The extracellular heparan sulfatase SULF2 limits myeloid IFNβ signaling and Th17 responses in inflammatory arthritis

Heparan sulfate (HS) proteoglycans are important regulators of cellular responses to soluble mediators such as chemokines, cytokines and growth factors. We profiled changes in expression of genes encoding HS core proteins, biosynthesis enzymes and modifiers during macrophage polarisation, and found that the most highly regulated gene was Sulf2, an extracellular HS 6-O-sulfatase that was markedly downregulated in response to pro-inflammatory stimuli. We then generated Sulf2+/− bone marrow chimeric mice and examined inflammatory responses in antigen-induced arthritis, as a model of rheumatoid arthritis. Resolution of inflammation was impaired in myeloid Sulf2+/− chimeras, with elevated joint swelling and increased abundance of pro-arthritic Th17 cells in synovial tissue. Transcriptomic and in vitro analyses indicated that Sulf2 deficiency increased type I interferon signaling in bone marrow-derived macrophages, leading to elevated expression of the Th17-inducing cytokine IL6. This establishes that dynamic remodeling of HS by Sulf2 limits type I interferon signaling in macrophages, and so protects against Th17-driven pathology

Monocytes induce STAT3 activation in human mesenchymal stem cells to promote osteoblast formation

A major therapeutic challenge is how to replace bone once it is lost. Bone loss is a characteristic of chronic inflammatory and degenerative diseases such as rheumatoid arthritis and osteoporosis. Cells and cytokines of the immune system are known to regulate bone turnover by controlling the differentiation and activity of osteoclasts, the bone resorbing cells. However, less is known about the regulation of osteoblasts (OB), the bone forming cells. This study aimed to investigate whether immune cells also regulate OB differentiation. Using in vitro cell cultures of human bone marrow-derived mesenchymal stem cells (MSC), it was shown that monocytes/macrophages potently induced MSC differentiation into OBs. This was evident by increased alkaline phosphatase (ALP) after 7 days and the formation of mineralised bone nodules at 21 days. This monocyte-induced osteogenic effect was mediated by cell contact with MSCs leading to the production of soluble factor(s) by the monocytes. As a consequence of these interactions we observed a rapid activation of STAT3 in the MSCs. Gene profiling of STAT3 constitutively active (STAT3C) infected MSCs using Illumina whole human genome arrays showed that Runx2 and ALP were up-regulated whilst DKK1 was down-regulated in response to STAT3 signalling. STAT3C also led to the up-regulation of the oncostatin M (OSM) and LIF receptors. In the co-cultures, OSM that was produced by monocytes activated STAT3 in MSCs, and neutralising antibodies to OSM reduced ALP by 50%. These data indicate that OSM, in conjunction with other mediators, can drive MSC differentiation into OB. This study establishes a role for monocyte/macrophages as critical regulators of osteogenic differentiation via OSM production and the induction of STAT3 signalling in MSCs. Inducing the local activation of STAT3 in bone cells may be a valuable tool to increase bone formation in osteoporosis and arthritis, and in localised bone remodelling during fracture repair

Thymosin beta 4 - a novel regulator of low density lipoprotein receptor related protein 1 (LRP1) in vascular disease

Vascular stability and tone are maintained by contractile smooth muscle cells (VSMCs). Injury-induced growth factors stimulate a phenotypic switching of VSMCs, from their quiescent contractile state to a more active synthetic phenotype that proliferate and migrate. Chronic VSMC dedifferentiation leads to vascular thickening and stiffness, exacerbates inflammation and promotes atherosclerotic lesion development and susceptibility to abdominal aortic aneurysm (AAA). Inhibiting VSMC phenotypic transformation has thus been shown to attenuate progression of vascular disease. We previously identified Thymosin β4 (Tβ4) as a key regulator of embryonic VSMC differentiation. TMSB4X, encoding Tβ4, is the most abundant transcript in healthy and AAA aorta, therefore we hypothesised that Tβ4 may additionally function to maintain vasculature and protect against disease throughout postnatal life. We identified an interaction between Tβ4 and Low density lipoprotein receptor related protein 1 (LRP1), an endocytic regulator of PDGFR? signalling which controls VSMC proliferation. LRP1 variants have been identified by GWAS as major risk loci for AAA and coronary artery disease. Adult Tβ4-null mice displayed aortic VSMC and elastin defects, phenocopying LRP1 mutants and suggesting compromised vascular integrity. During development, Tβ4 functions in a paracrine manner, secreted from endothelial cells (ECs) to induce mesoderm to VSMC differentiation. To distinguish between cell-autonomous and paracrine roles for Tβ4, and simultaneously discern adult maintenance versus developmental requirements, we selectively induced deletion of Tβ4 from VSMCs or ECs at 3 weeks of age. Histological assessment of aortas at 12 weeks demonstrated that VSMC-specific Tβ4 knockdown recapitulated the global KO phenotype, revealing a postnatal requirement for Tβ4 to maintain healthy vasculature. In keeping with this, we confirmed predisposition of these mice to disease in models of atherosclerosis and AAA. Aneurysmal aorta and plaques of Tβ4KO were characterised by accelerated contractile-synthetic VSMC switching, elastin degradation and augmented PDGFR? signalling. In vitro, enhanced sensitivity to PDGF-BB, upon loss of Tβ4, coincided with increased cell surface recycling of LRP1-PDGFR? complexes and reduced lysosomal targeting, suggesting that dysregulated endocytosis underlies enhanced phenotypic switching and proliferation. Given the VSMC differentiation, anti-inflammatory and anti-apoptotic roles of Tβ4, we sought to determine the vasculoprotective potential of exogenous Tβ4. In the AAA model, Tβ4 treatment significantly reduced aortic dilatation and rupture, and preserved VSMC phenotype and elastin integrity, associated with normalisation of PDGFR? signalling. Our study identifies Tβ4 as a key regulator of LRP1 for maintaining vascular health, providing insight that may reveal useful therapeutic targets for modulation of VSMC phenotypic switching and disease progression

Interaction of monodispersed strontium containing bioactive glass nanoparticles with macrophages

The cellular response of murine primary macrophages to monodisperse strontium containing bioactive glass nanoparticles (SrBGNPs), with diameters of 90 ± 10 nm and a composition (mol%) of 88.8 SiO2–1.8CaO-9.4SrO (9.4% Sr-BGNPs) was investigated for the first time. Macrophage response is critical as applications of bioactive nanoparticles will involve the nanoparticles circulating in the blood stream and macrophages will be the first cells to encounter the particles, as part of inflammatory response mechanisms. Macrophage viability and total DNA measurements were not decreased by particle concentrations of up to 250 μg/mL. The Sr-BGNPs were actively internalised by the macrophages via formation of endosome/lysosome-like vesicles bordered by a membrane inside the cells. The Sr-BGNPs degraded inside the cells, with the Ca and Sr maintained inside the silica network. When RAW264.7 cells were incubated with Sr-BGNPs, the cells were polarised towards the pro-regenerative M2 population rather than the pro-inflammatory M1 population. Sr-BGNPs are potential biocompatible vehicles for therapeutic cation delivery for applications in bone regeneration

Pathways identified by ontological analysis to be over-represented in MSC STAT3C versus MSC AdGFP.

<p>Pathways identified by ontological analysis to be significantly overrepresented in the differentially regulated genes of the STAT3C MSCs versus AdGFP MSCs analysis.</p

Selected genes associated with OB differentiation and/or function.

<p>Differentially regulated transcripts in MSC STAT3C compared to MSC AdGFP associated with osteogenic differentiation and function.</p

Genes down-regulated by STATC.

<p>The table lists the 50 most highly significantly down-regulated transcripts in MSC STAT3C compared to MSC AdGFP (with p<0.01 cutoff using one way ANOVA with Benjamini-Hochberg multiple testing correction). Values shown are the medians from the five donors.</p

OSM mediates monocyte osteogenic effect through STAT3 signaling.

<p>The levels of pSTAT3 and STAT3 were assessed using WB in MSCs infected either with STAT3C (50 M.O.I.) or STAT3DN (100 M.O.I.) adenoviruses in osteogenic media. GFP adenoviral infection was used as a control (100 M.O.I.) (A). MSC ALP activity was measured 7 days after infection either with STAT3DN, STAT3C or AdGFP (B). MSC infected with the STAT3C or AdGFP viruses were also kept for 21 days in osteogenic media when bone nodule formation was assessed using Alizarin Red S staining (C). MSC infected with either the STAT3DN, STAT3C or AdGFP were kept with or without conditioned supernatant from separate monocyte:MSC co-cultures (10∶1 s/n) for 7 days when ALP activity was quantified (D). Blots are representative of three independent experiments performed. Graphs show means ± SEM of three independent experiments performed in triplicate. Phase contrast pictures (10X) are representative of three independent experiments performed. *p≤0.05, ***p≤0.001.</p

Monocytes potently induce osteogenic differentiation of MSCs.

<p>1×10⁴ MSCs were cultured either alone or with increasing numbers of PBMCs (1,2,5 or 10×10⁵ PBMCs), in the presence or absence of osteogenic stimuli. After 7 days, MSC ALP was assessed by staining (A) and after 21 days, bone nodule formation was visualised by Alizarin Red S staining (B). ALP activity (C) and Alizarin Red content (D) were also quantified. Populations of T cells, B cells or monocytes within PBMCs were either depleted (E) or enriched (F) prior to culture with MSC (at 10∶1 ratio) and ALP activity was quantified after 7 days. Co-cultures of MSCs with enriched monocyte populations were also kept for 21 days in osteogenic media when bone nodule formation was assessed using Alizarin Red S staining (G) and quantification of dye content (H). Monocytes were differentiated to macrophages (Mφ) in either M-CSF or GM-CSF for 5 days prior to co-culture with MSCs at a ratio of 10∶1. After 7 days of co-culture, ALP activity was quantified (I). Phase contrast pictures (10X) are representative of three independent experiments performed. Graphs show means ± SEM of three independent experiments performed in triplicates. *p≤0.05, **p≤0.01, ***p≤0.001.</p