Arthritis in systemic lupus erythematosus is characterized by local IL-17A and IL-6 expression in synovial fluid

Abstract

Arthritis is a common clinical feature of systemic lupus erythematosus (SLE) and is usually non-erosive, as opposed to rheumatoid arthritis (RA). While RA synovial pathology has been extensively studied, little is known about the pathophysiology of lupus arthritis. Here, we aimed to explore the cytokine and cellular compartments in synovial fluids of SLE patients with arthritic manifestations. Acellular synovial fluid and paired serum samples from SLE patients (n = 17) were analyzed with cytokine bead array for T helper-associated cytokines. From two SLE patients, synovial fluid mononuclear cells (SFMC) could also be captured and were analyzed by multiparameter flow cytometry to dissect T cell, B cell, monocyte and dendritic cell phenotypes. SLE-derived SFMC were further stimulated in vitro to measure their capacity for producing interferon (IFN)-γ and interleukin (IL)-17A. All patients fulfilled the ACR 1982 classification criteria for SLE. Clinical records were reviewed to exclude the presence of co-morbidities such as osteoarthritis or overlap with RA. IL-17A and IL-6 levels were high in SLE synovial fluid. A clear subset of the synovial CD4⁺ T cells expressed CCR6⁺ , a marker associated with T helper type 17 (Th17) cells. IL-17A-production was validated among CD4⁺ CCR6⁺ T cells following in-vitro stimulation. Furthermore, a strong IFN-γ production was observed in both CD4⁺ and CD8⁺ cells. Our study shows high IL-17A and IL-6 levels in synovial fluids of patients with lupus arthritis. The Th17 pathway has been implicated in several aspects of SLE disease pathogenesis and our data also point to Th17 involvement for lupus arthritis.

Keywords: T cells; arthritis; cytokines; synovial fluid; systemic lupus erythematosus.

Fig. 1

Cytokine analysis of systemic lupus erythematosus synovial fluid (SLE SF) samples. (a) Interleukin (IL)‐17A, IL‐6 and IL‐10 cytokine levels were measured in SLE SF samples (n = 17), matched serum samples (n = 14, combined with lines) and SF RA samples (n = 10). Significant differences between groups were analyzed using the Mann–Whitney test. Bars indicate median. (b) Spearman’s correlations between IL‐17A, IL‐6 and IL‐10 levels in SF are displayed. (c) Spearman’s correlation between IL‐17A and IL‐6 levels in serum versus SF. (d) Immunoglobulin (Ig)A and IgM RF levels in serum and SF are shown. Red line indicates cut‐off: IgA RF = 18 IU/ml; IgM RF = 10 IU/ml).

Fig. 2

Longitudinal cytokine analysis and cellular composition in systemic lupus erythematosus synovial fluid (SLE SF). (a) Longitudinal cytokine analysis of SLE SF in one patient with multiple available time‐points. Change of medication and the time‐point of SF mononuclear cells (SFMC) acquired are indicated. (b) Proportions of cell subsets in SF and matched peripheral blood (PB) from the same patient described in (a). B cell subsets: plasmablasts (CD27⁺⁺CD38⁺⁺); naive (IgD⁺CD27⁻), double‐positive (IgD⁺CD27⁺), memory (IgD⁻CD27⁺), double‐negative (DN, IgD⁻CD27⁻). Monocyte subsets: non‐classical (CD14⁻C16⁺), intermediate (CD14⁺C16^−,+), classical (CD14⁺C16⁻) and non‐monocytes (CD14⁻CD16⁻). Dendritic cell subsets: classical DC (cDC, CD11c⁺), cDC1 (CD11c⁺CD1c⁺), cDC2 (CD11c⁺CD141⁺) and pDC (CD11c⁻CD123⁺CD303⁺). Frequencies < 4% are not displayed. (c) t‐Distributed stochastic neighbor‐embedding (tSNE) clustering of CD3⁺ cells from the two T cell panels (Supporting information, Table S4) and CD4⁺ and CD8⁺ T cell subsets in paired PB and SF (n = 1).

Fig. 3

T peripheral helper (Tph) cells in synovial fluid (SF) of systemic lupus erythematosus (SLE) patients. (a) Frequency of Tph cells in SF and serum samples in SLE (n = 2), spondyloarthritis (SpA) (n = 3) and age‐ and sex‐matched RA (n = 2) patients. (b) Gating plots of the SF samples of the two SLE patients. Tph cells are defined as CD4⁺ programmed cell death 1 (PD‐1^high) human leukocyte antigen D‐related (HLA‐DR)^high cells gated by quadrant gating. (c) Frequency of CCR6⁺ expression in Tph cells in the three different diseases. (d) SLE SF plots showing chemokine receptor (CCR6) gates after gating for CD4⁺ PD‐1^high HLA‐DR^high cells.

Fig. 4

T cell stimulation of synovial fluid mononuclear cells (SFMC) from systemic lupus erythematosus (SLE) and spondyloarthritis (SpA). Intracellular flow cytometry dot‐plots of (a) CCR6 versus IL‐17A and (b) interferon (IFN)‐γ versus interleukin (IL)‐17A in stimulated SFMC (one SLE and one SpA). The cells were stimulated overnight with anti‐CD3/CD28 beads and then stained for the intracellular cytokines IL‐17A and IFN‐γ. Gating strategies are shown in Supporting information, Fig. S4.