RESEARCH ARTICLE
Innate activation of human primary epithelial
cells broadens the host response to
Mycobacterium tuberculosis in the airways
Ann-Kathrin Reuschl1, Michael R. Edwards2, Robert Parker1, David W. Connell1,
Long Hoang1, Alice Halliday1, Hannah Jarvis1, Nazneen Siddiqui1, Corrina Wright3,
Samuel Bremang1, Sandra M. Newton4, Peter Beverley1, Robin J. Shattock5, Onn
Min Kon1, Ajit Lalvani1*
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1 Tuberculosis Research Centre, National Heart and Lung Institute, Imperial College London, St Mary’s
Campus, London, United Kingdom, 2 Department of Cytopathology, Imperial College London, St Mary’s
Hospital, Imperial College NHS Trust, London, United Kingdom, 3 Respiratory Medicine, National Heart and
Lung Institute, Imperial College London, St Mary’s Campus, Norfolk Place, London, United Kingdom,
4 Section of Paediatrics, Department of Medicine, St Mary’s Campus, Imperial College, London, United
Kingdom, 5 Department of Medicine, Imperial College London, St Mary’s Campus, London, United Kingdom
* a.lalvani@imperial.ac.uk
OPEN ACCESS
Citation: Reuschl A-K, Edwards MR, Parker R,
Connell DW, Hoang L, Halliday A, et al. (2017)
Innate activation of human primary epithelial cells
broadens the host response to Mycobacterium
tuberculosis in the airways. PLoS Pathog 13(9):
e1006577. https://doi.org/10.1371/journal.
ppat.1006577
Editor: Thomas R. Hawn, University of
Washington, UNITED STATES
Received: April 23, 2017
Accepted: August 11, 2017
Published: September 1, 2017
Copyright: © 2017 Reuschl et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which
permits unrestricted use, distribution, and
reproduction in any medium, provided the original
author and source are credited.
Abstract
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.
Data Availability Statement: All relevant data are
within the paper and its Supporting Information
files.
Funding: AKR was supported by a PhD
studentship from the National Heart and Lung
Institute (NHLI) Foundation (http://www.nhlif.info
registered charity: 1048073). AL was supported by
a Wellcome Senior Research Fellowship in Clinical
Science, National Institute for Health Research
Senior Investigator Award, and the National
Author summary
Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis, which remains a
major public health burden today. In the majority of cases, infection is acquired by inhalation of aerosolised bacteria. Mtb is thought to target alveolar macrophages in the lower
airways to establish infection. However, the cells predominantly lining the respiratory
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Human primary airway epithelial cells in the response to Mtb infection
Institute for Health Research Health Protection
Research Unit (NIHR HPRU) in Respiratory
Infections (Wellcome trust: https://wellcome.ac.uk
registered charity: 210183; National Institute for
Health Research: http://www.nihr.ac.uk). The
funders had no role in study design, data collection
and analysis, decision to publish, or preparation of
the manuscript.
Competing interests: The authors have declared
that no competing interests exist.
tract are epithelial cells and thus are likely crucial during the early host-pathogen interactions. We recovered primary human bronchial epithelial cells from healthy volunteers to
assess their global transcriptomic response to direct Mtb-exposure and exposure to Mtbinfected myeloid cells. Our analysis revealed that, while being inert to direct Mtb-infection, epithelial cells were highly responsive to soluble mediators released by infected macrophages. The epithelial response induced by this cellular cross-talk, promoted neutrophil
influx in vitro as well as the increase of antimycobaterial host responses. Our data provide
novel and unexpected insights into the role of the primary human airway epithelium and
define a non-redundant role for epithelial cells in shaping the local immunological environment at the site of initial Mtb infection.
Introduction
The first interactions between Mycobacterium tuberculosis (Mtb) and its human host occur in
the lungs after inhalation of aerosolised bacteria. Approximately fifty percent of individuals
exposed to Mtb remain uninfected [1] and host-determinants have been associated with this
resistance to or clearance of infection in humans [2]. The absence of a detectable peripheral
adaptive immune response in resistant individuals suggests that it is mediated by the local
innate immune system and that the respiratory mucosa plays a role in determining the outcome of exposure [3]. It is assumed that the primary target of Mtb, an intracellular pathogen,
is the alveolar macrophage in the lower airways. However, the majority of cells in the airway
lining are epithelial which constitute a surface of approximately 70 m2 [4] and are thus likely
to be the first point of contact for Mtb in the human host.
Epithelial cells are known to significantly contribute to the immune responses in the lungs
and can sense intra- and extracellular pathogens, such as viruses and bacteria, via a wide range
of pattern recognition receptors (PRR) [5]. In contrast to this, their responses in tuberculosis
(TB) are poorly defined and little investigated, which presents a surprising knowledge gap.
Recognition by, or infection of, epithelial cells in the human airways may shape early host
responses to the bacilli and act as an adjunct to the immune response against infections. Work
on human airway epithelial-like cell lines and murine airway epithelial cells has identified
potential mechanisms of recognition and response to Mtb-infection, including chemokine
release and antimicrobial action [6–8]. However, the response of primary human airway epithelial cells to Mtb infection has barely been investigated and has so far mainly concentrated
on isolated pathways rather than the global epithelial response to Mtb-infection [9–11]. The
need to investigate the interactions of Mtb with primary human airway epithelial cells is further underscored by detection of Mtb in these cells during infections in vivo. Previous studies
reported that in animal models of tuberculosis infection the bronchial and alveolar epithelium
is infected after instillation with Mtb [12,13] and Mtb DNA was detected in human epithelial
cells of histologically normal lung tissue in healthy latently infected individuals [14].
In this study, we hypothesised that human primary airway epithelial cells contribute to the
protective early host response to Mtb-infection in the human respiratory tract. To test this, we
interrogated the direct response of epithelial cells to Mtb as well as indirect effects mediated
via an immune-network comprising infected myeloid cells, by measuring the global epithelial
transcriptome. Through in vitro infection and transwell co-culture models, we identified primary bronchial epithelial cells to be inert to direct Mtb-infection, yet to be potent responders
within an Mtb-activated immune network, mediated by IL1 β and type I IFN, with the capacity
to shape the local antimicrobial and immunological environment.
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Human primary airway epithelial cells in the response to Mtb infection
Results
Bronchial epithelial cells dominate the airway lining and are resistant to
Mtb challenge in vitro
In healthy human airways, epithelial cells were the major cell type lining the respiratory tract
and harboured less than 10% of leukocyte subsets (Fig 1A), of which macrophages, lymphocytes and neutrophils contributed on average 86.4%, 12.5% and 1.1%, respectively. The abundance of epithelial cells increases the likelihood that Mtb interacts directly with the epithelial
lining after inhalation of aerosols. To assess whether human airway epithelial cells are permissive to intracellular Mtb-infection, primary bronchial epithelial cells (PBECs) recovered from
bronchial brushes were expanded in vitro and infected with Mtb. The total cell-associated
mycobacterial burden, after amikacin treatment, was over two logs less in PBECs than the
Mtb-burden in THP-1 macrophages after 24h (Fig 1B). The decreased association of Mtb
with PBECs was confirmed by microscopy in the presence of an increased multiplicity of
infection (MOI) (S1 Fig). The minimal level of epithelial infection was reflected by a lack of
Fig 1. Epithelial cells are the major subset in the airway lining and are not permissive to Mtb infection. (A) Presence
of total bronchial epithelial cells (BECs) and leukocytes (LEU) was measured ex vivo 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.
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Human primary airway epithelial cells in the response to Mtb infection
transcriptomic changes in PBECs after exposure to different MOIs of Mtb for 24h (Fig 1C).
Microarray analysis revealed no significant differences in gene expression after statistical
adjustment for multiple comparisons. Since PBECs were poor direct responders to Mtb exposure and infection, yet macrophages were readily infected, we reasoned that the role of PBECs
during the early host response to Mtb might be as part of the Mtb-activated immune network
with myeloid cells as the primary replicative niche for Mtb.
PBECs express antimicrobial peptides and immune mediators in
response to Mtb-induced myeloid activation
After infection with Mtb, macrophages, the largest leukocyte subset in the healthy airways
[15,16], can mount a pro-inflammatory response. To investigate if PBECs, whilst inert to direct
interaction with Mtb bacilli, are affected by these events, we established a contact-independent
co-culture system to measure the global epithelial response to myeloid-derived Mtb-induced
mediators (Fig 2A). THP-1 monocytic cells were used to mimic the myeloid compartment in
the airways and were infected with Mtb or remained uninfected. Microarrays were performed
on PBECs derived from eight healthy donors exposed to these two conditions to model crosstalk in the healthy and infected lungs (Fig 2B). Four-hundred-twenty-eight probesets representing 375 genes were differentially expressed in PBECs exposed to Mtb-infected THP-1 cells
compared to uninfected THP-1 cells (q-value<5%). Seventy of these were at least 1.5 fold differentially expressed (Fig 2B, see S1 Table for full list). The identified expression signature
included cytokines and antimicrobial peptides and 6 of these genes were selected for independent validation by RT-PCR (Fig 3): DEFB4 and S100A7A, which encode known antimicrobial
peptides β-defensin 2 (hBD2) and koebnerisin [17,18]; IL36G which is an emerging IL1-family
member and may be beneficial during tuberculosis infection [19] the chemokines IL8 and
C-X-C motif ligand (CXCL) 10; and two known type I IFN inducible targets interferoninduced protein with tetratricopeptide repeats (IFIT) 1 and interferon-induced protein (IFI)
44. The RT-PCR validation experiment confirmed the gene expression patterns identified by
microarray and showed that the signature was strongly dependent on exposure to infected
THP-1 cells and was not inducible by direct infection of PBECs with Mtb, which confirmed
the epithelial inertia to Mtb. While myeloid infection with virulent Mtb had profound effects
on epithelial gene expression, non-pathogenic M. bovis BCG infection of THP-1 cells at an
MOI of 10 only increased epithelial CXCL10 expression to 9 or 19% of the gene expression
induced by Mtb in two independent experiments, respectively.
THP-1 cells are a valuable model for in vitro studies of myeloid responses to Mtb, and we
confirmed that our model reflected responses in the lungs through the use of primary alveolar
macrophages (AMF). For this, PBECs were exposed to infected primary AMFs from independent donors in co-culture (n = 2, S2 Fig), which confirmed increased expression of DEFB4,
S100A7A, CXCL10 and IFIT1. Transwell co-culture with primary macrophages could only be
performed twice and cell-free supernatant of infected and uninfected AMFs was used to confirm expression patterns in depth (Fig 4). Soluble mediators secreted by AMFs induced similar
transcriptomic responses as observed when THP-1 cells were used. Thus, the epithelial
response to Mtb-induced inflammation we observed in our model mimicked paracrine AMFepithelial signalling that may occur early after exposure of the human airways to Mtb.
PBEC responses to Mtb-induced myeloid inflammation are driven
discretely by IL1β and type I IFN
To identify the Mtb-induced inflammatory signals which drove gene expression in PBECs, we
performed transcription factor binding site (TFBS) enrichment analysis with oPossum which
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Human primary airway epithelial cells in the response to Mtb infection
Fig 2. Whole transcriptome analysis of PBECs exposed to Mtb-infected myeloid cells. (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 poresized 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.
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Human primary airway epithelial cells in the response to Mtb infection
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).
https://doi.org/10.1371/journal.ppat.1006577.g002
Fig 3. PBEC gene expression in response to infection-driven inflammation is dependent on direct interaction between myeloid
cells and Mtb. 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.
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Human primary airway epithelial cells in the response to Mtb infection
Fig 4. Mtb-infected alveolar macrophages drives gene expression in PBECs. 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.
https://doi.org/10.1371/journal.ppat.1006577.g004
revealed that the epithelial expression signature was associated with nuclear factor kappalight-chain-enhancer of activated B cells (NFκB), including RelA and the p105 subunit
(NFKB1), and interferon regulatory factor (IRF)-family members 1 and 2 (Fig 5A). Exposure
of PBECs to myeloid Mtb-infection furthermore induced pathways involved in immune
response signalling, including interferon-driven pathways (Fig 5B). Similarly, gene ontology
was enriched for terms associated with cytokine and IFN signalling (Fig 5C).
It is well known that macrophages secrete several inflammatory mediators in response to
Mtb-infection, including IL1β and IFNβ [20–22]. We found that, IL1β levels in culture supernatants were over threefold enhanced when infected THP-1 cells were co-cultured with PBECs
(Fig 6A). A similar increase was not observed to the same extent when THP-1 cells were
infected with BCG in the presence of PBECs, which resulted in a release of only 28.9–81.2 pg/
ml IL1β during co-culture in three independent experiments. IFN signalling was indicated by
the pathway enrichment analysis and we confirmed IFN β expression and release from myeloid cells to identify whether type I IFNs may have been involved in epithelial activation. We
detected increases in gene expression and protein secretion during Mtb-infection in AMF and
THP-1 cells(Fig 6B and 6C). IFNβ induction co-incided with phosphorylation of STAT1, a
downstream mediator of type I IFN-signalling, in PBECs exposed to Mtb-infected THP-1 cells
(Fig 6D). IFNB was not induced in PBECs during co-culture (S3A Fig) and neither IFNγ nor
IFNλ could be detected by ELISA in culture supernatants of THP-1 cells 24h after Mtb-infection (S3B Fig).
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Human primary airway epithelial cells in the response to Mtb infection
Fig 5. PBEC responses to Mtb-driven inflammation are enriched for pro-inflammatory and interferon-mediated
signalling. 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.
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Stimulation with recombinant cytokines confirmed that IL1β and IFNβ were sufficient to
induce DEFB4 and CXCL10 expression respectively, but not vice versa (S3C Fig). To identify
whether IL1 β or type I IFN could in fact mediate the epithelial signature in response to myeloid Mtb-infection, we abrogated each pathway in co-culture. Neutralising IL1β prevented
upregulation of AMP genes DEFB4 and S100A7A (Fig 7A) as well as pro-inflammatory cytokines IL8 and IL36G (S3D and S3E Fig). Neutralisation of TNF in Mtb-infected co-cultures or
infected THP-1 macrophage monoculture, enhanced the IL1β mediated effects, likely reflecting autocrine activation of THP-1 cells (S4A–S4C Fig). In contrast, genes associated with interferon-signalling, CXCL10 and IFIT1, were unaffected by IL1β neutralisation (Fig 7A) and
dependent on IFNAR2 activation (Fig 7B), which mediates type I IFN signalling. Conversely,
abrogation of IFNAR2 signalling did not affect the expression of the tested IL1β -dependent
genes. Neutralisation of IFNγ had no effect on CXCL10 expression (S4D Fig).
PBECs can shape the antimycobacterial and cellular host response to
infection during Mtb-driven inflammation
DEFB4 and S100A7A, which were strongly expressed in PBECs in response to Mtb-driven
inflammation, were not expressed by Mtb-infected THP-1 cells. Mtb-infection also failed to
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Human primary airway epithelial cells in the response to Mtb infection
Fig 6. IL1β and IFN signalling are activated during Mtb-mediated inflammation. (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. IFNB expression was
measured by RT-PCR and is shown as relative expression (rE) over GAPDH and HPRT. 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.
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induce S100A7A expression in AMFs, while DEFB4 induction ranged from 0.84–14.08 fold in
three healthy donors. Since the release of AMPs may critically determine the outcome of exposure to Mtb, we tested whether the infection-induced defensins and S100A-family members
control free growing Mtb. Human β -defensin 2 (hBD2), which is encoded by DEFB4, has
been previously described to diminish Mtb growth in a resazurin assay [17] and was a potent
inhibitor of growth of Mtb H37Rv (Fig 8A) and clinical strains in our hands (S5A Fig).
Because, no bioactive form of S100A7A was available, we used the close homologue S100A7
[23], known as psoriasin, which was also induced in an IL1β-dependent manner in co-culture
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Human primary airway epithelial cells in the response to Mtb infection
Fig 7. Mtb-driven inflammation activates PBECs in an IL1β and type I IFN signalling dependent
manner. (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 Mtbinfected THP-1 cells (MOI5) in the presence of increasing concentrations of αIFNAR2 (5, 10 and 20 μg/ml) or
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Human primary airway epithelial cells in the response to Mtb infection
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 DEFB4 and S100A7A, respectively.
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(S5B and S5C Fig) and reflected the induction pattern observed for S100A7A (Fig 3). Psoriasin
decreased the median Mtb burden in liquid culture significantly by 62% (Fig 8B).
In addition to local responses to Mtb, influx of immune cells to the site of infection can
shape the human immune response in the lungs. As part of the myeloid-driven epithelial
expression signature identified here, chemokines including CXCL10 and IL8 were induced
through pro-inflammatory and IFN-driven pathways. Infected myeloid cells secrete chemokines which attract leukocytes [24,25] and in addition to this, constitutive or induced release
of mediators by epithelial cells may contribute or amplify timely immune cell recruitment to
the site of infection. To address this, we investigated whether the observed gene expression patterns in PBECs were associated with changes in the chemotactic environment during co-culture compared to infected THP-1 monoculture. THP-1 cells were infected with Mtb in the
presence or absence of PBECs and cell free culture supernatants were harvested after 48h. The
secreted protein content (secretome) was measured through LC-MS. We detected 654 proteins
at significantly different levels in co-culture compared to infected THP-1 monocultures (S2
Table). This included increased levels of IL1β in THP-1 PBEC co-culture, which confirmed
our previous ELISA data (Fig 6A). To identify which of these proteins were differentially
expressed at the gene level in PBECs during co-culture, the gene expression signature was converted into UniProt IDs, which mapped to 341 proteins of which 18 were detected in the transcriptomic and secretome analyses (Fig 9A). Three entities, the leukocyte chemoattractants
IL8, CXCL1 and CXCL10, associated with the GO term ‘chemotaxis’ (GO:0006935, AmiGO 2,
Gene Ontology Consortium). Myeloid-epithelial cross-talk significantly induced their expression in PBECs and the subsequent release into the culture supernatant, thus directly contributing to the chemotactic environment during Mtb-infection (Fig 9B). In fact, in a transwell
chemotaxis assay, confirmed, that the epithelial-driven differences in the mediator environment contributed to cellular influx towards the site of infection. The largest population in
peripheral blood are polymorphonuclear (PMN) cells (S6 Fig), of which neutrophils are rapid
responders during bacterial infections [26]. PMN migration towards cell free cell-culture
supernatants, which was generated in the same ways as the supernatants for secretome analysis, was measured. Supernatants from infected epithelial-myeloid co-cultures attracted on
average approximately 9-fold more PMNs than supernatants from infected THP-1 monocultures (Fig 9C–9F), suggesting that epithelial cells contribute crucially to the leukocyte recruitment to the site of infection.
Discussion
The first step in the natural history of tuberculosis infection, the host-pathogen interaction in
the human airways after Mtb inhalation, is likely shaped by the two dominant cell types in
the lungs: epithelial cells and alveolar macrophages. While macrophages are well known to
respond and interact with Mtb, airway epithelial cells are understudied. We present the first
global transcriptomic profiling of primary human airway epithelial cells in response to Mtb
and Mtb-driven inflammation and found that epithelial cells contribute distinctly to the
host response against Mtb through antimicrobial effectors as well as by amplifying the chemotactic environment initiated by infected macrophages to shape the local immunological
environment.
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Human primary airway epithelial cells in the response to Mtb infection
Fig 8. PBECs express antimycobacterial effectors DEFB4 and S100A7. Mtb H37Rv was grown in the
presence of the indicated concentrations of recombinant (A) hBD2, (B) psoriasin or vehicle control
(veh). 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 (OD595nm) 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.
https://doi.org/10.1371/journal.ppat.1006577.g008
To interrogate the epithelium in an in vitro setting which resembles early immune interactions in the human respiratory tract more physiologically, we established a co-culture model to
allow contact-independent communication between PBECs and infected myeloid cells in real
time. We used THP-1 cells to model myeloid responses in the co-culture system developed
here and confirmed that the epithelial expression patterns are the same in response to Mtbinfected alveolar macrophages or THP-1 cells.
Through biological over-representation analysis of the epithelial transcriptome, we have
uncovered the paracrine activation of epithelial cells by type I IFNs and IL1β. The interplay of
type I IFN and IL1 has recently been elegantly delineated within infected macrophages and has
been proposed as a point of therapeutic intervention during active tuberculosis [27]. While
the cross-regulation of IL1 and type I IFN signalling in the Mtb-infected macrophage is an
important well-documented rapidly-evolving area, our findings revealed for the first time that
these pathways also act on the epithelial lining. In contrast to the induction patterns of IL1 and
type I IFN within macrophages [20] and their respective cross-regulation, the paracrine epithelial activation induced by these two mediators occurred independently of each other with
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Human primary airway epithelial cells in the response to Mtb infection
Fig 9. PBECs upregulate chemokines and enhance neutrophil recruitment to the site of Mtb-infection. (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
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Human primary airway epithelial cells in the response to Mtb infection
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.
https://doi.org/10.1371/journal.ppat.1006577.g009
abrogation of either signalling pathway leaving gene expression of the opposite mediator
untouched.
The activation of epithelial cells through Mtb-driven myeloid-derived type I IFNs has not
previously been reported. Despite the association of type I IFNs with neutrophil-driven disease
severity of tuberculosis in humans [28] and mice [29], the effect of type I IFNs on the outcome
of Mtb-infection early after exposure is yet to be determined. Early myeloid-driven activation
of IFN-signalling may be beneficial for the host as evidenced by the finding that IFN I and
IFNγ are both required for optimal immune cell recruitment during the initial pre-adaptive
phase of Mtb-infection in a mouse model [30]. Our findings further support this concept and
showed that type I IFN signalling is required to induce epithelial transcription of the chemotactic factor CXCL10 during Mtb-driven inflammation in humans. This activation, together
with IL1β-mediated chemokine expression, amplifies cellular influx initiated by infected myeloid cells.
We observed that PBECs significantly enhance the neutrophil influx induced by infected
myeloid cells and suggest that they may support rapid recruitment to the initial site of infection
in vivo. Neutrophils are important early antimicrobial effectors with poor specificity. While
they can cause substantial tissue damage and inflammation, their antimicrobial properties are
crucial to control and prevent recurrent infections in humans [31]. Tissue neutrophilia is associated with pathogenesis during active TB; however, influx of neutrophils early after infection
has been suggested to be beneficial for host control of the pathogen. Absence of neutrophils
during early mycobacterial infection results in increased bacterial burden in mice and zebrafish [32,33] and growth control of mycobacteria by whole blood in vitro is strongly dependent
on neutrophils [34] which may be an important function at the site of infection.
Besides the release of chemokines, myeloid-derived IL1β mediated the antimicrobial
responses elicited in PBECs, including induction of DEFB4 expression, in accordance with
previously reported findings [9]. We further confirmed that DEFB4 was a potent direct antimycobacterial effector. While β-defensins are known to kill mycobacteria [17], IL1β-induced
S100-family members have not previously been considered to be active against Mtb. Whilst
both S100A7A (encoding koebnerisin) and S100A7 (encoding psoriasin) are antibacterial effectors in the skin, neither were known to have antimycobacterial activity prior to our current
discovery that psoriasin limited Mtb growth in liquid culture.
IL1β which mediated AMP-expression, is increased in the airways during active TB [35];
and may already be elevated early after Mtb-infection of alveolar macrophages before the
onset of detectable lung pathology. This would then result in the activation of epithelial antimicrobial responses at the earliest stages of the natural history of tuberculosis in the human airways. Interestingly, IL1β -dependent epithelial AMP-expression was further enhanced by TNF
in PBECs during co-culture with infected myeloid cells. While augmentation of TNF signalling
by IL1β has been described in macrophages before [36], we believe that this is the first time
that IL1β activation was shown to be enhanced by TNF in Mtb-infection. Epithelial antimicrobial peptides during infection may contribute to host defense against free extracellular mycobacteria thus preventing or diminishing cell to cell spread in early infection. They may also
be a promising therapeutic target, since their activity is not affected by multidrug resistance
of Mtb. Additionally, the identification of epithelial-specific AMP-expression with anti-
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mycobacterial activity may complement myeloid responses to Mtb in the airways, since monocytes and macrophages do not express high levels of the AMPs identified here [37,38].
Prior studies of macrophage-driven activation of epithelial cells focussed solely on specific
pathways [9,11], involving matrix metalloproteinases (MMPs) or DEFB4. Our data provide the
first global perspective on the epithelial response to Mtb infection, and reveal important new
targets in the epithelial response to Mtb-infection while simultaneously confirming the previously published findings. In transwell co-culture, a large proportion of the expression signature (112 genes) was associated with the innate immune response, including type I IFN and
cytokine-mediated signalling. IL36G, which is an emerging IL1-family member, was dependent on myeloid-derived IL1 β. It has recently been described to have a host-protective role
during Mtb-infection [39] and its epithelial release may support macrophage function further.
As part of the type I interferon-inducible mediators, the known antiviral factor ISG15 was
upregulated and its secretion during mycobacterial infection is involved in the appropriate
induction of IFNγ in humans [40]. These are only two of several genes uncovered by the coculture model we have established, which provide interesting targets for future investigations,
but where further analysis into their function was beyond the scope of this study.
In contrast to their multifaceted responses to myeloid Mtb-infection, PBECs were surprisingly inert to direct stimulation with Mtb. This was not due to a general non-responsiveness to
PRR-stimulation, as PBECs mount a substantial IL8 response towards synthetic TLR-agonists
and in response to Streptococcus pneumoniae (Reuschl et al, manuscript in preparation). The
global assessment of the transcriptomic response to direct stimulation with live Mtb revealed
that no genes were significantly upregulated after 24h and this was complemented by poor
invasion and adherence of Mtb to epithelial cells. This finding is consistent with reports of
poor invasion of Mtb into primary tracheal epithelial cells in comparison to dendritic cells
[41]. Interestingly, two days after intra tracheal Mtb-infection of mice, which results in locally
very high density of Mtb bacilli, the majority of bacilli are found in macrophages and only
approximately 10% of intracellular Mtb can be detected in epithelial cells [13]. Epithelial inertia to Mtb and the lack of uptake in humans may thus ensure the uptake of Mtb by local professional phagocytes, the preferred target host cell of Mtb after inhalation in the human lungs.
Our co-culture model did not include endothelial cells and it remains to be seen whether interaction with an endothelial layer would augment the epithelial response to Mtb. Our findings
contrast with previous studies, which described epithelial susceptibility to infection and upregulation of chemokines in A549 cells within 24h [10,42–44]. This most likely reflects differences between primary cells and cell lines, as A549 cells are adenocarcinoma-derived and may
not truly mimic the responses of healthy human respiratory epithelium [45]. Through the
recovery of PBECs from several donors, we have overcome the limitations of using a single cell
line and reflected true biological variation in humans.
In vivo, the airway epithelium is at an air-liquid-interface, polarised and differentiated.
However, in our model, the epithelial cells were undifferentiated and in submerged culture
which allowed for comparison with the existing literature. Mediator release of undifferentiated
epithelium to viral infection or air pollution particles surpasses the release by differentiated
epithelium [46,47] suggesting that the inert phenotype of PBECs in response to Mtb is not an
underestimate of their in vivo response and is moreover consistent with the lack of epithelial
infection observed in mice in vivo [13].
We expanded and studied human primary cells in vitro, as opposed to using respiratory epithelial cell lines or mouse models. A limitation of our study is the focus on primary bronchial
epithelial cells which, unlike alveolar epithelial cells, can be obtained by bronchoscopy. The
recovery of pure alveolar epithelial cells requires samples from resections of dissected lungs
or post-mortem cadaveric explants which may not reflect the responsiveness of healthy
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Human primary airway epithelial cells in the response to Mtb infection
epithelium, and their phenotype is difficult to maintain in vitro [48]. Primary alveolar epithelial
cells should be used to address whether our findings extend to human alveolar epithelial cells.
This would be of interest as differences in the responsiveness to pro-inflammatory mediators
between upper and lower airway epithelial cells have been previously described [49,50]. Myeloid-epithelial cross-talk was independent of cell-cell contact, providing evidence that the
bronchial epithelium is able to respond to Mtb-infection upon activation by secreted mediators which diffuse from the site of infection throughout the airspaces. In the future, it would be
important to assess the mechanisms identified in our study in PBECs derived from patients
with active pulmonary tuberculosis, where epithelial responsiveness may be altered. Most of
the epithelial cells within the human airways will not be in direct contact with the small number of infected alveolar macrophages. The majority of the initial epithelial response would
therefore be expected to be driven by soluble mediators. Our experimental rationale therefore
focussed on contact-independent interactions between infected myeloid and primary epithelial cells. Based on our findings, future work should also include direct interactions between
the myeloid and epithelial compartment during infection to assess how cell-cell contact modulates the response to Mtb.
Our findings shed light on the contribution of the respiratory epithelial lining to the
response against Mtb as well as the complexity of the immune response in the airways early
after infection. It is known from challenge studies with rhinovirus in humans and ex vivo infection of bronchial tissue that only a limited number of epithelial cells are actively infected
[51,52], yet potent immune activation occurs throughout the epithelial lining. Given that infection with Mtb requires only very few aerosolised mycobacteria [53,54], amplification of the
macrophage-response to infection by uninfected surrounding epithelium may very well be
decisive for the outcome of infection. Appropriate animal models of Mtb-infection should be
employed to assess the reported epithelial responses in vivo during early infection.
In summary, we show that PBECs are inert to direct early Mtb-infection, but potent
responders to infected alveolar macrophages. PBECs contributed to host defense as part of an
Mtb-activated immune network through pathways, which are likely protective early after
infection, by creating an extracellular antimicrobial milieu and promoting early neutrophil
influx. Through these cell-specific differential responses to myeloid-derived cytokines, airway
epithelial cells likely fulfil a non-redundant role in the human pulmonary host response to
Mtb as vital players with the potential to shape the human innate immune response to infection and influence the outcome of exposure to Mtb.
Methods
Recruitment and ethics statement
PBECs and AMFs were collected through bronchial brushings and bronchoalveolar lavage
from the airways of healthy volunteers during bronchoscopic procedure at St. Mary’s Hospital,
London, United Kingdom. All volunteers were adults and recruited through advertisements or
from Contact and TB clinics at Imperial College NHS Trust (St. Mary’s Hospital), London
North West Healthcare NHS Trust (Ealing Hospital, Northwick Park Hospital) and Barts
Health NHS Trust (Newham Chest Clinic). All samples were collected in accordance with the
Human Tissue Act 2004 and written informed consent was obtained from all participants
(National Research Ethics Service approval reference 07/H0712/85+5).
Cell culture
Bronchial epithelium was recovered from the right lower lobe of healthy volunteers through
cytological brushes (Olympus Keymed, Southend-on-Sea, UK). PBECs were cultured from up
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Human primary airway epithelial cells in the response to Mtb infection
to three brushes per volunteer and expanded in supplemented bronchial epithelial growth
medium (BEGM) (Lonza, Walkersville, USA) as previously described [55]. To prevent the outgrowth of recovered contaminating bacteria or fungi, the growth medium was supplemented
with gentamycin and amphothericin-B as per the manufacturer’s instruction. All cultures
showed the characteristic cobblestone appearance of bronchial epithelial cells. PBECs were
used in experiments after two passages. Cells were seeded at 8x104/ml in collagen/fibrinogencoated tissue culture plates and grown to confluence. BEGM was then changed to bronchial
epithelial basal medium (BEBM) over night before experimentation. To identify cell subsets in
the epithelial lining, ex vivo differential cell counts of one separate brush was obtained. Sample
sizes given in figure legends refer to biological replicates from independent donors.
Alveolar macrophages (AMF) were recovered from bronchoalveolar lavage and isolated
through overnight adherence to tissue culture plastic. AMF were cultured in RPMI with 10%
human serum and 50μg/ml gentamycin. For infection experiments, medium was changed to
medium with 5% human serum or BEBM. AMF were used at 106/ml for experiments.
Peripheral blood leukocytes (PBL) were isolated from heparinised whole blood. Red blood
cells were removed trough addition of 10 fold excess RBC lysis buffer (Biolegend, San Diego,
USA) for 10 min.
THP-1 cells were obtained from American Type Culture Collection (ATCC) and grown
RPMI with 10% FBS, 50000 U penicillin, 50 mg streptomycin (Sigma-Aldrich, Poole, UK) and
0.05 mM β-mercaptoethanol. For macrophage experiments, THP-1 cells were differentiated
with 50nM phorbol 12-myristate 13-acetate (PMA) for 24h, seeded in tissue culture plates and
rested overnight in complete medium without PMA. THP-1 cells were used at 106/ml for
experiments. Before infection experiments, culture medium was replaced with infection
medium containing 5% human serum or BEBM.
Mycobacterial culture
Mtb H37Rv, M. bovis BCG (SSI) or the clinical isolates Mtb CH [56] and Mtb NPH4216 [57]
were cultured in 7H9 Middlebrook medium supplemented with 10% OADC, 0.5% glycerol,
0.05% Tween-80 and 10 μ g/ml amphotericin. Cultures were harvested in mid-log phase and
frozen down in 15% glycerol. To standardise infection doses, cells were infected from frozen
stocks and multiplicity of infections determined based on the mycobacterial counts of the
stocks. For determination of colony forming units (CFU), mycobacteria were grown on 7H10
agar plates supplemented with 10% OADC, 0.5% glycerol and 0.05% Tween-80 and cultured
for 3–4 weeks at 37˚C. Antimycobacterial effects of selected peptides were performed in 96
well plates. hBD2 (PeproTech, London, UK), S100A7/Psoriasin (a kind gift from Prof. Joachim
Grötzinger [23]) and controls were diluted in fresh 7H9 and Mtb bacilli harvested in mid-log
phase was added. Plates were incubated shaking for up to 7 days at 36˚C. Culture growth was
monitored by measuring optical density at 595nm. OD-readings from 7H9-only controls were
subtracted from all culture conditions before the data was analysed. On day 7, cultures were
plated on 7H10 for CFU enumeration.
Mtb-infection experiments
For infections, Mtb bacilli were diluted in cell culture medium, washed by centrifugation and
taken up in cell culture medium for infection experiments. Mtb was sonicated for 40 seconds
in an ultrasonic waterbath (Grant) to disperse clumps before inoculation of cell culture. For
Mtb-uptake and adherence to THP-1 MF or PBECs. Mtb bacilli were added onto cells for
24h. Infection was determined by cell lysis with 0.1% Triton-X/PBS-Tween80. Lysates were
serially diluted in PBS-Tween80 and plated on 7H10 agar. Association of Mtb with PBECs was
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Human primary airway epithelial cells in the response to Mtb infection
visualised for representative experiments by Kinyoun stain with a Tb-color kit (Merck, Darmstadt, Germany) and images were acquired with an Axio Scope. A1 microscope (Zeiss, Rugby,
UK). For some experiments, 200 μg/ml amikacin (Sigma) was added for 2h before cell lysis. To
measure mediator release or gene expression, myeloid cells or PBECs were infected with Mtb
at the indicated MOI and supernatants or RNA harvested at the indicated time.
For transwell experiments, PBECs were seeded into tissue culture plates then 0.4 μm cell
culture inserts (Millipore) were placed on top with THP-1 cells added to them. Mtb was added
to either compartment as desired (MOI5 over THP-1). For blocking and neutralisation of
cytokine signalling, 20 μg/ml of antibodies (αIFNα/ β R2 (MMHAR-2, R&D Systems), α IFNγ
(K3.53, R&D Systems), α IL1β (2805, R&D Systems), α TNF (1825, R&D Systems), mIgG1
(11711, R&D), mIgG2A (20102, R&D Systems) were added to the bottom well 45 min before
transwell inserts and THP-1 cells were added. Culture supernatants were harvested from the
tissue culture wells after inserts were removed.
Supernatant transfer experiments
PBECs were exposed to soluble mediators released from uninfected or infected AMФ. Culture
supernatants from AMФ or cell-free incubation controls ± Mtb bacilli were harvested after
24h and 0.22 μm sterile filtered. Supernatants were diluted 1:10 in fresh BEBM, added to
PBECs and total RNA was harvested after 24h.
Microarrays and RT-PCR
RNA was extracted with the TRIzol Plus RNA Purification Kit (Ambion) and treated with
DNAse I (Thermo, Epsom, UK) to remove residual genomic DNA. For RT-PCR, RNA quality
and quantity was measured by Nanodrop. RNA was converted to cDNA using Maxima reverse
transcriptase and random hexamers. Solaris Gene Expression and Taqman Expression assays
were used to determine gene expression levels by RT-PCR. RT-PCR reactions were performed
in duplicate with 12.5 ng of cDNA. Expression data were analysed with Biogazelle qbase+ and
normalised to ACTB and PGK according to [58]. Solaris Gene Expression assays: ACTB (AX003451), CXCL10 (AX-007871), DEFB4A (AX-012997), IFIT1 (AX-019616), IFNB1 (AX019656), IL36G (AX-007959), IL8 (AX-004756), PGK (AX-006767), S100A7A (AX-027145).
Taqman Expression assays: S100A7 (Hs01923188_u1).
For microarrays, RNA quality was assessed by Bioanalyzer (Agilent, Stockport, UK)
(RIN > 9 for all samples) and RNA was quantified by Qubit (Thermo). 50-100ng total RNA
was prepared for GeneChip Human Transcriptome Array 2.0 with the GeneChip WT PLUS
Reagent Kit (Affymetrix, High Wycombe, UK). Samples were hybridized and scanned at the
MRC Genomics Laboratory (Hammersmith Campus, Imperial College London).
Soluble mediators
Soluble mediators were measured in cell culture supernatants after double-filtration through
0.22 μm centrifuge spin filters. Human IL1β, IFNγ and IFNλ were measured by DuoSet ELISAs (R&D Systems). IFNβ was measured by VeriKine Human IFN Beta ELISA kit (pbl interferon source).
Secretome analysis
The secretome of Mtb-infected THP-1 cells and Mtb-infected PBEC-THP-1 co-cultures was
measured by liquid chromatography—mass spectrometry (LC-MS). Six paired cell culture
supernatants were analysed using two independent PBEC cultures. Cell culture supernatants
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Human primary airway epithelial cells in the response to Mtb infection
were harvested at 48h and sterile filtered twice through 0.22 μm centrifuge filters. 500 μl of culture supernatants were buffer exchanged twice with 450 μl of 2 M urea, 100 mM Tris-HCL
(pH 8.0) using a 3 kDa nominal molecular weight limit cut-off spin filter (Millipore). A volume
of 80 μl of sample was reduced with 5 mM DTT (Sigma) for 30 min at 60˚C and alkylated with
10 mM of iodoacetamide (Sigma) in the dark at room temperature for 30 min. 1 μg Trypsin/
Lys-C mix (Promega) was added for 18 h at 37˚C. Samples were adjusted to 1% (v/v) trifluoroacetic acid (TFA) (Sigma)and peptide digests were purified using the C18 STop And Go
Extraction (STAGE) tips [PMID: 16602707] and resuspended in 2% (w/v) acetonitrile, 0.1%
(v/v) fomic acid for LC-MS. Samples were analysed using an EASY-nLC 1000 Liquid chromatography system coupled to a Q-Exactive mass spectrometer. The separation column and emitter was an EASY-Spray column, 50 cm x 75 μm ID, PepMap C18, 2 μm particles, 100 Å pore
size. Buffer A was 2% acetonitrile, 0.1% formic acid and buffer B 100% (v/v) acetonitrile, 0.1%
(v/v) formic acid. A gradient from 5% to 40% acetonitrile over 120 min was used to elute peptides for ionization by electrospray ionisation (ESI) and data dependent MS/MS acquisition
consisting of 1 full MS1 (R = 70K) scan acquisition from 350-1500m/z, and 10 HCD type MS2
scans (R = 15K). MS/MS charge targets were limited to 1E6 and iosolation window set to 2.0
m/z, monoisotopic precursor selection, charge state screening and dynamic exclusion were
enabled, charge states of +1, >4 and unassigned charge states were not subjected to MS2 fragmentation. Raw mass spectra were identified and quantified using Maxquant 1.5.15 using a 1%
peptide and protein FDR. Searches were conducted against the Uniprot SwissProt database
downloaded on 06/06/2014. The database was supplemented with common contaminant proteins introduced during proteomic experiments. Searches were specified as tryptic with 1
missed cleavage, 7 ppm precursor ion mass tolerance, 0.05 Da fragment ion mass tolerance,
fixed modifications of carbamidomethylation (C), and variable modification of oxidation (M),
acetylation (N-term, Protein). Protein Label free quantitation (LFQ) intensity measured in
medium alone was subtracted from all paired samples, and LFQ values were log2 transformed.
To identify both qualitative and quantitative effects of stimulation on extracellular all missing
values were replaced assuming a normal distribution (width 0.3, and a down shift of 1.8) for
only control/unstimulated samples as described in [59].
Western blot
For protein lysates PBECs were treated with RIPA buffer (Pierce) supplemented with 2x Halt
Protease and Phosphatase Inhibitor Cocktail (Pierce) and 250 U Benzonase (Sigma) for 15
min on ice. Lysates were centrifuged at 14000 rpm for 15 min at 4˚C. Total protein concentration was measured by BCA protein assay (Pierce). 12 μg of protein lysates lysates were mixed
with Laemmli buffer (BioRad, Hertfordshire, UK) and 25 mM DTT and boiled for 7 min. Proteins separated on 4%-20% Mini PROTEAN gels (BioRad) and transferred onto nitrocellulose
membranes by iBlot (Invitrogen). Membranes were blocked in 5% milk/TBST for 1h and
probed with primary antibodies (αbeta-Actin (D6A8, New England Biolabs), αPhospho-Stat1
(Tyr701) (D4A7, New England Biolabs), αStat1 (#9172, New England Biolabs)) at 1:1000 in
5% BSA/TBST over night at 4˚C. HRP-linked IgG (New England Biolabs) (1:2000) in 5%
BSA/TBST was added the next day for 1h. Membranes were developed with ECL Western blotting substrate (Pierce) and imaged with FUSION FX7 SPECTRA (Vilber).
PBL phenotyping by flow cytometry
PBLs were isolated and resuspended in PBS with 0.5% BSA and 2mM EDTA (FACS buffer).
Before staining, 10% human serum in FACS buffer was added to cells for 20 min to block Fc
receptors. For surface staining, PBLs were incubated with α CD14-Brillian Violet 421 (M5E2,
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Human primary airway epithelial cells in the response to Mtb infection
Biolegend), α CD15-Brillian Violet 605 (SSEA-1, Biolegend), α CD3-PE-CF594 (UCHT1, BD)
and α CD66b-PerCP/Cy5.5 (G10F5, Biolegend). Cells were treated with Cytofix Fixation
buffer, resuspendend in FACS buffer and left overnight before flow cytometric acquisition
using either a BD LSR II or BD Fortessa. Anti-Mouse Ig compensation beads (BD) were used
to determine compensation parameters.
PBL transwell migration assay
Cell free culture supernatants were generated through direct co-culture of Mtb-infected THP1 cells in the presence of absence of PBECs for 48h. BEBM was used as a background control
for unspecific migration. Conditioned or control medium was added to the bottom well of a
tissue culture plate and 0.5 μm transwell inserts (Corning) placed on top. The plates were
placed in a humified 37˚C CO2 incubator for 30 min to equilibrate. 2.5x105 PBLs were added
to the transwell insert and the plate was placed back into the incubator for cell migration. After
3h, 2mM EDTA was added to the bottom compartment to dislodge cells from the transwell
membrane. Cells were collected from the insert and bottom compartment, fixed and resuspended in equal volumes of FACS buffer. Cells in each sample were enumerated through
acquisition for 120 s using a BD LSR II flow cytometer.
Statistical analysis
Microarray data was normalised by Robust Multi-Array Average (RMA) using Partek Genomic Suite 6. All genes annotated by NCBI Reference Sequence Database (RefSeq), NIH GenBank or Ensembl were taken forward for statistical analysis. One-Way ANOVA on microarray
data was performed with PGS. For SAM analysis of expression and secretome data with TIGR
MultiExperiment Viewer 256 or 64 permutations were used, respectively. Pathway analysis
was performed with InnateDB for over-representation analysis [60]. Transcription factor binding site enrichment on differentially expressed genes was performed using oPossum [61].
Flow cytometry data was analysed using FlowJo v10. Statistical analysis of all other experiments was performed with GraphPad Prism 6 and is indicated where appropriate.
Supporting information
S1 Fig. Association of Mtb with PBECs visualised by Kinyoun stain. PBECs were infected
with Mtb H37Rv (MOI50) for 24h. The association of Mtb with PBECs was confirmed microscopically via Kinyoun stain after 24h of infection at MOI 50. Shown are representative images
from two donors at a 200x magnification. Arrowheads indicate Mtb.
(TIF)
S2 Fig. PBEC gene expression in response to AMФ in transwell co-culture. AMФ from two
healthy donors were co-cultured in the transwell model with PBECs and infected with Mtb
H37Rv (MOI5) as indicated. Gene expression was measured by RT-PCR.
(TIF)
S3 Fig. Interferon-release and gene expression in response to infection, transwell co-culture and recombinant cytokines.
A) PBECs were stimulated in the transwell co-culture system as indicated and IFNB expression
measured after 24h. Fold change was calculated over unstimulated cells. Boxplots show
median and range (n = 5). No significant differences were detected.
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Human primary airway epithelial cells in the response to Mtb infection
B) IFNγ and IFNλ release by THP-1 cells after 24h of Mtb-infection (MOI5) was measured by
ELISA in two independent experiments. Mean ± SD. n.d., not detected; φ, extrapolated values below the detection limit; horizontal lines indicate the detection limits of the assays.
C) PBECs were stimulated with 1 ng/ml IL1β or IFNβ for 24h and gene expression was measured by RT-PCR (n = 3). Mean ± SD are shown.
D) PBECs were co-cultured with Mtb-infected THP-1 cells in the presence of 20 μg/ml 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.
E) PBECs were co-cultured with Mtb-infected THP-1 cells in the presence of 20 μg/ml
αIFNAR2 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 control. n.s.,
not significant; , p<0.05.
(TIF)
S4 Fig. Effects of TNF and IFNψ on PBEC-myeloid co-culture.
(A) PBECs were co-cultured with Mtb-infected (MOI5) THP-1 cells with αTNF or IgG1 for
24h. DEFB4 expression was measured by RT-PCR and is shown as fold change over unstimulated PBECs (n = 3).
(B) IL1β release was measured in the co-culture supernatants of (A) by ELISA.
(C) THP-1 MFs were infected with Mtb (MOI5) in the presence of αTNF or IgG1 and IL1β
release measured after 24h. Cytokine levels are shown as % of IL1 β release during infection
in the presence of IgG1 (n = 5).
(D) PBECs were exposed to Mtb-infected THP-1 cells (MOI5) in co-culture in the presence of
αIFNγ or IgG2a. After 24h, CXCL10 expression was measured by RT-PCR and is shown as
fold change over unstimulated PBECs.
Mean ± SD are shown. (A, B and D) Wilcoxon signed rank test was used to compare
groups; (C) was compared by repeat-measure ANOVA with Holm-Sidak’s multiple comparisons test. , p<0.01 or exact p-values are given.
(TIF)
S5 Fig. Antimycobacterial effects of hBD2 and expression of S100A7 in PBECs during
transwell co-cultures.
(A) Clinical isolates Mtb NPH4216 and Mtb CH were incubated with 5 μg/ml recombinant
hBD2 or vehicle control as described in Fig 8. Colony forming units (CFU) were determined at day 7. Effects of hBD2 was compared with vehicle control by Student t-test.
Mean ± SD of triplicate measurements are shown. p<0.05; p<0.01
(B) In the transwell model, PBECs were exposed to THP-1 cells or Mtb H37Rv (MOI5 over
THP-1) for 24h as indicated. S100A7 expression in PBECs was measured by RT-PCR and is
shown as fold change over unstimulated PBECs (n = 5).
(C) PBECs were co-cultured with infected or uninfected THP-1 cells in the presence of αL1β
or IgG1 as indicated. After 24h, S100A7 expression was measured by RT-PCR and is shown
as fold change over unstimulated PBECs (n = 5).
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Human primary airway epithelial cells in the response to Mtb infection
Friedman test with Dunn’s post-test was used to compare expression with unstimulated or
respective isotype control. Boxplots show median and range. p<0.05; p<0.01.
(TIF)
S6 Fig. Gating strategy for PBL transwell migration experiments. PBLs were isolated from
whole blood and stained for CD3, CD14, CD15 and CD66b. Shown are representative plots
for the gating strategy from one of three donors. After gating for singlets, forward (FSC) and
side (SSC) scatter were used to define PBL subsets. PMN, polymorphonuclear cells.
(TIF)
S1 Table. Differentially expressed genes in PBECs exposed to Mtb-infected THP-1 cells in
transwell co-culture. Significantly differentially expressed genes at a q-value < 5% were determined by Significance Analysis of Microarrays.
(XLSX)
S2 Table. Secretome of Mtb-infected THP-1 monocultures and co-cultures with PBECs.
Significantly differentially secreted proteins identified in cell-free culture supernatants of Mtbinfected THP-1 cells co-cultured with PBECs compared to infected THP-1 monoculture at a
q-value < 5% (determined by SAM).
(XLSX)
Acknowledgments
We thank all volunteers for participating in the study. We thank the staff of the Endoscopy
Unit at St. Mary’s Hospital, the contact clinics at St. Mary’s Hospital and Ealing Hospital, especially O.M. Kon and H. Branley. We are grateful for the recruitment of participants by S. Bremang, L. Grass, H. Uzu, H. Piotrowski and A. Badhan. We are grateful to M. Noursadeghi for
critically reviewing the manuscript. I. Andrews (MRC Genomics Laboratory, Imperial College
London) for hybridizing and scanning the microarrays. Recombinant psoriasin was a kind gift
from J. Grötzinger, Univeristy of Kiel.
Author Contributions
Conceptualization: Ann-Kathrin Reuschl, Ajit Lalvani.
Formal analysis: Ann-Kathrin Reuschl, Robert Parker.
Funding acquisition: Ajit Lalvani.
Investigation: Ann-Kathrin Reuschl, Robert Parker, Alice Halliday.
Methodology: Ann-Kathrin Reuschl, Robert Parker, Long Hoang, Nazneen Siddiqui.
Resources: Michael R. Edwards, Robert Parker, David W. Connell, Long Hoang, Alice Halliday, Hannah Jarvis, Nazneen Siddiqui, Corrina Wright, Samuel Bremang, Sandra M. Newton, Onn Min Kon.
Supervision: Michael R. Edwards, Peter Beverley, Robin J. Shattock, Onn Min Kon, Ajit
Lalvani.
Visualization: Ann-Kathrin Reuschl, Long Hoang.
Writing – original draft: Ann-Kathrin Reuschl, Robert Parker, Peter Beverley, Robin J. Shattock, Onn Min Kon, Ajit Lalvani.
Writing – review & editing: Ann-Kathrin Reuschl, Ajit Lalvani.
PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1006577 September 1, 2017
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