Card9/neutrophil signalling axis promotes IL-17A-mediated ankylosing spondylitis

Abstract

Objective: Polymorphisms in the antifungal signalling molecule CARD9 are associated with ankylosing spondylitis (AS). Here, we investigated the cellular mechanism by which CARD9 controls pathogenic Th17 responses and the onset of disease in both experimental murine AS and patients.

Methods: Experiments in SKG, Card9^-/-SKG, neutrophil-deplete SKG mice along with in vitro murine, neutrophil and CD4⁺ T cell cocultures examined Card9 function in neutrophil activation, Th17 induction and arthritis in experimental AS. In AS patients the neutrophil: Bath Ankylosing Spondylitis Functional Index relationship was analysed. In vitro studies with autologous neutrophil: T cell cocultures examined endogenous CARD9 versus the AS-associated variant (rs4075515) of CARD9 in T cellular production of IL-17A.

Results: Card9 functioned downstream of Dectin-1 and was essential for induction of Th17 cells, arthritis and spondylitis in SKG mice. Card9 expression within T cells was dispensable for arthritis onset in SKG mice. Rather, Card9 expression controlled neutrophil function; and neutrophils in turn, were responsible for triggering Th17 expansion and disease in SKG mice. Mechanistically, cocultures of zymosan prestimulated neutrophils and SKG T cells revealed a direct cellular function for Card9 within neutrophils in the potentiation of IL-17 production by CD4⁺ T cells on TCR-ligation. The clinical relevance of the neutrophil-Card9-coupled mechanism in Th17-mediated disease is supported by a similar observation in AS patients. Neutrophils from HLA-B27⁺ AS patients expanded autologous Th17 cells in vitro, and the AS-associated CARD9^S12N variant increased IL-17A.

Conclusions: These data reveal a novel neutrophil-intrinsic role for Card9 in arthritogenic Th17 responses and AS pathogenesis. These data provide valuable utility in our future understanding of CARD9-specific mechanisms in spondyloarthritis .

Keywords: Arthritis; Arthritis, Experimental; Autoimmunity; Spondylitis, Ankylosing; T-Lymphocyte subsets.

Figure 1

Card9 is a genetic determinant of experimental AS in SKG mice. SKG and Card9^−/−SKG mice were injected with zymosan. (A) Mice were scored weekly for clinical arthritis. Data are combined from two experiments, n=11–14 total mice/group. (B) Visualisation and quantification of NIR signal within the legs. Regions of interest (ROIs) were quantified and mean differences between ROIs were normalised to those of corresponding ROI of healthy (non-arthritic), naïve WT-BALB/c mice. (C) Histopathology of the ankles at 8 weeks post-zymosan with corresponding photographical representation of ankles (inset). Data are combined from two experiments, n=11–14 total mice/group. (D) NIR quantification of the spine and (E) corresponding representative histopathology at 8 weeks post-zymosan. Data are mean±SEM, except for C, which shows medians with the 25th and 75th IQR (box) and min and max (whisker). Data were analysed with unpaired, two-tailed Student’s t-test or Mann-Whitney U test (C), *p<0.05, **p<0.01, ****p<0.0001. AS, ankylosing spondylitis; NIR, near-infrared.

Figure 2

Card9 promotes peripheral Th17 expansion independent of central tolerance mechanisms. (A) Serum cytokines were quantified by ELISA at 8 weeks post-zymosan. Data are combined from two experiments for n=6–11 total mice/group. The total number of live cells (B) and Thy1.2⁺ T cell subsets (C) in the popliteal draining lymph nodes (dLN) were evaluated by flow cytometry at 8 weeks post-zymosan. (D) The per cent of indicated cytokine⁺CD4⁺ T cells in the dLN was evaluated by intracellular cytokine staining and flow cytometry at 8 weeks post-zymosan. For (B–D), data were combined from three experiments, n=13 total mice/group. (E) IL-17A in the synovial fluid of ankles was measured by ELISA at 8weeks post-zymosan in SKG and Card9^−/−SKG mice or naïve (no zymosan) WT (BALB/c) mice. Data are combined from two experiments, n=5–6 total mice/group. (F) Arthritis scores of Rag1^−/− mice reconstituted with 10⁸ thymocytes from either naïve SKG or naive Card9^−/−SKG mice were evaluated weekly post-zymosan. (G) IL-17A and IL-22 expression by CD4⁺ T cells in dLN of naïve (no zymosan) SKG versus Card9^−/−SKG mice. For F–G, Data are combined from two experiments, n=6–7 mice/group. Data are shown as medians with the 25th and 75th IQR (box) and min and max (whisker); except for (F) which shows mean±SEM. Data were analysed with Mann-Whitney U test (C), *p<0.05, **p<0.01, ***p<0.001.

Figure 3

Card9 controls peripheral activation of neutrophils in SKG mice. (A) Total # of myeloid cells (Thy1.2^-B220^- that express CD11b, CD11c or Ly6g) in the dLN; and (B) frequency of myeloid subpopulations were evaluated in the dLN at 8 weeks post-zymosan. Neutrophils: CD11b⁺CD11c⁻Ly6g⁺, DCs: CD11b^variableCD11c⁺Ly6g⁻ and monocyte/macrophages: CD11b⁺CD11c⁻Ly6g⁻. (C) Neutrophil composition in bone marrow (BM) of naïve SKG or naïve Card9^−/−SKG mice. (D–G) Mice were injected with zymosan or saline and 4 hours later peritoneal lavage fluid was evaluated for (D) myeloid cell composition by flow cytometry and (E) levels of Cxcl1 and Cxcl2 by ELISA. Data are combined from two experiments, n=8–10 mice/group; and are shown as medians with the 25th and 75th IQR (box) and min and max (whisker). Data were analysed with unpaired, two-tailed Student’s t-test or Mann-Whitney U test (C), *p<0.05, **p<0.01. (F–G) Median fluorescence intensity (MFI) of indicated cell surface molecules on neutrophils in peritoneal lavage fluid were quantified by flow cytometry, with each dot representing an individual mouse. Representative data of one of three individual experiments (n=3 mice/genotype/experiment).

Figure 4

Neutrophils relate to increased AS disease burden and are necessary for induction of Th17 cells and arthritis in SKG mice. (A) A linear regression analysis was conducted with only the patients from online supplemental table 1, that were biologic-naïve and diagnosed with AS, to compare the disability index (BASFI score) with either the per cent of neutrophils or the absolute neutrophil count (ANC) in the blood. Data are displayed with best-fit line and 95% CIs for n=158 longitudinal laboratory observations, clustering of 72 patients. Per cent of neutrophil data correlation coefficient 0.059, p=0.005, cluster analysis (clustering 72 patients): correlation coefficient unchanged, p=0.042. ANC data correlation coefficient was 0.274, p=0.026, cluster analysis (clustering 72 patients); correlation coefficient unchanged, p=0.077. (B, C) SKG mice or SKG mice deplete of neutrophils via 1A8 mAb (anti-Ly6G) were injected with zymosan and 5 days later were evaluated for: (B) arthritis and (C) the percent of indicated cytokine+CD4⁺ T cells in the dLN by flow cytometry. Data are shown as medians with the 25th and 75th IQR (box) and min and max (whisker), and were analysed with Mann-Whitney U test, *p<0.05, **p<0.01. Shown is a representative experiment of three independent repeats with n=3–5 mice/condition/experiment. AS, ankylosing spondylitis; BASFI, Bath Ankylosing Spondylitis Functional Index; dLN, draining lymph nodes.

Figure 5

Card9-signalling within neutrophils promotes their activation and ability to expand Th17 cells. (A) The dLNs of mice were evaluated 5 days after zymosan injection by flow cytometry for the percentage of (A) neutrophils and (B) cytokine⁺ CD4⁺ T cells. (C) Early signs of arthritis were measured 5 days post-zymosan. Data shown are representative experiment of three independent repeats with n=5 mice/genotype/experiment. (D–H) Neutrophils purified from naïve SKG or naïve Card9^−/−SKG mice were stimulated with zymosan for 1 hour, washed, and then cocultured with SKG CD4+T cells (two neutrophils: 1 T cell) in the presence of mAb to CD3 and CD28 for 18 hours or 72 hours. (D) at 72 hours post-coculture, the total number of SKG CD4+T cells in each culture condition was quantified by flow cytometry. ^#p=0.03, comparison between SKG (SKG neutrophils: SKG T cells) versus none (SKG T cells only), and **p=0.001, comparison between Card9^−/−SKG (Card9^−/−SKG neutrophils: SKG T cells) versus none. (E) The total number of neutrophils/well and (F–G) cell surface expression of indicated markers was quantified on neutrophils freshly isolated (pre-zymosan stimulation and pre-coculture) from naïve SKG or Card9^−/−SKG mice (0 hour) or 18 hours post coculture by flow cytometry. (H) IL-17A was measured in culture supernatants at 18 hours and 72 hours post-coculture by ELISA. Data were analysed with Mann-Whitney U test (A–C) or unpaired, two-tailed Student’s t-test (D–H). *p<0.05, **p<0.01, *** p<0.001, ****p<0.0001, cumulative data from three independent experiments, 3–4 biological replicates/condition/experiment. dLN, draining lymph nodes.

Figure 6

IL-17 production by AS patient T cells is potentiated by neutrophils and is positively associated with expression of CARD9 rs4077515. (A) Autologous cocultures with CD4⁺ T cells and neutrophils derived from HLA-B27⁺ AS patients were carried out for 72 hours, and the percentage of CD4⁺ T cells producing IL-17A was determined by flow cytometry. Data were analysed by paired t test, ^#p<0.05, *p<0.05, where # indicates the comparison between T cells only and T cells+neutrophils for all patients and * for only patients diagnosed <5 years. Solid lines indicate patients diagnosed <5 years and dotted >5 years. (B) Serum IL-17A was quantified by ELISA in biological DMARD naïve, HLA-B27+AS patients (n=9) expressing either the wild-type CARD9 gene or the rs4077515 CARD9 variant. Mann-Whitney rank sum test, *p<0.05. AS, ankylosing spondylitis.