Effect of JAK Inhibition on the Induction of Proinflammatory HLA-DR+CD90+ Rheumatoid Arthritis Synovial Fibroblasts by Interferon-γ

Abstract

Objective: Findings from recent transcriptome analyses of the synovium of patients with rheumatoid arthritis (RA) have revealed that 15-fold expanded HLA-DR+CD90+ synovial fibroblasts potentially act as key mediators of inflammation. The reasons for the expansion of HLA-DR+CD90+ synovial fibroblasts are unclear, but genetic signatures indicate that interferon-γ (IFNγ) plays a central role in the generation of this fibroblast subset. The present study was undertaken to investigate the generation, function and therapeutically intended blockage of HLA-DR+CD90+ synovial fibroblasts.

Methods: We combined functional assays using primary human materials and focused bioinformatic analyses of mass cytometry and transcriptomics patient data sets.

Results: We detected enriched and activated Fcγ receptor type IIIa-positive (CD16+) NK cells in the synovial tissue from patients with active RA. Soluble immune complexes were recognized by CD16 in a newly described reporter cell model, a mechanism that could be contributing to the activation of natural killer (NK) cells in RA. In vitro, NK cell-derived IFNγ induced HLA-DR on CD90+ synovial fibroblasts, leading to an inflammatory, cytokine-secreting HLA-DR+CD90+ phenotype. HLA-DR+CD90+ synovial fibroblasts consecutively activated CD4+ T cells upon receptor crosslinking via superantigens. HLA-DR+CD90+ synovial fibroblasts also activated CD4+ T cells in the absence of superantigens, an effect that was initiated by NK cell-derived IFNγ and that was 4 times stronger in patients with RA compared to patients with osteoarthritis. Finally, JAK inhibition in synovial fibroblasts prevented HLA-DR induction and blocked proinflammatory signals to T cells.

Conclusion: The HLA-DR+CD90+ phenotype represents an activation state of synovial fibroblasts during the process of inflammation in RA that can be induced by IFNγ, likely generated from infiltrating leukocytes such as activated NK cells. The induction of these proinflammatory, interleukin-6-producing, and likely antigen-presenting synovial fibroblasts can be targeted by JAK inhibition.

Fig. 1. (A-C) Induction of HLA-DR on fibroblasts by recombinant and NK cell-derived IFNɣ.

SFs were cultured over 3 days in medium (w/o) +/− recombinant (r)IFNɣ or pooled NK-SN and analyzed by flow cytometry. FI, fluorescence intensity. (A) Example histograms and percentages of HLA-DR+ RA-SFs, p=0.028. (B) HLA-DR kinetic (n=7 experiments; 3 OA-SFs; 4 RA-SFs); p<0.0001. (C) Effect of anti(α)-IFNɣ antibody on HLA-DR induction. Left: titration, n=1 experiment; middle: IFNɣ concentration in NK-SNs; right: 50μg/ml α-IFNɣ; n=7 experiments with three different RA-SFs, p=0.003. Statistics: Friedman test; significant post-tests are indicated. (D-F) Synovial fluid from seropositive RA activates CD16 (FcγRIIIA). Mouse-CD16+BW5147 reporter cells were cultured with synovial fluid (SFl) from two joints from an active seropositive RA patient or from each one joint from two patients with seronegative joint swelling (seronegative chronic polyarthritis and OA). (D) CD16 expression on BW5147 cells. Empty histograms: autofluorescence. (E) CD16 activation by SFl at a dilution of 1:1200, assessed by mouse-IL-2-ELISA (A450nm) (1 experiment in duplicates). TNFα + infliximab (TNF/Ifx) served as positive control. (F) CD16-activation by titrated SFl (n=3 technical replicates, each performed in triplicates). Three-way ANOVA confirmed significant effects of dilution and seropositivity (p<0.0001 and p=0.002). Bars: means +/− standard deviations.

Fig. 2. Activated NK cells in synovial tissues.

(A) Flow cytometry analysis of synovial NK cells (CD45+CD3−CD56+CD19−CD14− lymphocytes). Left: gating strategy, right: percentage of CD69bright NK cells. (B-D) Mass cytometry data from synovial tissues from n=26 RA and n=15 OA patients. (B) Unbiased clustering with SPADE. Colors represent CD16 expression. Clustering channels are shown in Suppl. Fig. S3. Each one representative sample from leukocyte-rich or -poor RA and OA are shown. (C) Leveraging manual gating strategies, we defined leukocyte-poor RA (percentages of CD45+ live cells were similar to OA; n=17) and leukocyte-rich RA (percentages higher than the upper range of OA + 1 standard deviation; n=9) (left), identified HLA-DR+CD90+ cells among CD45-podoplanin(PDPN)+ fibroblasts (middle) and determined the ratio of NK cell / fibroblast counts (right). (D) CD69-positive CD16+ NK cells. Left: manual gating strategy; donors with less than twenty CD16+NK cells were excluded (n=9, 8 and 9 for OA, leukocyte-poor and -rich RA). CD69 on CD16+NK cells was controlled and visualized by SPADE; right: shows sections from clustering trees shown in (B) containing CD16+ NK cells. Box and whiskers: median and range. Significance was determined using Kruskal-Wallis test (p<0.003, in C and D, respectively); significant post tests are indicated.

Fig. 3. HLA-DRB1 is upregulated on fibroblasts in leukocyte-rich RA.

(A) HLA-DRB1 RNA expression profiles from sorted populations of T cells, B cells, monocytes and synovial fibroblasts (methods). Log2 fold change in expression of HLA-DRB1 RNA in leukocyte-poor (poor) and leukocyte-rich (rich) RA compared to osteoarthritis (OA). N for each cell type in the order of OA, RA poor and RA rich: T cells = 14; 13; 15. B cells = 7; 6; 12. Monocytes = 13; 14; 16. Fibroblasts = 12; 15; 14. Statistical analysis using Kruskal-Wallis test revealed significance only in the fibroblasts group (p=2.7×10⁻⁵). Post tests: ***, p=2.5×10⁻⁶; #, p=5.3×10^-5. (B, C) Protein expression of HLA-DR in the mass cytometry dataset introduced in fig. 3 (manual gating). Left graphs: Median fluorescence intensities (FI) of HLA-DR on CD45+CD14+ monocytes and CD45-PDPN+ synovial fibroblasts between patient groups (Fig. 3). Kruskal-Wallis test revealed significance in the fibroblasts group. Significant post tests are indicated by stars. ns, not significant. Right XY graphs: Median HLA-DR FI on monocytes and fibroblasts as well as percentage of HLA-DR+ fibroblasts in relation to the percentage of CD69+CD16+ NK cells. r, Spearman’s correlation coefficient. Percentages of HLA-DR+ monocytes are not shown (all cells were positive).

Fig. 4. Induction of HLA-DR and IL-6 in CD90+ synovial fibroblasts by activated NK cells in co-cultures.

(A-D) Synovial fibroblasts (SFs) from 3 OA patients were cultured in the absence or presence of IL-2-activated NK cells. The surface expressions of CD90, HLA-DR, HLA-ABC and CD54 (ICAM1) were analyzed by flow cytometry. Example histograms and means of median fluorescence intensities (FI) or means of percentages of positive cells and standard deviations are shown (n=3 identical experiments). Day 0: Untreated SFs. Statistical analysis: two-way ANOVA. *P<0.05, **P<0.01, ***P<0.001. Autofl.: autofluorescence. (E) Intracellular staining of IL-6 in OA-SF after direct co-culture with NK cells over three days. One of two similar experiments is shown. (F) For quantification of secreted IL-6, OA-SF and RA-SF (n=3 each) were cultured for 3 days +/− 20% pooled NK-SN. Supernatants were analyzed by ELISA. Significance was confirmed by Mann Whitney test (p=0.0022). In the same ELISA run, relevant amounts of IL-6 in NK-SN were excluded (n=6).

Fig. 5. Effect of upadacitinib on the induction of pro-inflammatory HLA-DR+CD90+ RA synovial fibroblasts by NK cell-derived IFNɣ.

(A) Relative mRNA expression of JAK1/2/3 in SFs in vivo. Heat map displaying log2 transformed transcripts per million of JAK1/2/3 in sorted SFs from OA, leukocyte-poor and leukocyte-rich RA (n=12;15;14) (methods). (B-F) SFs were cultured with pooled NK cell supernatant (NK-SN) +/− anti-IFNɣ or JAK1-inhibitor upadacitinib, and subsequently incubated with staphylococcus aureus enterotoxin B (SEB). After washing, freshly isolated CD4+ T cells were added overnight. CD69 as a correlate of CD4+ T cell activation was analyzed by flow cytometry. (B) Schematic experimental design. (C) HLA-DR fluorescence intensities (FI) on SFs depending on upadacitinib concentrations (n=2 RA and n=3 OA for 4µM; n=3 RA and n=4 OA in remaining concentrations). Recombinant (r)IFNɣ and anti(α)-IFNɣ (50μg/ml) served as controls. Statistics: Kruskal-Wallis test, significant post tests are indicated. (D-F) In n=6 parallel experiments, SFs from 6 OA and 4 RA patients were treated with NK-SN +/−4μM upadacitinib (Upa.). (D) Representative dot plots showing CD4+ T cells. SSC, sideward scatter. (E, F) HLA-DR on SFs (left); CD69 on CD4+ T cells (right). Groups of biological interest were compared using Wilcoxon test. *, p<0.05; ns, not significant.

Fig. 6. HLA-DR+CD90+ rheumatoid arthritis fibroblasts induced by NK-SN possess an intrinsically enhanced capacity to stimulate CD4+ T cells.

(A-D) For a summarizing statistical analysis and direct comparison of the effect of OA and RA SFs on CD4+ T cells, we pooled data derived from all series of SEB experiments with identical conditions (n=12). (A, B) percentage of CD69-positive CD4+ T cells after co-culture with OA-SF and RA-SF under variable conditions. (C, D) Direct comparison of the effect of OA-SF and RA-SF on CD4+ T cell activation in absence of SEB. (D) Percentage of CD69+ CD4+ T cells in relation to HLA-DR expression on OA-SF and RA-SF that were pre-treated with pooled NK-SN (OA: n=11, the outlier in C was excluded without relevant alteration of statistical results, r=0.160, p=0.6. RA: n=12, r=0.165, p=0.6). In A, B and C, Wilcoxon test was used to compare selected groups of biological interest. ***, p<0.001. (E, F) Pooled data across all conditions derived from all experiments in which the median fluorescence intensities (FI) of HLA-DR on SFs and the percentage of CD69-positive CD4+ T cells after co-culture overnight were determined in parallel. Correlation analyses were performed using Spearman’s test (r).