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Severe respiratory infections can result in acute respiratory distress syndrome (ARDS) 1 . There are no effective pharmacological therapies that have been shown to improve outcomes for patients with ARDS. Although the host inflammatory response limits spread of and eventually clears the pathogen, immunopathology is a major contributor to tissue damage and ARDS 1 , 2 . Here we demonstrate that respiratory viral infection induces distinct fibroblast activation states, which we term extracellular matrix (ECM)-synthesizing, damage-responsive and interferon-responsive states. We provide evidence that excess activity of damage-responsive lung fibroblasts drives lethal immunopathology during severe influenza virus infection. By producing ECM-remodelling enzymes—in particular the ECM protease ADAMTS4—and inflammatory cytokines, damage-responsive fibroblasts modify the lung microenvironment to promote robust immune cell infiltration at the expense of lung function. In three cohorts of human participants, the levels of ADAMTS4 in the lower respiratory tract were associated with the severity of infection with seasonal or avian influenza virus. A therapeutic agent that targets the ECM protease activity of damage-responsive lung fibroblasts could provide a promising approach to preserving lung function and improving clinical outcomes following severe respiratory infections. Viral infection of the respiratory system induces exuberant fibroblast activity, resulting in extensive remodelling of the extracellular matrix and cytokine release, which promote immune cell infiltration of the affected area at the expense of respiratory function.

Background/Aims: Aggrecanase-1 (ADAMTS-4) and aggrecanase-2 (ADAMTS-5) are secreted enzymes belonging to the ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) family that play significant roles in the progression of osteoarthritis (OA). Here, we aimed to determine whether the expression of ADAMTS-4/5 in chondrogenesis and inflammation is regulated by microRNA-92a-3p (miR-92a-3p). Methods: MiR-92a-3p and ADAMTS-4/5 expressions were determined by quantitative polymerase chain reaction (qPCR). To investigate the repressive effect of miR-92a-3p on ADAMTS-4/5 expression, chondrogenic human mesenchymal stem cells (hMSCs) and human chondrocytes were transfected with mature miR-92a-3p or an antisense inhibitor (anti-miR-92a-3p), respectively. ADAMTS-4/5 protein production was quantified by enzyme-linked immunosorbent assay (ELISA), and miR-92a-3p involvement in IL-1β-mediated catabolic effects was examined by immunoblotting. The roles of activated MAP kinases (MAPK) and nuclear factor (NF)-κB were evaluated by using specific inhibitors. Interaction between miR-92a-3p and its putative binding site in the 3′-untranslated region (3′-UTR) of ADAMTS-4/5 mRNA was confirmed by luciferase reporter assay. Results: miR-92a-3p expression was elevated in chondrogenic hMSCs, with significantly lower expression in OA cartilage than in normal cartilage. Stimulation with IL-1β significantly reduced miR-92a-3p expression in primary human chondrocytes (PHCs). Transfection of chondrocytes with miR-92a-3p downregulated IL-1β-induced ADAMTS-4/5 expression, and the activity of a reporter construct containing the 3′-UTR of human ADAMTS-4/5 mRNA. MiR-92a-3p expression was suppressed upon IL-1β-induced activation of MAPK and NF-κB in chondrocytes. Conclusion: MiR-92a-3p is an important regulator of ADAMTS-4/5 in human chondrocytes and may contribute to the development of OA.

A key feature of osteoarthritis is the gradual loss of articular cartilage and bone deformation, resulting in the impairment of joint function. The primary cause of cartilage destruction is considered to be the presence of elevated proteases, such as matrix metalloproteinases (MMPs) and a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTSs). However, clinically tested global MMP inhibitors have low efficacy that may be due to their lack of selectivity. We previously demonstrated in vitro that a variant of tissue inhibitor of metalloproteinase-3 ([-1A]TIMP3) inhibits ADAMTSs but not MMPs. In this study, we tested whether the selectivity of [-1A]TIMP3 is beneficial compared with that of the wild-type TIMP3 in preventing or delaying the onset of the degenerative effects in a mouse model of osteoarthritis. We generated transgenic mice that overexpressed TIMP3 or [-1A]TIMP3 driven by a chondrocyte-specific type II collagen promoter. TIMP3 transgenic mice showed compromised bone integrity as opposed to [-1A]TIMP3 mice. After surgically induced joint instability, TIMP3 overexpression proved to be less protective in cartilage destruction than [-1A]TIMP3 at late stages of OA. The selective inhibition of ADAMTSs provides the possibility of modifying TIMP3 to specifically target a class of cartilage-degrading proteinases and to minimize adverse effects on bone and possibly other tissues.

Background/Aims: ADAMTSs (A disintegrin and metalloprotease domains with thrombospondins motifs) are a family of extracellular proteases that have been related to both oncogenic and tumor-suppressive functions. The aim of the present study was to investigate: 1) the mutation, copy-number alterations, and expression profile of ADAMTSs in colorectal cancer and 2) whether ADAMTSs participate in colorectal cancer (CRC) progression and invasion. Methods: The mutation, copy-number alterations, and expression profile of ADAMTSs in CRC were analyzed in the TCGA cohort using cBioportal. ADAMTS4 expression in tumor tissues and cell lines were determined by immunostaining and real-time quantitative PCR. The role of ADAMTS-4 in CRC progression and the underlying mechanisms were studied by using short hairpin RNA-mediated knockdown of ADAMTS4. The effects of ADAMTS4 in cell proliferation and invasion were determined by clone formation assay and transwell migration assay, respectively. Macrophages were depleted by liposomal clodronate in immune-competent BALB/c mice and tumor growth was analyzed. Results: ADAMTS4 was differentially expressed in CRC and predicted a poor prognosis. Elevated ADAMTS4 expression was closely associated with larger tumor size, enhanced TNM stage, and a poor clinical outcome in patients with CRC. ADAMTS4 knockdown had no inhibitory implications on cell proliferation and invasion in vitro, but significantly attenuated tumor growth in vivo. Mechanistically, we revealed that ADAMTS4 was associated macrophages infiltration and polarization in the tumor microenvironment of CRC. Macrophage depletion largely abolished the promotive effect of ADAMTS4 on tumor growth in the immune competent BALB/c mice. Conclusion: ADAMTS4 seemed to be a promising prognostic indicator in CRC. The novel link between ADAMTS4 and macrophages mirrors the potential regulatory roles of ADAMTSs in the inflammatory microenvironment of cancers.

Inhibition of intervertebral disc (IVD) degeneration, which is often accompanied by painful inflammatory and immunopathological processes, is challenging. Current IVD gene therapeutic approaches are based on adenoviral gene delivery systems, which are limited by immune reactions to their viral proteins. Their applications in IVDs near to sensitive neural structure could provoke toxicity and immunological side-effects with neurological deficits. Self-complementary adeno-associated virus (scAAV) vectors, which do not express any viral gene and are not linked with any known disease in humans, are attractive therapeutic gene delivery vectors in degenerative IVDs. However, scAAV-based silencing of catabolic or inflammatory factor has not yet been investigated in human IVD cells. Therefore, we used scAAV6, the most suitable serotype for transduction of human nucleus pulposus (NP) cells, to knockdown the major catabolic gene (ADAMTS4) of IVD degeneration. IVD degeneration grades were determined by preoperative magnetic resonance imaging. Lumbar NP tissues of degeneration grade III were removed from 12 patients by nucleotomy. NP cells were isolated and cultured with low-glucose. Titre of recombinant scAAV6 vectors targeting ADAMTS4, transduction efficiencies, transduction units, cell viabilities and expression levels of target genes were analysed using quantitative PCR, fluorescence microscopy, fluorescence-activated cell sorting, 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide assays, quantitative reverse transcription PCR, western blot and enzyme-linked immunosorbent assays during 48 days of post-transduction. Transduction efficiencies between 98.2% and 37.4% and transduction units between 611 and 245 TU/cell were verified during 48 days of post-transduction (p<0.001). scAAV6-mediated knockdown of ADAMTS4 with maximum 87.7% and minimum 40.1% was confirmed on day 8 and 48 with enhanced the level of aggrecan 48.5% and 30.2% respectively (p<0.001). scAAV6-mediated knockdown of ADAMTS4 showed no impact on cell viability and expression levels of other inflammatory catabolic proteins. Thus, our results are promising and may help to design long-term and less immunogenic gene therapeutic approaches in IVD disorders, which usually need prolonged therapeutic period between weeks and months.

In the development of osteoarthritis, aggrecan degrades prior to cartilage destruction. Aggrecanase-1 (ADAMTS-4) is considered to be the major enzyme responsible for cleaving the Glu373–Ala374 bond in the interglobular domain of aggrecan in humans. Therefore, inhibitors of ADAMTS-4 have therapeutic potential in the treatment of osteoarthritis. In the present work, we developed a chemical feature based pharmacophore model of ADAMTS-4 inhibitors using the HipHop module within the Catalyst program package in order to elucidate the structure–activity relationship and to carry out in-silico screening. The Maybridge database was screened using Hypo1 as a 3D query, and the best-fit hits that followed Lipinski’s rule of five were subsequently screened to select the compounds. The hit compounds were then docked into the active site of ADAMTS-4, and interactions were visualized to determine the potential lead molecules. After subjecting all of the hits to various screening and filtering processes, 13 compounds were finally evaluated for their in vitro inhibitory activities. This study resulted in the identification of two lead compounds with potent inhibitory effects on ADAMTS-4 activity, with IC 50 values of 0.042 μM and 0.028 μM, respectively. These results provide insight into the pharmacophoric requirements for the development of more potent ADAMTS-4 inhibitors. Graphical Abstract The aggrecan-degrading metalloprotease ADAMTS-4 has been identified as a novel therapeutic target for osteoarthritis. In this work, we used HipHop-based pharmacophore modeling and virtual screening of the Maybridge database to identify novel ADAMTS-4 inhibitors. These novel lead compounds act as potent and specific inhibitors for the ADAMTS-4 enzyme and could have therapeutic potential in the treatment of OA

A central feature of osteoarthritis (OA) is the loss of articular cartilage, which is primarily attributed to cartilage breakdown. A group of metalloproteinases termed the A disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) family are reported to be important in cartilage breakdown. Recent studies have suggested that ADAMTS-4 is a major contributor to the pathogenesis of OA and that syndecan-1 is closely associated with activation of ADAMTS-4 in human chondrocytes. Accumulating evidence also suggests that cannabinoids have chondroprotective effects. The current study explored the effects of synthetic cannabinoid WIN-55,212-2 mesylate (WIN-55) on the expression of syndecan-1 and ADAMTS-4, as well as ADAMTS-4 activity, in unstimulated and interleukin (IL)-1β-stimulated OA chondrocytes. Primary human OA articular chondrocytes were treated with WIN-55 in the presence or absence of IL-1β and cannabinoid receptor antagonists. The results of the present study demonstrated that WIN-55 inhibited ADAMTS-4 activity in unstimulated and IL-1β-stimulated primary human OA articular chondrocytes in a concentration-dependent manner. Cannabinoid receptor type 1 (CB1) and 2 (CB2) were constitutively expressed in human OA articular chondrocytes. Furthermore, selective CB2 antagonist, JTE907, but not selective CB1 antagonist, MJ15, abolished the inhibitory effect of WIN-55 on ADAMTS-4 activity. WIN55 inhibited the expression of syndecan-1 but not ADAMTS-4, and overexpression of syndecan-1 reversed the inhibitory effect of WIN-55 on the ADAMTS-4 activity in unstimulated and IL-1β-stimulated human OA articular chondrocytes. Despite having no significant effect on syndecan-1 gene promoter activity, WIN-55 markedly decreased the stability of syndecan-1 mRNA via CB2. In conclusion, to the best of our knowledge, the present study provides the first in vitro evidence supporting that the synthetic cannabinoid WIN-55 inhibits ADAMTS-4 activity in unstimulated and IL-1β-stimulated human OA articular chondrocytes by decreasing the mRNA stability/expression of syndecan-1 via CB2. This suggests a novel mechanism by which cannabinoids may prevent cartilage breakdown in OA. In addition, it also provides novel insights into the pharmacological effects of synthetic cannabinoids on OA.

ObjectiveTo investigate the impact of systemic inhibition of interleukin 6 (IL-6) or signal transducer and activator of transcription (Stat3) in an experimental model of osteoarthritis (OA).MethodsExpression of major catabolic and anabolic factors of cartilage was determined in IL-6-treated mouse chondrocytes and cartilage explants. The anti-IL-6-receptor neutralising antibody MR16-1 was used in the destabilisation of the medial meniscus (DMM) mouse model of OA. Stat3 blockade was investigated by the small molecule Stattic ex vivo and in the DMM model.ResultsIn chondrocytes and cartilage explants, IL-6 treatment reduced proteoglycan content with increased production of matrix metalloproteinase (MMP-3 and MMP-13) and a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS-4 and ADAMTS-5). IL-6 induced Stat3 and extracellular signal-regulated kinase (ERK) 1/2 signalling but not p38, c-Jun N-terminal kinase or Akt. In the DMM model, Stat3 was activated in cartilage, but neither in the synovium nor in the subchondral bone. Systemic blockade of IL-6 by MR16-1 alleviated DMM-induced OA cartilage lesions, impaired the osteophyte formation and the extent of synovitis. In the same model, Stattic had similar beneficial effects on cartilage and osteophyte formation. Stattic, but not an ERK1/2 inhibitor, significantly counteracted the catabolic effects of IL-6 on cartilage explants and suppressed the IL-6-induced chondrocytes apoptosis.ConclusionIL-6 induces chondrocyte catabolism mainly via Stat3 signalling, a pathway activated in cartilage from joint subjected to DMM. Systemic blockade of IL-6 or STAT-3 can alleviate DMM-induced OA in mice.

Objectives Interleukin 1 (IL-1) is potentially important in the pathogenesis of intervertebral disc (IVD) degeneration; increasing production of matrix degradation enzymes and inhibiting matrix synthesis. Although IL-1 polymorphisms have been linked to increased risk of IVD degeneration, it is still unclear whether IL-1 drives IVD degeneration in vivo or is a secondary feature of degeneration. Here, we investigated whether IVD degeneration could be induced spontaneously by the removal of the natural inhibitor of IL-1 (IL-1 receptor antagonist) in mice that lack a functional IL-1rn gene. Methods Histological staining and immunohistochemistry was performed on BALB/c IL-1rn+/+ and IL-1rn−/− mice to examine degeneration and to localise and detect IL-1, matrix metalloproteinases (MMP)3, MMP7, a disintigrin and MMP with thrombospondin motifs (ADAMTS)4 protein production. In addition, IVD cells were isolated using collagenase and proliferation potential determined. Results IL-1rn−/− knockout mice displayed typical features of human disc degeneration: loss of proteoglycan and normal collagen structure and increased expression of matrix degrading enzymes: MMP3; MMP7 and ADAMTS4. Histological grade of degeneration increased in IL-1rn−/− mice which was more evident within older mice. In addition IVD cells isolated from IL-1rn−/− mice displayed reduced proliferation potential. Conclusions Here, we show that IL-1rn−/− mice develop spinal abnormalities that resemble characteristic features associated with human disc degeneration. The current evidence is consistent with a role for IL-1 in the pathogenesis of IVD degeneration. The imbalance between IL-1 and IL-1Ra which is observed during human IVD degeneration could therefore be a causative factor in the degeneration of the IVD, and as such, is an appropriate pharmaceutical target for inhibiting degeneration.

Objective To investigate the inhibitory effect of hyaluronan (HA) on mechanical stress- induced expression of a disintegrin and metalloproteinase with thrombospondin type1 motifs (ADAMTS)-4, -5 and matrix metalloproteinase (MMP)-13 by human chondrocytes. Materials and methods Normal human articular chondrocytes were pre-incubated with or without 1.0 mg/mL HA (2700 kDa) for 12 h at 37 °C in stretch chambers, then they were exposed to uni-axial cyclic tensile strain (CTS, 0.5 Hz, 10 % elongation). The expression of ADAMTS-4, -5, and MMP-13 were analyzed by real-time polymerase chain reaction and Immunocytochemistry. The concentration of IL-1β in the supernatant was measured using enzyme-linked immunosorbent assay (ELISA). The nuclear translocation of runt-related transcription factor 2 (RUNX-2) and nuclear factor-κB (NF-κB) was examined by ELISA and immunocytochemistry, and phosphorylation of NF-κB was examined by western blotting. Results HA inhibited mRNA expression of ADAMTS-4, -5, and MMP13 after 24 h CTS via inhibition of IL-1β secretion and NF-κB activation. However, HA failed to inhibit CTS-induced RUNX-2 expression and subsequent expression of ADAMTS-5 and MMP-13 1 h after CTS. Conclusions Our results demonstrated that HA significantly suppressed mechanical stress-induced expression of catabolic proteases by inhibition of the NF-κB–IL-1β pathway, but did not suppress mechanical stress-induced RUNX-2 signaling.