Synoviocyte-targeted therapy synergizes with TNF inhibition in arthritis reversal

Abstract

Fibroblast-like synoviocytes (FLS) are joint-lining cells that promote rheumatoid arthritis (RA) pathology. Current disease-modifying antirheumatic agents (DMARDs) operate through systemic immunosuppression. FLS-targeted approaches could potentially be combined with DMARDs to improve control of RA without increasing immunosuppression. Here, we assessed the potential of immunoglobulin-like domains 1 and 2 (Ig1&2), a decoy protein that activates the receptor tyrosine phosphatase sigma (PTPRS) on FLS, for RA therapy. We report that PTPRS expression is enriched in synovial lining RA FLS and that Ig1&2 reduces migration of RA but not osteoarthritis FLS. Administration of an Fc-fusion Ig1&2 attenuated arthritis in mice without affecting innate or adaptive immunity. Furthermore, PTPRS was down-regulated in FLS by tumor necrosis factor (TNF) via a phosphatidylinositol 3-kinase-mediated pathway, and TNF inhibition enhanced PTPRS expression in arthritic joints. Combination of ineffective doses of TNF inhibitor and Fc-Ig1&2 reversed arthritis in mice, providing an example of synergy between FLS-targeted and immunosuppressive DMARD therapies.

Fig. 1. Ig1&2 selectively inhibits the aggressive behavior of RA FLS but not OA FLS.

(A) Representative IF images of expression of PTPRS (green) and podoplanin (red) expression in RA (n = 5) and OA (n = 4) synovial tissue. Hoechst was used for nuclear stain and mouse IgG (mIgG) as control for PTPRS. Scale bar, 100 μm. (B) Expression of PTPRS by RNA-seq in lining (cadherin-11⁺CD34⁻THY⁻) and sublining (cadherin-11⁺CD34⁻THY⁺) layer FLS isolated from RA synovial tissue (n = 7). TPM, transcripts per million. (C) Scheme of human Fc-Ig1&2 fusion protein. (D) Invasion of RA FLS (n = 6) through type I collagen in response to 10% FBS in the presence or absence of Fc-Ig1&2 (20 nM). (E) Wound healing by RA (n = 10) and OA (n = 8) FLS in response to 1% FBS in the presence or absence of Fc-Ig1&2 (20 nM). ns, not significant. (F and G) Transwell migration (F) and invasion through Matrigel (G) by RA (migration, n = 9; invasion, n = 9) and OA (migration, n = 10; invasion, n = 8) FLS in response to 10% FBS in the presence or absence of Fc-Ig1&2. (H) Epigenomic landscape of the syndecan-4 (SDC4) locus in representative OA and RA FLS lines. (I and J) H3K4me3 signal in SDC4 (I) and SDC4 expression by RNA-seq (J) in RA (n = 10) and OA (n = 10) FLS. (D to G) Fold change relative to vehicle-treated controls. Graphs represent means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 by Mann-Whitney (B, I, and J) or paired ratio t test (D to G) calculated on non-normalized data.

Fig. 2. Fc-Ig1&2 accumulates in arthritic ankles and suppresses development of arthritis.

(A) BALB/c mice with established STIA were intravenously injected with the same amount of either ¹¹¹In-labeled Fc-mTNFR (280 μCi; n = 5) or Fc-Ig1&2 (165 μCi; n = 4). Representative images of measurement 6 hours after injection. (B) Comparison of accumulation of labeled proteins from (A) in hind paws of arthritic mice. (C) Clinical score of DBA/1J mice with CIA treated with either 0.5 mg of Fc-Ig1&2 (n = 6), Fc-mTNFR (4 mg/kg; n = 6), 0.5 mg of human IgG1-Fc control (Fc-hIgG1; n = 6), or vehicle (n = 6) by intraperitoneal injection once every other day for a total of five treatments as indicated. (D) Serum level of anti-collagen antibodies of mice in (C). OD, optical density. CII, type II collagen. (E and F) Representative Micro-CT, hematoxylin and eosin (H&E), and Safranin O (SO) images used for quantification (F) of synovial inflammation, bone erosion, and cartilage depletion of mice in (C). Scale bars, 500 μm. Bone erosions were evaluated by Micro-CT by measuring the change in bone surface (BS) divided by bone volume (BV) (1/mm). Graphs represent means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 by two-way analysis of variance (ANOVA) [(C) orange asterisks indicate significance for Fc-Ig1&2 versus Fc-IgG1 and vehicle, while blue stars indicate significance for Fc-mTNFR versus Fc-IgG1 and vehicle] or Kruskal-Wallis (D).

Fig. 3. The therapeutic effect of Ig1&2 in K/BxN arthritis is not mediated through the immune system.

(A) Top panel shows scheme for evaluating the effect of Ig1&2 on development of K/BxN STIA in WT mice reconstituted with either PTPRS WT or KO bone marrow (BM). Mice were treated with either vehicle or 0.5 mg of histidine tagged Ig1&2 (His-Ig1&2) by retro-orbital (r.o.) injection once every other day for a total of four treatments. Clinical score (left bottom panel) and change in ankle swelling (right bottom panel) is shown for groups; WT BM treated with vehicle (n = 4), WT BM treated with His-Ig1&2 (n = 5), KO BM treated with vehicle (n = 6), and KO BM treated with His-Ig1&2 (n = 7). (B) At 7 weeks of age, mice with active K/BxN arthritis were treated with 0.5 mg of His-Ig1&2 (n = 5) or vehicle (n = 5) r.o. every 2 days for a total of four treatments. After 10 days, sera were collected, and 100 μl was used to induce STIA in BALB/c mice (n = 5 per group). Arthritis in K/BxN and BALB/c mice was assessed by clinical scoring (top) and change in ankle thickness (bottom). Arthritis severity in (A) calculated using area under the curve. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 by Mann-Whitney (A) or two-way ANOVA (B).

Fig. 4. The therapeutic effect of Ig1&2 in CIA is not mediated through the immune system.

(A) Therapeutic treatment of DBA/1J mice with CIA by intraperitoneal injections of 0.5 mg of Fc-Ig1&2 (n = 9) or vehicle control (n = 8) every other day for a total of three injections. Mice were euthanized 4 days after the last treatment, which corresponds to 56 days after primary immunization. (B) Number of regulatory T cells (T_regs; Tcrβ⁺CD4⁺IL-17⁻FoxP3⁺) and T_H17 (Tcrβ⁺CD4⁺IL-17⁺FoxP3⁻) cells in arthritic ankles isolated from mice in (A). (C) MHCII⁺ and MHCII⁻ (tissue-resident) synovial macrophages (CD45⁺CD11b⁺Ly6G⁻Ly6C⁻CD64⁺) in arthritic ankles isolated from mice in (A). (D) Treatment of DBA/1J mice by intraperitoneal injections with either 0.5 mg of Fc-Ig1&2 (n = 9) or vehicle control (n = 10) during the primary and boost collagen immunization. Mice were treated every other day with one treatment the day before immunization and two treatments after immunization. (E to H) Number of follicular T cells [(Tfh); (E)], GC B cells (F), T_H1 and T_H17 cells (G), and T_regs (H) in joint draining lymph nodes of mice in (D). Graphs represent means ± SEM. **P < 0.01 and ***P < 0.001 by two-way ANOVA (A). PD-1, programmed cell death; PE, phycoerythrin.

Fig. 5. TNF down-regulates expression of PTPRS in RA FLS.

(A and B) Gene (A) and protein (B) expression of PTPRS in RA FLS (n = 13) stimulated with TNFα for 12 [gene, (A)] or 24 hours [protein, (B)]. RQ, relative quantification. (C) Time-dependent down-regulation of PTPRS in RA FLS (n = 7) stimulated with TNFα (5 ng/ml). (D) Expression of PTPRS in RA FLS (n = 6) stimulated with synovial fluid (SF) obtained from three individual patients with RA with active disease RA1 (squares; n = 3 FLS lines), RA2 (triangles; n = 2 FLS lines), and RA3 (diamond; n = 1 FLS line) for 12 hours. (E) Correlation between expression of TNF and PTPRS in RA synovial tissue (n = 42). Linear regression is shown. (F) RA FLS were stimulated with TNFα (50 ng/ml) for 24 hours, after which scratch-wounding healing was evaluated in response to 1% FBS and TNFα (50 ng/ml) in the presence or absence of 20 nM Fc-Ig1&2. (G) RA FLS were prestimulated with TNFα (50 ng/ml) for 24 hours, after which cells were subjected to transwell migration in response to 10% FBS in the presence or absence of TNFα (50 ng/ml) and Fc-Ig1&2. (H) Relative change in migration between RA FLS migrated in the presence or absence of TNFα and Fc-Ig1&2. Gene expression in (A), (C), (D), and (E) relative to GAPDH. Graphs represent means ± SEM. *P < 0.05, **P < 0.01, and ****P < 0.0001 by paired one-way ANOVA (A, C, and G), Mann-Whitney (H), Spearman correlation (E), or paired ratio t test (D) calculated on non-normalized data.

Fig. 6. TNF regulates PTPRS expression in RA FLS through the PI3K/GSK3β/USF2 pathway.

(A and B) Expression of PTPRS in RA FLS (n = 5) stimulated with TNFα (5 ng/ml) for 12 hours in the presence of NFκB or PI3K inhibitors (A) or after inhibition of GSK3β (B) in serum starved RA FLS (n = 5) using two different GSK3β inhibitors. (C) Epigenomic landscape of the PTPRS locus in one representative RA FLS line. Yellow box indicates PTPRS promoter region with highlight of DNase and transcription factor cluster around exon 1 obtained from the UCSC Genome Browser. (D) Expression of PTPRS in RA FLS (n = 4) cultured in normal growth media after transfection with either scrambled (siCtrl) or USF2-targeting (siUSF2) siRNA. (E) ChIP assay of USF2 binding to the promoter region of PTPRS. Representative of three independent experiments (n = 3) performed in technical triplicates. (F) Luciferase reporter assay of the promoter region of PTPRS in HEK293T cells after transfection with empty or USF2 expressing vector. Right panel shows expression of USF2 in transfected HEK293T cells. Representative of two independent experiments (n = 2). Graphs represent means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 by one-way ANOVA (E) or paired ratio t test (A, B, D, and F) calculated on non-normalized data.

Fig. 7. Therapeutic synergy between Fc-Ig1&2 and Fc-mTNFR in CIA.

(A) CIA was induced in male DBA/1J mice. Once arthritis was established, mice were treated intraperitoneally with either 0.1 mg of Fc-Ig1&2, Fc-mTNFR (2 mg/kg), combination of 0.1 mg of Fc-Ig1&2 and Fc-mTNFR (2 mg/kg), or vehicle control (n = 8 for each treatment group). Arthritis was evaluated by clinical scoring. (B and C) Representative images of H&E and Toluidine blue (TB) staining (B) used for histopathological evaluation of synovial inflammation, bone erosion, and cartilage depletion (C) in one hind paw and one front paw isolated from mice in (A). Scale bars, 500 μm. (D) Serum level of anti-collagen IgG at day 62 from mice in (A). Graphs represent means ± SEM. *P < 0.05, **P < 0.01, and ****P < 0.0001 by two-way ANOVA (A) or Kruskal-Wallis (C).