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

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), <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

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 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

The effect of <i>L</i>. <i>rahmonusus (</i>LR<i>) and B</i>. <i>breve</i> (BB) on cigarette smoke extract (CSE)-, lipopolysaccharide (LPS)- and CpG-induced CXCL-8 release by THP-1 macrophages.

<p>Cells 1x10⁶/ml were pretreated for 2hrs with 10 bacteria/cell (1), 20 bacteria/cell (2) and 50 bacteria/cell (3) LR and BB in the presence and absence of 1.5% CSE and CXCL-8 release (A) and mRNA expression (B) determined after 16 and 3hrs respectively. Expression of CXCL-8 mRNA is expressed as a ratio of the housekeeping gene GAPDH. Cell viability as assessed by Annexin V expression was not affected by any treatment (C). A similar effect of LR and BB on (D) LPS (1000ng/ml)-and (E) CpG oligonucleotide (3μM)-stimulated CXCL-8 release at 16h was also seen. (F) Cells were preincubated for 2 h with E.coli (at various ratios of bacteria to cells) and then stimulated with 1.5% CSE and CXCL-8 release determined after 16hrs. (G) U937 macrophages were stimulated with LR and BB in the presence and absence of 1.5% CSE and CXCL-8 release determined 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 CXCL-8.</p

CSE induces the expression of mRNA and the production of chemokines in cDCs.

<p>The supernatants of CSE-exposed cDCs were tested for the production and release of CCL3 (A) and CXCL2 (B) ELISA (upper panels) and cell pellets were tested for CCL3 and CXCL2 mRNA levels by real time PCR (upper panels). White bars represent cDCs treated with medium, black bars represent cDCs treated with CSE and gray bars cDCs treated with NAC and CSE. Data are representative of three independent experiments, showing the means±SEM from triplicate cultures. * represents significant differences compared with medium-treated cells (*p<0.05; ***p<0.001). ^# indicates significant differences between cells treated with CSE in combination with NAC and cells treated with CSE.</p

CSE increases cDC-induced CD8⁺T cell but inhibits CD4⁺T cell proliferation.

<p>cDCs from Balb/c mice were incubated with medium (white bars) or CSE ( black bars) and coincubated with allogenic T cells from C57BL/6 mice [CD8⁺ (A) and CD4⁺T cells (B)] in a MLR. Presented are pooled data from eight individual experiments using cDCs generated from eight isolations. Values are represented as mean±SEM. A statistically significant modulation of proliferation of T cells with CSE-primed cDCs occurred (^*<i>p</i><0.05 and ** p<0.01 when compared to medium-treated cDCs). The supernatants of MLR were collected for the measurement of IL-2 by ELISA (inserted graphs in A & B). Presented are pooled data from eight individual experiments using cDCs generated from eight isolations. Values are represented as mean±SEM. * Indicates significant differences between medium-treated cells (* p<0.05).</p

CSE increases the production of CCL3 and CXCL2 by TLR4 and MyD88 dependent manner.

<p>cDC were prepared by culturing BM cells from Balb/c mice preincubated with anti-TLR4 antibody (20 µg/ml) or isotype control (20 µg/ml) for 1 h and then stimulated with CSE for 16 h and amount of CCL3 (A) and CXCL2 ( B) were determined by ELISA. cDCs were prepared by culturing BM cells from Balb/c mice and age- and sex-matched MyD88-deficient mice. CSE or LPS were incubated for 16 h and supernatant were harvested for determination of CCL3 (C) and CXCL2 (D) by ELISA. White bars represent cDCs treated with medium, dotted bars are cDCs treated with LPS and black bars represent cDCs treated with CSE. Data are representative of three independent experiments, showing the means±SEM from triplicate cultures. * represent significant differences compared with medium-treated cells (***p<0.001).</p

CSE increases the production of intracellular ROS in cDC.

<p>cDCs were incubated with CSE , with or without NAC or PMA (as a control) and ROS generation was assayed by FACS analysis. The mean fluorescent intensity (MFI) of the following groups are indicated in the figure: control: unlabelled CSE-treated cells (green line), unstimulated: control labeled cells (red line), CSE: CSE-stimulated labeled cells (blue line), CSE+NAC: CSE-stimulated labeled cells treated with NAC (black line), PMA: PMA-stimulated labeled cells (light blue line).</p