Effects of adenovirus-mediated knockdown of IRAK4 on synovitis in the osteoarthritis rabbit model

Abstract

Background: The use of interleukin-1 receptor-associated kinase 4 (IRAK4) inhibitor as a treatment for the inflammatory joint disease is a promising method. However, its underlying mechanism in osteoarthritis (OA) remains unclear. The purpose of this study is to look into the effects of adenovirus-mediated knockdown of IRAK4 on synovitis in the OA rabbit model.

Methods: Ad-shIRAK4 was injected two weeks after anterior cruciate ligament resection. Six weeks later, the rabbits were killed. The expression of IRAK4, TNFR-associated factor 6(TRAF6), TGF-activated kinase 1(TAK1), p-IKB kinase (p-IKK), p-nuclear factor kappa-B (p-NFκB), p38, and p-p38 in the synovial membrane was detected by western blot, qRT-PCR, and immunohistochemistry analysis. Immunohistochemistry was to detect the expression of IRAK4 proteins in articular cartilage. H&E staining was to assess the pathological changes of synovium and cartilage. The levels of interleukin (IL)-1β, tumor necrosis factor-α(TNF-α), and MMP-13 in the synovial fluid were measured by ELISA. X-ray and micro-computerized tomography (μCT) scans were used to assess knee joint conditions and microstructure of subchondral bone.

Results: IRAK4 expression levels in synovial tissues of the OA model group exhibited a significant upward trend. Ad-shIRAK4 significantly reduced IRAK4 mRNA expression in synovium tissues. Notably, Ad-shIRAK4 suppressed the Toll-like receptor/interleukin-1 receptor (TLR/IL-1R) signaling. In addition, in the Ad-shIRAK4 treatment group, we can see less inflammatory cell infiltration and reduced hyperplasia and angiogenesis. The levels of IL-1β, TNF-α, and MMP-13 in the synovial fluid in the OA model group were significantly higher than that in the control group, which were reduced by Ad-shIRAK4 treatment. Finally, Results of HE stains, immunohistochemistry, and μCT showed that Ad-shIRAK4 treatment has a protective effect on cartilage damage.

Conclusions: IRAK4 is significantly upregulated in the synovium from the osteoarthritis rabbit model. In addition, Ad-shIRAK4 reduced the expression of IRAK4 and suppressed TLR/IL-1R signaling in the synovium from the osteoarthritis rabbit model. Ad-shIRAK4 could alleviate synovitis and cartilage degradation in the osteoarthritis rabbit model, and thus alleviate the symptoms of OA and prevent the progression of OA.

Keywords: Inflammation; Interleukin-1 receptor-associated kinase 4; Osteoarthritis; Synovitis; Toll-like receptor/IL-1 receptor.

Fig. 1

IRAK4 is upregulated in the synovium from the osteoarthritis rabbit model. A Anterior cruciate ligament resection was performed in New Zealand white rabbits (2.0–2.5 kg). B Cartilage damage was observed in rabbits 6 weeks after anterior cruciate ligament resection. Images of X-rays were obtained in rabbits 6 weeks after anterior cruciate ligament resection. C The protein expression levels of IRAK4 were detected by western blot. D Quantification of the relative protein expression via ImageJ software, β-actin was served as the internal control. E Macroscopic scoring of cartilage and K-L grading for X-ray. Data are expressed as the mean ± SD. Statistical analysis was performed by T tests, #P <0.05, compared between control and OA model groups. n=3 per group

Fig. 2

Ad-shIRAK4 alleviated the degree of synovitis in the osteoarthritis rabbit model. A, B H&E staining was applied to assess histopathological changes in the synovial membrane. Blue arrowheads indicate the enlarged lining cell layer, green arrowheads indicate angiogenesis and yellow arrowheads indicate inflammatory cell infiltration. The pathological score of synovitis was used to quantitatively assess the severity of synovitis. C The levels of IL-1β and TNF-α in the synovial fluid were measured by ELISA. Data are expressed as the mean ± SD. Statistical analysis was performed by one-way ANOVA, ^#P < 0.05 vs. control group; *P <0.05, **P <0.01, ***P <0.0001 vs. OA model group. n=5 per group

Fig. 3

Ad-shIRAK4 reduced the expression of IRAK4 and suppressed TLR/IL-1R signaling in the synovium from the osteoarthritis rabbit model. A IL-1β, IRAK4, TRAF6, IKK, and NFκB mRNA expression in synovial tissue was detected by qRT-PCR. B IRAK4, TRAF6, TAK1, p-IKK, p-NFκB, p38, and p-p38 protein expression in synovial tissue was detected by an automated western blot. Quantification of the relative protein expression via Image-J software, β-actin was served as the internal control. C Immunohistochemistry to identify IRAK4, TRAF6, and TAK1 in synovial tissue. D Positive staining for IRAK4, TRAF6, and TAK1were quantified by ImageJ software. Data are expressed as the mean ± SD. Statistical analysis was performed by one-way ANOVA, ^#P < 0.05 vs. control group; *P <0.05, **P <0.01, ***P <0.0001 vs. OA model group. n=3 per group. OA, OA model group; Ad-C, adenovirus empty vector group; Ad-shIRAK4-L, adenovirus low titer group; Ad-shIRAK4-H, adenovirus high titer group

Fig. 4

Ad-shIRAK4 limited OA development and delayed OA progression in the osteoarthritis rabbit model. A, B Cartilage damage was observed in rabbits 8 weeks after anterior cruciate ligament resection. the treatment with Ad-shIRAK4 significantly alleviated the cartilage damage. A, C, D Two-dimensional and three-dimensional images of micro-computerized tomography (μCT) scans were obtained 8 weeks after anterior cruciate ligament resection. A, E H&E stains were used to assess cartilage pathology. F ELISA was used to detect the level of MMP-13 in synovial fluid. Data are expressed as the mean ± SD. Statistical analysis was performed by one-way ANOVA, ^#P < 0.05 vs. control group; *P < 0.05, **P <0.01 vs. OA model group. n=5 per group

Fig. 5

Ad-shIRAK4 attenuates the expression of inflammatory signaling in articular cartilage from the osteoarthritis rabbit model. A Immunohistochemistry analysis was used to assess IRAK4 in cartilage. Data are expressed as the mean ± SD. Statistical analysis was performed by one-way ANOVA, ^#P < 0.05 vs. control group; *P < 0.05 vs. OA model group. n=3 per group