Innate activation of human primary epithelial cells broadens the host response to Mycobacterium tuberculosis in the airways

Early events in the human airways determining whether exposure to Mycobacterium tuberculosis (Mtb) results in acquisition of infection are poorly understood. Epithelial cells are the dominant cell type in the lungs, but little is known about their role in tuberculosis. We hypothesised that human primary airway epithelial cells are part of the first line of defense against Mtb-infection and contribute to the protective host response in the human respiratory tract. We modelled these early airway-interactions with human primary bronchial epithelial cells (PBECs) and alveolar macrophages. By combining in vitro infection and transwell co-culture models with a global transcriptomic approach, we identified PBECs to be inert to direct Mtb-infection, yet to be potent responders within an Mtb-activated immune network, mediated by IL1β and type I interferon (IFN). Activation of PBECs by Mtb-infected alveolar macrophages and monocytes increased expression of known and novel antimycobacterial peptides, defensins and S100-family members and epithelial-myeloid interactions further shaped the immunological environment during Mtb-infection by promoting neutrophil influx. This is the first in depth analysis of the primary epithelial response to infection and offers new insights into their emerging role in tuberculosis through complementing and amplifying responses to Mtb

Performance of Xpert MTB/RIF in the diagnosis of tuberculous mediastinal lymphadenopathy by endobronchial ultrasound

Rationale: The Xpert (GeneXpert) MTB/RIF, an integrated polymerase chain reaction assay, has not been systematically studied in extrapulmonary and in particular mediastinal tuberculosis (TB). Objectives: To investigate the performance of Xpert MTB/RIF in the diagnosis of intrathoracic nodal TB in a large tertiary urban medical center in the UK. Methods: We collected clinical, cytological, and microbiological data from two cohorts: 116 consecutive patients referred with mediastinal lymphadenopathy with detailed diagnostic information obtained, and an immediately subsequent second cohort of 52 consecutive patients with microbiologically confirmed mediastinal TB lymphadenopathy. All data were derived between January 2010 and October 2012. All patients underwent endobronchial ultrasound and transbronchial needle aspiration (TBNA). The performance of a single Xpert MTB/RIF assay alongside standard investigations, cytology, and microscopy/culture was evaluated against culture-confirmed TB. Measurements and Main Results: Microbiologically confirmed TB mediastinal lymphadenopathy was diagnosed in a total of 88 patients from both cohorts. Three culture-negative cases with associated caseating granulomatous inflammation on TBNA were given a probable diagnosis. A single Xpert MTB/RIF assay demonstrated overall sensitivity for culture-positive TB of 72.6% (62.3–81.0%). Xpert specificity from cohort 1 was 96.3% (89.1–99.1%). The positive predictive value was 88.9% (69.7–97.1%), negative predictive value was 86.5% (76.9–92.1%), and odds ratio was 51.3 (24.0–98.0) for correctly identifying culture-positive disease. Xpert captured all microscopy-positive cases (14 of 14) and the majority of microscopy-negative cases (48 of 71, 67.6%). Among the cases that were culture positive by TBNA, Xpert identified two-thirds of the multiple drug–resistant TB cases, leading to immediate regimen change up to 5 weeks ahead of positive cultures. The use of Xpert combined with cytology increased the sensitivity to 96.6%. Conclusions: Xpert MTB/RIF provides a rapid, useful, and accurate test to diagnose mediastinal nodal TB in intermediate-incidence settings. The additional use of TBNA cytology further enhances the sensitivity of Xpert. This combination can facilitate rapid risk assessment and prompt TB treatment

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

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

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 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

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