Mycobacteria Bypass Mucosal NF-kB Signalling to Induce an Epithelial AntiInflammatory IL-22 and IL-10 Response.
Lutay, Nataliya; Håkansson, Gisela; Alaridah, Nader; Hallgren, Oskar; Westergren-Thorsson,
Gunilla; Godaly, Gabriela
Published in:
PLoS ONE
DOI:
10.1371/journal.pone.0086466
2014
Link to publication
Citation for published version (APA):
Lutay, N., Håkansson, G., Alaridah, N., Hallgren, O., Westergren-Thorsson, G., & Godaly, G. (2014).
Mycobacteria Bypass Mucosal NF-kB Signalling to Induce an Epithelial Anti-Inflammatory IL-22 and IL-10
Response. PLoS ONE, 9(1), [e86466]. https://doi.org/10.1371/journal.pone.0086466
Total number of authors:
6
General rights
Unless other specific re-use rights are stated the following general rights apply:
Copyright and moral rights for the publications made accessible in the public portal are retained by the authors
and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the
legal requirements associated with these rights.
• Users may download and print one copy of any publication from the public portal for the purpose of private study
or research.
• You may not further distribute the material or use it for any profit-making activity or commercial gain
• You may freely distribute the URL identifying the publication in the public portal
Read more about Creative commons licenses: https://creativecommons.org/licenses/
Take down policy
If you believe that this document breaches copyright please contact us providing details, and we will remove
access to the work immediately and investigate your claim.
Mycobacteria Bypass Mucosal NF-kB Signalling to Induce
an Epithelial Anti-Inflammatory IL-22 and IL-10 Response
Nataliya Lutay1, Gisela Håkansson1, Nader Alaridah1, Oskar Hallgren2, Gunilla Westergren-Thorsson3,
Gabriela Godaly1*
1 Division of Laboratory Medicine, Department of MIG, Lund University, Lund, Sweden, 2 Division of Clinical Sciences, Department of Respiratory Medicine and
Allergology, Lund University, Lund, Sweden, 3 Division of Vascular- and Respiratory Research Unit of Lung Biology, Department of Experimental Medical Science, Lund
University, Lund, Sweden
Abstract
The mechanisms by which mycobacteria subvert the inflammatory defence to establish chronic infection remain an
unresolved question in the pathogenesis of tuberculosis. Using primary epithelial cells, we have analysed mycobacteria
induced epithelial signalling pathways from activation of TLRs to cytokine secretion. Mycobacterium bovis bacilli CalmetteGuerin induced phosphorylation of glycogen synthase kinase (GSK)3 by PI3K–Akt in the signalling pathway downstream of
TLR2 and TLR4. Mycobacteria did not supress NF-kB by activating the peroxisome proliferator-activated receptor c. Instead
the pro-inflammatory NF-kB was bypassed by mycobacteria induced GSK3 inhibition that promoted the anti-inflammatory
transcription factor CREB. Mycobacterial infection did not thus induce mucosal pro-inflammatory response as measured by
TNFa and IFNc secretion, but led to an anti-inflammatory IL-10 and IL-22 production. Apart from CREB, MAP3Ks p38 and
ERK1/2 activated the transcription factor AP-1 leading to IL-6 production. Interestingly, blocking of TLR4 before infection
decreased epithelial IL-6 secretion, but increased the CREB-activated IL-10 production. Our data indicate that mycobacteria
supress epithelial pro-inflammatory production by supressing NF-kB activation thereby shifting the infection towards an
anti-inflammatory state. This balance between the host immune response and the pathogen could determine the outcome
of infection.
Citation: Lutay N, Håkansson G, Alaridah N, Hallgren O, Westergren-Thorsson G, et al. (2014) Mycobacteria Bypass Mucosal NF-kB Signalling to Induce an
Epithelial Anti-Inflammatory IL-22 and IL-10 Response. PLoS ONE 9(1): e86466. doi:10.1371/journal.pone.0086466
Editor: Patricia T. Bozza, Fundação Oswaldo Cruz, Brazil
Received June 6, 2013; Accepted December 13, 2013; Published January 28, 2014
Copyright: ß 2014 Lutay 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.
Funding: This study was supported by a Research Scientist Grant from the Swedish Medical Research Council (2005-7364, 7364, 11550); the Crafoord Foundation;
the Heart and Lung Foundation; Evy and Gunnar Sandberg Foundation and the Medical Faculty of Lund University, Sweden. 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.
* E-mail: Gabriela.godaly@med.lu.se
lipoarabinomannan (LAM) and the cell wall-associated and
secreted 19-kDa glycolipoprotein, activate TLR signalling
[4,6,13,14]. TLR2 and TLR4 are also known to modulate the
activation of peroxisome proliferator-activated receptor (PPAR)c
[15] that mycobacteria utilize to affect the NF-kB activation
[16,17]. Ligand binding to TLR initiates a signalling cascade
through a MyD88-dependent and/or a MyD88-independent gene
expression [18]. The MyD88-dependent activation leads to a proinflammatory cytokine response by the IRAK-NF-kB pathway,
but also to chromatin remodelling by the MAPK kinases that
regulates extracellular signal-regulated kinase 1/2 (ERK1/2), p38
proteins and c-Jun N-terminal kinase (JNK) [18]. The cytosolic
domains of several TLRs bear also a conserved YxxM PI3K
binding motif and phosphorylation of Akt, a downstream kinase
activated by PI3K, is detected upon TLR stimulation [19].
Activation of Akt or p38 inactivates the glycogen synthase kinase 3
(GSK3) that is found further down the signalling pathway [20].
GSK3 is constitutively active in resting cells leading to the proinflammatory NF-kB transcription, but p38/Akt phosphorylation
of GSK3 switches the transcriptional activity to cAMP response
element-binding protein (CREB) [21]. TLR activation can thus
either lead to a pro-inflammatory cytokine response by activation
Introduction
Successful pathogen Mycobacterium tuberculosis (M. tuberculosis) use
intricate strategy to evade the immune response. This pathogen
invades the epithelial cells that cover the alveolar space of the lung
and modulate or fine-tune the immune responses to produce a
selective cytokine response [1–5]. The first phagocytes to be
attracted to the infectious foci are the neutrophils [1,2,6,7],
followed by monocytes, and these leukocytes cooperate in the
elimination of mycobacteria [8]. The extent of epithelial cytokine
secretion may lead to tissue damage and breakdown of extracellular matrix, thus favouring bacterial persistence and facilitating
mycobacterial transmission [9,10]. However, perturbed defence in
immune-compromised patients can tilt this balance leading to
active disease [11]. These initial innate events, depending on the
magnitude of the host immune responses, could thus determine
the outcome of mycobacterial infection.
Epithelial cells express molecular pattern associated receptors,
such as the Toll like receptors (TLRs) that interact with
mycobacteria [12]. TLR2 expression increases upon mycobacterial infection of alveolar epithelium and blocking of TLR2
decreases cytokine responsiveness [4]. Mycobacteria express
multiple ligands that bind to members of the TLR family,
especially TLR2 and TLR4. Mycobacterial products, such as
PLOS ONE | www.plosone.org
1
January 2014 | Volume 9 | Issue 1 | e86466
Mycobacteria Bypass Mucosal NF-kB Signalling
Infection and treatments of the cells
of NF-kB pathway, or an anti-inflammatory CREB-related
cytokine response.
The initial events of mycobacterial infections are not clear. The
first surface that the immobile bacterium will encounter after
inhalation into the lungs would most likely be epithelial. Several
groups have demonstrated that M. tuberculosis invades and survives
within human type II alveolar epithelial cells [1,22,23]. Previous
research revealed that the epithelia remain unresponsive to the
infection until the third day, when the cells secreted a distinct
pattern of cytokines [4,5]. There are conflicting reports regarding
the activation of NF-kB by pathogenic mycobacteria. In the
present study, we have analysed mycobacteria induced epithelial
signalling pathways from activation of TLRs to cytokine secretion.
Our data indicate that mycobacteria avoid epithelial proinflammatory production by bypassing NF-kB activation thus
balancing the infection towards an anti-inflammatory state.
For the infection experiments, primary cells were grown in 6well plates (2.06105 cells/well; Fisher Scientific, UK), infected
with BCG (one bacterium per cell; 1 MOI) or phenol purified LPS
(1 ng/ml; Sigma-Aldrich), lipoarabinomannan (LAM, 1 mg/ml,
Lionex GmbH) or 19-kDa glycolipoprotein (1 mg/ml, Lionex
GmbH) at 37uC for up to three days. For the blocking
experiments, monoclonal mouse anti-human TLR2 or monoclonal mouse anti-human TLR4 antibodies (R&D Systems) 10 mg/ml
were added to the epithelial cells 30 minutes before the addition of
bacteria.
For cytokine analysis, the samples were collected after 0, 6, 24,
48 and 72 hours and for western blot analysis, the cells were
detached by versene (140 mM NaCl, 2.4 mM KCl, 8 mM
Na2HPO4, 1.6 mM KH2PO4, 0.5 mM EDTA, pH 7.2) and
washed with PBS.
To investigate whether epithelial cells survive mycobacterial
infection, we analysed cell viability by trypan blue exclusion assay
according to manufactures instructions (Sigma Aldrich, Germany).
For analysis of bacterial survival within the epithelial cells, infected
epithelial cells were lysed in 300 ml of sterile distilled water for
15 minutes. 100 ml of the suspension was plated on Middlebrook
7H10 supplemented with 10% OADC Enrichment (Becton
Dickinson, Oxford, UK) and grown for 3 weeks.
Materials and Methods
Ethical Statement
The Swedish Research Ethical Committee in Lund (FEK 413/
2008) approved the isolation of the bronchial material for primary
cell cultures. Bronchial material for primary cell cultures was
obtained from lung explant from healthy donors with irreversible
brain damage and with no history of lung disease. Lungs were to
be used for transplantation but could instead be included in this
study as no matched recipients were available at that moment.
Written consent was obtained from their closest relatives.
Western Blot
The primary cells were washed with PBS containing 0.2 mM
phenylmethylsulfonyl fluoride (PMSF), 1 mg/ml PepstatinA, 5 mg/
ml Leupeptin (Sigma-Aldrich) and complete protease inhibitor
cocktail (Roche Diagnostics, Mannheim, Germany) and lysed with
modified Mammalian Protein Extraction Reagent (M-PER)
solution (50 mM HEPES, 150 mM NaCl, 2 mM EDTA,
50 mM ZnCl, 1% NP-40, 0.1% deoxycholate, 0.1% SDS; Pierce)
containing phosphatase (1:10) and the complete protease inhibitor
cocktail (1:25). The cells were then placed on a shaker for
5 minutes, collected and centrifuged at 10,0006g for 5 minutes.
Protein samples were used immediately for western blot analysis or
stored at 280uC.
Protein levels were measured in cells treated with BCG and cells
blocked for TLR2 or TLR4 with the NanoDropTM 8000
Spectrophotometer using the Pierce 660 nm assay (Thermo
Scientific). Medium alone, LPS, LAM and 19 kDa were used as
controls. Protein samples were mixed with PBS, 46 NuPAGE
LDS sample buffer (Life Technologies) and 1 M DTT and
incubated at 90uC for 10 minutes followed by centrifugation at
2186g for 5 minutes. Equal amounts of protein (10 mg/well) were
loaded on a NuPAGE 4%–12% Bis-Tris Gel (Life Technologies)
and separated by sodium dodecyl sulfate-PAGE. A molecular
weight marker (NovexH Sharp Prestained; Life Technologies) was
loaded onto each gel for protein band identification. After
separation, the proteins were transferred to a polyvinylidene
difluoride (PVDF) membrane (Healthcare Amersham). The
membrane was then blocked with either 5% dry-milk (Santa
Cruz Biotechnology, Santa Cruz, CA) or with 5% bovine serum
albumin (BSA; Santa Cruz Biotechnology) for 1 hour on a shaker
at room temperature. Membranes were then incubated on a
shaker overnight at 4uC with rabbit anti-human p-GSK-3a/b
(1:500; AF1590, R&D systems, Denmark), GSK-3a/b (1:250;
AF2157, R&D systems), p-CREB (1:1000; #9198 Cell Signalling
Technology, Inc., Danvers, MA), IkBa (1:1000; #4812 Cell
Signalling Technology), ERK1/2 (0.1 mg/mL, AF1018 R&D
systems), PPARc (1:1000; NBP1-61399 Novus Biologicals),
GAPDH (1:500; sc-25778 Santa Cruz Biotechnology) or mouse
anti-human NF-kB p65 (1:200; sc-8008 Santa Cruz Biotechnol-
Bacterial strains and growth conditions
Mycobacterium bovis bacillus Calmette-Guerin (BCG) Montreal
strain containing the pSMT1 shuttle plasmid was prepared as
previously described [24]. Briefly, the mycobacteria were grown in
Middlebrook 7H9 culture medium, supplemented with 10% ADC
enrichment (Becton Dickinson, Oxford, UK) and hygromycin
(50 mg/l; Roche, Lewes, UK), the culture was washed twice with
sterile PBS, and re-suspended in media again and then dispensed
into vials. Glycerol was added to a final concentration of 25% and
the vials were frozen at 280uC. Prior to each experiment, a vial
was defrosted, added to 9 ml of 7H9/ADC/hygromycin medium,
and incubated with shaking for 72 h at 37uC. Mycobacteria were
then centrifuged for 10 minutes at 30006 g, washed twice with
sterile PBS, and re-suspended in 2 ml of sterile PBS.
Cell Culture
Bronchial tissue was dissected from lungs and kept in
Dulbecco’s Modified Eagle Medium supplemented with gentamicin, penicillin, streptomycin, Fungizone and 10% fetal calf serum
(FCS) (all from Gibco, Paisley, UK) until further isolation. After
removing intraluminal mucus and surrounding tissue, bronchi
were digested in 0.1% Protease (Sigma St. Louis, MO) prepared in
Minimum Essential Medium Eagle Spinner Modification (SigmaAldrich) supplemented with gentamicin, penicillin, streptomycin
and Fungizone for 24 hours. Bronchial epithelial cells (HBEC)
were recovered by repeated intraluminar rinsing with Dulbecco’s
Modified Eagle Medium supplemented with gentamicin, penicillin, streptomycin, Fungizone and 10% FCS. Cells were filtered
through a 100 mm strainer (Falcon, Becton Dickinson) and seeded
in cell culture flasks coated with 1% Collagen-1 (PureCol, Inamed
Biomaterial, Freemont, CA) in Bronchial Epithelial Cell Growth
Medium (Clonetics). The following day cells were thoroughly
washed with a medium change every other day. Experiments were
performed in passage 3 and 4.
PLOS ONE | www.plosone.org
2
January 2014 | Volume 9 | Issue 1 | e86466
Mycobacteria Bypass Mucosal NF-kB Signalling
ogy, Heidelberg, Germany), or b-actin (1:10.000; Sigma-Aldrich)
primary antibody. Incubation was followed by washing 365 minutes with Tris-buffered saline (TBS)-Tween 20 and 165 minutes
TBS. The membrane was then incubated with goat-anti-rabbit
IgG HRP (1:2000; Santa Cruz Biotechnology) IgG secondary
antibody or with rabbit anti-mouse IgG1 HRP (1:4000; Dako)
secondary antibody for 2 hours on a shaker at room temperature
followed by washing with TBS-Tween 20 and TBS. The
housekeeping protein GAPDH and b-actin were used to confirm
equal loading on the wells. The membrane was developed using
Amersham ECL Plus Western Blotting Detection Reagents (GE
Healthcare, Little Chalfont, UK) and GelDoc equipment (Bio-Rad
Laboratories). Blot intensity was quantified using ImageJ software
28 and normalized against GAPDH or b-actin. If required,
membranes were stripped with Restore Western Blot Stripping
Buffer (Pierce, Rockford, IL), blocked and re-probed with new
antibodies.
in PBS and 5% FCS for 1 hour (shaking in dark) in room
temperature. After additional washing the cells was stained with
1 mg/ml of 49, 6-diamidino-2-phenylindole (DAPI) dissolved in
PBS for 5 minutes in dark and then washed again with PBS.
Finally the slides were mounted in fluoromount Aqueous
Mounting Medium (Sigma Aldrich, F4680). The slides were
examined with an inverted Nikon microscope (Nikon Diaphot
300) equipped with a 100 W mercury lamp (Osram, Berlin,
Germany) and Ploempac with the filter set for fluorescein
isothiocyanate and BioRad MRC 1024, controlled via LaserSharp
(version 5.2 for PC/Windows) and further examined with the
LSM 510 DUO confocal equipment with LSM software version
4.2 SP1 (Carl Zeiss, Jena, Germany). Sections incubated without
primary or secondary antibody were used as negative controls to
verify the lack of auto-fluorescence and unspecific secondary
antibody staining.
ELISA
Phospho-kinase array
IL-6 (D6050), TNFa (DTA00C), IFNc (DIF50), IL-10
(D1000B) and IL-22 (D2200) secretion by the infected cells were
quantified in supernatants by Human Quantikine ELISA Kits
(R&D Systems, Oxon, UK) according to manufactures instructions. NF-kB (EK1111) and AP-1 (c-Jun, EK1041) were quantified
with nuclear extraction kits containing ELISA-kit according to
manufacturers instructions (Affymetrix Panomics, UK).
Protein phosphorylation was examined with the Proteome
Human Phospho-Kinase Array Kit (Proteome Prolifer Array,
R&D Systems, Abingdon, Oxford, UK), which is a membrane
based sandwich immunoassay. The assay was performed according to the manufacturers’ instructions. Briefly, total cell extracts
were prepared from stimulated near-confluent cultures of normal
human primary epithelial cells grown in 6-well plates. Untreated
cells were used as control. The cell extracts containing 500 mg of
total protein were incubated with the Human Phospho-Kinase
Array. The proteins present in a lysate sample were captured by
discrete antibodies printed in duplicate across the nitrocellulose
membranes. The array was washed 36 with 1X Wash Buffer for
10 minutes on a rocking platform shaker to remove unbound
proteins. Washing was followed by incubation with a cocktail of
biotinylated detection antibodies (monoclonal anti-human of
phosphorylated Akt (S473), Akt (T308), AMPK alpha1 (T174),
AMPK alpha2 (T172), beta-Catenin, Chk-2 (T68), c-Jun (S63),
CREB (S133), EGF R (Y1086), eNOS (S1177), ERK1/2 (T202/
Y204, T185/Y187), FAK (Y397), Fgr (Y412), Fyn (Y420), GSK-3
alpha/beta (S21/S9), Hck (Y411), HSP27 (S78/S82), HSP60,
JNK pan (T183/Y185 T221/Y223), Lck (Y394), Lyn (Y397),
MSK1/2 (S376/S360), p27 (T198), p38 alpha (T180/Y182), p53
(S15), p53 (S392), p53 (S46), p70 S6 Kinase (T421/S424), PDGF
R beta (Y751), PLC gamma-1 (Y783), PRAS40 (T246), Pyk2
(Y402), RSK1/2/3 (S380/S386/S377), Src (Y419), STAT2
(Y689), STAT3 (S727), STAT3 (Y705), STAT5a (Y694),
STAT5a/b (Y694/Y699), STAT5b (Y699), STAT6 (Y641),
TOR (S2448), WNK-1 (T60), Yes (Y426) and subsequent
application of streptavidin-HRP conjugate. The signals were
detected with the ECL Plus Western Blotting Detection System
(GE Healthcare). Developed signals were analyzed using ImageJ
1.45s analysis software.
Statistics
The statistical program used was SigmaStat, version 3.5, for
Windows XP. The statistical difference between two groups was
investigated by Mann-Whitney test. Multiple comparisons were
done by one-way Analysis of Variance followed by Bonferroni test
or Dunnett’s test (***P#0.001, ** P,0.01, *P,0.05, ns = non
significant).
Results
Mycobacteria supress NF-kB and c-Jun
We used a low infection dose of 1:1 (bacterium:cell) [25,26] and
analysed alveolar nuclear extracts for NF-kB and c-Jun by ELISA.
BCG at low MOI was shown to invade and survives in alveolar
epithelial cells three days after infection without affecting epithelial
viability (Figures S1 and S2). The TLR4 agonist LPS was used as a
control. Infection of primary epithelial cells did not induce NF-kB
activation during the three days of infection (Figure 1a). However,
mycobacterial infection induced an early activation of c-Jun
proteins that was suppressed two days after infection (Figure 1b).
LPS induced an early NF-kB activation that was significantly
higher than medium control and BCG up to 48 hours after
addition to primary epithelial cells. Interestingly, BCG induced
significantly higher c-Jun protein activation at 6 hours than LPS
(p = 0.0177). We could confirm that BCG at low MOI invades and
survives in primary epithelial cells [5,27] three days after infection
(Figure S3).
Immunofluorescence microscopy
Expression of p-CREB and NF-kB in primary cells was detected
by immunofluorescence staining. After blocking and infection for
72 hours the cells were fixed with 3.7% formaldehyde and then
permeabilized in a mixture of PBS, 0.25% Triton X-100 and 5%
fetal calf serum (FCS) for 30 minutes shaking at room temperature. Specimens were then incubated for 2 hours shaking at room
temperature with PBS, 5% FCS, and the primary anti-rabbit pCREB-1 (Ser133) or anti-mouse NF-kB p65 antibodies (1:50;
Santa Cruz Biotechnology). The cells were washed two times with
PBS at 4006g for 5 minutes and then incubated with goat antirabbit or rabbit anti-mouse secondary antibody (1:100; Invitrogen)
PLOS ONE | www.plosone.org
Mycobacteria inactivates GSK3ab signalling pathways
GSK3 consist of the isoforms a and b. The un-phosphorylated
form of GSK3 promotes NF-kB activation, while phosphorylation
of GSK3 by p38 and Akt promotes CREB anti-inflammatory
activation. To investigate mycobacteria induced signalling pathways in primary epithelial cells, we analysed the GSK3ab-pathway
by Phospho-kinase array (Figure 1c–e). LAM and 19 kDa were
used as controls for mycobacterial virulence factors and are known
to signal through TLR2 [13,14], while LPS is a known TLR4
ligand. In the beginning of infection, live mycobacteria, induced
3
January 2014 | Volume 9 | Issue 1 | e86466
Mycobacteria Bypass Mucosal NF-kB Signalling
Figure 1. Mycobacteria bypass epithelial NF-kB signalling. (a) Infection of primary epithelial cells did not induce NF-kB activation quantified
by ELISA, but an early activation of c-Jun proteins in epithelial cells was observed. (b) Epithelial GSK3ab-pathway was analysed by Phospho-kinase
array upon mycobacteria infection. In the beginning of infection, live mycobacteria, the virulence factors LAM and 19 kDa, and the TLR4 agonist LPS,
induced comparable induction of p38, pAkt and pGSK3ab. During the first 24 h, LPS induced higher increase of pCREB protein levels than
mycobacteria (p = 0.0017). Third day of infection, mycobacteria significantly increased epithelial pCREB compared to medium control (p = 0.0357) or
LPS (p = 0.0089). Epithelial stimulation with LAM induced an increase in pGSK3ab and pAkt phosphorylation (p = 0.001 respectively p = 0.0196) during
the later stages of infection compared to the early time-point. Generally, mycobacteria induced a more persistent increase of the investigated
transcription factors three days after infection in primary epithelial cell than the controls LPS, 19 kDa and LAM. Data are presented as mean 6 SEM of
three separate experiments; **p,0.01 and ***p,0.001.
doi:10.1371/journal.pone.0086466.g001
pAkt phosphorylation (p = 0.001respectively p = 0.0196) during
the later stages of infection compared to the early time-point.
higher induction of p38 and pGSK3ab than the virulence factor
19 kDa and the TLR4 agonist LPS (Figure 1c–d). Interestingly,
mycobacterial virulence factor LAM significantly down-regulated
pGSK3ab after 24 hours of stimulation (Figure 1d). Mycobacteria
and LPS induced higher increase of Akt than LAM and 19 kDa,
but mycobacteria induced less pCREB protein levels during the
first 24 hours, compared to LPS (p = 0.0017) or medium control
(not significant) (Figure 1e–f). Three days after infection there was
a significant increase of pCREB in mycobacteria infected
epithelium compared to medium control (p = 0.0357) and LPS
(p = 0.0089) (Figure 1e). Generally, mycobacteria induced a more
persistent increase of the investigated transcription factors three
days after infection in primary epithelial cell than the controls
LPS, 19 kDa and LAM (Figure 1c–f). Interestingly, epithelial
stimulation with LAM induced a late increase in pGSK3ab and
PLOS ONE | www.plosone.org
Mycobacteria bypass NF-kB activation, but activated
ERK1/2 and cFos
To investigate mycobacteria induced epithelial signalling
pathways further we analysed several molecules in TLR-signalling
pathway by Western blotting. By comparing infected cells with uninfected cells during the investigated time-points, we could confirm
that mycobacterial infection did not induce higher NF-kB- or IkBactivation (Figure 2a,b) than medium control during infection.
Variations in suppression were observed during the time of
infection. Mycobacteria supressed epithelial IkB and pGSK3ab
proteins at the beginning of infection (Figure 2b; p = 0.002 and
Figure 2d; p = 0.0148 respectively), while the pGSK3ab and
4
January 2014 | Volume 9 | Issue 1 | e86466
Mycobacteria Bypass Mucosal NF-kB Signalling
Figure 2. Mycobacteria modulate epithelial signalling pathways. Several molecules in the TLR-signalling pathway were analysed by Western
blotting upon mycobacterial infection. (a–b) We could confirm that mycobacterial infection did not induce NF-kB- or IkB-activation. Mycobacterial
suppression of primary epithelial (b) (p = 0.002) IkB and (d) (p = 0.0148) pGSK3ab proteins were mostly pronounced at 24 hours of infection. The
phosphorylated forms of (c) (p = 0.0163) CREB and (d) (p = 0.0248) GSK3ab proteins reached highest levels 72 hours after infection. (e) Mycobacterial
infection increased the Fos family of AP-1 proteins, as c-Fos protein levels significantly increased 72 hours after infection (p = 0.0038). (f) Mycobacteria
induced two peaks of pERK1/2 protein levels, after 24 hours (p,0.001) and after 72 hours (p = 0.0034) of infection. (g) Epithelial cells express PPARc
protein, but mycobacterial infection did not significantly increase epithelial PPARc amount. Data are presented as mean 6 SEM of three experiments;
*p,0.05, **p,0.01 and ***p,0.001.
doi:10.1371/journal.pone.0086466.g002
previously reported to induce PPARc in order to modulate NF-kB
responses [16,17], but we could not observe that BCG significantly
affected epithelial PPARc protein concentration compared to
medium control (Figure 2g). The actin loading controls are shown
in the Figure S3.
pCREB proteins reached highest levels 72 hours after infection
(Figure 2c,d; p = 0.0163 and p = 0.0248 respectively). Mycobacteria affected both GSK3 isoforms similarly. Interestingly, mycobacterial infection increased the Fos family of AP-1 proteins, as cFos protein levels significantly increased 72 hours after infection
(Figure 2e; p = 0.0038). Mycobacteria induced two peaks of
pERK1/2 protein levels, after 24 hours (p,0.001) and after
72 hours (p = 0.0034) of infection (Figure 2f). Mycobacteria were
PLOS ONE | www.plosone.org
5
January 2014 | Volume 9 | Issue 1 | e86466
Mycobacteria Bypass Mucosal NF-kB Signalling
Mycobacterial infection controls epithelial cytokine
production
Generally, the pro-inflammatory cytokines, such as IFNc and
TNFa, orchestrate innate and adaptive host immune responses,
while anti-inflammatory cytokines, such as IL-10 and IL-22,
confine the inflammation and postpone the generation of adaptive
immunity [28]. Mycobacterial control of induced transcriptional
factors was analysed as epithelial cytokine secretion from six hours
up to three days after infection. Infection induced a significant IL6 and IL-10 secretion that peaked at 72 hours (Figure 3a,b). In
contrast, mycobacterial infection induced an early significant IL22 secretion from primary epithelial cells that ended 24 hours after
infection (Figure 3c). Mycobacterial infection did not induced
epithelial TNFa or IFNc secretion during the studied time interval
(data not shown).
Mycobacteria regulate TLR-induced inflammatory
response
TLR-induced CREB activation is important for IL-10 production [21]. To determine the impact of TLR2 and TLR4 on
mycobacteria induced pro- and anti-inflammatory cytokine
production, the receptors were blocked prior to mycobacterial
three-day infection of the primary epithelial cells (Figure 4).
Antibody blocking of TLR2 or TLR4 before infection decreased
epithelial IL-6 secretion (p = 0.0011 and p = 0.0047 respectively)
(Figure 4a). The blocking of TLR2 or TLR4 did not affect alveolar
survival during infection (Figure S1b). LPS induced a significantly
higher IL-6 response than BCG (p = 0.0063), while 19 kDa
induced a lower response compared to live mycobacteria
(p = 0.0029). Mycobacteria induced higher production of the
anti-inflammatory IL-10 production than LPS (p = 0.0032) in
human primary epithelial cells (Figure 4b). Blocking of TLR4 prior
to infection increased IL-10 secretion compared to unblocked
infection (p = 0.0399). Blocking with TLR2 or addition of 19-kDa
to the epithelial cells did not induce a significant change in
epithelial IL-10 production compared to mycobacteria.
Mycobacteria regulates CREB through TLRs
Mycobacterial infection was previously shown to increase
epithelial TLR2 and TLR4 [4]. The impact of TLR2 and
TLR4 were analysed by immuno-fluorescence staining of pCREB
and NF-kB expression in primary cells (Figure 5a). Mycobacterial
infection increased nuclear pCREB protein levels compared to
unstimulated cells, while the expression of NF-kB did not increase.
Blocking of TLR4 before mycobacterial infection resulted in a
granular cytoplasmic pCREB distribution, similar to pCREB
aggregation in 19 kDa-stimulated cells. TLR2 blocking and LAM
treatment induced similar pCREB distribution as live mycobacteria. Epithelial treatment with mycobacterial virulence factor 19kDa resulted in a granular cytoplasmic pCREB distribution, while
LAM treatment induced similar pCREB distribution as live
mycobacteria (Figure 5a). Further confirming our results, detection
of epithelial pCREB by confocal immuno-fluorescent microscopy
revealed that mycobacterial infection significantly increased
pCREB expression (p,0.001), but NF-kB expression was not
affected (Figure 5b). Blocking of TLR2 or TLR4 before
mycobacterial infection increased pCREB expression even further
(p = 0.0187 and p,0.001 respectively) compared to unstimulated
cells, but NF-kB expression was not affected (Figure 5b).
PLOS ONE | www.plosone.org
Figure 3. Controlled epithelial cytokine secretion. Mycobacterial
control of induced transcriptional factors was analysed as epithelial
cytokine secretion from six hours up to three days after infection.
Infection induced a significant (a) IL-6 and (b) IL-10 secretion that
peaked at 72 hours (p = 0.0425 and p = 0.0186 compared to LPS). (c)
Mycobacterial infection of primary epithelial cells induced an early
significant IL-22 secretion (p = 0.0463 compared to LPS) that ended
24 hours after infection. Data are presented as mean 6 SEM of four
separate experiments; *p,0.05 and **p,0.01.
doi:10.1371/journal.pone.0086466.g003
TLRs are involved in mycobacterial regulation of mucosal
inflammation
To further determine the impact of TLR2 and TLR4 on
mycobacteria induced cytokine production, the receptors were
blocked prior to mycobacterial infection and the impact of
modulated epithelial signalling was studied by Western blotting
three days after infection. Blocking of TLR2 (p = 0.0063) or TLR4
(p = 0.0047) prior to infection or stimulation with 19 kDa
6
January 2014 | Volume 9 | Issue 1 | e86466
Mycobacteria Bypass Mucosal NF-kB Signalling
We found that Mycobacterium bovis bacilli Calmette-Guerin
bypassed NF-kB activation during the first days of infection.
BCG is equipped with several genes coding for invasin/adhesinlike proteins [27,29–33] and the mycobacterial adhesion heparinbinding haemagglutinin [34] is believed to be involved in invasion
of human alveolar epithelial cells [35]. Activated NF-kB was
recently shown to be essential for mycobacterial elimination, since
blocking of this pathway prevented bacterial killing and allowed
the bacteria to grow in macrophages [36]. To date, reported data
regarding the activation of NF-kB by pathogenic mycobacteria are
conflicting. M. tuberculosis was shown to supress NF-kB pathway in
some studies [37,38], induce a transient NF-kB activation in other
studies and some studies observed activated NF-kB pathways
under some conditions [39–42]. However, several bacteria are
known to subvert the cell-intrinsic innate immunity by targeting
NF-kB. Salmonella, Shigella and enteropathogenic Escherichia coli
(EPEC) are known to supress the NF-kB pathway to counteract
the host defences [43,44]. Recent genetic studies revealed that
EPEC suppression of host NF-kB signalling and NF-kB dependent
anti-inflammatory cytokine production requires NleE, a type IIIsecreted effector that has homologues in Shigella and certain
Salmonella species [45–47]. Recently, genome-wide screens identified previously unidentified gene products for M. tuberculosis
persistence [48], but whether mycobacteria possess similar
elements are not known.
Innate recognition of mycobacteria involves the activation of
TLR2 and TLR4. Signalling through TLR activates the adaptor
protein MyD88 leading to NF-kB signalling and the activation of
ERK1/2, p38 and JNK [18]. Besides of MyD88, activation of
TLRs triggers also PI3K activation leading to subsequent Akt
phosphorylation. Akt and p38 phosphorylate the glycogen
synthase kinase 3 (GSK3), which switches the transcription from
the pro-inflammatory NF-kB to the anti-inflammatory CREB
activation [21]. We observed that mycobacteria induced the
MyD88 stimulated p38, ERK1/2 and AP-1 signalling. Interestingly, mycobacterial infection induced an early activation of the cJun family of AP-1 proteins in primary epithelial cells, and a late
activation of the AP-1 protein Fos. Mycobacterial activation of
PPARc is known to supress NF-kB in macrophages [16], but we
could not observe mycobacteria-induced PPARc activation in
primary epithelial cells. GSK3 regulates the transcriptional activity
of CREB and NF-kB by competing for the limited amount of
CREB-binding protein (CBP) [21]. TLR activation could therefore either lead to a pro-inflammatory cytokine response by
activation of NF-kB pathway, or an anti-inflammatory CREBrelated cytokine response. In this study, mycobacterial infection
induced increased GSK3 phosphorylation, switching thus the
transcriptional activity from NF-kB to CREB. Indeed, epithelial
cells responded early to mycobacterial infection by secreting IL-6
and the anti-inflammatory IL-22, while the anti-inflammatory IL10 increased two days after infection. The cytokine IL-6 is
transcribed by CREB, C/EBP, STAT3 and AP-1 [49,50], and can
act as both pro- and anti-inflammatory in many chronic
inflammatory diseases. IL-6 trans-signalling is critically involved
in the maintenance of a disease state by promoting transition from
acute to chronic inflammation [51]. In addition, IL-6 is required in
the rapid expression of an initial protective IFNc response during
M. tuberculosis infection [52]. However, concomitant IFN production can tilt the anti-inflammatory qualities of IL-10 and IL-22
towards a pro-inflammatory state [53]. We could not observe
epithelial IFNc production, suggesting that the secreted IL-10 and
IL-22 are produced to damper the inflammation. IL-10 modulates
the anti-inflammatory mechanisms by targeting NF-kB thereby
inhibiting cellular production of TNFa, which could be one of the
Figure 4. Mycobacterial regulation of TLR-induced cytokines.
To determine the impact of TLR2 and TLR4 on mycobacteria induced
pro- and anti-inflammatory cytokine production, the receptors were
blocked prior to mycobacterial infection of the primary epithelial cells.
(a) Blocking of TLR2 or TLR4 before infection decreased epithelial IL-6
secretion (p = 0.0011 and p = 0.0047 respectively) after three days. LPS
induced a significantly higher IL-6 response than BCG (p = 0.0063), while
19 kDa induced a lower response compared to live mycobacteria
(p = 0.0029). (b) Mycobacteria induced higher production of the antiinflammatory IL-10 production than LPS (p = 0.0032) in human primary
epithelial cells. Blocking of TLR4 prior to infection increased IL-10
secretion compared to unblocked infection (p = 0.0399). Blocking with
TLR2 or addition of 19-kDa to the epithelial cells did not induce a
significant change on epithelial IL-10 production compared to
mycobacteria. Data are presented as mean 6 SEM of three separate
experiments; *p,0.05 and **p,0.01.
doi:10.1371/journal.pone.0086466.g004
significantly increased epithelial pCREB production (p = 0.0163)
(Figure 6a). Blocking of TLRs or 19 kDa stimulation of epithelial
cells had a non-significant impact on pGSK3ba expression
(Figure 6b). Blocking of TLR2 or TLR4 before mycobacterial
infection of primary epithelial cells non-significantly restored the
NF-kB values to background levels (Figure 6c). The GADPH
loading controls are shown in the Figure S4.
Discussion
Functional NF-kB activation is essential for the maintenance of
physiological immune homeostasis and protective host defence.
PLOS ONE | www.plosone.org
7
January 2014 | Volume 9 | Issue 1 | e86466
Mycobacteria Bypass Mucosal NF-kB Signalling
Figure 5. TLR4 blocking results in cytoplasmic CREB aggregation. Mycobacterial modulation of TLR signalling pathways was confirmed by
immuno-fluorescence staining of pCREB and NF-kB expression in primary epithelial cells. (a) Mycobacterial infection increased nuclear pCREB protein
levels compared to unstimulated cells, while the expression of NF-kB did not increase. Blocking of TLR4 before mycobacterial infection resulted in a
granular cytoplasmic pCREB distribution, similar to pCREB aggregation in 19kDa-stimulated cells. TLR2 blocking and LAM treatment induced similar
pCREB distribution as live mycobacteria. (b) The results were further analysed by LSM software. Mycobacterial infection increased significantly
epithelial (p,0.001) pCREB expression as detected by confocal immuno-fluorescent microscopy, but NF-kB expression was not affected. Blocking of
TLR2 or TLR4 before mycobacterial infection increased pCREB expression even further (p = 0.0187 and p 0.001 respectively) compared to
unstimulated cells, but NF-kB expression was not affected. Original magnification 6300. Data are presented as representative images or mean 6 SEM
of three separate experiments; *p , 0.05 and ***p , 0.001.
doi:10.1371/journal.pone.0086466.g006
PLOS ONE | www.plosone.org
8
January 2014 | Volume 9 | Issue 1 | e86466
Mycobacteria Bypass Mucosal NF-kB Signalling
progression [57,58]. The role of IL-10 could be to limit
mycobacterial clearance during the early immune response
through the inhibition of IL-12p40 [59]. IL-22 is found in large
amounts in pleura from TB patients [60] and this cytokine is
primarily expressed by CD4+ T cells [61], but other leukocyte
subsets also express this cytokine [62]. IL-22 acts through the IL22 receptor complex expressed by epithelial cells and hepatocytes,
where it promotes regeneration and protects against tissue damage
[63,64], but accumulating evidence suggests that IL-22 can be
either pathogenic or protective depending on host conditions [65].
Using the TB mouse model, a recent study showed that
neutralization of IL-22 did not have any effect on the lung
bacterial burden or granuloma formation [66]. The mycobacterial
vaccine strain used in our study did not induce TNFa or IFNc
secretion. Interestingly, recent studies support the IL-17-CXCL13
pathway rather than the IFNc pathway as a new strategy to
improve mucosal vaccines against tuberculosis [67]. We are
currently investigating if alveolar epithelia induce IL-17 or
CXCL13 upon mycobacterial infection.
Blocking of epithelial TLR4 before mycobacterial infection
decreased the pro-inflammatory IL-6 secretion, but increased the
anti-inflammatory IL-10 secretion. TLR4 blocking prior to
mycobacterial infection resulted in a granular cytoplasmic pCREB
distribution similar to the 19-kDa stimulated cells. We could not
find any explanation of the cytoplasmic granular accumulation,
but granular accumulation of pERK in cytoplasm was shown to
alternate downstream signalling in Parkinson’s disease [68].
Normally, signals that induce NF-kB activity usually lead to IkB
phosphorylation by the IkB kinase (IKK) complex, and subsequent
multi-ubiquitination and degradation of this protein via proteasome, allowing NF-kB dimers’ translocation to nucleus [69]. We
observed that TLR4 blocking induced cytoplasmic accumulation
of NF-kB as well, although no increased NF-kB translocation to
epithelial nuclei was detected.
Mycobacteria cause persistent infections by minimizing the
degree of overt pathology, allowing long-term association with the
host. We have observed that mycobacterial infection of primary
epithelial cells supress NF-kB activation by increasing the
inhibitory GSK3, thereby supporting the production to the antiinflammatory cytokines IL-22 and IL-10. Production of antiinflammatory cytokines is known to impair antigen presentation,
which confines the inflammation and postpones the generation of
adaptive immunity resulting in antigen-specific anergy. These
events could lead to an impaired innate immune response by
which mycobacteria create a safe haven for chronic infection and
transmission to new hosts.
Figure 6. Mycobacteria control epithelial TLR responses. The
impact of TLRs on mycobacterial modulated epithelial signalling was
studied by Western blotting prior to infection and three days after
infection. (a) Blocking of TLR2 (p = 0.0063) or TLR4 (p = 0.0047) prior to
infection or stimulation with 19 kDa significantly increased epithelial
pCREB production (p = 0.0163). (b) Blocking of TLRs or 19 kDa
stimulation of epithelial cells had an non-significant impact on pGSK3ba
expression. (c) Blocking of TLR2 or TLR4 before mycobacterial infection
of primary epithelial cells non-significantly restored the NF-kB values to
background levels. Data are presented as mean 6 SEM of three
separate experiments; *p,0.05 and **p,0.01.
doi:10.1371/journal.pone.0086466.g006
Supporting Information
Figure S1
Intracellular viability of mycobacteria.
(TIF)
Figure S2 Epithelial viability visualized by trypan blue
exclusion assay three days after infection, with or
without blocking of TLR2 or TLR4.
(TIF)
Figure S3
Actin loading controls (Figure 2).
(TIF)
mechanisms of NF-kB suppression that we observed in our study
[54,55]. Mycobacteria was previously reported to induce IL-10
secretion from neutrophils through the phosphorylation of p38
and Akt kinases [56]. Mycobacterial infection of Il102/2 mice
show enhanced protection while showing no signs of aberrant
host-mediated pathology, which perhaps reflects the slow disease
PLOS ONE | www.plosone.org
Figure S4
GADPH loading controls (Figure 6).
(TIF)
Acknowledgments
We thank Märta Andersson for technical help with the manuscript.
9
January 2014 | Volume 9 | Issue 1 | e86466
Mycobacteria Bypass Mucosal NF-kB Signalling
reagents/materials/analysis tools: OH GWT. Wrote the paper: NL GH
GG.
Author Contributions
Conceived and designed the experiments: NL GH NA GG. Performed the
experiments: NL GH NA. Analyzed the data: NL GH GG. Contributed
References
26. Caceres N, Llopis I, Marzo E, Prats C, Vilaplana C, et al. (2012) Low dose
aerosol fitness at the innate phase of murine infection better predicts virulence
amongst clinical strains of Mycobacterium tuberculosis. PLoS One 7: e29010.
27. Florio W, Brancatisano FL, Bottai D, Esin S, Di Luca M, et al. (2009) The
BCG1619c gene is not essential for invasion and intracellular persistence of
Mycobacterium bovis BCG in human THP-1 and A549 cell lines.
Can J Microbiol 55: 975–982.
28. Mocellin S, Panelli MC, Wang E, Nagorsen D, Marincola FM (2003) The dual
role of IL-10. Trends Immunol 24: 36–43.
29. Cole ST, Brosch R, Parkhill J, Garnier T, Churcher C, et al. (1998) Deciphering
the biology of Mycobacterium tuberculosis from the complete genome sequence.
Nature 393: 537–544.
30. Garnier T, Eiglmeier K, Camus JC, Medina N, Mansoor H, et al. (2003) The
complete genome sequence of Mycobacterium bovis. Proc Natl Acad Sci U S A
100: 7877–7882.
31. Ahmad S, El-Shazly S, Mustafa AS, Al-Attiyah R (2005) The six mammalian
cell entry proteins (Mce3A-F) encoded by the mce3 operon are expressed during
in vitro growth of Mycobacterium tuberculosis. Scand J Immunol 62: 16–24.
32. Gao LY, Pak M, Kish R, Kajihara K, Brown EJ (2006) A mycobacterial operon
essential for virulence in vivo and invasion and intracellular persistence in
macrophages. Infect Immun 74: 1757–1767.
33. Brosch R, Gordon SV, Garnier T, Eiglmeier K, Frigui W, et al. (2007) Genome
plasticity of BCG and impact on vaccine efficacy. Proc Natl Acad Sci U S A 104:
5596–5601.
34. Menozzi FD, Reddy VM, Cayet D, Raze D, Debrie AS, et al. (2006)
Mycobacterium tuberculosis heparin-binding haemagglutinin adhesin (HBHA)
triggers receptor-mediated transcytosis without altering the integrity of tight
junctions. Microbes Infect 8: 1–9.
35. Pethe K, Puech V, Daffe M, Josenhans C, Drobecq H, et al. (2001)
Mycobacterium smegmatis laminin-binding glycoprotein shares epitopes with
Mycobacterium tuberculosis heparin-binding haemagglutinin. Mol Microbiol
39: 89–99.
36. Gutierrez MG, Mishra BB, Jordao L, Elliott E, Anes E, et al. (2008) NF-kappa B
activation controls phagolysosome fusion-mediated killing of mycobacteria by
macrophages. J Immunol 181: 2651–2663.
37. Pathak SK, Basu S, Basu KK, Banerjee A, Pathak S, et al. (2007) Direct
extracellular interaction between the early secreted antigen ESAT-6 of
Mycobacterium tuberculosis and TLR2 inhibits TLR signaling in macrophages.
Nat Immunol 8: 610–618.
38. Pathak SK, Basu S, Bhattacharyya A, Pathak S, Kundu M, et al. (2005)
Mycobacterium tuberculosis lipoarabinomannan-mediated IRAK-M induction
negatively regulates Toll-like receptor-dependent interleukin-12 p40 production
in macrophages. J Biol Chem 280: 42794–42800.
39. Toossi Z, Hamilton BD, Phillips MH, Averill LE, Ellner JJ, et al. (1997)
Regulation of nuclear factor-kappa B and its inhibitor I kappa B-alpha/MAD-3
in monocytes by Mycobacterium tuberculosis and during human tuberculosis.
J Immunol 159: 4109–4116.
40. Lee SB, Schorey JS (2005) Activation and mitogen-activated protein kinase
regulation of transcription factors Ets and NF-kappaB in Mycobacteriuminfected macrophages and role of these factors in tumor necrosis factor alpha
and nitric oxide synthase 2 promoter function. Infect Immun 73: 6499–6507.
41. Dhiman R, Raje M, Majumdar S (2007) Differential expression of NF-kappaB
in mycobacteria infected THP-1 affects apoptosis. Biochim Biophys Acta 1770:
649–658.
42. Loeuillet C, Martinon F, Perez C, Munoz M, Thome M, et al. (2006)
Mycobacterium tuberculosis subverts innate immunity to evade specific
effectors. J Immunol 177: 6245–6255.
43. Bhavsar AP, Guttman JA, Finlay BB (2007) Manipulation of host-cell pathways
by bacterial pathogens. Nature 449: 827–834.
44. Roy CR, Mocarski ES (2007) Pathogen subversion of cell-intrinsic innate
immunity. Nat Immunol 8: 1179–1187.
45. Nadler C, Baruch K, Kobi S, Mills E, Haviv G, et al. (2010) The type III
secretion effector NleE inhibits NF-kappaB activation. PLoS Pathog 6:
e1000743.
46. Newton HJ, Pearson JS, Badea L, Kelly M, Lucas M, et al. (2010) The type III
effectors NleE and NleB from enteropathogenic E. coli and OspZ from Shigella
block nuclear translocation of NF-kappaB p65. PLoS Pathog 6: e1000898.
47. Vossenkamper A, Marches O, Fairclough PD, Warnes G, Stagg AJ, et al. (2010)
Inhibition of NF-kappaB signaling in human dendritic cells by the enteropathogenic Escherichia coli effector protein NleE. J Immunol 185: 4118–4127.
48. Zhang YJ, Ioerger TR, Huttenhower C, Long JE, Sassetti CM, et al. (2012)
Global assessment of genomic regions required for growth in Mycobacterium
tuberculosis. PLoS Pathog 8: e1002946.
49. Hershko DD, Robb BW, Luo G, Hasselgren PO (2002) Multiple transcription
factors regulating the IL-6 gene are activated by cAMP in cultured Caco-2 cells.
Am J Physiol Regul Integr Comp Physiol 283: R1140–1148.
1. Lin Y, Zhang M, Barnes PF (1998) Chemokine production by a human alveolar
epithelial cell line in response to Mycobacterium tuberculosis. Infect Immun 66:
1121–1126.
2. Wickremasinghe MI, Thomas LH, Friedland JS (1999) Pulmonary epithelial
cells are a source of IL-8 in the response to Mycobacterium tuberculosis:
essential role of IL-1 from infected monocytes in a NF-kappa B-dependent
network. J Immunol 163: 3936–3947.
3. Mendez-Samperio P, Alba L, Perez A (2007) Mycobacterium bovis bacillus
Calmette-Guerin (BCG)-induced CXCL8 production is mediated through
PKCalpha-dependent activation of the IKKalphabeta signaling pathway in
epithelial cells. Cell Immunol 245: 111–118.
4. Andersson M, Lutay N, Hallgren O, Westergren-Thorsson G, Svensson M, et al.
(2012) Mycobacterium bovis bacilli Calmette-Guerin regulates leukocyte
recruitment by modulating alveolar inflammatory responses. Innate Immun
18: 531–540.
5. Hakansson G, Lutay N, Andersson M, Hallgren O, Westergren-Thorsson G, et
al. (2012) Epithelial G protein-coupled receptor kinases regulate the initial
inflammatory response during mycobacterial infection. Immunobiology.
6. Neufert C, Pai RK, Noss EH, Berger M, Boom WH, et al. (2001)
Mycobacterium tuberculosis 19-kDa lipoprotein promotes neutrophil activation.
J Immunol 167: 1542–1549.
7. Godaly G, Young DB (2005) Mycobacterium bovis bacille Calmette Guerin
infection of human neutrophils induces CXCL8 secretion by MyD88-dependent
TLR2 and TLR4 activation. Cell Microbiol 7: 591–601.
8. Silva MT, Silva MN, Appelberg R (1989) Neutrophil-macrophage cooperation
in the host defence against mycobacterial infections. Microb Pathog 6: 369–380.
9. Elkington PT, Emerson JE, Lopez-Pascua LD, O’Kane CM, Horncastle DE, et
al. (2005) Mycobacterium tuberculosis up-regulates matrix metalloproteinase-1
secretion from human airway epithelial cells via a p38 MAPK switch. J Immunol
175: 5333–5340.
10. Li Y, Wang Y, Liu X (2012) The role of airway epithelial cells in response to
mycobacteria infection. Clin Dev Immunol 2012: 791392.
11. Huynh KK, Joshi SA, Brown EJ (2011) A delicate dance: host response to
mycobacteria. Curr Opin Immunol 23: 464–472.
12. Brightbill HD, Libraty DH, Krutzik SR, Yang RB, Belisle JT, et al. (1999) Host
defense mechanisms triggered by microbial lipoproteins through toll-like
receptors. Science 285: 732–736.
13. Quesniaux VJ, Nicolle DM, Torres D, Kremer L, Guerardel Y, et al. (2004)
Toll-like receptor 2 (TLR2)-dependent-positive and TLR2-independent-negative regulation of proinflammatory cytokines by mycobacterial lipomannans.
J Immunol 172: 4425–4434.
14. Jo EK, Yang CS, Choi CH, Harding CV (2007) Intracellular signalling cascades
regulating innate immune responses to Mycobacteria: branching out from Tolllike receptors. Cell Microbiol 9: 1087–1098.
15. Necela BM, Su W, Thompson EA (2008) Toll-like receptor 4 mediates cross-talk
between peroxisome proliferator-activated receptor gamma and nuclear factorkappaB in macrophages. Immunology 125: 344–358.
16. Almeida PE, Silva AR, Maya-Monteiro CM, Torocsik D, D’Avila H, et al.
(2009) Mycobacterium bovis bacillus Calmette-Guerin infection induces TLR2dependent peroxisome proliferator-activated receptor gamma expression and
activation: functions in inflammation, lipid metabolism, and pathogenesis.
J Immunol 183: 1337–1345.
17. Mahajan S, Dkhar HK, Chandra V, Dave S, Nanduri R, et al. (2012)
Mycobacterium tuberculosis modulates macrophage lipid-sensing nuclear
receptors PPARgamma and TR4 for survival. J Immunol 188: 5593–5603.
18. Kawai T, Akira S (2010) The role of pattern-recognition receptors in innate
immunity: update on Toll-like receptors. Nat Immunol 11: 373–384.
19. Ruse M, Knaus UG (2006) New players in TLR-mediated innate immunity:
PI3K and small Rho GTPases. Immunol Res 34: 33–48.
20. Doble BW, Woodgett JR (2003) GSK-3: tricks of the trade for a multi-tasking
kinase. J Cell Sci 116: 1175–1186.
21. Martin M, Rehani K, Jope RS, Michalek SM (2005) Toll-like receptor-mediated
cytokine production is differentially regulated by glycogen synthase kinase 3. Nat
Immunol 6: 777–784.
22. Bermudez LE, Goodman J (1996) Mycobacterium tuberculosis invades and
replicates within type II alveolar cells. Infect Immun 64: 1400–1406.
23. Mehta PK, King CH, White EH, Murtagh JJ, Jr., Quinn FD (1996) Comparison
of in vitro models for the study of Mycobacterium tuberculosis invasion and
intracellular replication. Infect Immun 64: 2673–2679.
24. Snewin VA, Gares MP, Gaora PO, Hasan Z, Brown IN, et al. (1999) Assessment
of immunity to mycobacterial infection with luciferase reporter constructs. Infect
Immun 67: 4586–4593.
25. Saini D, Hopkins GW, Seay SA, Chen CJ, Perley CC, et al. (2012) Ultra-low
dose of Mycobacterium tuberculosis aerosol creates partial infection in mice.
Tuberculosis (Edinb) 92: 160–165.
PLOS ONE | www.plosone.org
10
January 2014 | Volume 9 | Issue 1 | e86466
Mycobacteria Bypass Mucosal NF-kB Signalling
60. Matthews K, Wilkinson KA, Kalsdorf B, Roberts T, Diacon A, et al.
Predominance of interleukin-22 over interleukin-17 at the site of disease in
human tuberculosis. Tuberculosis (Edinb) 91: 587–593.
61. Liang SC, Tan XY, Luxenberg DP, Karim R, Dunussi-Joannopoulos K, et al.
(2006) Interleukin (IL)-22 and IL-17 are coexpressed by Th17 cells and
cooperatively enhance expression of antimicrobial peptides. J Exp Med 203:
2271–2279.
62. Colonna M (2009) Interleukin-22-producing natural killer cells and lymphoid
tissue inducer-like cells in mucosal immunity. Immunity 31: 15–23.
63. Zenewicz LA, Yancopoulos GD, Valenzuela DM, Murphy AJ, Stevens S, et al.
(2008) Innate and adaptive interleukin-22 protects mice from inflammatory
bowel disease. Immunity 29: 947–957.
64. Aujla SJ, Chan YR, Zheng M, Fei M, Askew DJ, et al. (2008) IL-22 mediates
mucosal host defense against Gram-negative bacterial pneumonia. Nat Med 14:
275–281.
65. Rutz S, Eidenschenk C, Ouyang W (2013) IL-22, not simply a Th17 cytokine.
Immunol Rev 252: 116–132.
66. Wilson MS, Feng CG, Barber DL, Yarovinsky F, Cheever AW, et al. (2010)
Redundant and pathogenic roles for IL-22 in mycobacterial, protozoan, and
helminth infections. J Immunol 184: 4378–4390.
67. Gopal R, Rangel-Moreno J, Slight S, Lin Y, Nawar HF, et al. (2013) Interleukin17-dependent CXCL13 mediates mucosal vaccine-induced immunity against
tuberculosis. Mucosal Immunol.
68. Zhu JH, Kulich SM, Oury TD, Chu CT (2002) Cytoplasmic aggregates of
phosphorylated extracellular signal-regulated protein kinases in Lewy body
diseases. Am J Pathol 161: 2087–2098.
69. Caamano J, Hunter CA (2002) NF-kappaB family of transcription factors:
central regulators of innate and adaptive immune functions. Clin Microbiol Rev
15: 414–429.
50. Knight D, Mutsaers SE, Prele CM (2011) STAT3 in tissue fibrosis: is there a role
in the lung? Pulm Pharmacol Ther 24: 193–198.
51. Rose-John S, Scheller J, Elson G, Jones SA (2006) Interleukin-6 biology is
coordinated by membrane-bound and soluble receptors: role in inflammation
and cancer. J Leukoc Biol 80: 227–236.
52. Saunders BM, Frank AA, Orme IM, Cooper AM (2000) Interleukin-6 induces
early gamma interferon production in the infected lung but is not required for
generation of specific immunity to Mycobacterium tuberculosis infection. Infect
Immun 68: 3322–3326.
53. Muhl H (2013) Pro-Inflammatory Signaling by IL-10 and IL-22: Bad Habit
Stirred Up by Interferons? Front Immunol 4: 18.
54. de Waal Malefyt R, Abrams J, Bennett B, Figdor CG, de Vries JE (1991)
Interleukin 10(IL-10) inhibits cytokine synthesis by human monocytes: an
autoregulatory role of IL-10 produced by monocytes. J Exp Med 174: 1209–
1220.
55. Bhattacharyya S, Sen P, Wallet M, Long B, Baldwin AS, Jr., et al. (2004)
Immunoregulation of dendritic cells by IL-10 is mediated through suppression of
the PI3K/Akt pathway and of IkappaB kinase activity. Blood 104: 1100–1109.
56. Zhang X, Majlessi L, Deriaud E, Leclerc C, Lo-Man R (2009) Coactivation of
Syk kinase and MyD88 adaptor protein pathways by bacteria promotes
regulatory properties of neutrophils. Immunity 31: 761–771.
57. Murray PJ, Young RA (1999) Increased antimycobacterial immunity in
interleukin-10-deficient mice. Infect Immun 67: 3087–3095.
58. Jacobs M, Brown N, Allie N, Gulert R, Ryffel B (2000) Increased resistance to
mycobacterial infection in the absence of interleukin-10. Immunology 100: 494–
501.
59. Mazurek J, Ignatowicz L, Kallenius G, Svenson SB, Pawlowski A, et al. (2012)
Divergent effects of mycobacterial cell wall glycolipids on maturation and
function of human monocyte-derived dendritic cells. PLoS One 7: e42515.
PLOS ONE | www.plosone.org
11
January 2014 | Volume 9 | Issue 1 | e86466