Salt-inducible kinase 2 regulates fibrosis during bleomycin-induced lung injury.

Idiopathic pulmonary fibrosis is a progressive and normally fatal disease with limited treatment options. The tyrosine kinase inhibitor nintedanib has recently been approved for the treatment of idiopathic pulmonary fibrosis, and its effectiveness has been linked to its ability to inhibit a number of receptor tyrosine kinases including the platelet-derived growth factor, vascular endothelial growth factor, and fibroblast growth factor receptors. We show here that nintedanib also inhibits salt-inducible kinase 2 (SIK2), with a similar IC50 to its reported tyrosine kinase targets. Nintedanib also inhibited the related kinases SIK1 and SIK3, although with 12-fold and 72-fold higher IC50s, respectively. To investigate if the inhibition of SIK2 may contribute to the effectiveness of nintedanib in treating lung fibrosis, mice with kinase-inactive knockin mutations were tested using a model of bleomycin-induced lung fibrosis. We found that loss of SIK2 activity protects against bleomycin-induced fibrosis, as judged by collagen deposition and histological scoring. Loss of both SIK1 and SIK2 activity had a similar effect to loss of SIK2 activity. Total SIK3 knockout mice have a developmental phenotype making them unsuitable for analysis in this model; however, we determined that conditional knockout of SIK3 in the immune system did not affect bleomycin-induced lung fibrosis. Together, these results suggest that SIK2 is a potential drug target for the treatment of lung fibrosis.

miR-155-overexpressing monocytes resemble HLAhighISG15+ synovial tissue macrophages from patients with rheumatoid arthritis and induce polyfunctional CD4+ T-cell activation.

MicroRNAs (miRs) are known to regulate pro-inflammatory effector functions of myeloid cells, and miR dysregulation is implicated in rheumatoid arthritis (RA), a condition characterized by inflammation and destruction of the joints. We showed previously that miR-155 is increased in myeloid cells in RA and induces pro-inflammatory activation of monocytes and macrophages; however, its role at the interface between innate and adaptive immunity was not defined. Here, RNA-sequencing revealed that overexpression of miR-155 in healthy donor monocytes conferred a specific gene profile which bears similarities to that of RA synovial fluid-derived CD14+ cells and HLAhighISG15+ synovial tissue macrophages, both of which are characterized by antigen-presenting pathways. In line with this, monocytes in which miR-155 was overexpressed, displayed increased expression of HLA-DR and both co-stimulatory and co-inhibitory molecules, and induced activation of polyfunctional T cells. Together, these data underpin the notion that miR-155-driven myeloid cell activation in the synovium contributes not only to inflammation but may also influence the adaptive immune response.

ILDR2 Is a Novel B7-like Protein That Negatively Regulates T Cell Responses.

The B7-like protein family members play critical immunomodulatory roles and constitute attractive targets for the development of novel therapies for human diseases. We identified Ig-like domain-containing receptor (ILDR)2 as a novel B7-like protein with robust T cell inhibitory activity, expressed in immune cells and in immune-privileged and inflamed tissues. A fusion protein, consisting of ILDR2 extracellular domain with an Fc fragment, that binds to a putative counterpart on activated T cells showed a beneficial effect in the collagen-induced arthritis model and abrogated the production of proinflammatory cytokines and chemokines in autologous synovial-like cocultures of macrophages and cytokine-stimulated T cells. Collectively, these findings point to ILDR2 as a novel negative regulator for T cells, with potential roles in the development of immune-related diseases, including autoimmunity and cancer.

The role of microRNAs in glucocorticoid action.

Glucocorticoids (GCs) are steroids with profound anti-inflammatory and immunomodulatory activities. Synthetic GCs are widely used for managing chronic inflammatory and autoimmune conditions, as immunosuppressants in transplantation, and as anti-tumor agents in certain hematological cancers. However, prolonged GC exposure can cause adverse effects. A detailed understanding of GCs' mechanisms of action may enable harnessing of their desirable actions while minimizing harmful effects. Here, we review the impact on the GC biology of microRNAs, small non-coding RNAs that post-transcriptionally regulate gene expression. Emerging evidence indicates that microRNAs modulate GC production by the adrenal glands and the cells' responses to GCs. Furthermore, GCs influence cell proliferation, survival, and function at least in part by regulating microRNA expression. We propose that the beneficial effects of GCs may be enhanced through combination with reagents targeting specific microRNAs.

MicroRNA-146 and cell trauma down-regulate expression of the psoriasis-associated atypical chemokine receptor ACKR2.

Chemokines are the principal regulators of leukocyte migration and are essential for initiation and maintenance of inflammation. Atypical chemokine receptor 2 (ACKR2) binds and scavenges proinflammatory CC-chemokines, regulates cutaneous T-cell positioning, and limits the spread of inflammation in vivo Altered ACKR2 function has been implicated in several inflammatory disorders, including psoriasis, a common and debilitating T-cell-driven disorder characterized by thick erythematous skin plaques. ACKR2 expression is abnormal in psoriatic skin, with decreased expression correlating with recruitment of T-cells into the epidermis and increased inflammation. However, the molecular mechanisms that govern ACKR2 expression are not known. Here, we identified specific psoriasis-associated microRNAs (miRs) that bind ACKR2, inhibit its expression, and are active in primary cultures of human cutaneous cells. Using both in silico and in vitro approaches, we show that miR-146b and miR-10b directly bind the ACKR2 3'-UTR and reduce expression of ACKR2 transcripts and protein in keratinocytes and lymphatic endothelial cells, respectively. Moreover, we demonstrate that ACKR2 expression is further down-regulated upon cell trauma, an important trigger for the development of new plaques in many psoriasis patients (the Koebner phenomenon). We found that tensile cell stress leads to rapid ACKR2 down-regulation and concurrent miR-146b up-regulation. Together, we provide, for the first time, evidence for epigenetic regulation of an atypical chemokine receptor. We propose a mechanism by which cell trauma and miRs coordinately exacerbate inflammation via down-regulation of ACKR2 expression and provide a putative mechanistic explanation for the Koebner phenomenon in psoriasis.

The role of microRNA-155/liver X receptor pathway in experimental and idiopathic pulmonary fibrosis.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is progressive and rapidly fatal. Improved understanding of pathogenesis is required to prosper novel therapeutics. Epigenetic changes contribute to IPF; therefore, microRNAs may reveal novel pathogenic pathways. OBJECTIVES: We sought to determine the regulatory role of microRNA (miR)-155 in the profibrotic function of murine lung macrophages and fibroblasts, IPF lung fibroblasts, and its contribution to experimental pulmonary fibrosis. METHODS: Bleomycin-induced lung fibrosis in wild-type and miR-155-/- mice was analyzed by histology, collagen, and profibrotic gene expression. Mechanisms were identified by in silico and molecular approaches and validated in mouse lung fibroblasts and macrophages, and in IPF lung fibroblasts, using loss-and-gain of function assays, and in vivo using specific inhibitors. RESULTS: miR-155-/- mice developed exacerbated lung fibrosis, increased collagen deposition, collagen 1 and 3 mRNA expression, TGF-ß production, and activation of alternatively activated macrophages, contributed by deregulation of the miR-155 target gene the liver X receptor (LXR)α in lung fibroblasts and macrophages. Inhibition of LXRα in experimental lung fibrosis and in IPF lung fibroblasts reduced the exacerbated fibrotic response. Similarly, enforced expression of miR-155 reduced the profibrotic phenotype of IPF and miR-155-/- fibroblasts. CONCLUSIONS: We describe herein a molecular pathway comprising miR-155 and its epigenetic LXRα target that when deregulated enables pathogenic pulmonary fibrosis. Manipulation of the miR-155/LXR pathway may have therapeutic potential for IPF.

A Novel Allosteric Activator of Free Fatty Acid 2 Receptor Displays Unique Gi-functional Bias.

The short chain fatty acid receptor FFA2 is able to stimulate signaling via both Gi- and Gq/G11-promoted pathways. These pathways are believed to control distinct physiological end points but FFA2 receptor ligands appropriate to test this hypothesis have been lacking. Herein, we characterize AZ1729, a novel FFA2 regulator that acts as a direct allosteric agonist and as a positive allosteric modulator, increasing the activity of the endogenously produced short chain fatty acid propionate in Gi-mediated pathways, but not at those transduced by Gq/G11 Using AZ1729 in combination with direct inhibitors of Gi and Gq/G11 family G proteins demonstrated that although both arms contribute to propionate-mediated regulation of phospho-ERK1/2 MAP kinase signaling in FFA2-expressing 293 cells, the Gq/G11-mediated pathway is predominant. We extend these studies by employing AZ1729 to dissect physiological FFA2 signaling pathways. The capacity of AZ1729 to act at FFA2 receptors to inhibit ß-adrenoreceptor agonist-promoted lipolysis in primary mouse adipocytes and to promote chemotaxis of isolated human neutrophils confirmed these as FFA2 processes mediated by Gi signaling, whereas, in concert with blockade by the Gq/G11 inhibitor FR900359, the inability of AZ1729 to mimic or regulate propionate-mediated release of GLP-1 from mouse colonic preparations defined this physiological response as an end point transduced via activation of Gq/G11.

MicroRNA-146a governs fibroblast activation and joint pathology in arthritis.

Synovial fibroblasts are key cells orchestrating the inflammatory response in arthritis. Here we demonstrate that loss of miR-146a, a key epigenetic regulator of the innate immune response, leads to increased joint destruction in a TNF-driven model of arthritis by specifically regulating the behavior of synovial fibroblasts. Absence of miR-146a in synovial fibroblasts display a highly deregulated gene expression pattern and enhanced proliferation in vitro and in vivo. Deficiency of miR-146a induces deregulation of tumor necrosis factor (TNF) receptor associated factor 6 (TRAF6) in synovial fibroblasts, leading to increased proliferation. In addition, loss of miR-146a shifts the metabolic state of fibroblasts towards glycolysis and augments the ability of synovial fibroblasts to support the generation of osteoclasts by controlling the balance of osteoclastogenic regulatory factors receptor activator of NF-κB ligand (RANKL) and osteoprotegerin (OPG). Bone marrow transplantation experiments confirmed the importance of miR-146a in the radioresistant mesenchymal compartment for the control of arthritis severity, in particular for inflammatory joint destruction. This study therefore identifies microRNA-146a as an important local epigenetic regulator of the inflammatory response in arthritis. It is a central element of an anti-inflammatory feedback loop in resident synovial fibroblasts, who are orchestrating the inflammatory response in chronic arthritis. MiR-146a restricts their activation, thereby preventing excessive tissue damage during arthritis.

Proteinase-activated receptor 2 modulates OA-related pain, cartilage and bone pathology.

OBJECTIVE: Proteinase-activated receptor 2 (PAR2) deficiency protects against cartilage degradation in experimental osteoarthritis (OA). The wider impact of this pathway upon OA-associated pathologies such as osteophyte formation and pain is unknown. Herein, we investigated early temporal bone and cartilage changes in experimental OA in order to further elucidate the role of PAR2 in OA pathogenesis. METHODS: OA was induced in wild-type (WT) and PAR2-deficient (PAR2-/-) mice by destabilisation of the medial meniscus (DMM). Inflammation, cartilage degradation and bone changes were monitored using histology and microCT. In gene rescue experiments, PAR2-/- mice were intra-articularly injected with human PAR2 (hPAR2)-expressing adenovirus. Dynamic weight bearing was used as a surrogate of OA-related pain. RESULTS: Osteophytes formed within 7 days post-DMM in WT mice but osteosclerosis was only evident from 14 days post induction. Importantly, PAR2 was expressed in the proliferative/hypertrophic chondrocytes present within osteophytes. In PAR2-/- mice, osteophytes developed significantly less frequently but, when present, were smaller and of greater density; no osteosclerosis was observed in these mice up to day 28. The pattern of weight bearing was altered in PAR2-/- mice, suggesting reduced pain perception. The expression of hPAR2 in PAR2-/- mice recapitulated osteophyte formation and cartilage damage similar to that observed in WT mice. However, osteosclerosis was absent, consistent with lack of hPAR2 expression in subchondral bone. CONCLUSIONS: This study clearly demonstrates PAR2 plays a critical role, via chondrocytes, in osteophyte development and subchondral bone changes, which occur prior to PAR2-mediated cartilage damage. The latter likely occurs independently of OA-related bone changes.

MicroRNA-155 regulates monocyte chemokine and chemokine receptor expression in Rheumatoid Arthritis.

OBJECTIVE: To test the hypothesis that miR-155 regulates monocyte migratory potential via modulation of chemokine and chemokine receptor expression in RA, and thereby is associated with disease activity. METHODS: The miR-155 copy-numbers in monocytes from peripheral blood (PB) of healthy (n = 22), RA (n = 24) and RA SF (n = 11) were assessed by real time-PCR using synthetic miR-155 as a quantitative standard. To evaluate the functional impact of miR-155, human monocytes were transfected with control or miR-155 mimic, and the effect on transcript levels, and production of chemokines was evaluated by Taqman low-density arrays and multiplex assays. A comparative study evaluated constitutive chemokine receptor expression in miR-155-/- and wild-type murine (CD115 + Ly6C + Ly6G-) monocytes. RESULTS: Compared with healthy monocytes, the miR-155 copy-number was higher in RA, peripheral blood (PB) and SF monocytes (PB P < 0.01, and SF P < 0.0001). The miR-155 copy-number in RA PB monocytes was higher in ACPA-positive compared with ACPA-negative patients (P = 0.033) and correlated (95% CI) with DAS28 (ESR), R = 0.728 (0.460, 0.874), and with tender, R = 0.631 (0.306, 0.824) and swollen, R = 0.503 (0.125, 0.753) joint counts. Enforced-expression of miR-155 in RA monocytes stimulated the production of CCL3, CCL4, CCL5 and CCL8; upregulated CCR7 expression; and downregulated CCR2. Conversely, miR155-/- monocytes showed downregulated CCR7 and upregulated CCR2 expression. CONCLUSION: Given the observed correlations with disease activity, these data provide strong evidence that miR-155 can contribute to RA pathogenesis by regulating chemokine production and pro-inflammatory chemokine receptor expression, thereby promoting inflammatory cell recruitment and retention in the RA synovium.

IL-33 promotes ST2-dependent lung fibrosis by the induction of alternatively activated macrophages and innate lymphoid cells in mice.

BACKGROUND: The initiation and regulation of pulmonary fibrosis are not well understood. IL-33, an important cytokine for respiratory diseases, is overexpressed in the lungs of patients with idiopathic pulmonary fibrosis. OBJECTIVES: We aimed to determine the effects and mechanism of IL-33 on the development and severity of pulmonary fibrosis in murine bleomycin-induced fibrosis. METHODS: Lung fibrosis was induced by bleomycin in wild-type or Il33r (St2)(-/-) C57BL/6 mice treated with the recombinant mature form of IL-33 or anti-IL-33 antibody or transferred with type 2 innate lymphoid cells (ILC2s). The development and severity of fibrosis was evaluated based on lung histology, collagen levels, and lavage cytology. Cytokine and chemokine levels were quantified by using quantitative PCR, ELISA, and cytometry. RESULTS: IL-33 is constitutively expressed in lung epithelial cells but is induced in macrophages by bleomycin. Bleomycin enhanced the production of the mature but reduced full-length form of IL-33 in lung tissue. ST2 deficiency, anti-IL-33 antibody treatment, or alveolar macrophage depletion attenuated and exogenous IL-33 or adoptive transfer of ILC2s enhanced bleomycin-induced lung inflammation and fibrosis. These pathologic changes were accompanied, respectively, by reduced or increased IL-33, IL-13, TGF-ß1, and inflammatory chemokine production in the lung. Furthermore, IL-33 polarized M2 macrophages to produce IL-13 and TGF-ß1 and induced the expansion of ILC2s to produce IL-13 in vitro and in vivo. CONCLUSIONS: IL-33 is a novel profibrogenic cytokine that signals through ST2 to promote the initiation and progression of pulmonary fibrosis by recruiting and directing inflammatory cell function and enhancing profibrogenic cytokine production in an ST2- and macrophage-dependent manner.

Cutting edge: miR-223 and EBV miR-BART15 regulate the NLRP3 inflammasome and IL-1ß production.

Although microRNA (miRNA) regulation of TLR signaling is well established, this has not yet been observed for NLR proteins or the inflammasomes they form. We have now validated a highly conserved miR-223 target site in the NLRP3 3'-untranslated region. miR-223 expression decreases as monocytes differentiate into macrophages, whereas NLRP3 protein increases during this time. However, overexpression of miR-223 prevents accumulation of NLRP3 protein and inhibits IL-1ß production from the inflammasome. Virus inhibition of the inflammasome is an emerging theme, and we have also identified an EBV miRNA that can target the miR-223 binding site in the NLRP3 3'-untranslated region. Furthermore, this virus miRNA can be secreted from infected B cells via exosomes to inhibit the NLRP3 inflammasome in noninfected cells. Therefore, we have identified both the first endogenous miRNA that limits NLRP3 inflammatory capacity during myeloid cell development and also a viral miRNA that takes advantage of this, limiting inflammation for its own purposes.

MicroRNA-155 as a proinflammatory regulator in clinical and experimental arthritis.

MicroRNA (miRNA) species (miR) regulate mRNA translation and are implicated as mediators of disease pathology via coordinated regulation of molecular effector pathways. Unraveling miR disease-related activities will facilitate future therapeutic interventions. miR-155 recently has been identified with critical immune regulatory functions. Although detected in articular tissues, the functional role of miR-155 in inflammatory arthritis has not been defined. We report here that miR-155 is up-regulated in synovial membrane and synovial fluid (SF) macrophages from patients with rheumatoid arthritis (RA). The increased expression of miR-155 in SF CD14(+) cells was associated with lower expression of the miR-155 target, Src homology 2-containing inositol phosphatase-1 (SHIP-1), an inhibitor of inflammation. Similarly, SHIP-1 expression was decreased in CD68(+) cells in the synovial lining layer in RA patients as compared with osteoarthritis patients. Overexpression of miR-155 in PB CD14(+) cells led to down-regulation of SHIP-1 and an increase in the production of proinflammatory cytokines. Conversely, inhibition of miR-155 in RA synovial CD14(+) cells reduced TNF-α production. Finally, miR-155-deficient mice are resistant to collagen-induced arthritis, with profound suppression of antigen-specific Th17 cell and autoantibody responses and markedly reduced articular inflammation. Our data therefore identify a role of miR-155 in clinical and experimental arthritis and suggest that miR-155 may be an intriguing therapeutic target.

Liposomal AntagomiR-155-5p Restores Anti-Inflammatory Macrophages and Improves Arthritis in Preclinical Models of Rheumatoid Arthritis.

OBJECTIVE: We previously reported an increased expression of microRNA-155 (miR-155) in the blood monocytes of patients with rheumatoid arthritis (RA) that could be responsible for impaired monocyte polarization to anti-inflammatory M2-like macrophages. In this study, we employed two preclinical models of RA, collagen-induced arthritis and K/BxN serum transfer arthritis, to examine the therapeutic potential of antagomiR-155-5p entrapped within PEGylated (polyethylene glycol [PEG]) liposomes in resolution of arthritis and repolarization of monocytes towards the anti-inflammatory M2 phenotype. METHODS: AntagomiR-155-5p or antagomiR-control were encapsulated in PEG liposomes of 100 nm in size and -10 mV in zeta potential with high antagomiR loading efficiency (above 80%). Mice were injected intravenously with 1.5 nmol/100 µL PEG liposomes containing antagomiR-155-5p or control after the induction of arthritis. RESULTS: We demonstrated the biodistribution of fluorescently tagged PEG liposomes to inflamed joints one hour after the injection of fluorescently tagged PEG liposomes, as well as the liver's subsequent accumulation after 48 hours, indicative of hepatic clearance, in mice with arthritis. The injection of PEG liposomes containing antagomiR-155-5p decreased arthritis score and paw swelling compared with PEG liposomes containing antagomiR-control or the systemic delivery of free antagomiR-155-5p. Moreover, treatment with PEG liposomes containing antagomiR-155-5p led to the restoration of bone marrow monocyte defects in anti-inflammatory macrophage differentiation without any significant functional change in other immune cells, including splenic B and T cells. CONCLUSION: The injection of antagomiR-155-5p encapsulated in PEG liposomes allows the delivery of small RNA to monocytes and macrophages and reduces joint inflammation in murine models of RA, providing a promising strategy in human disease.

A single cell atlas of frozen shoulder capsule identifies features associated with inflammatory fibrosis resolution.

Frozen shoulder is a spontaneously self-resolving chronic inflammatory fibrotic human disease, which distinguishes the condition from most fibrotic diseases that are progressive and irreversible. Using single-cell analysis, we identify pro-inflammatory MERTKlowCD48+ macrophages and MERTK + LYVE1 + MRC1+ macrophages enriched for negative regulators of inflammation which co-exist in frozen shoulder capsule tissues. Micro-cultures of patient-derived cells identify integrin-mediated cell-matrix interactions between MERTK+ macrophages and pro-resolving DKK3+ and POSTN+ fibroblasts, suggesting that matrix remodelling plays a role in frozen shoulder resolution. Cross-tissue analysis reveals a shared gene expression cassette between shoulder capsule MERTK+ macrophages and a respective population enriched in synovial tissues of rheumatoid arthritis patients in disease remission, supporting the concept that MERTK+ macrophages mediate resolution of inflammation and fibrosis. Single-cell transcriptomic profiling and spatial analysis of human foetal shoulder tissues identify MERTK + LYVE1 + MRC1+ macrophages and DKK3+ and POSTN+ fibroblast populations analogous to those in frozen shoulder, suggesting that the template to resolve fibrosis is established during shoulder development. Crosstalk between MerTK+ macrophages and pro-resolving DKK3+ and POSTN+ fibroblasts could facilitate resolution of frozen shoulder, providing a basis for potential therapeutic resolution of persistent fibrotic diseases.

The liver X receptor pathway is highly upregulated in rheumatoid arthritis synovial macrophages and potentiates TLR-driven cytokine release.

OBJECTIVES: Macrophages are central to the inflammatory processes driving rheumatoid arthritis (RA) synovitis. The molecular pathways that are induced in synovial macrophages and thereby promote RA disease pathology remain poorly understood. METHODS: We used microarray to characterise the transcriptome of synovial fluid (SF) macrophages compared with matched peripheral blood monocytes from patients with RA (n=8). RESULTS: Using in silico pathway mapping, we found that pathways downstream of the cholesterol activated liver X receptors (LXRs) and those associated with Toll-like receptor (TLR) signalling were upregulated in SF macrophages. Macrophage differentiation and tumour necrosis factor α promoted the expression of LXRα. Furthermore, in functional studies we demonstrated that activation of LXRs significantly augmented TLR-driven cytokine and chemokine secretion. CONCLUSIONS: The LXR pathway is the most upregulated pathway in RA synovial macrophages and activation of LXRs by ligands present within SF augments TLR-driven cytokine secretion. Since the natural agonists of LXRs arise from cholesterol metabolism, this provides a novel mechanism that can promote RA synovitis.

Plasmacytoid dendritic cells play a key role in promoting atherosclerosis in apolipoprotein E-deficient mice.

OBJECTIVE: Clinical studies have identified that reduced numbers of circulating plasmacytoid dendritic cells (pDCs) act as a predictor of cardiovascular events in coronary artery disease and that pDCs are detectable in the shoulder region of human atherosclerotic plaques, where rupture is most likely to occur. Results from animal models are controversial, with pDCs seen to inhibit or promote lesion development depending on the experimental settings. Here, we investigated the role of pDCs in atherosclerosis in apolipoprotein E-deficient mice. METHODS AND RESULTS: We demonstrated that the aorta and spleen of both apolipoprotein E-deficient and C57BL/6 mice displayed similar numbers of pDCs, with similar activation status. In contrast, assessment of antigen uptake/presentation using the Eα/Y-Ae system revealed that aortic pDCs in apolipoprotein E-deficient(-) mice were capable of presenting in vivo systemically administered antigen. Continuous treatment of apolipoprotein E-deficient mice with anti-mouse plasmacytoid dendritic cell antigen 1 (mPDCA-1) antibody caused specific depletion of pDCs in the aorta and spleen and significantly reduced atherosclerosis formation in the aortic sinus (by 46%; P<0.001). Depletion of pDCs also reduced macrophages (by 34%; P<0.05) and increased collagen content (by 41%; P<0.05) in aortic plaques, implying a more stable plaque phenotype. Additionally, pDC depletion reduced splenic T-cell activation and inhibited interleukin-12, chemokine (C-X-C motif) ligand 1, monokine induced by interferon-γ, interferon γ-induced protein 10, and vascular endothelium growth factor serum levels. CONCLUSIONS: These results identify a critical role for pDCs in atherosclerosis and suggest a potential role for pDC targeting in the control of the pathology.

IL-33 shifts the balance from osteoclast to alternatively activated macrophage differentiation and protects from TNF-alpha-mediated bone loss.

IL-33 is a new member of the IL-1 family, which plays a crucial role in inflammatory response, enhancing the differentiation of dendritic cells and alternatively activated macrophages (AAM). Based on the evidence of IL-33 expression in bone, we hypothesized that IL-33 may shift the balance from osteoclast to AAM differentiation and protect from inflammatory bone loss. Using transgenic mice overexpressing human TNF, which develop spontaneous joint inflammation and cartilage destruction, we show that administration of IL-33 or an IL-33R (ST2L) agonistic Ab inhibited cartilage destruction, systemic bone loss, and osteoclast differentiation. Reconstitution of irradiated hTNFtg mice with ST2(-/-) bone marrow led to more bone loss compared with the chimeras with ST2(+/+) bone marrow, demonstrating an important endogenous role of the IL-33/ST2L pathway in bone turnover. The protective effect of IL-33 on bone was accompanied by a significant increase of antiosteoclastogenic cytokines (GM-CSF, IL-4, and IFN-γ) in the serum. In vitro IL-33 directly inhibits mouse and human M-CSF/receptor activator for NF-κB ligand-driven osteoclast differentiation. IL-33 acts directly on murine osteoclast precursors, shifting their differentiation toward CD206(+) AAMs via GM-CSF in an autocrine fashion. Thus, we show in this study that IL-33 is an important bone-protecting cytokine and may be of therapeutic benefit in treating bone resorption.

Three-dimensional, in-vitro approaches for modelling soft-tissue joint diseases.

Diseases affecting the soft tissues of the joint represent a considerable global health burden, causing pain and disability and increasing the likelihood of developing metabolic comorbidities. Current approaches to investigating the cellular basis of joint diseases, including osteoarthritis, rheumatoid arthritis, tendinopathy, and arthrofibrosis, involve well phenotyped human tissues, animal disease models, and in-vitro tissue culture models. Inherent challenges in preclinical drug discovery have driven the development of state-of-the-art, in-vitro human tissue models to rapidly advance therapeutic target discovery. The clinical potential of such models has been substantiated through successful recapitulation of the pathobiology of cancers, generating accurate predictions of patient responses to therapeutics and providing a basis for equivalent musculoskeletal models. In this Review, we discuss the requirement to develop physiologically relevant three-dimensional (3D) culture systems that could advance understanding of the cellular and molecular basis of diseases that affect the soft tissues of the joint. We discuss the practicalities and challenges associated with modelling the complex extracellular matrix of joint tissues-including cartilage, synovium, tendon, and ligament-highlighting the importance of considering the joint as a whole organ to encompass crosstalk across tissues and between diverse cell types. The design of bespoke in-vitro models for soft-tissue joint diseases has the potential to inform functional studies of the cellular and molecular mechanisms underlying disease onset, progression, and resolution. Use of these models could inform precision therapeutic targeting and advance the field towards personalised medicine for patients with common musculoskeletal diseases.