Production of sTLR2 involves a post-translation mechanism.

<p>(<b>A</b>) THP-1 cells were pretreated or not with cycloheximide (100 µg/mL) for 30 min and stimulated with Pam₃CSK₄ (1 µg/mL) for 5 h and the amount of sTLR2 was quantified in the cell culture supernatants by ELISA. Student t test, * p<0.05. (<b>B</b>) Detection of sTLR2 in 18 h culture supernatant (SN) of THP-1 cells by Western blotting using a N-terminal anti-TLR2 antibody. One representative of three experiments is shown. SN: cell culture supernatant; rhTLR2: recombinant human TLR2 protein. (<b>C</b>) Cell surface TLR2 levels evaluated by flow cytometry after stimulation with Pam₃CSK₄ (1, 10 or 20 µg/mL) for 2 h. ***, p<0.0001. (<b>D</b>) Cell surface TLR2 levels evaluated by flow cytometry after treatment with cycloheximide and then stimulation for 2 h with Pam₃CSK₄. MFI =  mean fluorescence intensity. *, p<0.05; **, p = 0.009.</p

Metalloproteinase activity regulates sTLR2 release and TLR2 surface content from THP-1 cells.

<p>(<b>A</b>) Cells were treated with APMA (10 µM), vehicle (DMSO) or left untreated (-) for 5 h and the concentration of sTLR2 in the supernatants was determined by ELISA. Student t-test, **, p = 0.003. (<b>B</b>) Cell surface TLR2 was examined on these cells after 2 h addition of APMA (10 µM), vehicle (DMSO) or left untreated (-) using anti-TLR2-FITC conjugated antibody and analysis by flow cytometry. ***, p<0.0001. MFI =  mean fluorescence intensity. A representative histogram is shown using cells stained with isotype control antibody (filled histogram), untreated cells (dot line) and APMA-treated cells (black line). (<b>C</b>) Cells were treated for 18 h with EDTA (2 mM), TAPI-1 (25 µM or 75 µM), GI254023X (5 µM) (GI) or left untreated (-) and the sTLR2 concentration in the cell supernatants was determined by ELISA. *, p<0.05; **, p = 0.003. Westernblot (10% SDS-PAGE reducing gel) shows sTLR2 release in cell supernatant of TAPI-1 or left untreated (-) treated THP-1 cells using N-terminal anti-TLR2 antibody. One representative of three experiments is shown. (<b>D</b>) Cells treated with TAPI-1 (25 µM or 75 µM), GI254023X (GI) (5 µM) or left untreated (-) were added 30 min prior to cell stimulation with Pam₃CSK₄ (1 µg/mL) for 18 h and the concentration of sTLR2 in cell supernatants was assayed by ELISA. Data represent the mean ± SE of three independent determinations. Student t test **, p = 0.001; ***, p = 0.0001.</p

TLR2 shedding is involved in sTLR2 generation in human peripheral monocytes.

<p>(<b>A</b>) Isolated human peripheral CD14⁺ cells were treated with APMA (10 µM), vehicle (DMSO) or left untreated (-) for 5 h. *, p<0.05. (<b>B</b>) Cells were incubated for 18 h with TAPI-1 (25 µM or 75 µM), GM6001 (10 µM) or left untreated (-).*, p<0.05.(<b>C</b>) Cells treated with TAPI-1 (25 µM or 75 µM), GI254023X (GI) (5 µM) or left untreated (-) were added 30 min prior to cell stimulation with Pam₃CSK₄ (1 µg/mL) for 18 h. Supernatants were harvested and soluble receptor was measured by ELISA. *, p<0.05; ** p<0.01. Data are express as percentage of maximal release ± SE of two independent determinations using four different healthy donors.</p

ADAM10 and ADAM17 are involved in TLR2 shedding.

<p>MEF cells were transfected with an expression plasmid encoding human TLR2 or with an empty vector; sTLR2 content in cell supernatants was analyzed by ELISA. sTLR2 content (pg/mL) was normalized to total TLR2 cell levels (ng/mg total protein). Data represent the mean ± SE of three independent determinations. *, p<0.05.</p

Additional file 1: Figure S1. of Soluble ST2 is a sensitive clinical marker of ulcerative colitis evolution

Distribution of serum IL33 according to response to therapy. Serum IL33 levels at baseline and 6 months in responders (R) and non-responders (NR) in relation to therapy without significant differences (p > 0.05). Differences were assessed using Wilcoxon signed rank test. (TIFF 25551 kb