PBECs express antimycobacterial effectors <i>DEFB4</i> and <i>S100A7</i>. Mtb H37Rv was grown in the presence of the indicated concentrations of recombinant (A) hBD2, (B) psoriasin or vehicle control (veh).

<p>Amikacin and gentamycin were used as positive controls at 200 μg/ml and 100 μg/ml respectively. Mtb growth was measured by optical density at 595nm (OD_595nm) over time. Mean readings of one representative experiment are shown. At day 7, cultures were plated to measure the bacterial burden by CFU (n = 3 representative for two independent H37Rv cultures). Groups were compared against vehicle control at day 7 by one-way ANOVA or Mann-Whitney test (pooled normalised CFU data from three (psoriasin) or two (hBD2) independent experiments are shown). Median is shown. ϕ, p<0.001;**, p<0.01; ***, p<0.001.</p

PBEC gene expression in response to infection-driven inflammation is dependent on direct interaction between myeloid cells and Mtb.

<p>In the transwell model, PBECs were exposed to THP-1 cells or Mtb H37Rv bacilli (MOI5) for 24h as indicated. Gene expression in PBECs was measured by RT-PCR and is shown as fold change over unstimulated PBECs (n = 5). Friedman test with Dunn’s post-test was used to compare expression with unstimulated controls (fold change = 1, not displayed). Boxplots show median and range. * p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001.</p

Epithelial cells are the major subset in the airway lining and are not permissive to Mtb infection.

<p>(A) Presence of total bronchial epithelial cells (BECs) and leukocytes (LEU) was measured <i>ex vivo</i> by differential cell counts of bronchial brushings derived from the airway lining. Subsets are shown as % of total cells recovered from healthy volunteers (n = 17). Wilcoxon signed rank test was used to compare groups. Boxplots show median and range. **** p<0.0001. (B) PBECs (n = 4) and THP-1 MΦ (mean ± SD of 2 independent experiments) were infected with Mtb H37Rv for 24h. To remove extracellular bacteria, 200 μg/ml amikacin was added for an additional 2h where indicated. Mtb bacilli were enumerated by colony forming units (CFU). (C) Affymetrix HTA2.0 arrays were performed on PBECs (uninfected, MOI10 or MOI50, 24h) from 4 donors. The log₂ fold change compared to unstimulated PBECs and the associated FDR-adjusted q-value are shown in volcano plots for MOI10 (left) and MOI50 (right). The solid black lines intersect at q-value of 0.05.</p

PBEC responses to Mtb-driven inflammation are enriched for pro-inflammatory and interferon-mediated signalling.

<p>All DE genes at a q-value < 5% were used for statistical over-representation analysis (ORA) by InnateDB and oPOSSUM. The most significant (A) Transcription Factor Binding Sites (TFBS), (B) Pathways and (C) Gene ontology terms are shown. Red dashed line indicates a z-score of 10 or a corrected p-value of 0.05. For (A), IRF- and NFκB-family members are highlighted in blue and red respectively.</p

Mtb-infected alveolar macrophages drives gene expression in PBECs.

<p>Cell-free conditioned medium from Mtb-infected and uninfected AMΦ, alongside the appropriate culture medium controls was added to PBECs. After 24h gene expression was measured by RT-PCR. Gene expression is shown as fold change over unstimulated PBECs (n = 5). Friedman test with Dunn’s post-test was used to compare stimulations. Boxplots show median and range. * p<0.05; **, p<0.01 or the exact p-value are given.</p

Whole transcriptome analysis of PBECs exposed to Mtb-infected myeloid cells.

<p>(A) To interrogate the effect of myeloid-epithelial cross-talk during Mtb infection, a transwell co-culture system was established. Separated by a 0.4 μm pore-sized transwell membrane to allow exchange of soluble mediator only, PBECs were seeded in the bottom well of a tissue culture plate and THP-1 cells were added into the transwell insert. Cells were infected as indicated. (B) In the transwell co-culture model, PBECs from eight donors were exposed to uninfected (THP, T) or Mtb-infected THP-1 (THP + Mtb, TM) cells for 24h. Affymetrix HTA2.0 microarrays were performed on PBECs. Hierarchical clustering of all significantly differentially expressed genes at a fold change of > 1.5 and a q-value < 5% was performed using average linkage and Euclidean distance. Expression range from low (green) to high (red).</p

PBECs upregulate chemokines and enhance neutrophil recruitment to the site of Mtb-infection.

<p>(A) The Venn diagramme shows the overlap between significantly induced genes in PBECs exposed to Mtb-infected THP-1 cells at 24h (Expression) and significantly increased secreted proteins during infected PBEC-THP-1 co-culture over infected THP-1 monoculture at 48h (Secretome). Three of the overlapping proteins were associated with chemotaxis (GO:0006935) and their fold changes in their respective comparisons are shown in (B). PBLs were added into a transwell insert on top of a culture well containing conditioned medium from Mtb-infected THP-1 cells ± PBECs or medium control (BEBM). Supernatants were generated from five different PBEC donors (SN1-5). After 3 h cells from the insert and the bottom well were collected and enumerated by flowcytometry. (C) Shows representative plots of the gating strategy to define migrated subsets by forward scatter (FSC) and side scatter (SSC). (D) As a background control, migration across the transwell membrane towards medium (BEBM) is shown as total number of recovered PMNs. (E) Number of migrated PMNs towards cell-free conditioned medium derived from Mtb-infected THP-1 cells ± PBECs is shown. (F) Shows PMN migration as % of total cells. Groups were compared by (n = 12). Horizontal bars indicate the median. *, p<0.05; **, p<0.01; ***, p<0.001.</p

Mtb-driven inflammation activates PBECs in an IL1β and type I IFN signalling dependent manner.

<p>(A) PBECs were co-cultured with infected or uninfected THP-1 cells in the presence of αL1β or IgG₁ (isotype control) as indicated. After 24h, gene expression was measured by RT-PCR. Expression is shown as fold change over unstimulated (n = 6). Boxplots show median and range. (B) PBECs were exposed to Mtb-infected THP-1 cells (MOI5) in the presence of increasing concentrations of αIFNAR2 (5, 10 and 20 μg/ml) or IgG₂ (isotype control). After 24h, gene expression was measured by RT-PCR and is shown as fold change over unstimulated (n = 3). Median is shown. Friedman test with Dunn’s post-test was used to compare groups against isotype controls. n.s., not significant; *, p<0.05; **, p<0.01. ϕ, baseline expression not detected in two or one donor for <i>DEFB4</i> and <i>S100A7A</i>, respectively.</p

IL1β and IFN signalling are activated during Mtb-mediated inflammation.

<p>(A) In co-culture, IL1β release from Mtb-infected THP-1 cells (MOI5) was measured in the presence or absence of PBECs at 24h (n = 5). (B) AMФs from three donors were infected with Mtb H37Rv (MOI5) for 24h. <i>IFNB</i> expression was measured by RT-PCR and is shown as relative expression (rE) over <i>GAPDH</i> and <i>HPRT</i>. ϕ, not detected in one donor. (C) IFNβ release by THP-1 cells after 24h of infection was measured by ELISA. Mean ± SD are shown (n = 3). (D) Western blot for STAT1 and phospho-STAT1 on PBECs stimulated for 24h with medium (1), transwell co-culture with uninfected (THP) (2) or Mtb-infected (THP + Mtb) (3) THP-1 cells or stimulated with 10 ng/ml recombinant IL1β (5h, 4) or IFNβ (5h, 5 and 24h, 6). Shown is one representative blot of three independent experiments. One-tailed Wilcoxon signed rank test was used to compare groups in (A) and (B). n.d., not detected; *, p<0.05.</p

Modelled temporal changes in gene expression.

<p><b>A</b>. Heat map showing modelled changes in expression of the significant gene transcripts in TBM patients from the time of diagnosis (0) to 180 days. Green represents lower transcript abundance, red represents higher transcript abundance and black represents no difference in expression as compared to healthy children with a past history of TB sampled at least one year after diagnosis and treatment. The relative degree of transcript abundance is indicated by the colour intensity derived from the fitted mean expression levels over time (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0185973#sec002" target="_blank">methods</a>). Genes showing similar temporal patterns of expression have been clustered together. The apparent linear change in colour is derived from the statistical model that interpolates the observed time points and can therefore be represented as a continuum. <b>B and C</b>. Example plots of two significantly differentially expressed gene transcripts. Expression levels for each TBM patient (red circles n = 9) are shown from diagnosis (time 0) to day 180. Blue circles are expression levels for healthy children (n = 9) with a past history of TB sampled at least one year after diagnosis and treatment. M = “minus” and denotes the log₂ ratio of the red and green channels. The line represents the fitted mean gene expression level over time, from linear mixed-effects model (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0185973#sec002" target="_blank">methods</a>). 1b = <i>TARP</i>; 1c = <i>IL1R2</i>.</p