleen, peripheral blood (PB), local lymph nodes (LNs) and joints in normal and CIA model mice. ILC1s, ILC2s, CCR6⁺ILC3s and NCR⁺ILC3s were determined by flow cytometry using surface markers, NK1.1 (ILC1), IL-33R (ILC2), CCR6 and NKp46 (ILC3). Gene expression of the TFs and cytokines were­­ measured by quantitative real-time PCR (qPCR) to confirm that ILC subsets are precisely classified by these surface markers. Finally, we compared the absolute cell number, ratio, gene expressions of TFs and cytokines of each ILC sunset in normal and in CIA model mice.

Results: CCR6⁺ILC3s showed high gene expression of RORγt, IL-17, IL-22 and TNF-α as reported previously. In CIA model mouse, in addition to the increase of ratio of CCR6⁺ILC3s in PB and joints, the levels of gene expression of TFs and cytokines were significantly elevated. Almost all of NKp46⁺ILC3s expressed NK1.1, which is a marker for ILC1s. NKp46⁺ILC3s showed lower gene expression of RORγt, IL-17 and IL22 than CCR6⁺ILC3, while they showed high gene expression of T-bet and Interferon-γ(IFN-γ), suggesting that NKp46⁺ ILC3s in our purification method may include a fraction of ILC1s. Another marker which can further subdivide NKp46⁺ILC3 population is required. 

Conclusion: CCR6⁺ILC3 produce IL-17, IL-22 and TNF-α, which are essential cytokines for the development of CIA. CCR6⁺ILC3s may play roles in pathogenesis of RA through its production of cytokines.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/functional-and-quantitative-changes-of-ccr6-type3-innate-lymphoid-cells-in-murine-collagen-induced-arthritis/