Suppression of the inflammatory response by disease-inducible interleukin-10 gene therapy in a three-dimensional micromass model of the human synovial membrane

Abstract

Background: Gene therapy has the potential to provide long-term production of therapeutic proteins in the joints of osteoarthritis (OA) patients. The objective of this study was to analyse the therapeutic potential of disease-inducible expression of anti-inflammatory interleukin-10 (IL-10) in the three-dimensional micromass model of the human synovial membrane.

Methods: Synovial tissue samples from OA patients were digested and the cells were mixed with Matrigel to obtain 3D micromasses. The CXCL10 promoter combined with the firefly luciferase reporter in a lentiviral vector was used to determine the response of the CXCL10 promoter to tumour necrosis factor alpha (TNF-α), interleukin-1β (IL-1β) and lipopolysaccharide (LPS). The effects of recombinant IL-10 on gene expression were determined by quantitative PCR. The production of IL-10 from the CXCL10p-IL10 vector and the effects on pro-inflammatory cytokine production were assessed by multiplex ELISA.

Results: Micromasses made from whole synovial membrane cell suspensions form a distinct surface composition containing macrophage and fibroblast-like synoviocytes thus mimicking the synovial lining. This lining can be transduced by lentiviruses and allow CXCL-10 promoter-regulated transgene expression. Adequate amounts of IL-10 transgene were produced after stimulation with pro-inflammatory factors able to reduce the production of synovial IL-1β and IL-6.

Conclusions: Synovial micromasses are a suitable model to test disease-regulated gene therapy approaches and the CXCL10p-IL10 vector might be a good candidate to decrease the inflammatory response implicated in the pathogenesis of OA.

Keywords: Cytokines; Gene therapy; Inflammation; Micromasses; Osteoarthritis; Synovium.

Fig. 1

Immunohistochemical detection of fibroblasts and macrophages in synovial micromasses. Synovial micromasses were generated from digested synovial tissue cell suspension and cultured for 7 days. a IgG control antibody for 11-Fibrau staining. b 11-fibrau (brown) staining for synovial fibroblasts. c IgG control antibody for CD68 staining. d CD68 staining (brown) for macrophages. e Confocal fluorescent image of the micromass after transduction with lentiviral PGK-GFP. Side view with DAPI (blue) and GFP (green) staining. The micromasses used in Fig. 1a-d were derived from RA material and similar results were obtained staining OA micromass sections

Fig. 2

Activation of the CXCL10 promoter in synovial cell micromasses by LPS, TNF-α and IL-1β. Synovial micromasses of four OA patients were transduced with the CXCL10p-fluc-RPL22p-rluc dual-luciferase construct and stimulated for 6 h with (a) 100 ng/ml LPS, (b) 10 ng/ml TNF-α or (c) 10 ng/ml IL-1β and compared to the unstimulated medium condition. The signal ratio was calculated as fireflyRLU/renillaRLU +/- SD. Micromasses are depicted as individual points and different colours represent different patients. Statistical analysis between medium and stimulated condition was performed by Student’s t test and comparisons within individual patient samples were calculated by two-way ANOVA. *P < 0.05, **P < 0.01. IL-1β interleukin-1 beta, LPS lipopolysaccharide, TNF-α tumour necrosis factor alpha

Fig. 3

Gene expression in synovial micromasses after stimulation with LPS and TNF-α in the absence or presence of IL-10. Micromasses from synovial cell suspensions (three per group) were cultured until lining formation was evident. Subsequently, the micromasses were stimulated for 2 h or 4 h with medium containing 100 ng/ml LPS, 10 ng/ml TNF-α, 10 ng/ml IL-1β in the absence and presence of 10 ng/ml IL-10. Gene expression levels of SOCS3 at 2 h (a), TNF-α at 4 h (b), IL-1β at 4 h (c) and IL-6 at 4 h (d) were measured. Expression levels are depicted as threshold cycle (Ct) +/- SD, corrected for GAPDH expression. Statistical comparison within stimulation groups was performed by two-way ANOVA and between groups by one-way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001. IL-1β interleukin-1 beta, IL-6 interleukin-6, LPS lipopolysaccharide, SOCS3 suppressor of cytokine signaling 3, TNF-α tumour necrosis factor alpha

Fig. 4

Production of IL-10 by transduced micromasses. Micromasses from synovial cell suspensions were transduced with lentiviral vectors coding for PGK-luciferase, PGK-IL10 or CXCL10p-IL10 after formation of a synovial lining. Subsequently, the micromasses were stimulated with medium containing 100 ng/ml LPS (a) or 10 ng/ml TNF-α (b). The IL-10 concentration in the supernatant after 24 h was measured using a multiplex ELISA assay. Concentrations below 1 pg/ml were included in the statistical analysis, but are shown as 1 pg/ml in the Figure. An insufficient number of micromasses could be generated from patients 10 and 12 to determine the response to TNF-α. The medium, LPS- and TNF-α-stimulated groups were compared by one-way ANOVA. *P < 0.05, **P < 0.01. IL-10 interleukin-10, LPS lipopolysaccharide, TNF-α tumour necrosis factor alpha

Fig. 5

Cytokine production by stimulated micromasses treated with IL-10 viral vectors. Micromasses from synovial cell suspensions of five patients were transduced with lentiviral vectors coding for PGK-luciferase, PGK-IL10 or CXCL10p-IL10 after formation of a synovial lining. Subsequently, the micromasses were stimulated with medium containing 100 ng/ml LPS (all patients) or 10 ng/ml TNF-α (three patients). The concentrations of IL-1β (a), IL-6 (b) and TNF-α (c) in the supernatant after 24 h were measured using a multiplex ELISA assay. IL-1β and TNF-α could only be quantified in three patients. Because of high variations in IL-6 production between patients, the values were first normalized for every individual patient for PGK-luc unstimulated. The basal values (pg/ml) were 2.7 × 10⁵, 6.5 × 10⁵, 2.7 × 10⁴, 5.1 × 10⁴ and 3.3 × 10⁵ respectively. The medium, LPS- and TNF-α-stimulated groups were compared by t test. Significancies without a capped line were compared to PGK-luc. *P < 0.05, **P < 0.01, ***P < 0.001. IL-1β interleukin-1 beta, IL-6 interleukin-6, LPS lipopolysaccharide, TNF-α tumour necrosis factor alpha