Adult and neonatal lymphocytes mixed leukocyte reaction (MLR) assay.

<p>BDCs isolated from adult and neonatal pigs were cultured during 5 days with allogeneic CD14⁻ CD172⁻ PBMCs isolated from either adult (A) or neonatal (B) pigs. The neonatal (white boxes) and adult (black boxes) BDCs were cultured with the CD14⁻ CD172⁻ at two different ratios (1∶2 and 1∶10) and the lymphoproliferation was assessed by tritiated thymidine incorporation using a liquid scintillation counter. Each dot represents cells coming from one animal (n = 6 animals) and the horizontal bar represents the median. (<i>*p<0.05</i> Mann-Whitney U test).</p

Cytokine production by activated adult and neonatal BDCs.

<p>BDCs were stimulated 24 h with LPS (1 µg/ml), poly I∶C (10 µg/ml), imiquimod (10 µg/ml), class A or class C CpG ODN (5 µg/ml), then the supernatants were harvested and the concentration of the different cytokines determined by ELISA. The upper panel shows the results for neonatal BDCs and the bottom panel the ones for adult BDCs. We show here the concentration of TNF-α (A, D), IL-12p40 (B, E) and IFN-α (C, F) in the supernatants from stimulated (black boxes) or non-stimulated (white boxes) BDCs. Results show the mean cytokine production in pg/ml ± SD from 5 or 6 animals. (*<i>p<0.05</i>, Wilcoxon signed rank test).</p

Adult and neonatal monocyte stimulation by TLR ligands.

<p>(A) Expression of TLR3, 4, 7 and 9 by non-stimulated adult and neonatal monocytes was assessed using RT-qPCR and is expressed as relative mRNA expression using the 2^−ΔCt calculation method. Data represent the mean relative mRNA expression ± SD from 4 different animals. (B) Adult (black boxes) and neonatal (white boxes) monocytes were stimulated 24 h with LPS (1 µg/ml), poly I∶C (10 µg/ml), imiquimod (10 µg/ml), class A or class C CpG ODN (5 µg/ml) or left non-stimulated (control) and were then harvested and prepared for co-stimulatory molecules staining. The percentage of adult and neonatal monocytes expressing CD80/86 was determined using flow cytometry. For each of the TLR stimulation and the corresponding control, data show the mean percentage of positive cells ± SD obtained for cells isolated from 4 animals. (C) and (D) Monocytes were stimulated with TLR ligands 24 h as in (B), then the supernatants were harvested and the concentration of the different cytokines determined by ELISA. We show here the concentration of TNF-α (C) and IL-12p40 (D) in the supernatants from adult (black boxes) or neonatal (white boxes) monocytes. Each data point represents one animal with median value for each group represented by the horizontal bar.</p

Chemokine receptors expression by adult and neonatal BDCs following TLR stimulation.

<p>(A) Expression of CCR2, 5 and 7 by non-stimulated adult and neonatal BDCs was assessed using RT-qPCR and is expressed as relative mRNA expression using the 2^−ΔCt calculation method. Data represent the mean relative mRNA expression ± SD from 5 different animals. (*<i>p<0.05</i>, Mann-Whitney U test). (B, C, D) Adult and neonatal BDCs were stimulated 6 h with LPS (1 µg/ml), poly I∶C (10 µg/ml), imiquimod (10 µg/ml), class A or class C CpG ODN (5 µg/ml) or left non-stimulated and then harvested in TRIZOL®. After mRNA extraction, the expression of CCR2 (B), CCR5 (C) and CCR7 (D) was assessed by RT-qPCR. Results show the fold of increase following stimulation compared to the non-stimulated cells using the 2^−ΔΔCt and show the mean ± SD from 5 animals. (<i>*p<0.05</i> Mann-Whitney U test).</p

Chemokines expression and production by stimulated adult and neonatal BDCs.

<p>Porcine BDCs from 8-week old adults and 1-week old neonates were cultured 6 h with LPS (1 µg/ml), poly I∶C (10 µg/ml), imiquimod (10 µg/ml), class A or class C CpG ODN (5 µg/ml) and were then harvested in TRIzol. After mRNA extraction, the gene expression of CCL2, CCL3, CCL4 and CXCL10 by stimulated adult (▴) and neonatal (▵) BDCs was determined. Each of the data point corresponds to one animal and displays the fold of increase compared to unstimulated cells with the median, using the 2^−ΔΔCt calculation method. (<i>*p<0.05</i> Mann-Whitney U test). (B) Adult and neonatal BDCs were stimulated 24 h with the different TLR ligands and the supernatants were harvested. The concentration of CXCL8 was assessed by ELISA, showing the production of the chemokine after stimulation (black boxes) or without stimulation (white boxes). Results show the mean production of CXCL8 in pg/ml ± SD from 5 or 6 animals. (*<i>p<0.05</i>, Wilcoxon signed rank test).</p

TLRs and co-stimulatory molecules expression by adult and neonatal BDCs.

<p>(A) Expression of TLR3, 4, 7 and 9 by non-stimulated adult and neonatal BDCs was assessed using RT-qPCR and is expressed as relative mRNA expression using the 2^−ΔCt calculation method. Data represent the mean relative mRNA expression ± SD from 6 different animals. (*<i>p<0.05</i>, Mann-Whitney U test). (B, C) Adult and neonatal BDCs were stimulated (▴) 24 h with LPS (1 µg/ml), poly I∶C (10 µg/ml), imiquimod (10 µg/ml), class A or class C CpG ODN (5 µg/ml) or left non-stimulated (▵). Cells were then harvested and prepared for co-stimulatory molecules staining. The percentage of adult (B) and neonatal (C) BDCs expressing CD80/86 was determined using flow cytometry. Each data point represents one animal with median value for each group represented by the horizontal bar. (<i>*p<0.05, **p<0.01</i>, paired t test).</p

Cytokine (IFN-γ, TNF-α, IL-12p70, IL-6, IL-4 and IL-17) and chemokine (MCP-1) concentrations in whole lung homogenates following c-di-GMP treatment and intranasal challenge infection with <i>B. pertussis</i>.

<p>Mice were treated with c-di-GMP (black bars) or control c-GMP (white bars) at 24 hr prior to challenge infection. Whole lungs were removed and cytokine concentrations determined in lung homogenate supernatants at 24 hr post treatment (24PT) and days two, four and six post challenge (Day 2–6). The results shown are as the means ± SD of cytokine and chemokine concentrations detected by ELISA from 3 separate experiments and nine to twelve animals per group.</p

Nitric oxide production following c-di-GMP treatment.

<p>Nitrite production, as a measure of NO expression, was assessed at 24 hr after treatment with c-di-GMP and 24 hr after infection with <i>B. pertussis</i> in lungs of mice. Control animals were treated with PBS. Values shown represent the means ± SD from two independent experiments (10 animals per group).</p

MIP-2 mRNA levels in lung tissues following c-di-GMP treatment. Mice were treated intranasally with c-di-GMP or control c-GMP at 24 hr prior to challenge infection.

<p>MIP-2 mRNA levels in lung were determined prior to challenge and at days 2, 4 and 6 post challenge by real-time PCR. Values shown represent the fold increase of the mean compared to non-infected control mice (five to eight animals per group).</p

Infiltration of neutrophils and macrophages in c-di-GMP treated animals.

<p>(A) Photomicrograph of lung biopsies taken from a control mouse at 24 hr post treatment with c-GMP, obtained with a 10 X objective plus digital zoom. (B-D) Photomicrographs of lung biopsies taken from three different mice at 24 h post treatment with c-di-GMP (10 X objective plus digital zoom).</p