Additional file 1: Figure S1. of Cigarette smoke differentially modulates dendritic cell maturation and function in time

BMDCs (control, CSE co-cultured, or LPS co-cultured) were mixed with CFSE-labeled DO11.10 T cells (CD4 KJ1-26) in ratio of 1:10 and 1:20 in the presence of OVA peptide for 72 h. After 72 h the CSFE dilution profile were analyzed by flow cytometry. (DOCX 196 kb

Image_3.TIF

<p>Background and Aim: Endothelial activation is characterized by excessive production of cytokines and chemokines as well as adhesion molecules expression which is involved in the development of atherosclerosis. The aim of our study is to investigate the effects of short chain fatty acids (SCFA) on lipopolysaccharide (LPS) or tumor necrosis factor alpha (TNFα)-induced endothelial activation.</p><p>Methods and Results: Human umbilical vein endothelial cells (HUVEC) were pre-treated with acetate (10 mM), butyrate (0.1 mM) or propionate (0.3 mM) for 1, 16, or 24 h and then stimulated with LPS (1 or 10 μg/ml) or TNFα (100 pg/ml or 1 ng/ml) for 6, 12, or 24 h. Cytokines in the supernatant were measured by ELISA. HUVEC were pre-treated with acetate (10 mM), butyrate (5 mM) or propionate (10 mM) for 24 h and then stimulated with LPS (1 μg/ml) or TNFα (1 ng/ml) for 8 h. The expression of the adhesion molecules intracellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) was detected by flow cytometry. The human blood mononuclear cell adhesive level to HUVEC monolayer was measured. LPS and TNFα induced a significant increase in the release of interleukin-6 (IL-6) and IL-8. Acetate, butyrate and propionate reduced IL-6 and IL-8 levels and the magnitude was dependent on the incubation times. LPS or TNFα increased ICAM-1 and VCAM-1 expression. Pre-incubation with acetate had no effect. In contrast, butyrate and propionate decreased VCAM-1 expression in TNFα stimulated cells but showed no effects on ICAM-1 expression. Butyrate significantly inhibited the adhesion of mononuclear cells to an endothelial monolayer and propionate was less effective.</p><p>Conclusion: SCFA, including acetate, butyrate and propionate, influenced LPS- or TNFα-induced endothelial activation by inhibiting the production of IL-6 and IL-8, and reducing the expression of VCAM-1 and subsequent cell adhesion. Results were dependent on the concentrations and pre-incubation time of each SCFA and stimulation time of LPS or TNFα.</p

Image_1_The Anti-inflammatory Effects of Short Chain Fatty Acids on Lipopolysaccharide- or Tumor Necrosis Factor α-Stimulated Endothelial Cells via Activation of GPR41/43 and Inhibition of HDACs.TIF

<p>Background and Aim: Previously, we found that short chain fatty acids (SCFA) inhibit LPS or TNFα-induced endothelial inflammatory responses and excessive vascular cell adhesion molecule-1 (VCAM-1) expression, two important steps in the development of atherosclerosis. However, the mechanisms involved are still unclear. We hypothesized that the effects of SCFA are associated with activation of G-protein coupled receptor 41/43 (GPR41/43) and/or inhibition of histone deacetylases (HDACs).</p><p>Methods: The expression and location of GPR41/43 and HDAC3 in human umbilical vein endothelial cells (HUVEC) were confirmed. HUVEC were pre-incubated with acetate, butyrate or propionate alone or in combination with GLPG0974 (GLPG, antagonist of GPR43) or β-hydroxybutyrate (SHB, antagonist of GPR41) and then exposed to LPS or TNFα. Interleukin (IL)-6 and IL-8 levels and VCAM-1 expression were measured. HDAC activity was measured after treatment with butyrate, propionate and trichostatin A (TSA, HDAC inhibitor). The peripheral blood mononuclear cell (PBMC) adhesive level was also determined after TSA treatment.</p><p>Results: GPR41/43 were expressed on the membrane of HUVEC and HDAC3 was located in cytoplasm and nucleus. The GLPG and/or SHB treatments restored the inhibitory effects of acetate on IL-6 and IL-8 production and the inhibitory effects of butyrate or propionate on IL-6 production, but not on IL-8. In contrast, GLPG and/or SHB treatments did not affect the inhibitory effects of butyrate or propionate on TNFα-induced VCAM-1 expression. TSA showed similar effects on IL-8 production and VCAM-1 expression as butyrate and propionate. In addition, TSA significantly inhibited the adhesion of PBMC to an endothelial monolayer.</p><p>Conclusion: Activation of GPR41/43 mediates the effects of acetate on IL-6 and IL-8 production and the effects of butyrate and propionate on IL-6 production. Furthermore, inhibition of HDACs mediates the effects of butyrate and propionate on IL-8 production, VCAM-1 expression, and PBMC adhesion to an endothelial monolayer. These data indicate the beneficial roles of SCFA in preventing vascular inflammation and relevant diseases by activation of GPR41/43 and inhibition of HDACs.</p

Image_2_The Anti-inflammatory Effects of Short Chain Fatty Acids on Lipopolysaccharide- or Tumor Necrosis Factor α-Stimulated Endothelial Cells via Activation of GPR41/43 and Inhibition of HDACs.TIF

<p>Background and Aim: Previously, we found that short chain fatty acids (SCFA) inhibit LPS or TNFα-induced endothelial inflammatory responses and excessive vascular cell adhesion molecule-1 (VCAM-1) expression, two important steps in the development of atherosclerosis. However, the mechanisms involved are still unclear. We hypothesized that the effects of SCFA are associated with activation of G-protein coupled receptor 41/43 (GPR41/43) and/or inhibition of histone deacetylases (HDACs).</p><p>Methods: The expression and location of GPR41/43 and HDAC3 in human umbilical vein endothelial cells (HUVEC) were confirmed. HUVEC were pre-incubated with acetate, butyrate or propionate alone or in combination with GLPG0974 (GLPG, antagonist of GPR43) or β-hydroxybutyrate (SHB, antagonist of GPR41) and then exposed to LPS or TNFα. Interleukin (IL)-6 and IL-8 levels and VCAM-1 expression were measured. HDAC activity was measured after treatment with butyrate, propionate and trichostatin A (TSA, HDAC inhibitor). The peripheral blood mononuclear cell (PBMC) adhesive level was also determined after TSA treatment.</p><p>Results: GPR41/43 were expressed on the membrane of HUVEC and HDAC3 was located in cytoplasm and nucleus. The GLPG and/or SHB treatments restored the inhibitory effects of acetate on IL-6 and IL-8 production and the inhibitory effects of butyrate or propionate on IL-6 production, but not on IL-8. In contrast, GLPG and/or SHB treatments did not affect the inhibitory effects of butyrate or propionate on TNFα-induced VCAM-1 expression. TSA showed similar effects on IL-8 production and VCAM-1 expression as butyrate and propionate. In addition, TSA significantly inhibited the adhesion of PBMC to an endothelial monolayer.</p><p>Conclusion: Activation of GPR41/43 mediates the effects of acetate on IL-6 and IL-8 production and the effects of butyrate and propionate on IL-6 production. Furthermore, inhibition of HDACs mediates the effects of butyrate and propionate on IL-8 production, VCAM-1 expression, and PBMC adhesion to an endothelial monolayer. These data indicate the beneficial roles of SCFA in preventing vascular inflammation and relevant diseases by activation of GPR41/43 and inhibition of HDACs.</p

Image_1.TIF

<p>Background and Aim: Endothelial activation is characterized by excessive production of cytokines and chemokines as well as adhesion molecules expression which is involved in the development of atherosclerosis. The aim of our study is to investigate the effects of short chain fatty acids (SCFA) on lipopolysaccharide (LPS) or tumor necrosis factor alpha (TNFα)-induced endothelial activation.</p><p>Methods and Results: Human umbilical vein endothelial cells (HUVEC) were pre-treated with acetate (10 mM), butyrate (0.1 mM) or propionate (0.3 mM) for 1, 16, or 24 h and then stimulated with LPS (1 or 10 μg/ml) or TNFα (100 pg/ml or 1 ng/ml) for 6, 12, or 24 h. Cytokines in the supernatant were measured by ELISA. HUVEC were pre-treated with acetate (10 mM), butyrate (5 mM) or propionate (10 mM) for 24 h and then stimulated with LPS (1 μg/ml) or TNFα (1 ng/ml) for 8 h. The expression of the adhesion molecules intracellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) was detected by flow cytometry. The human blood mononuclear cell adhesive level to HUVEC monolayer was measured. LPS and TNFα induced a significant increase in the release of interleukin-6 (IL-6) and IL-8. Acetate, butyrate and propionate reduced IL-6 and IL-8 levels and the magnitude was dependent on the incubation times. LPS or TNFα increased ICAM-1 and VCAM-1 expression. Pre-incubation with acetate had no effect. In contrast, butyrate and propionate decreased VCAM-1 expression in TNFα stimulated cells but showed no effects on ICAM-1 expression. Butyrate significantly inhibited the adhesion of mononuclear cells to an endothelial monolayer and propionate was less effective.</p><p>Conclusion: SCFA, including acetate, butyrate and propionate, influenced LPS- or TNFα-induced endothelial activation by inhibiting the production of IL-6 and IL-8, and reducing the expression of VCAM-1 and subsequent cell adhesion. Results were dependent on the concentrations and pre-incubation time of each SCFA and stimulation time of LPS or TNFα.</p

THP-1 cells efficiently phagocytose FITC-labeled-<i>L. rhamnosus</i> and <i>B. breve</i>.

<p>Data are presented as median (95% confidence intervals) of n = 4 independent experiments.</p><p>THP-1 cells efficiently phagocytose FITC-labeled-<i>L. rhamnosus</i> and <i>B. breve</i>.</p

Effects of <i>L</i>. <i>rhamnosus</i> (LR) and <i>B</i>. <i>breve</i> (BB) on the release of cytokines induced by cigarette smoke extract (CSE).

<p>THP-1 cells (1x10⁶/ml) were preincubated for 2h with LR or BB (both at 10 bacteria/cell) in the presence or absence of 1.5% CSE and the release of IL-23 (A), IL-10 (B), IL-1β (C), TNF-α (D) and IL-6 (E) assessed after 16hrs. Data are presented as mean ± S.E.M. of three independent experiments. *p < 0.05; **p < 0.01 versus control and ^#p<0.05 versus CSE-stimulated mediator release.</p

Effects of <i>L</i>. <i>rhamnosus</i> (LR) and <i>B</i>. <i>breve</i> (BB) on cigarette smoke extract (CSE)-induced NF-κB activation.

<p>Cells 5x10⁴ /well were preincubated for 2h with LR or BB (both at 10 bacteria/cell) and then incubated for 24h in the presence or absence of 1.5% CSE. SEAP activity was measured in cell culture supernatant (A). *p<0.05 compared to the control and ^#p< 0.05 compared to CSE alone. The effect of RL and BB on NF-κB p65 nuclear import was also assessed by Western blotting with histone H1 (H1) as a nuclear loading control (B). A representative blot of 3 independent experiments is shown (upper panel) with the mean±S.E.M. data presented graphically (lower panel). *p < 0.05; **p < 0.01 versus control and ^#p<0.05 versus CSE-stimulated p65 nuclear import.</p

Concentrations of: IFN-γ (pg/ml) MIP-2 (pg/ml) TNF-α (pg/ml) in the bronchoalveolar lavage fluid 2, 7, 14, and 21 days after infection of mice

<p><b>Copyright information:</b></p><p>Taken from "induces a sustained airway hyperresponsiveness and inflammation in mice"</p><p>http://respiratory-research.com/content/8/1/83</p><p>Respiratory Research 2007;8(1):83-83.</p><p>Published online 19 Nov 2007</p><p>PMCID:PMC2194694.</p><p></p> Data are presented as mean ± SEM, n = 7–8. P < 0.005 ***, p < 0.0001 compared to the saline groups. #p < 0.01 compared to the group on day 2

Effects of <i>L</i>. <i>rhamnosus</i> (LR), <i>B</i>. <i>breve</i> (BB) and cigarette smoke extract (CSE) on expression and release of HMGB1.

<p>Cells were preincubated with LR or BB (both at 10 bacteria/cell) for 2h before stimulation with 1.5% CSE for 5h for analysis of intracellular HMGB1 by Western blotting (A) and 16h for detection of HMGB1 release by ELISA (B). A representative blot of the results from 3 independent experiments is shown with histone H1 (H1) as a housekeeping protein. Data in B are presented as the mean±S.E.M. of 3 independent experiments. *p < 0.05; **p < 0.01 versus control and ^#p<0.05, ^##p<0.01 versus CSE-stimulated HMGB1 release.</p