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P. B-eatrice-demoures, . Laurent, . Val-erie-jolivel, I. Phd, ;. Douchet et al., PhD: CNRS-UMR 5164 Immuno ConcEpT, Bordeaux University, vol.2

J. E. Guimaraes,

M. D. Bertrand, J. Seneschal, . Md, M. Phd, M. D. Doutre et al., Demoures, Mr. Guimaraes, and Dr. Bertrand contributed equally to this work, Address correspondence to C ecile Contin-Bordes

F. Bordeaux, vasculopathy: the presence of digital ulcers, scleroderma renal crisis, and/or PAH on right-sided heart catheterization (mean 6 SEM 19, cecile.bordes@chu-bordeaux.fr. Submitted for publication October, vol.9, 2015.

, Results are representative of 5 healthy donors, 8 patients with lcSSc, and 10 patients with dcSSc. Original magnification 3 40. Bottom, Higher-magnification views of immunohistochemical staining for TSLP in the same subjects as in the top panel, showing the perivascular distribution of the TSLP-positive cells. Arrowheads indicate positive TSLP staining. Original magnification 3 100. B, Percentage of TSLP-positive cells among the total number of cells in the analyzed section of the dermis in healthy donors and patients with SSc. Two separate slides for each of 5 healthy donors and 18 SSc patients were analyzed. Symbols represent individual samples; horizontal lines and error bars show the mean 6 SEM. *** 5 P , 0.0001 by unpaired t-test. C, Correlation between percentage of TSLP-expressing dermal cells and the modified Rodnan skin thickness score (MRSS). D, Correlation between the percentage of TSLP-expressing dermal cells and disease duration, Overexpression of TSLP in the skin of SSc patients compared to healthy donors, and correlation of TSLP with fibrosis. A, Immunohistochemical staining to identify ex vivo expression of TSLP in human skin. Top

, TSLP-positive dermal cells was significantly increased in SSc compared to healthy donor skin

, A trend toward a higher percentage of TSLPpositive cells in dcSSc than in lcSSc was observed, Figure 2B)

, Both a-smooth muscle actin (a-SMA)-positive myofibroblasts and CD31-positive ECs stained positively for TSLP in SSc patients and negatively in healthy donors (Figure 3A). Notably, perivascular a-SMA-positive pericytes were negative for TSLP in both SSc patients and healthy donors (Figure 3A). To confirm that ECs are a potent TSLP producer in skin, we cultured HDMECs in vitro and in the presence or absence of profibrotic cytokines, Dermal TSLP expression correlated with skin TSLP, we performed immunofluorescence experiments

T. Il-1b and I. , Only IL-1b any effect, except for LPS, which slightly but reproducibly induced TSLP production (Figure 3B). Taken together, these results demonstrate that HDMECs are potent TSLP producers in SSc and that IL-1b is a main inducer of its secretion. Activated platelets promote the secretion of TSLP by ECs via an IL-1b-and serotonin-dependent mechanism. Among the IL-1b-secreting cells activated in SSc patients, platelets were of significant interest, vol.24, issue.10

P. Tslp, the addition of anti2IL-1b-blocking antibody induced an incomplete inhibition of TSLP production (for a 100:1 ratio of platelets to ECs, mean 6 SEM 173.1 6 0.4 pg/ml in stimulated cells versus 369.6 6 12.7 pg/ml in unstimulated cells; for a 200:1 ratio of platelets to ECs, mean 6 SEM 357.7 6 28.4 pg/ml in stimulated cells versus 648.5 6 13.8 pg/ml in unstimulated cells), SSc FIBROSIS 2789 and a 200:1 ratio of platelets to ECs

, HDMECs were purified from normal skin (obtained from patients undergoing plastic surgery) and stimulated for 24 hours with either cytokines potentially involved in SSc or the Toll-like receptor 4 agonist lipopolysaccharide (LPS) (B) or increasing amounts of IL-1b (0-50 ng/ml) in MV2 complete medium (M) (C). The supernatants were harvested, Figure 3. Endothelial cell (EC) production of TSLP ex vivo in SSc and in vitro. A

, Quantification of TSLP mRNA in IL-1b-activated HDMECs by real-time quantitative polymerase chain reaction. HDMECs were stimulated with 50 ng/ml of IL-1b

, to a lesser extent, serotonin. TSLP induction of fibrosis in humans via ECM production and collagenase inhibition. TSLP has been implicated in the fibrotic process in mouse models, but its role in humans remains unclear (16,17). Therefore, we assessed whether recombinant TSLP directly affects the activation and ECM production of dermal fibroblasts from healthy donors. Healthy donor dermal fibroblasts were stimulated with IL-1b, *** 5 P 5 0.0004 by Kruskal-IL-1b and

, TSLP) production by human dermal microvascular endothelial cells (HDMECs) in an interleukin-1b (IL-1b)-dependent manner. A, TSLP levels in HDMECs cultured in MV2 complete medium with increasing amounts of purified nonactivated platelets (10-200 platelets per HDMEC) (open bars) and in HDMECs stimulated with purified ADP-activated platelets (solid bars). A total of 70,000 HDMECs were cultured for 24 hours in a 24-well culture plate

. ***, 5 P , 0.0001 versus unstimulated cells (except where indicated otherwise), by Kruskal-Wallis nonparametric test followed by multiple comparison post hoc test

B. , TSLP levels in HDMECs stimulated with increasing ratios of ADP-activated platelets alone or with increasing ratios of ADP-activated platelets and an anti-IL-1b-blocking antibody

, Wallis nonparametric test followed by multiple comparison post hoc test. C, TSLP levels in HDMECs stimulated with increasing doses of serotonin (0-90 ng/ml). * 5 P , 0.05 versus unstimulated cells, by paired t-test. D, TSLP levels in HDMECs stimulated with ADP-activated platelets (at a 1:200 ratio of HDMECs to platelets) with or without anti-IL-1b-blocking antibody and with or without 5HT receptor-blocking antibodies (antibodies blocking 5HTR2a and 5HTR2b). The supernatants were harvested, and TSLP content was measured using a dedicated enzyme-linked immunosorbent assay, *** 5 P , 0.0001, without anti-IL-1b antibody versus with anti-IL-1b antibody

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