Induction of IFN-III genes by different bacterial species in epithelial cells.

<p><b>A.</b> LoVo intestinal cells infected with <i>S. enterica</i> at multiplicity of infection (MOI) of 20 for the indicated time, were lysed and processed for quantification of intracellular bacterial by counting colony-forming units (CFU) on agar plates, or for total cellular RNA extraction and quantification of IFN gene expression, as described for <i>L. monocytogenes</i> in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039080#pone-0039080-g001" target="_blank">Figure 1A</a>. Values from three independent experiments are expressed as mean ± S.D. of the fold change relative to uninfected control cell values at the beginning of the experiment. <b>B.</b> LoVo cells, uninfected or infected for 18 h with different bacterial species, were processed for quantification of bacterial load and mRNA. Quantification of internalized bacteria: data are means ± S.D. of CFU for 10⁵ cells (<i>n</i> = 3) at the indicated MOI, except for <i>C. </i><i>trachomatis,</i> for which the percentage of infected cells (<i>n</i> = 4) were determined by flow cytometry after antibody labelling (see text). <i>IFN-λ1</i> and <i>IFN-λ2</i> transcript levels were determined by qRT-PCR and normalized to <i>GAPDH</i> transcript levels. Values are expressed as mean ± S.D. of the fold change relative to that in uninfected cells (<i>n</i> = 3 to 5). <b>C.</b> Quantification of IFN-λ1/λ3 and IL-8 production were done by ELISA, using culture supernatants of LoVo cells infected for 28 h with the indicated bacterium at a MOI of 50. Experiments were done in triplicates and reproduced once. <b>D</b>. Quantification of IFN-λ mRNAs in A549 lung epithelial cells infected with <i>GFP</i>-expressing <i>M. tuberculosis</i> (<i>n = 4</i>). The percentage of infected cells were determined by flow cytometry (see text).</p

Response to IFN-III in mouse placenta and fetal membrane.

<p>Pregnant B6.A2G-Mx1-IFNAR1^0/0 mice were treated subcutaneously with 5 µg of mouse IFN-λ2 or PBS (mock) at 24 and 12 h prior to sacrifice. Embryos <i>in utero</i> were extracted and fixed with formaldehyde. Sections of paraffin-embedded tissue were immuno-stained for IFN-inducible Mx1 protein. IFN-λ response was monitored by nuclear Mx1 staining (red) in epithelial cells of the fetal membrane and various regions of the placenta. Counterstaining: background auto-fluorescence (white). Zooms of squared regions are shown below. Scale bar: 50 µm.</p

List of bacterial strains used in this study and their effect on IFN-III genes in epithelial cells.

<p>(a) Reference of the strain.</p><p>(b) Effect on the expression of IFN-III. “++”: high induction; “+”: induction; “+/−”: weak induction; “–”: no induction.</p><p>(c) Reference for the observed effect on IFN-III genes.</p><p>(d) Data not shown.</p

Induction of IFN-III genes by <i>L. monocytogenes</i> in HepG2 hepatocytes and BeWo trophoblastic cells.

<p>Quantification of bacterial loads (CFU) and IFN-λ mRNAs levels in <i>Listeria</i>-infected HepG2 or BeWo cells. <i>IFN-λ1</i> and <i>IFN-λ2</i> transcript levels were determined by qRT-PCR and normalized to <i>GAPDH</i> transcript levels. Values are expressed as mean ± S.D. of the fold change relative to that in uninfected cells (<i>n</i> = 3). <i>IFN-λ2</i> levels in uninfected HepG2 cells were below the detection threshold, preventing measures of fold change. <i>L. monocytogenes</i> (<i>L. m.</i>), <i>L. innocua</i> (<i>L. in.</i>), <i>L. </i><i>innocua</i> expressing <i>inlA</i> (<i>L. in. (inlA)</i>).</p

Induction of IFN-III and IFN-stimulated genes in mouse placenta.

<p>Pregnant E16P^+/+ mice were inoculated intravenously with 4×10⁴ CFUs <i>L. monocytogenes</i> in PBS (<i>L.m.</i>) or with PBS only (<i>n.i.</i>) in two independent experiments (exp. A and B). Bacterial numbers were determined in livers and placentas 72 h post-infection. Placentas (<i>n</i> = 7 in exp.A; <i>n</i> = 8 in exp.B) from the two mice that displayed the highest bacterial loads in liver lysates, and placentas (<i>n</i> = 3) from two uninfected mice (<i>n.i.</i>), were processed for RNA extraction and bacterial quantifications. <b>A.</b> Quantification of <i>L. monocytogenes</i> loads and IFN-λ transcripts in placenta homogenates. <b>B.</b> Quantification of <i>IFN-λ2</i> and <i>IFN-λ3</i> transcript levels in placentas by qRT-PCR, with normalization to <i>GAPDH</i> transcripts. Values are expressed as mean ± S.D. of the fold change relative to that in placenta of uninfected mouse A1 or mouse B1 ( = 1). There is no significant change in uninfected mouse A2 or mouse B2 (<i>n.i. A2</i>, <i>n.i. B2</i>)<b>. C.</b> Quantification of transcript levels of IFN-responsive genes (<i>IFIT1, Mx1, Mx2)</i> and a control gene (<i>IGF2</i>) in placentas were determined by qRT-PCR and normalized to <i>YWHAZ</i> housekeeping gene. Values are expressed as mean ± S.D. of the fold change relative to that in placenta of all uninfected mice of exp.A or exp.B. (*<i>p</i><0.05; **<i>p</i><0.005, Student <i>t</i> test).</p

Changes in relative miRNA arm expression upon infection.

<p>(A) Detection and relative expression of miRNA hairpin arms. Stacked bar plot shows the proportion of detected miRNA hairpins for which either one or both mature miRNA sequences were detected. Density plot shows the distribution of log₂ expression ratios for hairpins where both arms were expressed. (B) An example of one miRNA, miR-361, which showed a strong infection-dependent change in relative arm expression. Axes show the expression of the 3p and 5p arms of all sequenced individuals at each time point. In each panel, colours denote the different time points, and infection conditions are plotted in separate panels. The displacement of points towards the right of the plot in infected samples at later time points shows that the change in the expression ratio of the two arms is due to the increased expression of the 3p arm that, in some conditions, becomes dominant.</p

Differential expression of miRNAs in DCs upon infection with a panel of bacteria.

<p>(A) Numbers of significantly differentially expressed miRNAs upon infection at each time point for each bacterium. As we did not have expression profiles for the 48h time point for STP infection, this point is missing from the plot. (B) Heatmap illustrating the hierarchical clustering of experimental conditions based on the mean expression levels of the 50 most variable miRNAs. (C) Overlap of differentially expressed miRNAs between bacteria using two different significance cut-offs. Left-bar shows overlap using a single cut-off of FDR-adjusted p<0.01 and |log₂ fold change|>1, while the right bar shows the overlap using a secondary cut-off where a miRNA was called as significant if the absolute log₂ fold change was less than 1, if it passed the first more stringent fold-change cut-off upon infection with at least one of the six bacteria. (D and E) Venn diagrams showing the overlap of significantly differentially expressed miRNAs between bacteria of the MTBC (D) and between MTB-Rv and all other non-mycobacterial infections (E).</p

Shared and specific miRNA responses to bacterial infection.

<p>(A) Plots of the temporal dynamics of two core response miRNAs. As we did not have expression profiles for the 48h time point for STP infection, this bacterium was excluded from the analysis. (B) Multidimensional scaling analysis (MDS) representing the distances between the temporal miRNA responses to different bacterial infections. Distances were based on the sum of edit distances, for each miRNA, between bacteria using STEM-assigned model temporal profiles. (C) The expression of miR-132-3p increased following infection with MTB-Rv and MTB-Bj but not BCG. Transformation of BCG with RD1 from MTB resulted in a significant increase in miR-132-3p expression, not significantly different to that induced by MTB-Rv. Both MTB-Ra and MTB-Hk showed significantly lower miR-132-3p expression than virulent mycobacteria. (D) The induction of miR-212-3p was significantly higher following BCG::RD1 infection, compared to infection with control BCG, and was not significantly different to the response to MTB-Rv. Though the difference between the induction of this miRNA upon infection with virulent or avirulent MTB strains was not significant, the tendencies observed were consistent with miR-132-3p. Significance was calculated using a Mann-Whitney test (* = p < 0.05).</p

Alveolar CD11b⁺ Cells Over-Express DC-SIGN in Patients with TB

<div><p>(A) BAL cells from a patient with TB (upper four panels) and from a patient with sarcoidosis (lower two panels) were analyzed by flow cytometry. Expression of CD3 and CD4 was analyzed on cells from R1. CD11b and DC-SIGN expression was analyzed on cells from R2.</p> <p>(B) Distribution of the proportion of CD11b⁺DC-SIGN⁺ cells in BALs according to pathology and age. Black circles indicate ≤15 y of age; black triangles indicate ≥20 y; NC, no case.</p> <p>(C) DC-SIGN (upper panels) and <i>M. tuberculosis</i> (lower panels) immunodetection in serial sections of a lung biopsy from a patient with TB. The pictures are representative of results obtained with samples from a total of four patients. G, granuloma.</p> <p>(D) DC-SIGN immunodetection in a lung biopsy from a patient with sarcoidosis. The pictures are representative of results obtained with samples from a total of three patients.</p> <p>In (C) and (D), magnification in left panels is 100×, and regions in squares are shown at higher magnification in right panels.</p></div

Alveolar DC-SIGN⁺ Cells in Patients with TB Are Mϕs

<div><p>(A) Total BAL cells from a patient with TB were allowed to adhere to the plastic for 1 h at 37 °C in complete medium. CD11b and DC-SIGN expression was analyzed by flow cytometry before (left) and after (right) adherence.</p> <p>(B) Surface and intracellular DC-SIGN (red) expression by an adherent alveolar cell examined under the confocal microscope.</p> <p>(C) Flow cytometry analysis of surface expression of BDCA-1 (CD1c), BDCA-2, BDAC-3, CD1a, CD11b, CD11c, CD14, CD68, CD83, and CD123 in DC-SIGN⁺ BAL cells from a patient with TB.</p> <p>(D) Flow cytometry analysis of surface expression of CD40, CD86, HLA-DR, CD11b, CD11c, CD206, CD16, CD32, CD40, CD64, TLR2, TLR4, and TLR9 in DC-SIGN⁺ BAL cells from a patient with TB.</p> <p>In (C) and (D), analysis was performed on DC-SIGN–expressing cells in R2, as shown in <a href="http://www.plosmedicine.org/article/info:doi/10.1371/journal.pmed.0020381#pmed-0020381-g001" target="_blank">Figure 1</a>.</p></div