A Tam

Connective tissue growth factor causes EMT-like cell fate changes in vivo and in vitro

Summary Connective tissue growth factor (CTGF) plays an important role in the pathogenesis of chronic fibrotic diseases. However, the mechanism by which paracrine effects of CTGF control the cell fate of neighboring epithelial cells is not known. In this study, we investigated the paracrine effects of CTGF overexpressed in fibroblasts of Col1a2-CTGF transgenic mice on epithelial cells of skin and lung. The skin and lungs of Col1a2-CTGF transgenic mice were examined for phenotypic markers of epithelial activation and differentiation and stimulation of signal transduction pathways. In addition to an expansion of the dermal compartment in Col1a2-CTGF transgenic mice, the epidermis was characterized by focal hyperplasia, and basal cells stained positive for &agr;SMA, Snail, S100A4 and Sox9, indicating that these cells had undergone a change in their genetic program. Activation of phosphorylated p38 and phosphorylated Erk1/2 was observed in the granular and cornified layers of the skin. Lung fibrosis was associated with a marked increase in cells co-expressing epithelial and mesenchymal markers in the lesional and unaffected lung tissue of Col1a2-CTGF mice. In epithelial cells treated with TGF&bgr;, CTGF-specific siRNA-mediated knockdown suppressed Snail, Sox9, S100A4 protein levels and restored E-cadherin levels. Both adenoviral expression of CTGF in epithelial cells and treatment with recombinant CTGF induced EMT-like morphological changes and expression of &agr;-SMA. Our in vivo and in vitro data supports the notion that CTGF expression in mesenchymal cells in the skin and lungs can cause changes in the differentiation program of adjacent epithelial cells. We speculate that these changes might contribute to fibrogenesis.

FRI0406 Increased frequency of circulating cd163+ non-classical monocytes in scleroderma and enhanced dual polarisation towards m1 and m2-like phenotypes in monocyte-derived macrophages

Background Scleroderma (SSc) is an autoimmune connective tissue disease involving complex interactions between various cell types leading to organ-based tissue fibrosis. Emergence of the monocytes (Mo)/macrophages (Mφ) lineage(s) as key contributors to inflammation, vascular dysfunction and scarring in scleroderma1,2 have led to increased scrutiny of their phenotype and function. Objectives To determine the circulating Mo subpopulations and phenotypes of Mφ in SSc. Methods PBMC were collected from healthy (HC) and SSc donors, and analysed by flow cytometry using Mo phenotypic antibodies or purified and cultured in vitro. For flow cytometry immunophenotyping, Mo were gated on CD3-CD19-CD56-HLA-DR+populations, and subsets defined by CD14, CD16, CD163 and CD206 expression. For Mφ cultures, Mo were negatively selected from PBMCs, cultured for 7 days, and treated with IFN-γ(5 ng/ml) or IL-4(20 ng/ml) for 24 hours. Cytokine levels in the conditioned media were evaluated by MSD analyses and normalised to total protein levels. Results The frequency of circulating CD163+ non-classical Mo (CD14loCD16hi) was 2-fold higher in SSc patients than in HC (unpaired t-test, p=0.026). No difference was found in the frequency of CD206+ monocyte subsets between HC and SSc. In vitro, unstimulated SSc Mφ (M0) secreted higher levels of classically-activated pro-inflammatory (M1) and alternatively-activated pro-regenerative (M2) cytokines. Compared to HC cells, SSc Mφ were more readily polarised towards an M1 phenotype or an M2 phenotype, when cultured in the presence of IFN-γ or IL-4, respectively. Th17 markers and MMPs were significantly increased in SSc Mφ (table 2).Abstract FRI0406 – Table 1 Demographics. Mo (flow cytometry) Mφ supernatant (cytokine assay) n HC n=9 SSc n=10 HC n=13 SSc n=27 Age (years) 56.7±14.3 50.7±5.7 60.6±16.7 52.1±13.0 Female : Male 7:2 8:2 6:7 26:5 SSc subtype - dcSSc(10 - dcSSc(27 Disease duration - ≤5 years(10 - ≤5 years(,15>5 year(12Abstract FRI0406 – Table 2 Cytokines significantly increased in SSc vs control. Unpaired t-tests, *p<0.05, **P<0.01. Conclusions Studies exploring Mo have revealed distinct populations with selective biological functions. Our observation of an increased number of CD163+ non-classical Mo in SSc suggests that this subpopulation may play a key role in inflammatory-driven fibrosis and act as an important source of pro-fibrotic cytokines. This data is consistent with previous reports of elevated serum levels of CD163 and increased CD163 secretion by SSc PBMCs3. SSc Mφ showed a pronounced and enhanced dual M1 and M2 polarisation basally compared to HC, indicating cells were ‘primed’ to undergo phenotypic polarisation. Our studies support the notion that Mφ cytokine secretion generates a pro-fibrotic milieu in scleroderma tissues, playing a prominent role in dysregulated tissue repair in fibrosis. References [1] Chia JJ, Lu TT. Curr Opin Rheumatol2015Nov;27(6):530–536. [2] Christmann RB, Lafyatis R. Arthritis Res Ther. 2010;12(5):146. [3] Hassan WASE, et al. Eur J Rheumatol2016Sep;3(3):95–100. Disclosure of Interest None declared

OP0213 Macrophages from a scleroderma subgroup with higher skin scores express activation markers and induce fibroblasts in co-culture

Background Scleroderma (SSc) is characterized by pathological fibrosis. The mechanisms by which fibrosis occurs in SSc are not fully understood. Alternatively activated M2-like macrophages are associated with fibrosis and have been found to have an important role in pathological fibrosis in humans. Therefore, there is interest in elucidating their role in SSc. M2 macrophages express mannose receptor CD206 and are known to secrete a number of soluble factors to establish a pro-fibrotic milieu when present in damaged tissues. Furthermore, we have shown adenosine tri-phosphate (ATP) concentration is increased in the skin of patients with SSc. Within the extra-cellular environment, ATP is a Damage-Associated Molecular Pattern (DAMP), binding the P2X class of purinergic receptors. Such mechanisms may contribute to SSc pathology. Objectives In this study, we explore the relationship of macrophage CD206 and P2X7 expression to Rodnan Skin Score. The role of these cells in establishing fibrosis was also examined in vitro. Methods 17 SSc patients and 9 controls were consented and their skin score recorded. Macrophages were derived from peripheral blood mononuclear cells (PBMCs) and identified through CD14 expression by FACS. CD206 and P2X7 co-expression was quantified. CD206 immunofluorescence of skin biopsies was also performed. Macrophages were co-cultured with 8x104 and 2x104 fibroblasts in a collagen matrix and within a monolayer respectively. Collagen gel contraction was quantified as a measure of fibrotic activity. CTGF and Collagen mRNA expression from gel matrices and cellular monolayers was quantified by qPCR. Results CD206 and P2X7 expression is higher on SSc PBMC-derived macrophages (mean fluorescence 776.1 SD=409.1, 724.4 SD=455.3) compared to healthy controls (mean fluorescence 632.2 SD=73.7, 472.9 SD=25.4). There is significant correlation of CD206 expression to P2X7 expression (p<0.001, r2=0.76) and CD206 expression is significantly correlated to Rodnan skin score (p<0.05, r2=0.26). P2X7 expression is positively correlated to skin score. Double positive P2X7 and CD206 cells were seen in a subgroup with higher skin scores. Healthy fibroblasts co-cultured with scleroderma macrophages showed increased collagen mRNA by qPCR compared to co-culture with healthy macrophages (p<0.01). CTGF mRNA was positively correlated with macrophage P2X7 (r2=0.23) and CD206 (r2=0.81) expression. Preliminary work suggests contraction of collagen discs in fibroblast and macrophage co-culture is increased with SSc macrophages compared to healthy controls. Conclusions Data indicates a correlation between disease severity and CD206 expression by macrophages. Upregulation of CTGF and collagen expression in fibroblasts co-cultured with macrophages expressing high CD206 suggests a role for these cells in pathogenic fibrosis. The co-expression of high levels of P2X7 with CD206 also indicates a possible role for the purinergic pathway in SSc fibrosis. Future work will examine the mechanism of macrophage-fibroblast cross-talk and investigate the effect of inhibitors of CD206. Acknowledgements Rosetrees Trust Arthritis Research UK Scleroderma and Raynauds UK Royal Free Charity Disclosure of Interest None declared

SAT0193 Macrophages Responding To Mechanical Stress in Scleroderma

Background Inflammation, vasculopathy and tissue fibrosis are key features of scleroderma (SSc). While healthy forearm skin which has a Youngs modulus of 4–12kPa, SSc fibrotic skin measures 50–80kPa with its increased mechanical stiffness1. We have shown that mechano-sensing properties of fibroblasts in SSc are mediated by myocardin-related transcription factor A (MRTF-A)3. Monocytes/macrophages are likely to be involved in SSc pathogenesis but the effect of mechanical stress on these cells remain to be elucidated3,4. Objectives To investigate if a mechanically-stressed microenvironment like that in SSc tissue, promotes macrophages towards a pathogenic phenotype, and whether MRTF-A is involved in this process. Methods Control and scleroderma skin sections were immunostained with anti-CD68 and anti-MRTF-A antibodies (n=3). Human PBMC-derived macrophages were cultured in RPMI/M-CSF on 4kPa and 50kPa collagen-fibronectin-coated plates to mimic “soft”/healthy and “stiff”/fibrotic skin, and activated with LPS (10ng/ml) or IL-10 (10ng/ml) for macrophages designated M(LPS) and M(IL-10) (n=4). MRTF-A expression was assessed by qPCR and conditioned media profiled by Luminex array for inflammatory cytokines. Mouse bone marrow-derived macrophages (BMDMs) of wildtype and MRTF-A-null mice were maintained in RPMI/M-CSF on soft and stiff substrates. The data were analysed by two-way ANOVA and Tukey test (p<0.05, CI 95%). Results We observed a greater number of CD68+ macrophages in diffuse SSc skin compared to control skin, mainly around perivascular regions. These macrophages also expressed MRTF-A. Human macrophages expressed MRTF-A mRNA and showed differential cytokine expression when cultured on soft and stiff substrates. M(LPS) on soft matrix expressed IFN-γ, which was undetectable with M(LPS) on stiff substrate (mean difference 0.2075±0.1576pg/ml, p<0.01). LPS- and IL-10 activation on soft substrate increased MCP-3 expression compared to controls (mean difference 68.51±49.19pg/ml, p<0.01, 92.88±49.22pg/ml, p<0.0001, respectively). M(LPS) on stiff compared to soft substrate showed lower MCP-3 expression (mean difference 57.01±49.22pg/ml, p<0.05). M(IL-10) on soft substrate showed higher MCP-1 expression compared to controls (mean difference 2448±2232pg/ml, p<0.05). M(IL-10) on stiff substrate decreased expression of MCP-1 (mean difference 2590±2233pg/ml, p<0.05) and increased fractalkine levels compared to soft substrate (mean difference 51.22±36.28pg/ml, p<0.01). Wildtype BMDMs on stiff compared to soft substrate displayed a more elongated morphology. MRTF-A-null BMDMs remained rounded on stiff substrate. Conclusions MRTF-A is a mechanical stress-responsive factor which co-activates transcription of cytoskeletal and extracellular matrix-modifying genes. MRTF-A may couple mechanical stress to macrophage activation in SSc, where stiff matrix promotes macrophage secretion of cytokines and growth factors that exacerbate fibrosis. References Sacksen et al., Arthritis Rheum 2013. Shiwen et al., PLoS One 2015. Stifano et al., Curr Rheumatol Rep 2015. Chia et al., Curr Opin Rheumatol 2015. Disclosure of Interest None declared