Increased IL-6 receptor expression and signaling in ageing cartilage can be explained by loss of TGF-ß-mediated IL-6 receptor suppression.

OBJECTIVE: Osteoarthritis (OA) development is strongly associated with ageing, possibly due to age-related changes in transforming growth factor-ß (TGF-ß) signaling in cartilage. Recently, we showed that TGF-ß suppresses interleukin (IL)-6 receptor (IL-6R) expression in chondrocytes. As IL-6 is involved in cartilage degeneration, we hypothesized that age-related loss of TGF-ß signaling results in increased IL-6R expression and signaling in ageing cartilage. DESIGN: Bovine articular cartilage was collected and immediately processed to study age-related changes in IL-6R expression using qPCR and IHC (age-range: 0.5-14 years). Moreover, cartilage from young and aged cows was stimulated with rhIL-6 and/or rhTGF-ß1 to measure IL-6-induced p-STAT3 using Western blot. Expression of STAT3-responsive genes was analyzed using qPCR. RESULTS: Expression of IL-6 receptor (bIL-6R) significantly increased in cartilage upon ageing (slope: 0.32, 95%CI: 0.20-0.45), while expression of glycoprotein 130 (bGP130) was unaffected. Cartilage stimulation with IL-6 showed increased induction of p-STAT3 upon ageing (slope: 0.14, 95%CI: 0.08-0.20). Furthermore, IL-6-mediated induction of STAT3-responsive genes like bSOCS3 and bMMP3 was increased in aged compared to young cartilage. Interestingly, the ability of TGF-ß to suppress bIL6R expression in young cartilage was lost upon ageing (slope: 0.21, 95%CI: 0.13-0.30). Concurrently, an age-related loss in TGF-ß-mediated suppression of IL-6-induced p-STAT3 and bSOCS3 expression was observed. CONCLUSIONS: Ageing results in enhanced IL-6R expression and subsequent IL-6-induced p-STAT3 signaling in articular cartilage. This is likely caused by age-related loss of protective TGF-ß signaling, resulting in loss of TGF-ß-mediated IL-6R suppression. Because of the detrimental role of IL-6 in cartilage, this mechanism may be involved in age-related OA development.

IL-37 diminishes proteoglycan loss in human OA cartilage: donor-specific link between IL-37 and MMP-3.

OBJECTIVE: A hallmark of osteoarthritis (OA) is degradation of articular cartilage proteoglycans. In isolated human OA chondrocytes, the anti-inflammatory cytokine Interleukin-37 (IL-37) lowers the expression of the proteolytic MMP and ADAMTS enzymes, which mediate this degradation. Therefore, we investigated if IL-37 protects against proteoglycan loss in freshly obtained human OA explants. MATERIAL AND METHODS: Human OA cartilage explants were incubated with IL-37. Release of sulphated proteoglycans (sGAGs) was measured with the dimethylmethylene-blue assay. Production and degradation of newly synthesized proteoglycans was measured using 35S-sulphate. Proteoglycan and proteolytic enzyme expression were analyzed by qPCR and Western Blot. Proteolytic activity was determined by measuring MMP- and ADAMTS-generated aggrecan neo-epitopes with ELISA and by using MMP-3-, MMP-13- or ADAMTS-5-inhibitors. RESULTS: Over time, a linear release of sGAGs from OA cartilage was measured. IL-37 reduced this release by 87 µg/ml (24%) 95%CI [21.04-141.4]. IL-37 did not affect 35S-sulphate incorporation or proteoglycan gene expression. In contrast, IL-37 reduced loss of 35S-sulphate labeled GAGs and reduced MMP-3 protein expression, indicating that IL-37 inhibits proteoglycan degradation. Remarkably, we observed two groups of patients; one group in which MMP-3-inhibition lowered sGAG release, and one group in which ADAMTS5-inhibition had this effect. Remarkably, IL-37 was only functional in the group of patients that responded to MMP-3-inhibition. CONCLUSION: We identified a relationship between IL-37 and reduced sGAG loss in OA cartilage. Most likely, this effect is mediated by inhibition of MMP-3 expression. These results suggest that IL-37 could be applied as therapy in a subgroup of OA patients, in which cartilage degradation is mediated by MMP-3.

Osteoarthritis year in review 2016: biology.

This review highlights a selection of literature in the area of osteoarthritis biology published between the 2015 and 2016 Osteoarthritis Research Society International (OARSI) World Congress. Highlights were selected from a pubmed search covering cartilage, bone, inflammation and pain. A personal selection was made based, amongst other things, on topics presented during the 2015 conference. This covers circadian rhythm, TGF-ß signaling, autophagy, SIRT6, exercise, lubricin, TLR's, pain and NGF. Furthermore, in this review we have made an effort to connect these seemingly distant topics into one scheme of connections between them, revealing a theoretical big picture underneath.

TGF-ß is a potent inducer of Nerve Growth Factor in articular cartilage via the ALK5-Smad2/3 pathway. Potential role in OA related pain?

OBJECTIVE: Pain is the main problem for patients with osteoarthritis (OA). Pain is linked to inflammation, but in OA a subset of patients suffers from pain without inflammation, indicating an alternative source of pain. Nerve Growth Factor (NGF) inhibition is very efficient in blocking pain during OA, but the source of NGF is unclear. We hypothesize that damaged cartilage in OA releases Transforming Growth Factor-ß (TGF-ß), which in turn stimulates chondrocytes to produce NGF. DESIGN: Murine and human chondrocyte cell lines, primary bovine and human chondrocytes, and cartilage explants from bovine metacarpal joints and human OA joints were stimulated with TGF-ß1 and/or Interleukin-1 (IL-1)ß. We analyzed NGF expression on mRNA level with QPCR and stained human OA cartilage for NGF immunohistochemically. Cultures were additionally pre-incubated with inhibitors for TAK1, Smad2/3 or Smad1/5/8 signaling to identify the TGF-ß pathway inducing NGF. RESULTS: NGF expression was consistently induced in higher levels by TGF-ß than IL-1 in all of our experiments: murine, bovine and human origin, in cell lines, primary chondrocytes and explants cultures. TAK1 inhibition consistently reduced TGF-ß-induced NGF whereas it fully blocked IL-1ß-induced NGF expression. In contrast, ALK5-Smad2/3 inhibition fully blocked TGF-ß-induced NGF expression. Despite the large variation in basal NGF in human OA samples (mRNA and histology), TGF-ß exposure led to a consistent high level of NGF induction. CONCLUSION: We show for the first time that TGF-ß induces NGF expression in chondrocytes, in a ALK5-Smad2/3 dependent manner. This reveals a potential alternative non-inflammatory source of pain in OA.

Inducible chondrocyte-specific overexpression of BMP2 in young mice results in severe aggravation of osteophyte formation in experimental OA without altering cartilage damage.

OBJECTIVES: In osteoarthritis (OA) chondrocytes surrounding lesions express elevated bone morphogenetic protein 2 (BMP2) levels. To investigate the functional consequence of chondrocyte-specific BMP2 expression, we made a collagen type II dependent, doxycycline (dox)-inducible BMP2 transgenic mouse and studied the effect of elevated BMP2 expression on healthy joints and joints with experimental OA. METHODS: We cloned a lentivirus with BMP2 controlled by a tet-responsive element and transfected embryos of mice containing a collagen type II driven cre-recombinase and floxed rtTA to gain a mouse expressing BMP2 solely in chondrocytes and only upon dox exposure (Col2-rtTA-TRE-BMP2). Mice were treated with dox to induce elevated BMP2 expression. In addition, experimental OA was induced (destabilisation of the medial meniscus model) with or without dox supplementation and knee joints were isolated for histology. RESULTS: Dox treatment resulted in chondrocyte-specific upregulation of BMP2 and severely aggravated formation of osteophytes in experimental OA but not in control mice. Moreover, elevated BMP2 levels did not result in alterations in articular cartilage of young healthy mice, although BMP2-exposure did increase VDIPEN expression in the articular cartilage. Strikingly, despite apparent changes in knee joint morphology due to formation of large osteophytes there were no detectible differences in articular cartilage: none with regard to structural damage nor in Safranin O staining intensity when comparing destabilisation of the medial meniscus with or without dox exposure. CONCLUSIONS: Our data show that chondrocyte-specific elevation of BMP2 levels does not alter the course of cartilage damage in an OA model in young mice but results in severe aggravation of osteophyte formation.