Heterogeneous expression pattern of interleukin 17A (IL-17A), IL-17F and their receptors in synovium of rheumatoid arthritis, psoriatic arthritis and osteoarthritis: possible explanation for nonresponse to anti-IL-17 therapy?

Abstract

Introduction: Accumulating evidence suggests an important role for interleukin 17 (IL-17) in the pathogenesis of several inflammatory diseases, including rheumatoid arthritis (RA) and psoriatic arthritis (PsA). Accordingly, clinical trials aimed at blocking IL-17 have been initiated, but clinical results between patients and across different diseases have been highly variable. The objective was to determine the variability in expression of IL-17A, IL-17F and their receptors IL-17RA and IL-17RC in the synovia of patients with arthritis.

Methods: Synovial biopsies were obtained from patients with RA (n = 11), PsA (n = 15) and inflammatory osteoarthritis (OA, n = 14). For comparison, synovia from noninflamed knee joints (n = 7) obtained from controls were included. Frozen sections were stained for IL-17A, IL-17F, IL-17RA and IL-17RC and evaluated by digital image analysis. We used confocal microscopy to determine which cells in the synovium express IL-17A and IL-17F, double-staining with CD4, CD8, CD15, CD68, CD163, CD31, von Willebrand factor, peripheral lymph node address in, lymphatic vessel endothelial hyaluronan receptor 1, mast cell tryptase and retinoic acid receptor-related orphan receptor γt (RORγt).

Results: IL-17A, IL-17F, IL-17RA and IL-17RC were abundantly expressed in synovial tissues of all patient groups. Whereas IL-17RA was present mostly in the synovial sublining, IL-17RC was abundantly expressed in the intimal lining layer. Digital image analysis showed a significant (P < 0.05) increase of only IL-17A in arthritis patients compared to noninflamed control tissues. The expression of IL-17A, IL-17F and their receptors was similar in the different patient groups, but highly variable between individual patients. CD4+ and CD8+ cells coexpressed IL-17A, and few cells coexpressed IL-17F. IL-17A and IL-17F were not expressed by CD15+ neutrophils. Mast cells were only occasionally positive for IL-17A or IL-17F. Interestingly, IL-17A and IL-17F staining was also observed in macrophages, as well as in blood vessels and lymphatics. This staining probably reflects receptor-bound cytokine staining. Many infiltrated cells were positive for the transcription factor RORγt. Colocalisation between RORγt and IL-17A and IL-17F indicates local IL-17 production.

Conclusions: Increased expression of IL-17A is not restricted to synovial tissues of RA and PsA patients; it is also observed in inflammatory OA. The heterogeneous expression levels may explain nonresponse to anti-IL-17 therapy in subsets of patients.

Figure 1

Expression of interleukins 17A and 17F and their receptors in synovial tissue. The expression of interleukin 17A (IL-17A), IL-17F, IL-17RA and IL-17RC in rheumatoid arthritis (RA), osteoarthritis (OA), psoriatic arthritis (PsA) and noninflamed synovial tissue was determined by immunohistochemistry using monoclonal antibodies. For each marker, a representative microscopic picture is given in synovial tissue of a RA patient (left) and a PsA patient (right) (A). The intensity of staining was analysed using digital image analysis, and levels were compared between the noninflammatory and arthritis groups using a Mann-Whitney U test (B) and between OA, PsA and RA patients using a Kruskal-Wallis test with post hoc Dunn’s multiple-comparisons tests (C). *P < 0.05. IOD, Integrated optical density.

Figure 2

Colocalisation between interleukins 17A, 17F and cell-specific markers. The colocalisation between interleukin 17A (IL-17A) or IL-17F and markers for T cells, neutrophils, mast cells, macrophages, blood vessels and lymphatics was examined using double-immunofluorescence labelling and visualised by confocal microscopy. White arrowheads indicate colocalisation. Representative pictures are presented. Type 17 helper T (Th17) cells were used to show that the IL-17A and IL-17F antibodies used can detect Th17-produced IL-17. Lyve-1, Lymphatic vessel endothelial hyaluronan receptor 1; MCT, Mast cell tryptase; PNAd, Peripheral lymph node addressin; vWF, von Willebrand factor. Pictures illustrating the separate channels for CD4, CD8, CD68 and CD163 staining with IL-17 are shown in Additional file 1: Figure S1 and Additional file 2: Figure S2.

Figure 3

Retinoic acid receptor–related orphan receptor γt–positive cells in synovium. Immunohistochemistry showed an abundant nuclear expression of the IL-17-specific transcription factor retinoic acid receptor–related orphan receptor γt (RORγt) (shown in red) in synovial tissue biopsies (A). Immunofluorescence showed that RORγt-positive cells (red nuclei) in synovial tissue were indeed positive for interleukin 17A (IL-17A) (B) or IL-17F (C).