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

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

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

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

image_2_Glucocorticoids Impair Phagocytosis and Inflammatory Response Against Crohn’s Disease-Associated Adherent-Invasive Escherichia coli.PDF

<p>Crohn’s disease (CD) is a chronic inflammatory bowel disorder characterized by deregulated inflammation triggered by environmental factors. Notably, adherent-invasive Escherichia coli (AIEC), a bacterium with the ability to survive within macrophages is believed to be one of such factors. Glucocorticoids are the first line treatment for CD and to date, it is unknown how they affect bactericidal and inflammatory properties of macrophages against AIEC. The aim of this study was to evaluate the impact of glucocorticoid treatment on AIEC infected macrophages. First, THP-1 cell-derived macrophages were infected with a CD2-a AIEC strain, in the presence or absence of the glucocorticoid dexamethasone (Dex) and mRNA microarray analysis was performed. Differentially expressed mRNAs were confirmed by TaqMan-qPCR. In addition, an amikacin protection assay was used to evaluate the phagocytic and bactericidal activity of Dex-treated macrophages infected with E. coli strains (CD2-a, HM605, NRG857c, and HB101). Finally, cytokine secretion and the inflammatory phenotype of macrophages were evaluated by ELISA and flow cytometry, respectively. The microarray analysis showed that CD2-a, Dex, and CD2-a + Dex-treated macrophages have differential inflammatory gene profiles. Also, canonical pathway analysis revealed decreased phagocytosis signaling by Dex and anti-inflammatory polarization on CD2-a + Dex macrophages. Moreover, amikacin protection assay showed reduced phagocytosis upon Dex treatment and TaqMan-qPCR confirmed Dex inhibition of three phagocytosis-associated genes. All bacteria strains induced TNF-α, IL-6, IL-23, CD40, and CD80, which was inhibited by Dex. Thus, our data demonstrate that glucocorticoids impair phagocytosis and induce anti-inflammatory polarization after AIEC infection, possibly contributing to the survival of AIEC in infected CD patients.</p

image_5_Glucocorticoids Impair Phagocytosis and Inflammatory Response Against Crohn’s Disease-Associated Adherent-Invasive Escherichia coli.PDF

<p>Crohn’s disease (CD) is a chronic inflammatory bowel disorder characterized by deregulated inflammation triggered by environmental factors. Notably, adherent-invasive Escherichia coli (AIEC), a bacterium with the ability to survive within macrophages is believed to be one of such factors. Glucocorticoids are the first line treatment for CD and to date, it is unknown how they affect bactericidal and inflammatory properties of macrophages against AIEC. The aim of this study was to evaluate the impact of glucocorticoid treatment on AIEC infected macrophages. First, THP-1 cell-derived macrophages were infected with a CD2-a AIEC strain, in the presence or absence of the glucocorticoid dexamethasone (Dex) and mRNA microarray analysis was performed. Differentially expressed mRNAs were confirmed by TaqMan-qPCR. In addition, an amikacin protection assay was used to evaluate the phagocytic and bactericidal activity of Dex-treated macrophages infected with E. coli strains (CD2-a, HM605, NRG857c, and HB101). Finally, cytokine secretion and the inflammatory phenotype of macrophages were evaluated by ELISA and flow cytometry, respectively. The microarray analysis showed that CD2-a, Dex, and CD2-a + Dex-treated macrophages have differential inflammatory gene profiles. Also, canonical pathway analysis revealed decreased phagocytosis signaling by Dex and anti-inflammatory polarization on CD2-a + Dex macrophages. Moreover, amikacin protection assay showed reduced phagocytosis upon Dex treatment and TaqMan-qPCR confirmed Dex inhibition of three phagocytosis-associated genes. All bacteria strains induced TNF-α, IL-6, IL-23, CD40, and CD80, which was inhibited by Dex. Thus, our data demonstrate that glucocorticoids impair phagocytosis and induce anti-inflammatory polarization after AIEC infection, possibly contributing to the survival of AIEC in infected CD patients.</p

image_7_Glucocorticoids Impair Phagocytosis and Inflammatory Response Against Crohn’s Disease-Associated Adherent-Invasive Escherichia coli.PDF

<p>Crohn’s disease (CD) is a chronic inflammatory bowel disorder characterized by deregulated inflammation triggered by environmental factors. Notably, adherent-invasive Escherichia coli (AIEC), a bacterium with the ability to survive within macrophages is believed to be one of such factors. Glucocorticoids are the first line treatment for CD and to date, it is unknown how they affect bactericidal and inflammatory properties of macrophages against AIEC. The aim of this study was to evaluate the impact of glucocorticoid treatment on AIEC infected macrophages. First, THP-1 cell-derived macrophages were infected with a CD2-a AIEC strain, in the presence or absence of the glucocorticoid dexamethasone (Dex) and mRNA microarray analysis was performed. Differentially expressed mRNAs were confirmed by TaqMan-qPCR. In addition, an amikacin protection assay was used to evaluate the phagocytic and bactericidal activity of Dex-treated macrophages infected with E. coli strains (CD2-a, HM605, NRG857c, and HB101). Finally, cytokine secretion and the inflammatory phenotype of macrophages were evaluated by ELISA and flow cytometry, respectively. The microarray analysis showed that CD2-a, Dex, and CD2-a + Dex-treated macrophages have differential inflammatory gene profiles. Also, canonical pathway analysis revealed decreased phagocytosis signaling by Dex and anti-inflammatory polarization on CD2-a + Dex macrophages. Moreover, amikacin protection assay showed reduced phagocytosis upon Dex treatment and TaqMan-qPCR confirmed Dex inhibition of three phagocytosis-associated genes. All bacteria strains induced TNF-α, IL-6, IL-23, CD40, and CD80, which was inhibited by Dex. Thus, our data demonstrate that glucocorticoids impair phagocytosis and induce anti-inflammatory polarization after AIEC infection, possibly contributing to the survival of AIEC in infected CD patients.</p

image_6_Glucocorticoids Impair Phagocytosis and Inflammatory Response Against Crohn’s Disease-Associated Adherent-Invasive Escherichia coli.PDF

<p>Crohn’s disease (CD) is a chronic inflammatory bowel disorder characterized by deregulated inflammation triggered by environmental factors. Notably, adherent-invasive Escherichia coli (AIEC), a bacterium with the ability to survive within macrophages is believed to be one of such factors. Glucocorticoids are the first line treatment for CD and to date, it is unknown how they affect bactericidal and inflammatory properties of macrophages against AIEC. The aim of this study was to evaluate the impact of glucocorticoid treatment on AIEC infected macrophages. First, THP-1 cell-derived macrophages were infected with a CD2-a AIEC strain, in the presence or absence of the glucocorticoid dexamethasone (Dex) and mRNA microarray analysis was performed. Differentially expressed mRNAs were confirmed by TaqMan-qPCR. In addition, an amikacin protection assay was used to evaluate the phagocytic and bactericidal activity of Dex-treated macrophages infected with E. coli strains (CD2-a, HM605, NRG857c, and HB101). Finally, cytokine secretion and the inflammatory phenotype of macrophages were evaluated by ELISA and flow cytometry, respectively. The microarray analysis showed that CD2-a, Dex, and CD2-a + Dex-treated macrophages have differential inflammatory gene profiles. Also, canonical pathway analysis revealed decreased phagocytosis signaling by Dex and anti-inflammatory polarization on CD2-a + Dex macrophages. Moreover, amikacin protection assay showed reduced phagocytosis upon Dex treatment and TaqMan-qPCR confirmed Dex inhibition of three phagocytosis-associated genes. All bacteria strains induced TNF-α, IL-6, IL-23, CD40, and CD80, which was inhibited by Dex. Thus, our data demonstrate that glucocorticoids impair phagocytosis and induce anti-inflammatory polarization after AIEC infection, possibly contributing to the survival of AIEC in infected CD patients.</p

table_2_Glucocorticoids Impair Phagocytosis and Inflammatory Response Against Crohn’s Disease-Associated Adherent-Invasive Escherichia coli.PDF

<p>Crohn’s disease (CD) is a chronic inflammatory bowel disorder characterized by deregulated inflammation triggered by environmental factors. Notably, adherent-invasive Escherichia coli (AIEC), a bacterium with the ability to survive within macrophages is believed to be one of such factors. Glucocorticoids are the first line treatment for CD and to date, it is unknown how they affect bactericidal and inflammatory properties of macrophages against AIEC. The aim of this study was to evaluate the impact of glucocorticoid treatment on AIEC infected macrophages. First, THP-1 cell-derived macrophages were infected with a CD2-a AIEC strain, in the presence or absence of the glucocorticoid dexamethasone (Dex) and mRNA microarray analysis was performed. Differentially expressed mRNAs were confirmed by TaqMan-qPCR. In addition, an amikacin protection assay was used to evaluate the phagocytic and bactericidal activity of Dex-treated macrophages infected with E. coli strains (CD2-a, HM605, NRG857c, and HB101). Finally, cytokine secretion and the inflammatory phenotype of macrophages were evaluated by ELISA and flow cytometry, respectively. The microarray analysis showed that CD2-a, Dex, and CD2-a + Dex-treated macrophages have differential inflammatory gene profiles. Also, canonical pathway analysis revealed decreased phagocytosis signaling by Dex and anti-inflammatory polarization on CD2-a + Dex macrophages. Moreover, amikacin protection assay showed reduced phagocytosis upon Dex treatment and TaqMan-qPCR confirmed Dex inhibition of three phagocytosis-associated genes. All bacteria strains induced TNF-α, IL-6, IL-23, CD40, and CD80, which was inhibited by Dex. Thus, our data demonstrate that glucocorticoids impair phagocytosis and induce anti-inflammatory polarization after AIEC infection, possibly contributing to the survival of AIEC in infected CD patients.</p