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The Journal of Immunology, 2007, 179, 1178-1189
Copyright © 2007 by The American Association of Immunologists, Inc.
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IL-1 Receptor-Mediated Signal Is an Essential Component of MyD88-Dependent Innate Response to Mycobacterium tuberculosis Infection

Cecile M. Fremond*, Dieudonnée Togbe*, Emilie Doz*, Stephanie Rose*, Virginie Vasseur*, Isabelle Maillet*, Muazzam Jacobs{dagger}, Bernhard Ryffel1,2,*,{dagger} and Valerie F. J. Quesniaux1,2,*,{dagger}

* University of Orleans and Centre National de la Recherche Scientifique, Molecular Immunology and Embryology, Orleans, France; and {dagger} Division of Immunology, Institute of Infectious Disease and Molecular Medicine, Health Sciences Faculty, University of Cape Town, Cape Town, South Africa


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
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.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Tuberculosis is a highly infectious respiratory infection caused by Mycobacterium tuberculosis (MTB).3 Although one-third of the world’s population has been in contact with the pathogen, only 10% develop overt clinical symptoms, whereas roughly 90% of the infected persons contain the infection (1). Prominent 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 receptor, 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 several MTB cell wall and several secreted components such as 19-kDa lipoprotein, lipomannan, and mannosylated phosphatidylinositol, activate macrophages and dendritic cells (DC) through TLR2 (5, 6, 7, 8, 9, 10, 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, translocation of NF-{kappa}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 resistance to MTB (14, 15), as it compromised resistance to several infectious agents (16, 17, 18, 19, 20, 21, 22, 23). MyD88-deficient mice succumbed within 4 wk of aerogenic MTB infection with acute, necrotic pulmonary infection despite their ability to mount an adaptive immune 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, 25, 26, 27, 28, 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 TLR2 plus TLR9 double-deficient mice (13), and would be even more pronounced in the absence of MyD88 adaptor, invalidating simultaneously 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 contribution of these signals in the defective response to MTB observed in MyD88-deficient mice cannot be excluded. Previous reports showed rather limited host resistance defects upon MTB infection of IL-18 (31), IL-1beta 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 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 signaling 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
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

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 National 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-Guérin (BCG) (Pasteur strain 1173P2) were grown to mid-log phase in Middlebrook 7H9 liquid medium (Difco Laboratories), supplemented with 10% oleic acid-albumin-dextrose-catalase (Difco Laboratories) at 37°C. Aliquots were prepared and frozen at –80°C. Before use, an aliquot was thawed, briefly vortexed, and diluted in sterile saline containing 0.04% Tween 20 and clumping was disrupted by 20 repeated aspirations through a 30-gauge needle (Omnican). Pulmonary infection with M. tuberculosis H37Rv was performed by delivering ~200 bacteria into both nasal cavities (20 µl 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 evaluated at different time points after infection with M. tuberculosis H37Rv as described (37). Organs were weighed, and defined aliquots were homogenized 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 log10 CFU per organ.

Preparation and analyses of lung homogenates for cytokine/chemokine determination

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-µm pore size filter (Costar-Corning), and stored at –80°C until determination of IL-1{alpha}, IL-1beta, IL-6, IL-12p40, TNF, MCP-1, and IFN-{gamma} 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 paraffin. Sections of 2–3 µm 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, formalin-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 diaminobenzidine 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-mm3 pieces and was incubated in RPMI 1640 (Invitrogen Life Technologies) containing 5% FCS, antibiotics (penicillin 100 U/ml-streptomycin 100 µg/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% NaN3 and 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-{gamma} secretion by CD4+ or CD8+ cells was detected using a capture anti-IFN-{gamma} rat IgG1 mAb conjugated to a CD4- or CD8-specific rat IgG2b mAb, according to the manufacturer’s recommendations (mouse IFN-{gamma} secretion assay; Miltenyi Biotec). After restimulation of the lung-infiltrating cells with a lyophilized preparation of BCG culture supernatant (supBCG at 10 µg/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-{gamma} was detected with a second anti-IFN-{gamma} 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 106 cells/ml for 7 days in DMEM (Sigma-Aldrich) supplemented with 2 mM L-glutamine and 2 x 10–5 M 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 macrophages. Alternatively, murine bone marrow cells were differentiated into myeloid DCs after culturing (change on days 3, 6, and 8) at 2 x 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 microculture plates (at 105 cells/well in DMEM supplemented with 2 mM L-glutamine and 2 x 10–5 M 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 Pam3CSK4 (S-[2,3-bis-(palmitoyloxy)-(2-RS)-propyl]-N-palmitoyl-(R)-Cys- (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 lyophilized 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 µg/ml). Cell supernatants were harvested after 24 or 48 h of stimulation in the presence of IFN-{gamma} (100 U/ml) for TNF, IL-1{alpha}, IL-1beta, 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 105 cells in 0.2 ml of DMEM supplemented with 2 mM L-glutamine and 2 x 10–5 M 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 expressing 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 medium was removed, and the cultures were washed once with warm PBS and fixed with paraformaldehyde 4% in PBS. After overnight fixation, macrophages 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 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 beta-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 IRBE microscope (x100 oil immersion objective) by counting the macrophages containing one or two isolated intracellular bacteria and the noninfected macrophages over 10 different observation fields per slide (2 slides per mouse). Cells derived from two individual mice were tested per genotype in each experiment. The slides were counted blindly by two observers.

Statistical analysis

Analysis was performed using Student’s t and ANOVA tests and values of p ≤ 0.05 were considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
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 mycobacterial infection is MyD88 dependent (14, 23). Because MyD88 signals 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-{alpha}, 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 (data not shown). We further tested the IL-1R1 dependence of mycobacterial induced iNOS activation and demonstrate unaffected nitrite production in IL-1R1-deficient macrophages compared 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 shown).


Figure 1
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  		FIGURE 1. Proinflammatory cytokine and NO production in IL-1R1- and MyD88-deficient macrophages and DCs. Bone marrow-derived macrophages (A–F) and DCs (G and H) prepared from IL-1R1-deficient ({square}), MyD88-deficient (Figure 1), and wild-type ({blacksquare}) 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 µg/ml), a lyophilized preparation of BCG culture supernatant (supBCG, 10 µg/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-1beta (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.

 
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 sustantially 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 mycobacteria, we determined the levels of IL-1{alpha} and IL-1beta. Mycobacterial stimulation of wild-type macrophages yielded a secretion of IL-1{alpha} and IL-1beta in the 100- to 600-ng/ml range that was strongly reduced or absent in MyD88-deficient cells but was similar in IL-1R-deficient macrophages (data not shown) or DC culture supernatants (Fig. 1H).

IL-18R seemed to play no role in the in vitro response to mycobacteria 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{alpha}, IL-1beta, 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, mediated via IL-1/IL-1R signaling in bone marrow-derived macrophages. 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 signaling might be critical for the innate immune response to MTB infection.

Lethal M. tuberculosis infection in the absence of IL-1R1

We showed earlier that MyD88-deficient mice are extremely sensitive 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, 25, 26, 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 experiment, similar to wild-type mice. Both IL-1R1- and MyD88-deficient mice exhibited substantially increased lung weight (Fig. 2B) and >2 log10 higher bacterial load in the lungs as compared with wild-type controls 3 wk after infection (Fig. 2C). This defective response was not observed in mice deficient for IL-18R, which exhibited no overt lung inflammation (Fig. 2E) and controlled bacterial load (Fig. 2F), even 95 days after infection. Therefore, IL-1R1-deficient mice are highly susceptible to MTB infection, similar to MyD88-deficient mice, whereas absence of IL-18R was not detrimental.


Figure 2
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  		FIGURE 2. IL-1R1-deficient mice are unable 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 experiments). Lung wet weight (B) and the number of viable bacteria present in the lungs (C) of IL-1R1-deficient mice ({square}), MyD88-deficient mice (Figure 2) and wild-type controls ({blacksquare}) were measured 24 days postinfection. Lung weight (D) and bacterial load (E) were measured 95 days postinfection for IL-18R-deficient (Figure 2) and wild-type controls ({blacksquare}). Mean ± SD of n = 3–4 lung from 1 representative experiment of 3 and 2 independent experiments at day 24 and 95, respectively; *, p ≤ 0.05; **, p ≤ 0.01.

 
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 mycobacterial growth. In view of the increased lung weight indicative 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 macrophages accompanied by lymphocytic perivascular and peribronchiolar cuffing. The lung lesions observed in IL-1R1-deficient mice were reminiscent of those induced by mycobacterial infection in the absence of functional MyD88 or TNF pathways (14, 44, 45, 46). Thus, the absence of IL-1R1 pathway alone accounts for most of the massive necrosis and infiltration of inflammatory cells in the lungs with uncontrolled MTB growth seen in MyD88-deficient mice.


Figure 3
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  		FIGURE 3. IL-1R1-deficient mice exhibit acute necrotic pneumonia 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, magnification x50 for low power and x400 for details) with abundant mycobacteria in the extracellular space (C; Ziehl-Neelsen stain; x1000). Lungs of IL-18R-deficient mice exhibited well-defined granuloma with few mycobacteria, comparable with results in wild-type lungs (A–C).

 
The production of NO and related nitrogen intermediates by macrophages is a major effector mechanism responsible for antimycobacterial activity. We next investigated the extent of pulmonary 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 residing in well-defined granulomas, whereas IL-1R1 and MyD88-deficient mice showed massive iNOS expression throughout the pulmonary tissue.


Figure 4
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  		FIGURE 4. iNOS induction in lungs of IL-1R1-deficient mice in response to M. tuberculosis infection. Expression of iNOS in the lung tissue from IL-1R1 and MyD88-deficient mice and wild-type controls was analyzed 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. x100.

 
Therefore, iNOS expression is independent of the IL-1R/MyD88 pathway but appears to be unable to control the MTB infection.

Efficient recruitment of lymphocytes and myeloid cells in IL-1R1-deficient mice

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 previously 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-{gamma} in MTB-infected IL-1R1-deficient lungs. Using conjugated reagents designed to stain freshly produced IFN-{gamma} 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-{gamma} similar to those of wild-type mice upon Ag restimulation. This response was specific because essentially no IFN-{gamma} 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 cellular IFN-{gamma} production, estimated by the mean fluorescence intensity, was similar in the three mouse lines.


Figure 5
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  		FIGURE 5. Inflammatory cell recruitment, activation, and priming in the lung of IL-1R1-deficient infected mice. Infiltrating cells from the lungs of IL-1R1-deficient ({square}), MyD88-deficient (Figure 5) and wild-type ({blacksquare}) 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-{gamma} secretion by lung infiltrating CD4+ or CD8+ cells was detected using a capture anti-IFN-{gamma} 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-{gamma} was detected with a second, labeled anti-IFN-{gamma} 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-{gamma} producing CD4+ and CD8+ cells from two to three mice per genotype are shown in K and L, respectively.

 
However, there was a markedly increased number of CD11b+ cells in the lung of both IL-1R1- and MyD88-deficient as compared with wild-type mice, associated with up-regulated expression 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 macrophages 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-1R1-deficient mice

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 cytokines 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{alpha}, IL-1beta, IL-6, TNF, MCP-1, and IFN-{gamma} 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{alpha}, IL-1beta, IL-6, TNF-{alpha}, MCP-1, and IFN-{gamma} but not IL-12p40, as compared with control mice, in line with the strong local inflammatory cell infiltration seen in these mice.


Figure 6
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  		FIGURE 6. High cytokine and chemokine pulmonary levels in MTB-infected IL-1R1-deficient mice. Cytokine and chemokine concentrations were determined in lung homogenates from IL-1R1-deficient and control mice 4 wk after MTB infection. IL-1{alpha} (A), IL-1beta (B), IL-6 (C), TNF-{alpha} (D), IFN-{gamma} (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.

 
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 involved in the internalization of mycobacteria, we infected macrophages 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 internalization was reduced by one-half in MyD88-deficient macrophages, 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 macrophages 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.


Figure 7
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  		FIGURE 7. Mycobacterial internalization in IL-1R1-deficient macrophages. Internalization of fluorescent M. bovis BCG expressing GFP (BCG-GFP) by wild-type, IL-1R1-deficient, or MyD88-deficient macrophages was quantified after incubation at 37°C for 2 h, overnight fixation, and staining of F-actin using beta-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 percentage of macrophages infected with isolated BCG-GFP (n = 2 mice per mutated genotype) and are from 1 representative of 2 independent experiments.

 
Minor contribution of TLR2/4 TIRAP pathway in the MyD88 phenotype

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 of these TLR pathways in the MyD88-dependent response to mycobacteria, 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 partially 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 unresponsive (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. Microscopic 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-deficient mice (Fig. 8F). Therefore, impairment of TLR2 plus TLR4 pathways, two of the most prominent TLRs involved in mycobacterial recognition, in TIRAP-deficient mice did not compromise their control of early in vivo MTB infection, as seen in MyD88-deficient mice.


Figure 8
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  		FIGURE 8. Control of M. tuberculosis infection in TIRAP-deficient mice. TIRAP-deficient (open symbols) and wild-type mice (closed symbols) 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, x100) with few mycobacteria in TIRAP-deficient mice (F; Ziehl-Neelsen stain, x400).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
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 sensitivity 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 because mice doubly deficient for TLR2 and TLR9 were more susceptible to MTB infection than either of the single TLR-deficient mice. However, the phenotype of TLR2 and TLR9 double-deficient mice was not as drastic as that of MyD88-deficient mice (13), questioning the role of other MyD88-dependent but TLR-independent 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 pathway 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 recently described such as the involvement of MyD88 in cross-talk with the focal adhesion kinase pathway (48) or with others members of the IL-1R family (49). In cutaneous Staphylococcus aureus infection, the extreme sensitivity of MyD88-deficient mice has recently been ascribed, at least in part, to deficient IL-1R/IL-18R signaling pathways (50). IL-1{alpha} and IL-1beta 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 decoy 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-1beta or IL-1R1-deficient mice showed less striking 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 105 CFU of H37Rv in IL-1R1-deficient mice.

The involvement of IL-1R pathway in the control of MTB infection 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 internalization, 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 adaptive response, or on effector mechanisms. We addressed these different aspects below.

In vivo, both MyD88- and IL-1R1-deficient mice showed massive 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 macrophages, and that a dominant negative MyD88 mutant did not impair the iNOS promoter in murine macrophages (51). However, the strong iNOS expression was not sufficient to control MTB bacterial growth, and abundant acid-fast resistant bacilli were present in the lung of both MyD88- and IL-1R1-deficient mice. Although IFN-{gamma}-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 susceptible to MTB infection (53). LRG-47 is important for the maturation of the phagosome, and autophagy of infected cells represents 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 activation 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 phenotype 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 inflammatory cell infiltration in the lung of MTB-infected IL-1R1-deficient mice. All cytokines tested, IL-1{alpha}, IL-1beta, IL-6, TNF, and IFN-{gamma}, 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{alpha}, IL-1beta, IL-6, and NO secretion, but it severely impaired IL-12p40 production by macrophages. Such decreased IL-12p40 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, associated 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{alpha} and IL-1beta 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 pathway 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-{gamma} 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-deficient 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-specific Abs of all isotypes and ex vivo Ag specific CD4+ T cell proliferation (59).

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 component of the TLR4 and TLR2 signaling cascade to LPS and mycobacterial 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 response 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 proinflammatory cytokines such as TNF, IL-12, or IFN-{gamma} in the host response 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-{gamma}, in mounting an efficient immune response to acute MTB infection. In humans, genetic polymorphism analysis in different tuberculosis patient populations revealed that functional polymorphism in the IL-1 or IL-1R genes influences the susceptibility to tuberculosis (62, 63, 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 arthritis. 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 atypical forms of tuberculosis, more so with long-lasting Abs than with soluble TNF receptor (66, 67, 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 infections 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 established MTB infections, in both mice (38) and humans (66, 67, 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.


   Acknowledgments
 
We acknowledge the fruitful collaboration with Dr. Brigitte Gicquel (Pasteur Institute, Paris, France).


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
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.

1 B.R. and V.F.J.Q. contributed equally to this study as senior authors. Back

2 Address correspondence and reprint requests to Drs. Valerie Quesniaux and Bernhard Ryffel, Transgenose Institute, 3B rue de la Férollerie, 45071 Orléans, France. E-mail addresses: bryffel{at}cnrs-orleans.fr and quesniaux{at}cnrs-orleans.fr Back

3 Abbreviations used in this paper: MTB, Mycobacterium tuberculosis; DC, dendritic cell; TIRAP, Toll-IL-1R domain-containing adaptor protein; iNOS, inducible NO synthase; BCG, bacillus Calmette-Guérin; HKLM, heat-killed Listeria monocytogenes; MOI, multiplicity of infection; i.n., intranasally; BLP, bacterial lymphopeptide. Back

Received for publication August 30, 2006. Accepted for publication May 12, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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