IL-1 Receptor-Mediated Signal Is an Essential Component
of MyD88-Dependent Innate Response to Mycobacterium
Cecile M. Fremond,* Dieudonne ´e Togbe,* Emilie Doz,* Stephanie Rose,* Virginie Vasseur,*
Isabelle Maillet,* Muazzam Jacobs,†Bernhard Ryffel,1,2*†and Valerie F. J. Quesniaux1,2*†
MyD88, the common adapter involved in TLR, IL-1, and IL-18 receptor signaling, is essential for the control of acute Mycobacterium
tuberculosis (MTB) infection. Although TLR2, TLR4, and TLR9 have been implicated in the response to mycobacteria, gene disruption
for these TLRs impairs only the long-term control of MTB infection. Here, we addressed the respective role of IL-1 and IL-18 receptor
pathways in the MyD88-dependent control of acute MTB infection. Mice deficient for IL-1R1, IL-18R, or Toll-IL-1R domain-containing
adaptor protein (TIRAP) were compared with MyD88-deficient mice in an acute model of aerogenic MTB infection. Although primary
MyD88-deficient macrophages and dendritic cells were defective in cytokine production in response to mycobacterial stimulation,
IL-1R1-deficient macrophages exhibited only a reduced IL-12p40 secretion with unaffected TNF, IL-6, and NO production and up-
regulation of costimulatory molecules CD40 and CD86. Aerogenic MTB infection of IL-1R1-deficient mice was lethal within 4 wk with
2-log higher bacterial load in the lung and necrotic pneumonia but efficient pulmonary CD4 and CD8 T cell responses, as seen in
MyD88-deficient mice. Mice deficient for IL-18R or TIRAP controlled acute MTB infection. These data demonstrate that absence of
IL-1R signal leads to a dramatic defect of early control of MTB infection similar to that seen in the absence of MyD88, whereas IL-18R
and TIRAP are dispensable, and that IL-1, together with IL-1-induced innate response, might account for most of MyD88-dependent
host response to control acute MTB infection. The Journal of Immunology, 2007, 179: 1178–1189.
the pathogen, only 10% develop overt clinical symptoms, whereas
roughly 90% of the infected persons contain the infection (1). Prom-
inent mechanisms of the host leading to protective immunity include
secretion of several cytokines and chemokines, together with APCs
and T cells for mounting an adaptive immunity (2).
Several pattern recognition receptors have been involved in
MTB recognition, including scavenger receptor, complement re-
ceptor, and more recently dendritic cell (DC)-specific C-type lectin
ICAM3-grabbing nonintegrin (DC-SIGN) (3). The contribution in
MTB recognition of the TLRs, involved in pathogen recognition
and innate immune cells activation, thereby linking innate and
adaptive immunity (4), was investigated. Live MTB, but also sev-
eral MTB cell wall and several secreted components such as 19-
kDa lipoprotein, lipomannan, and mannosylated phosphatidylino-
uberculosis is a highly infectious respiratory infection
caused by Mycobacterium tuberculosis (MTB).3Although
one-third of the world’s population has been in contact with
sitol, activate macrophages and dendritic cells (DC) through TLR2
(5–11), and the contribution of other TLRs such as TLR4 or TLR9
in MTB motives recognition has been reported (12, 13).
Most TLRs, with the exception of TLR3, use the intracellular
adaptor protein MyD88 to link receptor recognition with activation
of IL-1R-associated kinase and TNFR-associated factor, translo-
cation of NF-?B and gene transcription (4). We showed recently
that MyD88 was essential in the control of MTB infection. Indeed,
absence of MyD88 resulted in a dramatic reduction of host resis-
tance to MTB (14, 15), as it compromised resistance to several
infectious agents (16–23). MyD88-deficient mice succumbed
within 4 wk of aerogenic MTB infection with acute, necrotic pul-
monary infection despite their ability to mount an adaptive im-
mune response (14). Therefore, the MyD88 signaling pathway is
crucial for the early control of acute MTB infection.
TLR2, TLR4, or TLR6 seemed to play no or only a minor role in
the early host response to MTB infection in vivo (24–29), although
TLR2 (30) and TLR4 (24) were required to control the chronic stage
of infection. Therefore, a partial redundancy of the TLRs involved in
MTB recognition might explain a rather modest phenotype observed
in the absence of single TLR pathways, that was more pronounced in
pronounced in the absence of MyD88 adaptor, invalidating simulta-
neously the signaling of most TLRs.
However, MyD88 is involved not only in TLRs, but also in both
IL-1 and IL-18R/IL-1R-associated kinase signaling and the con-
tribution of these signals in the defective response to MTB ob-
served in MyD88-deficient mice cannot be excluded. Previous re-
ports showed rather limited host resistance defects upon MTB
infection of IL-18 (31), IL-1? or IL-R1-deficient mice (32, 33).
To address the relative contribution of IL-1 and IL-18 vs TLRs
signal in the substantial impairment of host response to acute MTB
infection seen in the absence of MyD88, we investigated side by
*University of Orleans and Centre National de la Recherche Scientifique, Molecular
Immunology and Embryology, Orleans, France; and†Division of Immunology, In-
stitute of Infectious Disease and Molecular Medicine, Health Sciences Faculty, Uni-
versity of Cape Town, Cape Town, South Africa
Received for publication August 30, 2006. Accepted for publication May 12, 2007.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1B.R. and V.F.J.Q. contributed equally to this study as senior authors.
2Address correspondence and reprint requests to Drs. Valerie Quesniaux and Bern-
hard Ryffel, Transgenose Institute, 3B rue de la Fe ´rollerie, 45071 Orle ´ans, France.
E-mail addresses: email@example.com and firstname.lastname@example.org
3Abbreviations used in this paper: MTB, Mycobacterium tuberculosis; DC, dendritic
cell; TIRAP, Toll-IL-1R domain-containing adaptor protein; iNOS, inducible NO syn-
thase; BCG, bacillus Calmette-Gue ´rin; HKLM, heat-killed Listeria monocytogenes; MOI,
multiplicity of infection; i.n., intranasally; BLP, bacterial lymphopeptide.
Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00
The Journal of Immunology
side IL-1R1 and IL-18R-deficient mice, which cannot respond to
IL-1 or IL-18, respectively, and Toll-IL-1R domain-containing
adaptor protein (TIRAP)-deficient mice that have an impaired sig-
naling for both TLR2 and TLR4, and MyD88-deficient in an acute
model of aerogenic MTB infection. The current data demonstrate
that absence of IL-1R signal recapitulated most of the dramatic
defect of early control of MTB infection seen in the absence of
MyD88, whereas IL-18R and TIRAP were dispensable for host
response to acute MTB infection.
Materials and Methods
Mice deficient for IL-1R1 (34), IL-18R (35), MyD88 (16), or TIRAP (36)
were bred in our animal facility at the Transgenose Institute (Centre Na-
tional de la Recherche Scientifique). MyD88-deficient mice were back-
crossed 10 times on the C57BL/6 genetic background, 7 times for IL-1R1-
deficient mice, and 6 times for TIRAP-deficient mice. For experiments, adult
(8- to 15-wk-old) animals were kept in sterile isolators in a biohazard animal
unit. The infected mice were monitored regularly for clinical status and
weighed weekly. All animal experiments complied with the ethical and animal
experiment regulations of the French Government.
Bacteria and infection
M. tuberculosis H37Rv (Pasteur) and Mycobacterium bovis bacillus
Calmette-Gue ´rin (BCG) (Pasteur strain 1173P2) were grown to mid-log
phase in Middlebrook 7H9 liquid medium (Difco Laboratories), supple-
mented with 10% oleic acid-albumin-dextrose-catalase (Difco Laborato-
ries) at 37°C. Aliquots were prepared and frozen at ?80°C. Before use, an
aliquot was thawed, briefly vortexed, and diluted in sterile saline contain-
ing 0.04% Tween 20 and clumping was disrupted by 20 repeated aspira-
tions through a 30-gauge needle (Omnican). Pulmonary infection with M.
tuberculosis H37Rv was performed by delivering ?200 bacteria into both
nasal cavities (20 ?l each) under xylazine-ketamine anesthesia, and the
inoculum size was verified by sacrificing mice 48 h after infection and
determining bacterial load in the lungs of infected mice.
Bacterial load in tissues
Bacterial loads in the lung, liver, and spleen of infected mice were eval-
uated at different time points after infection with M. tuberculosis H37Rv as
described (37). Organs were weighed, and defined aliquots were homog-
enized in 0.04% Tween 20 containing PBS. Tenfold serial dilutions of
organ homogenates were plated in duplicates onto Middlebrook 7H10 agar
plates containing 10% oleic acid-albumin-dextrose-catalase and incubated
at 37°C. Colonies were enumerated at 3 wk, and results are expressed as
log10CFU per organ.
Preparation and analyses of lung homogenates for cytokine/
Mice were deeply anesthetized with xylazine-ketamine and perfused with
0.02% EDTA-PBS until the lung tissue turned white. Whole lungs were
harvested, weighed, placed in 1 ml of 4°C PBS solution in a specific sterile
plastic tube, and homogenized in Dispomix homogenizer (Medic Tools;
Axonlab) for 20 s at 6000 rpm. Homogenates were centrifuged at 14,000
rpm, sterilized by filtration through 0.22-?m pore size filter (Costar-Corning),
and stored at ?80°C until determination of IL-1?, IL-1?, IL-6, IL-12p40,
TNF, MCP-1, and IFN-? levels by multiplex cytokine detection assays using
Luminex technology (Cytokine Profiler from Millipore, Upstate).
Histopathological and immunohistochemical analysis
For histological analysis lungs were removed at different time points of
infection, fixed in 4% phosphate-buffered formalin, and embedded in par-
affin. Sections of 2–3 ?m were stained with H&E and a modified Ziehl-
Neelsen method, staining in a prewarmed (60°C) carbol-fuchsin solution
for 10 min followed by destaining in 20% sulfuric acid and 90% ethanol
before counterstaining with methylene blue. For immunostaining, forma-
lin-fixed paraffin-embedded sections were deparaffinized and rehydrated
and stained with rabbit anti-mouse Ab specific for inducible NO synthase
(iNOS; BD Pharmingen). The tissue sections were then washed in PBS and
incubated for 30 min at room temperature with the biotinylated secondary
Ab. The sections were then incubated with avidin-biotin complexes (ABC
Vector Kit; Vector) for 30 min, washed, and incubated with diaminoben-
zidine substrate (Dako). After a rinsing in PBS, the tissue sections were
mounted in Eukitt (Kindler).
FACS of infiltrating cells from infected lung
FACS analysis of inflammatory cells from infected lung was performed as
described (38, 39). In brief, mice were deeply anesthetized with xylazine-
ketamine and perfused with 0.02% EDTA-PBS until the tissue turned
white. After removal, lung tissue was sliced into 1- to 2-mm3pieces and
was incubated in RPMI 1640 (Invitrogen Life Technologies) containing
5% FCS, antibiotics (penicillin 100 U/ml-streptomycin 100 ?g/ml), 10 mM
HEPES (Invitrogen Life Technologies), and collagenase (150 U/ml),
DNase (50 U/ml; Sigma-Aldrich). After 1.5 h of incubation at 37°C, a
single-cell suspension was obtained by vigorous pipetting. Cells were
washed three times in PBS containing 0.01% NaN3and 0.5% BSA and
were then stained according to Ab manufacturer protocols. Rat anti-mouse
CD4-PerCP (clone RM4-5), CD8-APC (clone 53-6.7), CD11b-PerCP
(clone M1/70), Ly6G-PE (clone RD6-8C5), CD40-PE (clone 3/23), CD86-
FITC (clone GL1), IA/IE-FITC (clone 2G9) were purchased from BD
Pharmingen. IFN-? secretion by CD4?or CD8?cells was detected using
a capture anti-IFN-? rat IgG1 mAb conjugated to a CD4- or CD8-specific
rat IgG2b mAb, according to the manufacturer’s recommendations (mouse
IFN-? secretion assay; Miltenyi Biotec). After restimulation of the lung-
infiltrating cells with a lyophilized preparation of BCG culture supernatant
(supBCG at 10 ?g/ml), irrelevant Ag heat-killed Listeria monocytogenes
(HKLM; at a multiplicity of infection (MOI) of 100) or medium for 16 h
at 37°C, the cells were incubated with the capture conjugate for 45 min at
37°C, and captured IFN-? was detected with a second anti-IFN-? rat IgG1-
PE. Stained cells were washed twice, fixed with 1% paraformaldehyde
(FACS Lysing solution; BD Pharmingen), and analyzed by flow cytometry
on a LSR analyzer (BD Biosciences). Data were processed with CellQuest
software (BD Immunocytometry Systems).
Primary macrophage and DC cultures
Murine bone marrow cells were isolated from femurs and differentiated
into macrophages after culturing at 106cells/ml for 7 days in DMEM
(Sigma-Aldrich) supplemented with 2 mM L-glutamine and 2 ? 10?5M
2-ME, 20% horse serum, and 30% L929 cell-conditioned medium as a
source of M-CSF (40). Three days after washing and reculturing in fresh
medium, the cell preparation contained a homogeneous population of mac-
rophages. Alternatively, murine bone marrow cells were differentiated into
myeloid DCs after culturing (change on days 3, 6, and 8) at 2 ? 106
cells/ml for 10 days in RPMI supplemented with 10% FCS and 4% J558L
cell-conditioned medium as a source of GM-CSF as described (41).
Stimulation of macrophages and DCs
Bone marrow-derived macrophages and DCs were plated in 96-well mi-
croculture plates (at 105cells/well in DMEM supplemented with 2 mM
L-glutamine and 2 ? 10?5M 2-ME) and stimulated with TLR4 agonist
LPS (Escherichia coli, serotype O111:B4, Sigma-Aldrich, at 100 ng/ml),
TLR2 agonist bacterial lymphopeptide (BLP), the synthetic BLP
(S)-Ser-Lys4-OH, trihydrochloride (EMC Microcollections) at 500 ng/ml),
TLR9 agonist CpG (ODN1826 at 125 nM), M. tuberculosis H37Rv (heat
killed for 40 min at 80°C; 2 bacteria per cell), M. bovis BCG (from Pasteur
Institute, Paris, France; at a MOI of 2 bacteria per cell), or with a lyoph-
ilized preparation of BCG culture supernatant (supBCG), heat-killed M.
bovis BCG, or lyophilized/extended freeze-dried BCG (42) (BCG lyoph),
gifts from Professor G. Marchal, Pasteur Institute, Paris, France; at 10
?g/ml). Cell supernatants were harvested after 24 or 48 h of stimulation in
the presence of IFN-? (100 U/ml) for TNF, IL-1?, IL-1?, IL-12p40, and
IL-6 quantification using commercial ELISA (R&D Duoset) and nitrite
measurements by Griess reagents as described (43).
Confocal microscopy of mycobacterial internalization
Macrophage monolayers were established by plating 105cells in 0.2 ml of
DMEM supplemented with 2 mM L-glutamine and 2 ? 10?5M 2-ME onto
sterile glass coverslips in 24-well microtiter plates and incubated overnight
at 37°C in humidified air containing 5% CO2.
BCG internalization was studied using fluorescent M. bovis BCG ex-
pressing GFP (gift from Dr. V. Snewin, Wellcome Trust London, U.K.).
BCG-GFP stored at ?80°C was rapidly thawed, passed 30-fold through a
25-gauge needle and then 10-fold through a 30-gauge needle, sonicated six
times for 15 s, and immediately added to the cultures at a MOI of 1. After
2 h at 37°C under a humidified atmosphere containing 5% CO2, the me-
dium was removed, and the cultures were washed once with warm PBS and
fixed with paraformaldehyde 4% in PBS. After overnight fixation, macro-
phages on coverslips were washed once in warm PBS for 10 min. Cells
were then permeabilized for 3 min with 0.1% Triton X-100 in PBS, washed
for 10 min with PBS, quenched with 50 mM NH4Cl for 30 min, washed for
1179The Journal of Immunology
10 min in water, preincubated for 30 min with 1% BSA in PBS, and
washed again in PBS. To stain F-actin, macrophages were incubated for 20
min with ?-phalloidin conjugated to rhodamine at 5 U/ml (Molecular
Probes) followed by two 5-min washes in PBS. Coverslips were mounted
using DAKO mounting medium with 4?,6?-diamidino-2-phenylindole.
BCG-GFP internalization was assessed using a fluorescence Leica DM
(A–F) and DCs (G and H) prepared from IL-1R1-deficient (?), MyD88-deficient (u), and wild-type (f) mice were incubated with LPS (100 ng/ml), BLP
(500 ng/ml), CpG (125 nM), M. bovis BCG (at a MOI of 2), heat-killed BCG (HKBCG at a MOI of 2), lyophilized BCG (BCG lyoph 10 ?g/ml), a
lyophilized preparation of BCG culture supernatant (supBCG, 10 ?g/ml) or M. tuberculosis H37Rv (at a MOI of 2). After 24 h, the production of TNF
(A), IL-6 (B), IL-12p40 (C and G), IL-1? (H), or nitrite (D) was determined in the supernatants by ELISA or Griess reaction. Up-regulation of CD40 (E)
and CD86 (F) expression by macrophages stimulated with LPS, M. bovis BCG, or H37Rv was analyzed by FACS. Data are from one experiment
representative of three independent experiments with n ? 2 mice per genotype; values are means ? SD.
Proinflammatory cytokine and NO production in IL-1R1- and MyD88-deficient macrophages and DCs. Bone marrow-derived macrophages
M. tuberculosis INFECTION IN IL-1R1- VS MyD88-DEFICIENT MICE
IRBE microscope (?100 oil immersion objective) by counting the mac-
rophages containing one or two isolated intracellular bacteria and the non-
infected macrophages over 10 different observation fields per slide (2 slides
per mouse). Cells derived from two individual mice were tested per geno-
type in each experiment. The slides were counted blindly by two observers.
Analysis was performed using Student’s t and ANOVA tests and values of
p ? 0.05 were considered significant.
Selective reduction in cytokine production, but normal
up-regulation of costimulatory molecules in Mycobacterium-
activated macrophages from IL-1R1 vs MyD88-deficient mice
We showed previously that the production of cytokines, but not the
expression of costimulatory molecules in response to mycobacte-
rial infection is MyD88 dependent (14, 23). Because MyD88 sig-
nals both TLRs, including TLR2, TLR4, and TLR9 that have been
involved in mycobacterial recognition, as well as IL-1R1 and IL-
18R, we further investigated the specific role of these different
pathways in MyD88-dependent responses to mycobacterial Ags.
Bone marrow-derived macrophages from IL-1R1- or MyD88-
deficient mice were stimulated with mycobacteria in vitro, and
their ability to secrete TNF-?, IL-6 and IL-12p40 was determined.
After stimulation with MTB H37Rv or M. bovis BCG either live or
killed by heat or lyophilization, TNF (Fig. 1A) and IL-6 (Fig. 1B)
production were unaffected, whereas IL-12p40 production was
drastically reduced in IL-1R1-deficient macrophages compared
with wild-type controls (Fig. 1C). As expected, TNF, IL-6, and
IL-12p40 production was strongly reduced in MyD88-deficient
macrophages. Similar results were obtained after 24 or 48 h of
stimulation, excluding that the low IL-12p40 levels seen in IL-
1R1-deficient macrophages were merely due to delayed kinetics
to control acute M. tuberculosis infection. Mice
deficient for IL-1R1, MyD88, or IL-18R and
wild-type mice were exposed to a low aerogenic
dose of M. tuberculosis H37Rv (200 CFU/
mouse i.n.) and monitored for body weight (A;
mean values of n ? 6–8 mice per group from 1
representative experiment of 4 independent ex-
periments). Lung wet weight (B) and the number
of viable bacteria present in the lungs (C) of IL-
1R1-deficient mice (?), MyD88-deficient mice
(u) and wild-type controls (f) were measured
24 days postinfection. Lung weight (D) and bac-
terial load (E) were measured 95 days postinfec-
tion for IL-18R-deficient (o) and wild-type con-
trols (f). Mean ? SD of n ? 3–4 lung from 1
representative experiment of 3 and 2 indepen-
dent experiments at day 24 and 95, respectively;
?, p ? 0.05; ??, p ? 0.01.
IL-1R1-deficient mice are unable
1181The Journal of Immunology
(data not shown). We further tested the IL-1R1 dependence of
mycobacterial induced iNOS activation and demonstrate unaf-
fected nitrite production in IL-1R1-deficient macrophages com-
pared with wild-type controls, whereas the nitrite production at
24 h was strongly reduced in MyD88-deficient macrophages (Fig.
1D). Nitrite production was only delayed and was largely restored
after 48 h of culture of MyD88-deficient macrophages (data not
In contrast, the expression of the costimulatory molecules CD40
and CD86 was up-regulated in both IL-1R1 or MyD88-deficient
macrophages in response to mycobacteria (Fig. 1, E and F) as in
wild-type control cells. The expression of MHC class II IA/IE,
down-regulated after LPS stimulation in wild-type control cells,
was up-regulated in MyD88-deficient macrophages but much less
in IL-1R1-deficient macrophages in response to mycobacteria
(data not shown).
The reduced IL-12p40 production consistently seen in IL-1R1-
deficient macrophages was not observed in bone marrow-derived
DCs. IL-1R1-deficient DCs stimulated with MTB H37Rv or M.
bovis BCG produced normal levels of NO, TNF, and IL-6 (data not
shown) but also high levels of IL-12p40 comparable with those of
wild-type controls (Fig. 1G), whereas these secretions were sus-
tantially reduced in MyD88-deficient DCs. The reduced IL-12p40
secretion in IL-1R1-deficient macrophages was suggestive of an
indirect, IL-1-mediated mechanism. To further assess the direct
involvement of IL-1R1 in macrophages and DC response to my-
cobacteria, we determined the levels of IL-1? and IL-1?. Myco-
bacterial stimulation of wild-type macrophages yielded a secretion
of IL-1? and IL-1? in the 100- to 600-ng/ml range that was
strongly reduced or absent in MyD88-deficient cells but was sim-
ilar in IL-1R-deficient macrophages (data not shown) or DC cul-
ture supernatants (Fig. 1H).
IL-18R seemed to play no role in the in vitro response to my-
cobacteria analyzed, because macrophages and DCs from IL-18R-
deficient mice produced normal levels of TNF, IL-6, IL-12p40,
and NO after stimulation with MTB H37Rv or M. bovis BCG as
above (data not shown).
Therefore, the production of TNF, IL-1?, IL-1?, IL-6, and NO,
as well as the expression of costimulatory molecules in response to
mycobacterial stimulation, is independent of IL-1R1 and IL-18R
signal, although IL-12p40 production seems to be indirect, medi-
ated via IL-1/IL-1R signaling in bone marrow-derived macro-
phages. On the basis of these results, we hypothesized that the
IL-1R pathway might account for part for the defective cytokine
production observed in MyD88-deficient cells and that IL-1R1 sig-
naling might be critical for the innate immune response to MTB
Lethal M. tuberculosis infection in the absence of IL-1R1
We showed earlier that MyD88-deficient mice are extremely sen-
sitive to virulent M. tuberculosis H37Rv infection and die within
4 wk postinfection (14). Because the contribution of individual
TLRs, namely TLR2, TLR4, and TLR9, to MTB response seems
rather modest, with only delayed increase in sensitivity after 4–6
mo of infection (Refs. 13, 24–27, and 30 and unpublished data),
we now addressed the contribution of the MyD88-dependent, non-
TLR pathways. IL-1R1-deficient mice infected with a low dose
(200 CFU/mouse) of virulent M. tuberculosis H37Rv started to
lose body weight around 3 wk and died ?4 wk postinfection (Fig.
2A), very similar to the MyD88-deficient mice. In contrast, IL-
18R-deficient mice survived for ?3 mo, the duration of the ex-
periment, similar to wild-type mice. Both IL-1R1- and MyD88-
deficient mice exhibited substantially increased lung weight (Fig.
2B) and ?2 log10higher bacterial load in the lungs as compared
with wild-type controls 3 wk after infection (Fig. 2C). This defec-
tive response was not observed in mice deficient for IL-18R, which
exhibited no overt lung inflammation (Fig. 2E) and controlled bac-
terial load (Fig. 2F), even 95 days after infection. Therefore, IL-
1R1-deficient mice are highly susceptible to MTB infection, sim-
ilar to MyD88-deficient mice, whereas absence of IL-18R was not
Necrotic pneumonia develops rapidly after M. tuberculosis
infection in the absence of IL-1R1
The establishment of well-defined granulomas, the result of a
structured cell-mediated immune response, is crucial for inhibiting
with large nodules but defective granuloma formation in response to M.
tuberculosis infection, similar to MyD88-deficient mice. Lung tissue from
IL-1R1-, MyD88-, or IL-18R-deficient mice and wild-type controls was
analyzed on day 24 after M. tuberculosis H37Rv infection (200 CFU i.n.).
Lungs of IL-1R1-deficient mice showed large and confluent nodules in
comparison with wild-type mice, which were similar to MyD88-deficient
lungs (A). Microscopic examination showing extensive inflammation and
necrosis in infected IL-1R1 and MyD88-deficient lungs (B; H&E, magni-
fication ?50 for low power and ?400 for details) with abundant myco-
bacteria in the extracellular space (C; Ziehl-Neelsen stain; ?1000). Lungs
of IL-18R-deficient mice exhibited well-defined granuloma with few my-
cobacteria, comparable with results in wild-type lungs (A–C).
IL-1R1-deficient mice exhibit acute necrotic pneumonia
sponse to M. tuberculosis infection. Expression of iNOS in the lung tissue
from IL-1R1 and MyD88-deficient mice and wild-type controls was ana-
lyzed by immunostaining on day 24 after M. tuberculosis H37Rv infection
(200 CFU i.n.). Both IL-1R1- and MyD88-deficient mice showed a strong
induction of iNOS in comparison with wild-type mice. ?100.
iNOS induction in lungs of IL-1R1-deficient mice in re-
M. tuberculosis INFECTION IN IL-1R1- VS MyD88-DEFICIENT MICE
deficient (?), MyD88-deficient (u) and wild-type (f) mice were isolated on day 26 after M. tuberculosis H37Rv infection and analyzed by flow cytometry for
the expression of CD4?and CD8?(M) and CD11b, Ly-6G, costimulatory molecules CD40, CD86, and MHC class II IA-IE(N). Results, expressed as percent
of positive cells, are mean ? SD from two mice per genotype, from one representative of two independent experiments. IFN-? secretion by lung infiltrating CD4?
or CD8?cells was detected using a capture anti-IFN-? Ab conjugated to a CD4 (A–E)- or CD8 (F–J)-specific Ab, after restimulation of the cells with medium
(A and F), an irrelevant Ag HKLM (B and G), or a lyophilized preparation of BCG culture supernatant (supBCG; C–E and H–J) for 16 h at 37°C. After
restimulation and incubation with the capture conjugate for 45 min at 37°C of the cells from wild-type mice (A–C and F–H), or mice deficient for IL-1R1 (D and
I) or MyD88 (E and J), captured IFN-? was detected with a second, labeled anti-IFN-? Ab. Cells were gated on typical lymphocyte forward light scatter/side light
scatter and further gated for CD4?or CD8?cells. Individual, representative dot plots are shown in A–J, and bar graphs showing the mean ? SD percentage of
IFN-? producing CD4?and CD8?cells from two to three mice per genotype are shown in K and L, respectively.
Inflammatory cell recruitment, activation, and priming in the lung of IL-1R1-deficient infected mice. Infiltrating cells from the lungs of IL-1R1-
1183The Journal of Immunology
mycobacterial growth. In view of the increased lung weight indic-
ative of a strong local inflammation, we next examined the lung
morphology and asked whether the IL-1R1 pathway is essential for
granuloma formation upon MTB infection. Macroscopically, the
lungs of IL-1R1-deficient mice displayed pleural adhesions and
effusions and large subpleural and confluent nodules, similar to
MyD88-deficient mice, whereas mice lacking IL-18R behaved like
wild-type controls (Fig. 3A). Microscopic investigation of the
lungs of IL-1R1-deficient mice revealed severe inflammation with
important reduction of ventilated alveolar spaces and massive
mononuclear and neutrophil infiltrations with extensive confluent
necrosis in the absence of proper granuloma formation at 24 days
(Fig. 3B) and abundant mycobacteria within macrophages and also
in the extracellular space (Fig. 3C), similar to MyD88-deficient
mice. The pathology was more controlled in IL-18R-deficient
mice, similar to wild-type controls which developed typical
granulomatous lesions characterized by epithelioid macro-
phages accompanied by lymphocytic perivascular and peribron-
chiolar cuffing. The lung lesions observed in IL-1R1-deficient
mice were reminiscent of those induced by mycobacterial in-
fection in the absence of functional MyD88 or TNF pathways
(14, 44–46). Thus, the absence of IL-1R1 pathway alone ac-
counts for most of the massive necrosis and infiltration of in-
flammatory cells in the lungs with uncontrolled MTB growth
seen in MyD88-deficient mice.
The production of NO and related nitrogen intermediates by
macrophages is a major effector mechanism responsible for anti-
mycobacterial activity. We next investigated the extent of pulmo-
nary macrophage activation in IL-1R1- and MyD88-deficient mice
by iNOS immunostaining (Fig. 4). On day 24 postinfection, iNOS
expression in wild-type mice was confined to macrophages resid-
ing in well-defined granulomas, whereas IL-1R1 and MyD88-de-
ficient mice showed massive iNOS expression throughout the pul-
Therefore, iNOS expression is independent of the IL-1R/
MyD88 pathway but appears to be unable to control the MTB
Efficient recruitment of lymphocytes and myeloid cells in IL-
In view of the acute and uncontrolled pulmonary infection in both
IL-1R1- and MyD88-deficient mice, we asked how the immune
response and the recruitment of immunoinflammatory cells in the
lung parenchyma was modulated in these mice. The total number
of cells recovered from the lungs of both groups was comparable
with those of wild-type mice (data not shown). We showed pre-
viously that MyD88-deficient T cell recruitment and activation
was similar to that of wild-type controls after MTB infection.
Here, flow cytometric analysis revealed similar amounts of CD4?
and CD8?T cells and activated CD44?subsets in IL-1R1 or
MyD88-deficient and control lungs at 4 wk of infection (Fig. 5).
To further assess Th1 immune response in IL-1R1-deficient mice,
we measured the expression of CD4?and CD8?T cell-derived
IFN-? in MTB-infected IL-1R1-deficient lungs. Using conjugated
reagents designed to stain freshly produced IFN-? by CD4?or
CD8?cells and flow cytometry analysis, we showed that the lung-
infiltrating CD4?(Fig. 5, A–E) and CD8?(Fig. 5, F–J) cells of
both IL-1R1 and MyD88-deficient mice produced levels of IFN-?
similar to those of wild-type mice upon Ag restimulation. This
response was specific because essentially no IFN-? secretion was
detected in cells treated with medium only or incubated with an
irrelevant Ag, HKLM (Fig. 5, K and L). Further, the level of cel-
lular IFN-? production, estimated by the mean fluorescence inten-
sity, was similar in the three mouse lines.
However, there was a markedly increased number of CD11b?
cells in the lung of both IL-1R1- and MyD88-deficient as com-
pared with wild-type mice, associated with up-regulated expres-
sion of IA/IE and CD86, and CD11b?cells expressing high levels
of Ly6G were particularly increased (Fig. 5N), consistent with the
strong neutrophil pulmonary infiltration seen in these mice after
MTB infection (Fig. 3).
Thus, the data demonstrate that the high infectious burden in
MTB-infected IL-1R1 or MyD88-deficient mice is accompanied
by a vigorous lung inflammatory response with increased macro-
phages and neutrophils recruitment and cell activation in terms of
expression of MHC class II and costimulation molecules, and a
normal recruitment, activation, and priming of effector CD4?and
CD8?T lymphocytes, that occurred in the absence of IL-1R1
and/or MyD88 signaling.
Cytokine and chemokine pulmonary levels in MTB-infected IL-
IL-1R1-deficient macrophages and dendritic cells show a normal
cytokine production in response to mycobacteria in vitro but for a
defect in IL12p40 production by macrophages (Fig. 1). However,
the marked leukocyte infiltration in the lungs at 4 wk of MTB-
infected IL-1R1-deficient mice suggested an increase of cyto-
kines and/or chemokines levels. We thus measured the local
cytokine and chemokine concentrations in the lungs of infected
wild-type and IL-1R-deficient mice (Fig. 6). IL-1R1-deficient
mice exhibited elevated levels of IL-1?, IL-1?, IL-6, TNF,
MCP-1, and IFN-? levels 4 wk after MTB infection, which may
be linked to the high bacterial burden in these mice. IL-12p40
levels were very low in wild-type mice and below the detection
limit in IL-1R1-deficient mice (Fig. 6G), reminiscent of the reduced
IL-12p40 production by IL-1R1-deficient macrophages. Therefore,
MTB-infected IL-1R1-deficient mice showed increased pulmonary
levels of IL-1?, IL-1?, IL-6, TNF-?, MCP-1, and IFN-? but not IL-
12p40, as compared with control mice, in line with the strong local
inflammatory cell infiltration seen in these mice.
Limited role of IL-1R1 on mycobacterial internalization and
growth control by macrophages
We showed earlier that uptake of fluorescently labeled M. bovis
BCG was reduced in the absence of functional MyD88 pathway,
but less so in the absence of TLR4 and TLR2 (23, 47), suggesting
the contribution of other MyD88-dependent mechanisms besides
TLR2 and TLR4. To investigate whether IL-1R1 might be in-
volved in the internalization of mycobacteria, we infected macro-
phages from wild-type mice, IL-1R1-deficient mice, and MyD88-
deficient mice with GFP-transfected BCG and analyzed bacteria
internalization by confocal microscopy. Although BCG-GFP in-
ternalization was reduced by one-half in MyD88-deficient macro-
phages, bacteria uptake by IL-1R1-deficient macrophages was
70% of that seen with wild-type macrophages (Fig. 7). Also, the
control of MTB growth was impaired in MyD88-deficient macro-
phages as compared with wild-type cells, but IL-1R1-deficient
macrophages controlled MTB growth (data not shown), in line
with the efficient NO production seen in these cells (Fig. 1D).
Therefore, absence of MyD88 signaling is associated with a defect
in internalizing and controlling mycobacterial growth in vitro
which is not recapitulated in IL-1R1-deficient macrophages.
Minor contribution of TLR2/4 TIRAP pathway in the MyD88
We showed earlier that absence of TLR2, and to a lesser extent
TLR4, increased murine susceptibility to MTB in the late, chronic
phase of the infection (24, 30). To further assess the contribution
M. tuberculosis INFECTION IN IL-1R1- VS MyD88-DEFICIENT MICE
of these TLR pathways in the MyD88-dependent response to my-
cobacteria, we compared the response of mice deficient for MyD88
with mice deficient for TIRAP, the adapter restricted to TLR2 and
TLR4 pathways. In vitro, TIRAP-deficient macrophages had a par-
tially impaired response to mycobacteria, similar to that seen with
TLR4 agonist LPS and TLR2 agonist Pam3CSK4(data not shown).
whereas MyD88-deficient macrophages were essentially unre-
sponsive (as shown in Fig. 1). In vivo, the phenotype of TIRAP-
deficient mice was not as marked as that of MyD88- or IL-1R1-
deficient mice (Fig. 8A). All TIRAP-deficient mice survived for at
least 7 wk after infection with 200 CFU of H37Rv, whereas
MyD88- and IL-1R1-deficient mice died within 4 wk. TIRAP-
deficient mice exhibited no overt lung inflammation (Fig. 8B) and
controlled bacterial load (Fig. 8C) on day 26 postinfection, at a
time point where both MyD88- and IL-1R1-deficient mice showed
exacerbated lung inflammation and high bacterial burden. Micro-
scopic examination revealed well defined granuloma in TIRAP-
deficient mice (Fig. 8E), similar to wild-type controls (Fig. 8D),
with few, intracellular mycobacteria in the lung of TIRAP-defi-
cient mice (Fig. 8F). Therefore, impairment of TLR2 plus TLR4
pathways, two of the most prominent TLRs involved in mycobac-
terial recognition, in TIRAP-deficient mice did not compromise
their control of early in vivo MTB infection, as seen in
We showed earlier that the MyD88-mediated signaling pathway is
critically involved in the development of innate, but not adaptive,
immunity in response to MTB infection and led to a strong sen-
sitivity to acute MTB infection in MyD88-deficient mice (14, 15).
Because absence of either TLR2 or TLR4 signaling affected
mostly the long-term control of chronic MTB infection, with little
effect on the early response to acute MTB infection in TLR2 or
TLR4-deficient mice (24, 30), we proposed that other TLRs or
pattern recognition receptors may be involved in this response
(14). A certain TLR redundancy could not be excluded, and the
involvement TLR9 in the response to MTB has recently been
reported (13). TLR9 seemed to act in conjunction with TLR2 be-
cause mice doubly deficient for TLR2 and TLR9 were more sus-
ceptible to MTB infection than either of the single TLR-deficient
mice. However, the phenotype of TLR2 and TLR9 double-defi-
cient mice was not as drastic as that of MyD88-deficient mice (13),
determined in lung homogenates from IL-1R1-deficient and control mice 4 wk after MTB infection. IL-1? (A), IL-1? (B), IL-6 (C), TNF-? (D), IFN-? (E),
MCP-1 (F), and IL-12p40 (G) were quantified by multiplex cytokine detection assays using Luminex technology. Results are expressed as mean ? SD from
three mice per genotype run in duplicate.
High cytokine and chemokine pulmonary levels in MTB-infected IL-1R1-deficient mice. Cytokine and chemokine concentrations were
phages. Internalization of fluorescent M. bovis BCG expressing GFP
(BCG-GFP) by wild-type, IL-1R1-deficient, or MyD88-deficient macro-
phages was quantified after incubation at 37°C for 2 h, overnight fixation,
and staining of F-actin using ?-phalloidin conjugated to rhodamine (A).
BCG-GFP internalization was assessed using a fluorescence Leica DM
IRBE microscope. Results are expressed as the mean ? SD of the per-
centage of macrophages infected with isolated BCG-GFP (n ? 2 mice per
mutated genotype) and are from 1 representative of 2 independent
Mycobacterial internalization in IL-1R1-deficient macro-
1185The Journal of Immunology
questioning the role of other MyD88-dependent but TLR-indepen-
dent pathways in the response to MTB infection.
We thus investigated the contribution of the IL-1 and/or IL-18
signals in the defective control of MTB infection observed in
MyD88-deficient mice. We show here that absence of IL-1R1 led
to a strong defect of response to acute MTB infection, similar to
that seen in MyD88-deficient mice, and propose that IL-1R path-
way is an essential component of the MyD88-mediated signaling
leading to the development of innate response to MTB infection.
MyD88 is at the crossroad of multiple TLR-dependent and
TLR-independent signaling pathways, including IL-1R and
IL-18R. In addition, other MyD88-dependent pathways were re-
cently described such as the involvement of MyD88 in cross-talk
with the focal adhesion kinase pathway (48) or with others mem-
bers of the IL-1R family (49). In cutaneous Staphylococcus aureus
infection, the extreme sensitivity of MyD88-deficient mice has re-
cently been ascribed, at least in part, to deficient IL-1R/IL-18R
signaling pathways (50). IL-1? and IL-1? bind to receptors termed
the type 1 and type 2 IL-1 receptors. IL-1R1 is responsible for
specific signaling, whereas IL-1R2 functions as a nonsignaling de-
coy receptor. To determine the effect of a defect in IL-1-mediated
signaling, mice have been produced with a genetically disrupted
type I IL-1 receptor gene (34). Previous reports on MTB infection
of IL-18 (31) or IL-1? or IL-1R1-deficient mice showed less strik-
ing defects in host resistance (32, 33) than those seen in MyD88-
deficient mice (14). However, in our hands, mice deficient for IL-
1R1 succumb within 3–4 wk to severe and uncontrolled infection
after exposure to 200 CFU intranasally (i.n.), which is the earliest
time point at which severely immunocompromised mice such as
MyD88- or TNF-deficient mice develop fatal MTB infection (14,
45, 46). Differences in experimental conditions might explain the
milder results reported in the high dose model used by Juffermans
et al. (32) that led to progressive fatal infection 3-?20 wk after
i.n. application of an inoculum of 105CFU of H37Rv in
The involvement of IL-1R pathway in the control of MTB in-
fection could be multifold, IL-1 and/or IL-1R1 potentially exerting
direct or indirect effects. No clear direct mycobactericidal effect of
IL-1, nor indirect IL-1R1 mediated control of bacterial growth
through IL-1 autocrine activation could be evidenced in IL-1R1-
deficient macrophages nor after addition of exogenous IL-1 (10–
100 ng/ml) or, conversely, of IL-1Ra- or IL-1-neutralizing Abs to
MTB-infected macrophages (data not shown). Also, there was no
drastic direct effect of IL-1R pathway on mycobacterial internal-
ization, as reported for MyD88 (23), because we show here that
internalization was less affected by the absence of IL-1R1 than in
the absence of MyD88, likely due to the contribution of TLRs in
this process. Therefore, the effect of IL-1 on MTB infection control
seems to be indirect, by acting on the innate/inflammatory or adap-
tive response, or on effector mechanisms. We addressed these dif-
ferent aspects below.
In vivo, both MyD88- and IL-1R1-deficient mice showed mas-
sive lung pathology with inflammatory cell infiltration, necrosis,
and iNOS expression at 4 wk, at a late stage of infection. This was
in line with the fact that nitrite production was unaffected and only
delayed, respectively, in IL-1R1- and MyD88-deficient macro-
phages, and that a dominant negative MyD88 mutant did not im-
pair the iNOS promoter in murine macrophages (51). However, the
strong iNOS expression was not sufficient to control MTB bacte-
rial growth, and abundant acid-fast resistant bacilli were present in
the lung of both MyD88- and IL-1R1-deficient mice. Although
IFN-?-iNOS2 is considered a principal effector mechanism in
MTB control, other pathways exist such as the p47 GTP family
member, LRG-47, that acts independently of iNOS to protect
against MTB infection (52). LRG-47-deficient mice are highly sus-
ceptible to MTB infection (53). LRG-47 is important for the mat-
uration of the phagosome, and autophagy of infected cells repre-
sents an alternative mechanism for the intracellular MTB
elimination (54). This could be an alternative effector mechanism
involved in the increased susceptibility of MyD88- and IL-1R1-
deficient mice. The IL-1 or MyD88 dependence of LRG-47 acti-
vation has not yet been investigated.
Insufficient cell replacement in the face of massive programmed
cell death in granuloma that results in necrosis and uncontrolled
MTB proliferation (55) may also contribute to the severe pheno-
type of MTB-infected IL-1R1-deficient mice. However, the high
bacterial burden was associated with elevated levels of cytokines
and chemokines such as MCP-1, likely to trigger local inflamma-
tory cell infiltration in the lung of MTB-infected IL-1R1-deficient
mice. All cytokines tested, IL-1?, IL-1?, IL-6, TNF, and IFN-?,
were elevated in the lung of infected IL-1R1-deficient mice, with
the notable exception of IL-12p40 which was reduced as compared
with wild-type mice.
Indeed, in vitro IL-1R1 deficiency had no influence on TNF,
IL-1?, IL-1?, IL-6, and NO secretion, but it severely impaired
IL-12p40 production by macrophages. Such decreased IL-12p40
mice. TIRAP-deficient (open symbols) and wild-type mice (closed sym-
bols) were exposed to a low aerogenic dose of M. tuberculosis H37Rv (200
CFU/mouse i.n.) and monitored for body weight (A; mean values of n ?
6–8 mice per group from 1 representative experiment of 2 independent
experiments). Lung wet weight (B) and the number of viable bacteria
present in the lungs (C) of TIRAP-deficient mice (spotted bar) and wild-
type controls (solid bar) were measured 26 days post infection (B and C;
results are mean ? SD of n ? 3–4 mice per group, from one representative
experiment of 2 independent experiments). Microscopic examination
showing well-defined granuloma in TIRAP-deficient (E) and wild-type
mice (D; H&E, ?100) with few mycobacteria in TIRAP-deficient mice (F;
Ziehl-Neelsen stain, ?400).
Control of M. tuberculosis infection in TIRAP-deficient
M. tuberculosis INFECTION IN IL-1R1- VS MyD88-DEFICIENT MICE
secretion was not observed in IL-1R1-deficient DCs, which may
partly be explained by the very robust IL-12p40 expression in bone
marrow-derived DC, often 5- to 10-fold higher than what is seen
in bone marrow-derived macrophages, or by different regulatory
mechanisms in these cells. Disparity in IL-12p40 release in DC
and macrophages to MTB has recently been documented, associ-
ated with rapid and extensive remodeling at nucleosome 1 of the
p40 promoter in DC, and with different TLR use (56). Here, the
reduced IL-12p40 secretion by IL-1R1-deficient macrophages, but
not DC, is suggestive of an autocrine IL-1 activation loop in this
process. Indeed, mycobacterial stimulation yielded a secretion of
IL-1? and IL-1? by wild-type macrophages and DCs that was
severely impaired in the absence of MyD88 but unaffected in IL-
1R1-deficient cells. Therefore, the intrinsic defect in the IL-1 path-
way in IL-1R1-deficient mice might be associated with an indirect,
IL-1 mediated, effect on local IL-12p40 secretion likely to further
compromise their resistance to MTB infection (57, 58).
Because we showed previously that MyD88-deficient mice are
able to mount a protective Th1 immune response to mycobacteria
upon BCG vaccination (14), and because MyD88-deficient mice
have a disrupted IL-1R signal pathway, we anticipated that Th1
immunity would not be impaired in IL-1R1-deficient mice. Indeed,
similar levels of lung-infiltrating CD4?or CD8?cells producing
IFN-? upon mycobacterial Ag restimulation were found in IL-
1R1- and MyD88-deficient mice as in wild-type mice. Therefore,
the local Th1 immune response seemed unaffected in IL-1R1-de-
ficient mice. These results are in line with the normal pulmonary
immune responses of IL-1R1-deficient mice reported in a severe
model of asthma using alum adjuvant, in terms of inflammatory
cell recruitment including eosinophils in the airways, OVA-spe-
cific Abs of all isotypes and ex vivo Ag specific CD4?T cell
The fact that mice deficient for either IL-1R1 or MyD88 were
highly sensitive upon MTB infection strongly supported the notion
that the implication of MyD88 in this response was due to its
contribution to IL-1 signaling. To further establish this point we
ruled out a contribution of MyD88 through either IL-18 or TIRAP
signaling. We and others have shown that TIRAP is a critical com-
ponent of the TLR4 and TLR2 signaling cascade to LPS and my-
cobacterial lipomannan in isolated cells (Ref. 60 and unpublished
data), but less is known about the role of TIRAP in tuberculosis in
vivo. Here we show that mice deficient for IL-18R or TIRAP that
have an impaired signaling through TLR2 and TLR4 had a normal
response to MTB infection, much in contrast to the abrogated re-
sponse seen in MyD88- or IL-1R1-deficient mice. Our data clearly
established that IL-18R and TIRAP are dispensable for the early
response to acute MTB infection. Therefore, IL-1R is involved,
together with MyD88, in the early innate response to aerogenic
MTB, but the MyD88-dependent, TLR2/TLR4/TIRAP, and IL-
18R signaling are dispensable for this response.
Experimental tuberculosis infection of gene-deficient mice has
demonstrated the nonredundant contribution of several proinflam-
matory cytokines such as TNF, IL-12, or IFN-? in the host re-
sponse to MTB infection (61). Here, we show that the IL-1R1
pathway is also nonredundant and essential, in addition to TNF,
IL-12, and IFN-?, in mounting an efficient immune response to
acute MTB infection. In humans, genetic polymorphism analysis
in different tuberculosis patient populations revealed that func-
tional polymorphism in the IL-1 or IL-1R genes influences the
susceptibility to tuberculosis (62–64), further supporting a role for
IL-1 in the pathogenesis of tuberculosis.
Inhibiting IL-1 secretion or IL-1 signal has been a long quest for
the treatment of inflammatory diseases such as rheumatoid arthri-
tis. Anakinra, a recombinant human IL-1R antagonist, is thus far
the only approved biological drug for neutralization of IL-1 in the
clinic (65). TNF neutralization therapies prove highly effective in
patients with rheumatoid arthritis, Crohn’s disease, and psoriasis
but revealed serious adverse effects including reactivation of atyp-
ical forms of tuberculosis, more so with long-lasting Abs than with
soluble TNF receptor (66–68). The strong involvement of IL-1R1
pathway in the host response to acute MTB infection that we report
here suggests that IL-1 blockade may also lead to uncontrolled
mycobacterial infections. Although recent reports on the adverse
events associated with anakinra treatment mention no tuberculosis
cases, serious infections including upper respiratory tract infec-
tions did occur (69, 70). The short half-life of anakinra, requiring
daily injections, may preclude full IL-1 neutralization and allow
some control of MTB infection. Alternatively, the role of IL-1 on
preventing tuberculosis reactivation is not yet established, whereas
TNF was shown to be essential for keeping under control estab-
lished MTB infections, in both mice (38) and humans (66–68).
Additional experiments will be required to assess the effect of
complete IL-1 pathway blockade on tuberculosis reactivation.
In conclusion, IL-1R signaling is an essential component of the
MyD88-mediated pathway leading to the development of innate
response that control acute aerosol MTB infection.
We acknowledge the fruitful collaboration with Dr. Brigitte Gicquel (Pas-
teur Institute, Paris, France).
The authors have no financial conflict of interest.
1. Dye, C., B. G. Williams, M. A. Espinal, and M. C. Raviglione. 2002. Erasing the
world’s slow stain: strategies to beat multidrug-resistant tuberculosis. Science
2. Flynn, J. L., and J. Chan. 2001. Immunology of tuberculosis. Annu. Rev. Immu-
nol. 19: 93–129.
3. Tailleux, L., O. Schwartz, J. L. Herrmann, E. Pivert, M. Jackson, A. Amara,
L. Legres, D. Dreher, L. P. Nicod, J. C. Gluckman, et al. 2003. DC-SIGN is the
major Mycobacterium tuberculosis receptor on human dendritic cells. J. Exp.
Med. 197: 121–127.
4. Akira, S. 2003. Mammalian Toll-like receptors. Curr. Opin. Immunol. 15: 5–11.
5. Aliprantis, A. O., R. B. Yang, M. R. Mark, S. Suggett, B. Devaux, J. D. Radolf,
G. R. Klimpel, P. Godowski, and A. Zychlinsky. 1999. Cell activation and
apoptosis by bacterial lipoproteins through Toll-like receptor-2. Science 285:
6. Thoma-Uszynski, S., S. Stenger, O. Takeuchi, M. T. Ochoa, M. Engele,
P. A. Sieling, P. F. Barnes, M. Rollinghoff, P. L. Bolcskei, M. Wagner, et al.
2001. Induction of direct antimicrobial activity through mammalian Toll-like
receptors. Science 291: 1544–1547.
7. Underhill, D. M., A. Ozinsky, K. D. Smith, and A. Aderem. 1999. Toll-like
receptor-2 mediates mycobacteria-induced proinflammatory signaling in macro-
phages. Proc. Natl. Acad. Sci. USA 96: 14459–14463.
8. Gilleron, M., V. F. Quesniaux, and G. Puzo. 2003. Acylation state of the phos-
phatidylinositol hexamannosides from Mycobacterium bovis bacillus Calmette
Gue ´rin and Mycobacterium tuberculosis H37Rv and its implication in Toll-like
receptor response. J. Biol. Chem. 278: 29880–29889.
9. Guerardel, Y., E. Maes, V. Briken, F. Chirat, Y. Leroy, C. Locht, G. Strecker, and
L. Kremer. 2003. Lipomannan and lipoarabinomannan from a clinical isolate of
Mycobacterium kansasii: novel structural features and apoptosis-inducing prop-
erties. J. Biol. Chem. 278: 36637–36651.
10. Quesniaux, V. J., D. M. Nicolle, D. Torres, L. Kremer, Y. Guerardel,
J. Nigou, G. Puzo, F. Erard, and B. Ryffel. 2004. Toll-like receptor 2 (TLR2)-
dependent-positive and TLR2-independent-negative regulation of proinflam-
matory cytokines by mycobacterial lipomannans. J. Immunol. 172: 4425–
11. Gilleron, M., J. Nigou, D. Nicolle, V. Quesniaux, and G. Puzo. 2006. The acy-
lation state of mycobacterial lipomannans modulates innate immunity response
through Toll-like receptor 2. Chem. Biol. 13: 39–47.
12. Means, T. K., S. Wang, E. Lien, A. Yoshimura, D. T. Golenbock, and
M. J. Fenton. 1999. Human Toll-like receptors mediate cellular activation by
Mycobacterium tuberculosis. J. Immunol. 163: 3920–3927.
13. Bafica, A., C. A. Scanga, C. G. Feng, C. Leifer, A. Cheever, and A. Sher.
2005. TLR9 regulates Th1 responses and cooperates with TLR2 in mediating
optimal resistance to Mycobacterium tuberculosis. J. Exp. Med. 202:
1187The Journal of Immunology
14. Fremond, C. M., V. Yeremeev, D. M. Nicolle, M. Jacobs, V. F. Quesniaux, and
B. Ryffel. 2004. Fatal Mycobacterium tuberculosis infection despite adaptive
immune response in the absence of MyD88. J. Clin. Invest. 114: 1790–1799.
15. Scanga, C. A., A. Bafica, C. G. Feng, A. W. Cheever, S. Hieny, and A. Sher.
2004. MyD88-deficient mice display a profound loss in resistance to Mycobac-
terium tuberculosis associated with partially impaired Th1 cytokine and nitric
oxide synthase 2 expression. Infect. Immun. 72: 2400–2404.
16. Kawai, T., O. Adachi, T. Ogawa, K. Takeda, and S. Akira. 1999. Unresponsive-
ness of MyD88-deficient mice to endotoxin. Immunity 11: 115–122.
17. Takeuchi, O., S. Sato, T. Horiuchi, K. Hoshino, K. Takeda, Z. Dong,
R. L. Modlin, and S. Akira. 2002. Cutting edge: role of Toll-like receptor 1 in
mediating immune response to microbial lipoproteins. J. Immunol. 169: 10–14.
18. Muraille, E., C. De Trez, M. Brait, P. De Baetselier, O. Leo, and Y. Carlier. 2003.
Genetically resistant mice lacking MyD88-adapter protein display a high suscep-
tibility to Leishmania major infection associated with a polarized Th2 response.
J. Immunol. 170: 4237–4241.
19. Schnare, M., G. M. Barton, A. C. Holt, K. Takeda, S. Akira, and R. Medzhitov.
2001. Toll-like receptors control activation of adaptive immune responses. Nat.
Immunol. 2: 947–950.
20. Mun, H. S., F. Aosai, K. Norose, M. Chen, L. X. Piao, O. Takeuchi, S. Akira,
H. Ishikura, and A. Yano. 2003. TLR2 as an essential molecule for protective
immunity against Toxoplasma gondii infection. Int. Immunol. 15: 1081–1087.
21. Scanga, C. A., J. Aliberti, D. Jankovic, F. Tilloy, S. Bennouna, E. Y. Denkers,
R. Medzhitov, and A. Sher. 2002. Cutting edge: MyD88 is required for resistance
to Toxoplasma gondii infection and regulates parasite-induced IL-12 production
by dendritic cells. J. Immunol. 168: 5997–6001.
22. Feng, C. G., C. A. Scanga, C. M. Collazo-Custodio, A. W. Cheever, S. Hieny,
P. Caspar, and A. Sher. 2003. Mice lacking myeloid differentiation factor 88
display profound defects in host resistance and immune responses to Mycobac-
terium avium infection not exhibited by Toll-like receptor 2 (TLR2)- and TLR4-
deficient animals. J. Immunol. 171: 4758–4764.
23. Nicolle, D. M., X. Pichon, A. Bouchot, I. Maillet, F. Erard, S. Akira, B. Ryffel,
and V. F. Quesniaux. 2004. Chronic pneumonia despite adaptive immune re-
sponse to Mycobacterium bovis BCG in MyD88-deficient mice. Lab. Invest. 84:
24. Abel, B., N. Thieblemont, V. J. Quesniaux, N. Brown, J. Mpagi, K. Miyake,
F. Bihl, and B. Ryffel. 2002. Toll-like receptor 4 expression is required to control
chronic Mycobacterium tuberculosis infection in mice. J. Immunol. 169:
25. Reiling, N., C. Holscher, A. Fehrenbach, S. Kroger, C. J. Kirschning, S. Goyert,
and S. Ehlers. 2002. Cutting edge: Toll-like receptor (TLR)2- and TLR4-medi-
ated pathogen recognition in resistance to airborne infection with Mycobacterium
tuberculosis. J. Immunol. 169: 3480–3484.
26. Sugawara, I., H. Yamada, C. Li, S. Mizuno, O. Takeuchi, and S. Akira. 2003.
Mycobacterial infection in TLR2 and TLR6 knockout mice. Microbiol. Immunol.
27. Heldwein, K. A., M. D. Liang, T. K. Andresen, K. E. Thomas, A. M. Marty,
N. Cuesta, S. N. Vogel, and M. J. Fenton. 2003. TLR2 and TLR4 serve distinct
roles in the host immune response against Mycobacterium bovis BCG. J. Leu-
kocyte Biol. 74: 277–286.
28. Shim, T. S., O. C. Turner, and I. M. Orme. 2003. Toll-like receptor 4 plays no role
in susceptibility of mice to Mycobacterium tuberculosis infection. Tuberculosis
29. Shi, S., A. Blumenthal, C. M. Hickey, S. Gandotra, D. Levy, and S. Ehrt. 2005.
Expression of many immunologically important genes in Mycobacterium tuber-
culosis-infected macrophages is independent of both TLR2 and TLR4 but de-
pendent on IFN-?? receptor and STAT1. J. Immunol. 175: 3318–3328.
30. Drennan, M. B., D. Nicolle, V. J. Quesniaux, M. Jacobs, N. Allie, J. Mpagi,
C. Fremond, H. Wagner, C. Kirschning, and B. Ryffel. 2004. Toll-like receptor 2-
deficient mice succumb to Mycobacterium tuberculosis infection. Am. J. Pathol.
31. Sugawara, I., H. Yamada, H. Kaneko, S. Mizuno, K. Takeda, and S. Akira. 1999.
Role of interleukin-18 (IL-18) in mycobacterial infection in IL-18-gene-disrupted
mice. Infect. Immun. 67: 2585–2589.
32. Juffermans, N. P., S. Florquin, L. Camoglio, A. Verbon, A. H. Kolk, P. Speelman,
S. J. van Deventer, and T. van Der Poll. 2000. Interleukin-1 signaling is essential
for host defense during murine pulmonary tuberculosis. J. Infect. Dis. 182:
33. Yamada, H., S. Mizumo, R. Horai, Y. Iwakura, and I. Sugawara. 2000. Protective
role of interleukin-1 in mycobacterial infection in IL-1 ?/? double-knockout
mice. Lab. Invest. 80: 759–767.
34. Labow, M., D. Shuster, M. Zetterstrom, P. Nunes, R. Terry, E. B. Cullinan,
T. Bartfai, C. Solorzano, L. L. Moldawer, R. Chizzonite, and K. W. McIntyre.
1997. Absence of IL-1 signaling and reduced inflammatory response in IL-1 type
I receptor-deficient mice. J. Immunol. 159: 2452–2461.
35. Hoshino, K., O. Takeuchi, T. Kawai, H. Sanjo, T. Ogawa, Y. Takeda, K. Takeda,
and S. Akira. 1999. Cutting edge: Toll-like receptor 4 (TLR4)-deficient mice are
hyporesponsive to lipopolysaccharide: evidence for TLR4 as the Lps gene prod-
uct. J. Immunol. 162: 3749–3752.
36. Horng, T., G. M. Barton, R. A. Flavell, and R. Medzhitov. 2002. The adaptor
molecule TIRAP provides signalling specificity for Toll-like receptors. Nature
37. Jacobs, M., N. Brown, N. Allie, and B. Ryffel. 2000. Fatal Mycobacterium bovis
BCG infection in TNF-LT-?-deficient mice. Clin. Immunol. 94: 192–199.
38. Botha, T., and B. Ryffel. 2003. Reactivation of latent tuberculosis infection in
TNF-deficient mice. J. Immunol. 171: 3110–3118.
39. Lyadova, I. V., E. B. Eruslanov, S. V. Khaidukov, V. V. Yeremeev,
K. B. Majorov, A. V. Pichugin, B. V. Nikonenko, T. K. Kondratieva, and
A. S. Apt. 2000. Comparative analysis of T lymphocytes recovered from the
lungs of mice genetically susceptible, resistant, and hyperresistant to Mycobac-
terium tuberculosis-triggered disease. J. Immunol. 165: 5921–5931.
40. Muller, M., H. P. Eugster, M. Le Hir, A. Shakhov, F. Di Padova, C. Maurer,
V. F. Quesniaux, and B. Ryffel. 1996. Correction or transfer of immunodeficiency
due to TNF-LT ? deletion by bone marrow transplantation. Mol. Med. 2:
41. Lutz, M. B., N. Kukutsch, A. L. Ogilvie, S. Rossner, F. Koch, N. Romani, and
G. Schuler. 1999. An advanced culture method for generating large quantities of
highly pure dendritic cells from mouse bone marrow. J. Immunol. Methods 223:
42. Hubeau, C., M. Singer, M. Lagranderie, G. Marchal, and B. Vargaftig. 2003.
Extended freeze-dried Mycobacterium bovis bacillus Calmette-Gue ´rin induces
the release of interleukin-12 but not tumour necrosis factor-? by alveolar mac-
rophages, both in vitro and in vivo. Clin. Exp. Allergy 33: 386–393.
43. Green, L. C., D. A. Wagner, J. Glogowski, P. L. Skipper, J. S. Wishnok, and
S. R. Tannenbaum. 1982. Analysis of nitrate, nitrite, and [15N]nitrate in biolog-
ical fluids. Anal. Biochem. 126: 131–138.
44. Flynn, J. L., M. M. Goldstein, J. Chan, K. J. Triebold, K. Pfeffer,
C. J. Lowenstein, R. Schreiber, T. W. Mak, and B. R. Bloom. 1995. Tumor
necrosis factor-? is required in the protective immune response against Myco-
bacterium tuberculosis in mice. Immunity 2: 561–572.
45. Bean, A. G., D. R. Roach, H. Briscoe, M. P. France, H. Korner, J. D. Sedgwick,
and W. J. Britton. 1999. Structural deficiencies in granuloma formation in TNF
gene-targeted mice underlie the heightened susceptibility to aerosol Mycobacte-
rium tuberculosis infection, which is not compensated for by lymphotoxin. J. Im-
munol. 162: 3504–3511.
46. Jacobs, M., M. W. Marino, N. Brown, B. Abel, L. G. Bekker, V. J. Quesniaux,
L. Fick, and B. Ryffel. 2000. Correction of defective host response to Mycobac-
terium bovis BCG infection in TNF-deficient mice by bone marrow transplanta-
tion. Lab. Invest. 80: 901–914.
47. Nicolle, D., C. Fremond, X. Pichon, A. Bouchot, I. Maillet, B. Ryffel, and
V. J. Quesniaux. 2004. Long-term control of Mycobacterium bovis BCG infection
in the absence of Toll-like receptors (TLRs): investigation of TLR2-, TLR6-, or
TLR2-TLR4-deficient mice. Infect. Immun. 72: 6994–7004.
48. Zeisel, M. B., V. A. Druet, J. Sibilia, J. P. Klein, V. Quesniaux, and
D. Wachsmann. 2005. Cross talk between MyD88 and focal adhesion kinase
pathways. J. Immunol. 174: 7393–7397.
49. Martin, M. U., and H. Wesche. 2002. Summary and comparison of the signaling
mechanisms of the Toll/interleukin-1 receptor family. Biochim. Biophys. Acta
50. Gamero, A. M., and J. J. Oppenheim. 2006. IL-1 can act as number one. Immunity
51. Means, T. K., B. W. Jones, A. B. Schromm, B. A. Shurtleff, J. A. Smith, J. Keane,
D. T. Golenbock, S. N. Vogel, and M. J. Fenton. 2001. Differential effects of a
Toll-like receptor antagonist on Mycobacterium tuberculosis-induced macro-
phage responses. J. Immunol. 166: 4074–4082.
52. MacMicking, J. D., G. A. Taylor, and J. D. McKinney. 2003. Immune control of
tuberculosis by IFN-?-inducible LRG-47. Science 302: 654–659.
53. Feng, C. G., C. M. Collazo-Custodio, M. Eckhaus, S. Hieny, Y. Belkaid,
K. Elkins, D. Jankovic, G. A. Taylor, and A. Sher. 2004. Mice deficient in
LRG-47 display increased susceptibility to mycobacterial infection associated
with the induction of lymphopenia. J. Immunol. 172: 1163–1168.
54. Singh, S. B., A. S. Davis, G. A. Taylor, and V. Deretic. 2006. Human IRGM
induces autophagy to eliminate intracellular mycobacteria. Science 313:
55. Riley, L. W. 2006. Of mice, men, and elephants: Mycobacterium tuberculosis cell
envelope lipids and pathogenesis. J. Clin. Invest. 116: 1475–1478.
56. Pompei, L., S. Jang, B. Zamlynny, S. Ravikumar, A. McBride, S. P. Hickman,
and P. Salgame. 2007. Disparity in IL-12 release in dendritic cells and macro-
phages in response to Mycobacterium tuberculosis is due to use of distinct TLRs.
J. Immunol. 178: 5192–5199.
57. Holscher, C., R. A. Atkinson, B. Arendse, N. Brown, E. Myburgh, G. Alber, and
F. Brombacher. 2001. A protective and agonistic function of IL-12p40 in myco-
bacterial infection. J. Immunol. 167: 6957–6966.
58. Cooper, A. M., A. Kipnis, J. Turner, J. Magram, J. Ferrante, and I. M. Orme.
2002. Mice lacking bioactive IL-12 can generate protective, antigen-specific cel-
lular responses to mycobacterial infection only if the IL-12p40 subunit is present.
J. Immunol. 168: 1322–1327.
59. Schmitz, N., M. Kurrer, and M. Kopf. 2003. The IL-1 receptor 1 is critical for
Th2 cell type airway immune responses in a mild but not in a more severe asthma
model. Eur. J. Immunol. 33: 991–1000.
60. Yamamoto, M., S. Sato, H. Hemmi, H. Sanjo, S. Uematsu, T. Kaisho,
K. Hoshino, O. Takeuchi, M. Kobayashi, T. Fujita, et al. 2002. Essential role for
TIRAP in activation of the signalling cascade shared by TLR2 and TLR4. Nature
61. Flynn, J. L. 2006. Lessons from experimental Mycobacterium tuberculosis in-
fections. Microbes Infect. 8: 1179–1188.
62. Awomoyi, A. A., M. Charurat, A. Marchant, E. N. Miller, J. M. Blackwell,
M. tuberculosis INFECTION IN IL-1R1- VS MyD88-DEFICIENT MICE
K. P. McAdam, and M. J. Newport. 2005. Polymorphism in IL-1?: IL1?-511
association with tuberculosis and decreased lipopolysaccharide-induced
IL-1? in IFN-? primed ex-vivo whole blood assay. J. Endotoxin. Res. 11:
63. Gomez, L. M., J. F. Camargo, J. Castiblanco, E. A. Ruiz-Narvaez, J. Cadena, and
J. M. Anaya. 2006. Analysis of IL1B, TAP1, TAP2 and IKBL polymorphisms on
susceptibility to tuberculosis. Tissue Antigens 67: 290–296.
64. Amirzargar, A. A., N. Rezaei, H. Jabbari, A. A. Danesh, F. Khosravi,
M. Hajabdolbaghi, A. Yalda, and B. Nikbin. 2006. Cytokine single nucleotide
polymorphisms in Iranian patients with pulmonary tuberculosis. Eur. Cytokine
Network 17: 84–89.
65. Bresnihan, B., J. M. Alvaro-Gracia, M. Cobby, M. Doherty, Z. Domljan,
P. Emery, G. Nuki, K. Pavelka, R. Rau, B. Rozman, et al. 1998. Treatment of
rheumatoid arthritis with recombinant human interleukin-1 receptor antagonist.
Arthritis Rheum. 41: 2196–2204.
66. Keane, J., S. Gershon, R. P. Wise, E. Mirabile-Levens, J. Kasznica,
W. D. Schwieterman, J. N. Siegel, and M. M. Braun. 2001. Tuberculosis asso-
ciated with infliximab, a tumor necrosis factor ?-neutralizing agent. N. Engl.
J. Med. 345: 1098–1104.
67. Mohan, A. K., T. R. Cote, J. A. Block, A. M. Manadan, J. N. Siegel, and
M. M. Braun. 2004. Tuberculosis following the use of etanercept, a tumor ne-
crosis factor inhibitor. Clin. Infect. Dis. 39: 295–299.
68. Keane, J. 2005. TNF-blocking agents and tuberculosis: new drugs illuminate an
old topic. Rheumatology 44: 1205–1206.
69. Fleischmann, R. M., J. Tesser, M. H. Schiff, J. Schechtman, G. R. Burmester,
R. Bennett, D. Modafferi, L. Zhou, D. Bell, and B. Appleton. 2006. Safety of
extended treatment with anakinra in patients with rheumatoid arthritis. Ann.
Rheum. Dis. 65: 1006–1012.
70. Konttinen, L., V. Honkanen, T. Uotila, J. Pollanen, M. Waahtera, M. Romu,
K. Puolakka, M. Vasala, A. Karjalainen, R. Luukkainen, and D. C. Nordstrom.
2006. Biological treatment in rheumatic diseases: results from a longitudinal
surveillance: adverse events. Rheumatol. Int. 26: 916–922.
1189 The Journal of Immunology