Autoantibodies Which Bind to and Activate Keratinocytes in Systemic Sclerosis

Systemic sclerosis (SSc) is a multisystem connective tissue disease characterised by pathological processes involving autoimmunity, vasculopathy and resultant extensive skin and organ fibrosis. Recent studies have demonstrated activation and aberrant wound healing responses in the epithelial layer of the skin in this disease, implicating the epithelial keratinocytes as a source of pro-fibrotic and inflammatory mediators. In this paper, we investigated the role of Immunoglobulin G (IgG) autoantibodies directed against epithelial cells, as potential initiators and propagators of pathological keratocyte activation and the ensuing SSc fibrotic cascade. A keratinocyte cell-based ELISA is used to evaluate the binding of SSc IgG. SSc skin biopsies were stained by immunofluorescence for the presence of IgG in the keratinocyte layer. Moreover, IgG purified from SSc sera was evaluated for the potential to activate keratinocytes in tissue culture and to induce TLR2 and 3 signalling in reporter cell lines. We demonstrate enhanced binding of SSc IgG to keratinocytes and the activation of these cells leading to the release of IL-1α, representing a potential initiating pathway in this disease

Pathogenic Activation of Mesenchymal Stem Cells Is Induced by the Disease Microenvironment in Systemic Sclerosis

Objective: In systemic sclerosis (SSc), a persistent tissue repair process leads to progressive fibrosis of the skin and internal organs. The role of mesenchymal stem cells (MSCs), which characteristically initiate and regulate tissue repair, has not been fully evaluated. We undertook this study to investigate whether dividing metakaryotic MSCs are present in SSc skin and to examine whether exposure to the disease microenvironment activates MSCs and leads to transdifferentiation. Methods: Skin biopsy material from patients with recent-onset diffuse SSc was examined by collagenase spread of 1-mm–thick surface-parallel sections, in order to identify dividing metakaryotic stem cells in each tissue plane. Adipose-derived MSCs from healthy controls were treated with dermal blister fluid (BF) from patients with diffuse SSc and profiled by next-generation sequencing, or they were evaluated for phenotypic changes relevant to SSc. Differential responses of dermal fibroblasts were studied in parallel. Results: MSC-like cells undergoing active metakaryotic division were identified in SSc sections (but not control sections) most prominently in the deep dermis and adjacent to damaged microvessels, in both clinically involved and uninvolved skin. Furthermore, exposure to SSc BF caused selective MSC activation, inducing a myofibroblast signature, while reducing signatures of vascular repair and adipogenesis and enhancing migration and contractility. Microenvironmental factors implicated in inducing transdifferentiation included the profibrotic transforming growth factor β, the presence of lactate, and mechanosensing, while the microenvironment Th2 cytokine, interleukin-31, enhanced osteogenic commitment (calcinosis). Conclusion: Dividing MSC-like cells are present in the SSc disease microenvironment where multiple factors, likely acting in concert, promote transdifferentiation and lead to a complex and resistant disease state

Scleroderma and related disorders: 223. Long Term Outcome in a Contemporary Systemic Sclerosis Cohort

Background: We have previously compared outcome in two groups of systemic sclerosis (SSc) patients with disease onset a decade apart and we reported data on 5 year survival and cumulative incidence of organ disease in a contemporary SSc cohort. The present study examines longer term outcome in an additional cohort of SSc followed for 10 years. Methods: We have examined patients with disease onset between years 1995 and 1999 allowing for at least 10 years of follow-up in a group that has characteristics representative for the patients we see in contemporary clinical practice. Results: Of the 398 patients included in the study, 252 (63.3%) had limited cutaneous (lc) SSc and 146 (36.7%) had diffuse cutaneous (dc) SSc. The proportion of male patients was higher among the dcSSc group (17.1% v 9.9%, p = 0.037) while the mean age of onset was significantly higher among lcSSc patients (50 ± 13 v 46 ± 13 years ± SD, p = 0.003). During a 10 year follow-up from disease onset, 45% of the dcSSc and 21% of the lcSSc subjects developed clinically significant pulmonary fibrosis, p < 0.001. Among them approximately half reached the endpoint within the first 3 years (23% of dcSSc and 10% of lcSSc) and over three quarters within the first 5 years (34% and 16% respectively). There was a similar incidence of pulmonary hypertension (PH) in the two subsets with a steady rate of increase over time. At 10 years 13% of dcSSc and 15% of lcSSc subjects had developed PH (p=0.558), with the earliest cases observed within the first 2 years of disease. Comparison between subjects who developed PH in the first and second 5 years from disease onset demonstrated no difference in demographic or clinical characteristics, but 5-year survival from PH onset was better among those who developed this complication later in their disease (49% v 24%), with a strong trend towards statistical significance (p = 0.058). Incidence of SSc renal crisis (SRC) was significantly higher among the dcSSc patients (12% v 4% in lcSSc, p = 0.002). As previously observed, the rate of development of SRC was highest in the first 3 years of disease- 10% in dcSSc and 3% in lcSSc. All incidences of clinically important cardiac disease developed in the first 5 years from disease onset (7% in dcSSc v 1% in lcSSc, p < 0.001) and remained unchanged at 10 years. As expected, 10-year survival among lcSSc subjects was significantly higher (81%) compared to that of dcSSc patients (70%, p = 0.006). Interestingly, although over the first 5 years the death rate was much higher in the dcSSc cohort (16% v 6% in lcSSc), over the following years it became very similar for both subsets (14% and 13% between years 5 and 10, and 18% and 17% between years 10 and 15 for dcSSc and lcSSc respectively). Conclusions: Even though dcSSc patients have higher incidence for most organ complications compared to lcSSc subjects, the worse survival among them is mainly due to higher early mortality rate. Mortality rate after first 5 years of disease becomes comparable in the two disease subsets. Disclosure statement: The authors have declared no conflicts of interes

Stiff surfaces and TGFβ induce MRTF-A nuclear accumulation.

<p>SSc and control dermal fibroblast lines (both N = 3) were cultured on 6 well plates with collagen type I coated soft substrates (5 kPa Softwell), or hard substrate (50 kPa). <b>B. Induction of collagen transcription on hard surfaces requires MRTF-A.</b> Mouse fibroblasts from wild type (WT) and loss-of-function (KO) mice with collagen promoter driving GFP were cultured on fibronectin coated soft and hard surfaces. Pictures taken 24 hours after plating. Fluorescence was quantified using ImageJ. The numbers of cells in each image was counted (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0126015#pone.0126015.s004" target="_blank">S4 Fig</a>) to determine the luminance per cell. <b>C.&D.</b> Normal control fibroblasts and SSc fibroblasts were cultured without serum for 16 hours then treated with TGFβ (4 ng/ml) for 0, 8 and 24 hours or treated with either saline or TGFβ (4 ng/ml) with or without CCG1423 (10μM) or NSC2376 (50 μM). Proteins (20 μg) from cytoplasm and nuclei were extracted using NE-PER Nuclear Protein Extraction Kit and separated on 4–12% gradient gel and visualized using MRTF-A antibody.</p

Nuclear MRTF-A expression is more prominent in SSc skin then healthy control.

<p><b>A.</b> Representative pictures of healthy control and SSc sections of biopsy. Histological samples of human skin were stained with MRTF-A antibody (1:2000). Higher magnification of epithelium and small vessels (20X) in papillary dermis. Brown arrows = MRTF-A nuclei, Blue arrow = hematoxylin stained nuclei without MRTF-A. Graphical representations of % nuclei in epidermis, vasculature, and interstitial cells in papillary dermis. Histological samples of 5 healthy control and 9 scleroderma human skin were evaluated for nuclear staining. B. Total cells with MRTF-A nuclear localization in the epidermis, vasculature, and interstitial papillary dermal layers were counted and compared with the total amount of nuclei in the epidermal/papillary dermal layer. White bars = healthy controls, Black bars = SSc (* = p<0.01 using nonparametric Mann-Whitney U, two-tailed).</p

Increased expression of MRTF-A in the SSc epidermis and at the epidermal dermal junction in established SSc correlates with increased intracellular procollagen, and SMA.

<p>The expression of MRTF-A, procollagen type I, and SMA was detected by immunohistochemical staining in the epidermis and papillary dermis of early and established SSc patients and controls healthy control patients. Arrows point to staining in vascular cells (V), epidermis (e), and fibroblast (F).</p

Fibril diameters in the KO reticular dermis are smaller and more uniform than WT fibrils.

<p><b>A.</b> Representative micrographs of skin in deep dermal areas near subcutaneous fat. (Scale bar = 0.5 μm) <b>B.</b> Histogram of the frequency of collagen fibrils with a given diameter range from WT (white bars) and KO (black bars). At least 200 fibrils from 3 WT and 3 KO animals at 5 months of age were used for analysis. P<0.05.</p

Mouse KO skin and lung are less stiff than WT skin and lung.

<p>A 3x1x1 mm strip of dorsal skin or lung tissue was placed longitudinally into a computer-controlled dual-mode lever arm force transducer system and stretched intermittently. <b>A.</b> Young’s module plotted at each strain. The Young’s module is the slope of the stress-strain curve. <b>B.</b> Dynamic stretch storage modulus describes the ability of the material to store elastic energy during the loading phase of a cyclic stretch <b>C</b>. Dynamic stretch loss modulus—The amount of energy lost (usually as heat) during a cycle. Vertical brackets denote the overall group differences using 2-way repeated measure ANOVA (*: <i>p</i><0.05, **: <i>p</i><0.01 and ***: <i>p</i><0.001) whereas horizontal brackets show Tukey’s post hoc differences between WT and KO at the same strain (Panel A) or frequency (Panels B and C). For the Young’s and loss moduli of lung tissue, there are also significant interactions between strain and group (panel A) as well as frequency and group (panel C, p<0.05). For the skin, there is a significant interaction between frequency and group only for the loss modulus (p<0.05).</p

Enhanced type I collagen and CCN2 expression as well as collagen gel contraction in scleroderma fibroblasts is dependent on MRTF-A pathway.

<p><b>A. The MRTF-A inhibitor, CCG-1423, blocks collagen and CCN2 synthesis.</b> Healthy control fibroblasts (Control) and scleroderma fibroblasts (SSc) from 3 independent isolates were cultured with or without the MRTF-A inhibitor CCG-1423 (10 μM). Basal CCN2 and type I collagen was increased in scleroderma cells and inhibited by CCG-1423. <b>B. Knockdown of MRTF-A by siRNA blocks collagen and CCN2 synthesis in SSc fibroblasts.</b> Control and SSc fibroblasts were treated with MRTF-A siRNA, (siMRTFA), a non-target siRNA (siNT), or vehicle (Cont). <b>C. Loss or inhibition of MRTF-A blocks contraction of collagen floating gels.</b> Knockdown (siMRTFA) and inhibition (CCG-1423) of MRTF-A partially blocks collagen floating gel contraction by SSc fibroblasts. * = p<0.05.</p