RSV- and BPZE1-induced signaling pathways in MDDC.

<p><b>A</b>: Total cell lysates were prepared from MDDC treated with BPZE1 (100∶1), RSV (MOI of 1) or both at the indicated time-points. Mock-infected cells were added as control. Proteins were resolved by 10% SDS-PAGE and Western blot analysis was performed to detect the phosphorylation of STAT1, IkBα, ERK1/2 and p38. Human phosphorylated β actin was used to normalize the results. Data are from one representative of three independent experiments performed with MDDC obtained from different donors. <b>B</b>: Histograms represent means of the relative optical density of phosphorylated proteins from three independent experiments. Error bars represent SEM.</p

Impact of BPZE1 on RSV-induced T lymphocyte polarization.

<p>MDDC were treated with BPZE1 (bacteria/cell ratio of 100∶1), RSV (MOI of 1) or both. Mock-infected cells were added as control. After 48 h, treated MDDC were cultured with purified allogeneic CD3 T cells. On day12, supernatants were collected, and IFN-γ (Th1), IL-5 (Th2) and IL-17 (Th17) were measured by ELISA. Values are expressed as medians with interquartile range of 13 independent experiments performed with MDDC obtained from different donors and expressed as ng/ml (IFNγ) or pg/ml (IL-5 and IL-17). Statistical significant differences are indicated by * (<i>P</i>≤0.0083).</p

Impact of BPZE1 on phenotypic maturation of RSV-infected MDDC.

<p>MDDC were treated with BPZE1 (bacteria/cell ratio 100∶1), RSV (MOI of 1) or both. After 24 h, cells were analyzed for the indicated surface markers associated with a mature phenotype. Mock-infected cells were used as control. Fluorescence data are reported as median fluorescence intensity (MedFI) for CD80 and CD38, and as percentage of positive cells for CD83. Values are expressed as medians with interquartile range of seven independent experiments performed with MDDC obtained from different donors. Statistical significant differences are indicated by * (<i>P</i>≤0.0083).</p

RSV- and BPZE1-induced IL-6, IFNβ, CCL5, IL-12p40 and IL-12p35 gene expression in MDDC.

<p>MDDC were treated with BPZE1 (100∶1), RSV (MOI of 1) or both. Mock-infected cells were added as control. Total RNA was extracted at the indicated time points. Kinetics of mRNA expression for p40 and p35 subunits of IL-12p70, IL-6, IFNβ and CCL5 was evaluated by real-time quantitative RT-PCR. The mRNA transcripts were normalized with respect to the endogenous reference (human β actin) sample. Data were expressed as fold increase (mean ± SEM of four experiments) with respect to mock-treated cells at 5 h.</p

Impact of BPZE1 on IL-10, IL-12p70 and IL-23 production of RSV-infected MDDC.

<p>MDDC were treated with BPZE1 (bacteria/cell ratio of 100∶1), RSV (MOI of 1) or double infected with BPZE1 and RSV. Mock-infected cells were used as control. After 24 h, cytokines released in the culture media were measured by ELISA. Values are expressed as medians with interquartile range from 13 (IL-10 and IL-23) and from 15 (IL-12p70) independent experiments performed with MDDC obtained from different donors and expressed as ng/ml (IL-10) or pg/ml (IL-12p70 and IL-23). Statistical significant differences are indicated by * (<i>P</i>≤0.0083).</p

Impact of BPZE1 on RSV-induced allogeneic T cell proliferation.

<p>MDDC were infected with RSV (MOI of 1), BPZE1 (bacteria/cell ratio of 100∶1), or both. Mock-infected cells were added as control. After 48 h, MDDC were co-cultured with allogeneic purified T cells at increasing T cells/MDDC ratios (5/1, 10/1, 20/1, 40/1) for 6 d. Results are reported as percentages of BrdU positive cells (mean ± SEM of 4 independent experiments performed with MDDC obtained from different donors).</p

Lack of IL-10 increases chemokine and cytokine production in the airways during RSV infection.

<p>BAL samples from BALB/c and IL-10^−/− mice were analyzed for indicated cytokines and chemokines on day 4 and 8 after RSV infection using Luminex. A group of naive BALB/c controls are included in the day 4 data. Error bars indicate the SEM. The data are pooled from two independent experiments with n = 4–5 mice per group.</p

Lack of IL-10 increases cellular influx into the lungs and delays recovery during RSV infection.

<p>BALB/c mice and IL-10^−/− mice were infected with 10⁶ PFU RSV i.n. (day 0). (A) Illness was monitored daily by changes in weight for 14 days after RSV infection; percentage of original weight (day 0) is shown. (B) Copies of the RSV L gene were quantified in the lung on day 4 post infection using qPCR. (C) Total numbers of cells in the lung and BAL were enumerated on day 4 and 8 from naïve or RSV infected mice. (D) Total numbers of neutrophils in the BAL were quantified using differential cell counting of H&E stained cytospins slides on day 4 and 8 post infection. (E) Total numbers of NK cells (CD3⁻ NKp46⁺) and CD3 gated CD4⁺Foxp3⁻ and CD8⁺ T cells in the lung were quantified using flow cytometry on day 4 and 8 post RSV infection. Error bars indicate the SEM. The data is representative of three independent experiments with n = 4–5 mice per group.</p

Blocking IL-10R signalling increases cellular influx into the lungs and delays recovery during RSV infection.

<p>BALB/c mice were infected with 10⁶ PFU RSV i.n. (day 0). Where indicated, mice were injected with anti-IL-10R antibody on day -1 (i.p.), 3 (i.p. and i.n) and 6 (i.p.) post RSV infection. Control groups were injected with rat IgG. (A) Illness was monitored daily by changes in weight for 8 days after RSV infection; the percentage of original weight is shown. (B) Viral titer was measured in the lung on day 4 post infection by quantifying RSV L gene copies by qPCR. (C) Total numbers of cells in the lung and BAL were enumerated on day 4 and 8 from naïve or RSV infected mice. (D) Total numbers of neutrophils in the BAL were quantified using differential cell counting of H&E stained cytospins slides on day 4 and 8 post infection. (E) Total numbers of NK cells (CD3⁻ NKp46⁺) and CD3-gated, CD4⁺Foxp3⁻ and CD8⁺ T cells in the lung were quantified using flow cytometry on day 4 and 8 post RSV infection. Error bars indicate the SEM. The data are representative of two independent experiments with n = 4–5 mice per group.</p

Increased frequency of IFN-γ-expressing T cells in IL-10^−/− mice after RSV infection.

<p>BALB/c mice were infected with 10⁶ PFU RSV i.n. (day 0). CD3⁺ gated, CD4⁺ and CD8⁺ T cells from lungs on day 8 post RSV infection were analyzed by flow cytometry. Representative plots of intracellular IFN-γ expression after a 3 hr PMA/ionomycin restimulation are shown. In addition, quantifications of the frequencies of IFN-γ expression in CD4⁺ T cells and CD8⁺ T cells are shown. The data are representative of three independent experiments with n = 4–5 mice per group.</p