Differentiation Potential of CD14+ Monocytes into Myofibroblasts in Patients with Systemic Sclerosis

BACKGROUND: Circulating monocytes are a highly plastic and functionally heterogeneic cell type with an activated phenotype in patients with systemic sclerosis (SSc). CD14(+) monocytes have the potential to differentiate into extra-cellular matrix (ECM) producing cells, possibly participating in fibrogenesis. AIM: To study the effect of GM-CSF, IL-4 and endothelin -1 (ET-1) alone or in combination on monocyte differentiation into myofibroblasts. METHODS: CD14(+) cells were isolated from peripheral blood from 14 SSc patients and healthy controls by positive selection and incubated with different combinations of GM-CSF, IL-4 and ET-1 for 14 days. Type-1 collagen and α-SMA were detected by Western blot, qPCR and confocal microscopy. HLA-DR, CD11c and CD14 expression was analysed by flow cytometry. A collagen gel contraction assay was performed for functional myofibroblast assessment. RESULTS: GM-CSF both induced collagen and α-SMA expression after 14 days. ET-1 further increased GM-CSF-induced collagen expression in a dose dependent manner up to 30-fold. IL-4/GM-CSF combination leads to a more DC-like phenotype of monocytes associated with reduced collagen and α-SMA expression compared to GM-CSF alone. Collagen and α-SMA expression was higher in monocytes from SSc patients and monocytes were more prone to obtain a spindle form. In contrast to controls, ET-1 and IL-4 alone were sufficient to induce α-SMA expression in monocytes from SSc patients. Despite the induction of α-SMA expression, monocyte-derived myofibroblasts only had a moderate capability of contraction in functional analyses. CONCLUSION: SSc monocytes display increased maturation towards myofibroblasts demonstrated by their phenotype and α-SMA expression when compared to monocytes from healthy controls, however only with minor functional contraction properties

Rapid Analyses of Proteomes and Interactomes Using an Integrated Solid-Phase Extraction–Liquid Chromatography–MS/MS System

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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

Einfluss der Estrogen Rezeptor α-Expression [Estrogen-Rezeptor alpha-Expression] auf die leptininduzierte Stat3-Aktivierung in Brustkrebszellen

Brustkrebs repräsentiert die häufigste Krebserkrankung bei Frauen. Der Estrogen Rezeptor &#945; (ER&#945;) ist hier als ein wichtiger prognostischer Marker für Mammakarzinome etabliert, der die Homonsensitivität des Tumors determiniert. Dieser impliziert den Einsatz von Anti-Estrogenen, die die wachstumsfördenden Funktionen von ER&#945; blockieren können. Übergewicht erhöht deutlich das Risiko einer Brustkrebserkrankung bei postmenopausalen Frauen. Übergewicht geht generell mit einem erhöhten Serumleptinspiegel einher. Das Zytokinhormon Leptin ist ein zentraler humoraler Faktor bei der Wahrnehmung und Steuerung der körpereigenen Energiereserven im Gehirn, das darüber hinaus auch pleiotrope Funktionen in der Angiogenese, Hämatopoese, Reproduktion und im Immunsystem ausübt. Leptin bindet an den Transmembranrezeptor Ob-RL (Obese-Rezeptor, lange Isoform) und aktiviert unter anderem den Januskinase (Jak)- „signal transducer and activator of transcription“ (Stat)-Signalweg, der mit neoplastischen Veränderungen assoziiert ist. Weil der kausale Zusammenhang zwischen Übergewicht und Krebserkrankungen gut belegt ist, während die molekularen Mechanismen jedoch noch nicht vollständig aufgeklärt sind, sollte daher in der vorliegenden Arbeit die mögliche Wechselwirkung des leptininduzierten Jak-Stat-Signalwegs und des ER&#945;´s untersucht werden. Ein Zusammenspiel der Ob-RL- und ER&#945;-abhängigen Signalwege wurde einerseits mit Luziferase-Reporter-Konstrukten untersucht, die unter der Kontrolle eines Stat3-responsiven Elements standen. Dabei konnten zelltyp-spezifsche Unterschiede in der leptinabhängigen Stat3-Aktivierung beobachtet werden. Die endogen ER&#945;-exprimierenden Brustkrebszellen MCF-7 wiesen eine wesentlich stärkere Stat3-Aktiverung und Phosphorylierung nach Leptinapplikation auf, als ER&#945;-negative HeLa Zervixkarzinomzellen oder die Brustkrebslinie MDA-MB-231. Dieser Effekt konnte durch die Hemmung der ER&#945;-Expression mittels siRNA in MCF-7 Zellen revertiert werden. Die ER&#945; Überexpression in HeLa Zellen resultierte in einer verstärkten Stat3-Transaktiverung nach Leptinbehandlung. Diese Zunahme in der Stat3-Aktivierung konnte nicht durch eine Kostimulation der Zellen mit einem ER&#945;-Agonisten oder Antagonisten beeinflusstwerden. Weiterhin konnte eine gesteigerte Zellviabilität nach Leptinstimulation in ER&#945;-positiven Zellen detektiert werden. Durch Koimmunpräzipitationsstudien konnte direkte Interaktion von ER&#945;, Jak2 und Stat3 nachgewiesen werden, was auf einen zytoplasmatisch lokalisierten Komplex hindeutet. Eine Mikroarray-Analyse von leptinstimulierten Zellen ergab eine differentielle Regulation von 133 Genen in Abhängigkeit ihres ER&#945;-Expressionstatus. Von den 133 Zielgene sind 42 in der Literatur bereits in Zusammenhang mit malignem Wachstum beschrieben und könnten somit die erhöhte Viabilität der ER&#945;-exprimierenden Zellen in Reaktion auf Leptin erklären. Die in dieser Arbeit gewonnen Erkenntnisse deuten auf eine entscheidende Rolle des ER&#945;´s in der Verstärkung der leptininduzierten Stat3-Aktiverung hin. Diese Daten zur Interaktion von ER&#945; mit einem wachstumsfördernden Signalweg können zur Entwicklung neuartiger Behandlungsmöglichkeiten in der Brustkrebstherapie von übergewichtigen Patientinnen beitragen

Phosphoproteomics Reveals Regulatory T Cell-Mediated DEF6 Dephosphorylation That Affects Cytokine Expression in Human Conventional T Cells

Regulatory T cells (Tregs) control key events of immune tolerance, primarily by suppression of effector T cells. We previously revealed that Tregs rapidly suppress T cell receptor (TCR)-induced calcium store depletion in conventional CD4+CD25− T cells (Tcons) independently of IP3 levels, consequently inhibiting NFAT signaling and effector cytokine expression. Here, we study Treg suppression mechanisms through unbiased phosphoproteomics of primary human Tcons upon TCR stimulation and Treg-mediated suppression, respectively. Tregs induced a state of overall decreased phosphorylation as opposed to TCR stimulation. We discovered novel phosphosites (T595_S597) in the DEF6 (SLAT) protein that were phosphorylated upon TCR stimulation and conversely dephosphorylated upon coculture with Tregs. Mutation of these DEF6 phosphosites abrogated interaction of DEF6 with the IP3 receptor and affected NFAT activation and cytokine transcription in primary Tcons. This novel mechanism and phosphoproteomics data resource may aid in modifying sensitivity of Tcons to Treg-mediated suppression in autoimmune disease or cancer

Characterization and usage of the EASY-spray technology as part of an online 2D SCX-RP ultra-high pressure system

Ultra-high pressure liquid chromatography (UHPLC) systems combined with state-of-the-art mass spectrometers have pushed the limit of deep proteome sequencing to new heights making it possible to identify thousands of proteins in a single LC-MS experiment within a few hours. The recently released EASY-spray technology allows one to implement nano-UHPLC with straightforwardness. In this work we initially characterized the EASY-spray containing a 50 cm column containing 7500 proteins. We report, here, that with this fast online SCX-RP UHPLC-MS/MS workflow, the proteome coverage can be substantially extended without significantly compromising the analysis time and sample usage

Characterization and usage of the EASY-spray technology as part of an online 2D SCX-RP ultra-high pressure system

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Patient monocytes need less stringent stimulation to upregulate α-SMA.

<p>A) Monocytes from healthy controls or SSc patients were cultured for 14 days with GM-CSF, IL-4 or ET-1. α-SMA levels were detected by Western blot analysis. GAPDH was used as loading control. B) Quantification of Western blots from 8 patients and 8 healthy controls showed stronger expression of α-SMA in SSc monocytes compared to healthy controls despite no significant difference could be detected. C) qPCR analysis of the procollagen expression in GM-CSF treated monocytes from healthy controls (HC) (n = 4) or SSc patients (n = 8). Data were normalized to expression of 18S. (p = 0.481). D) and E) Monocytes from HC (n = 4) and SSc patients (n = 3) were treated with various combinations of GM-CSF, IL-4 and ET-1. Procollagen expression was measured by qPCR. Data were normalized to expression of 18S. The concentration of ET-1 in all experiments was 100 ng/ml. * Statistically significant (P-value<0.05).</p

Healthy monocytes can differentiate into α-SMA and collagen expressing cells upon GM-CSF stimulation.

<p>A) CD14⁺ monocytes from healthy donors were cultured for 14 days with GM-CSF, IL-4 or ET-1. Western blot analysis showed expression of type-1 collagen and α-SMA under distinct conditions. Fibroblasts were used as a positive control, GAPDH was used as loading control. B) Monocytes were treated with GM-CSF and 0–500 ng/ml of ET-1 over 14 days. Western Blot analysis for type-1 collagen and α-SMA was conducted. GAPDH was used as loading control. C) and D) Quantification of the western blots from 3 independent experiments are shown. E) Expression of CD14, HLA-DR and CD11c was assessed by flow cytometry.</p