Burkholderia Cenocepacia Triggered by the Opportunistic Bacteria Host Against Fatal Lung Inflammation Lack of Myd88 Protects

Lack of MyD88 Protects the Immunodeficient Host Against Fatal Lung Inflammation Triggered by the Opportunistic Bacteria Burkholderia cenocepacia This information is current as of September 24, 2021. Grasiella M. de C. Ventura, Viviane Balloy, Reuben Ramphal, Huot Khun, Michel Huerre, Bernhard Ryffel, Maria-Cristina M. Plotkowski, Michel Chignard and Mustapha Si-Tahar

J Immunol 2009; 183:670-676; Prepublished online 17 June Downloaded from 2009; doi: 10.4049/jimmunol.0801497 http://www.jimmunol.org/content/183/1/670 http://www.jimmunol.org/

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2009 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Lack of MyD88 Protects the Immunodeﬁcient Host Against Fatal Lung Inﬂammation Triggered by the Opportunistic Bacteria Burkholderia cenocepacia1

Grasiella M. de C. Ventura,2*† Viviane Balloy,2*† Reuben Ramphal,‡ Huot Khun,§ Michel Huerre,§ Bernhard Ryffel,¶ Maria-Cristina M. Plotkowski,# Michel Chignard,*† and Mustapha Si-Tahar3*†

Burkholderia cenocepacia is an opportunistic pathogen of major concern for cystic fibrosis patients as well as immunocompromised cancer patients and transplant recipients. The mechanisms by which B. cenocepacia triggers a rapid health deterioration of the susceptible host have yet to be characterized. TLR and their key signaling intermediate MyD88 play a central role in the detection of microbial molecular patterns and in the initiation of an effective immune response. We performed a study to better understand the role Downloaded from of TLR-MyD88 signaling in B. cenocepacia-induced pathogenesis in the immunocompromised host, using an experimental murine model. The time-course of several dynamic parameters, including animal survival, bacterial load, and secretion of critical inflammatory mediators, was compared in infected and immunosuppressed wild-type and MyD88؊/؊ mice. Notably, when compared with wild-type ,mice, infected MyD88؊/؊ animals displayed significantly reduced levels of inflammatory mediators (including KC, TNF-␣, IL-6, MIP-2 and G-CSF) in blood and lung airspaces. Moreover, despite a higher transient bacterial load in the lungs, immunosuppressed mice deficient in MyD88 had an unexpected survival advantage. Finally, we showed that this B. cenocepacia-induced life-threatening infection http://www.jimmunol.org/ of wild-type mice involved the proinflammatory cytokine TNF-␣ and could be prevented by corticosteroids. Altogether, our findings demonstrate that a MyD88-dependent pathway can critically contribute to a detrimental host inflammatory response that leads to fatal pneumonia. The Journal of Immunology, 2009, 183: 670–676.

he increasing number of immunodeficient patients and the which B. cenocepacia triggers a rapid health deterioration of the sus- growing resistance of bacteria to commonly used antibiotics ceptible host have yet to be characterized. T have made infections in the compromised host one of the TLRs represent a conserved family of innate immune recogni- most persistent medical issues. Burkholderia cenocepacia is a multi- tion receptors that play key roles in detecting microbes, initiating by guest on September 24, 2021 drug resistant Gram-negative bacillus that is widely distributed in the innate immune responses, and linking innate and adaptive immu- natural environment. This opportunistic pathogen is of particular con- nity (8–11). The interaction of a microbial-associated molecular cern for patients with cystic fibrosis (CF),4 in whom it can cause pattern with its specific TLR triggers the binding of the adapter respiratory infection and has been associated with increased rates of molecule MyD88 to the intracellular carboxyterminus domain of morbidity and mortality (1–3). B. cenocepacia is also a well-known the receptor (10). This is the first step of the signaling cascade that nosocomial pathogen among non-CF patient populations, including eventually activates NF-␬B and other nuclear factors that regulate immunocompromised cancer patients and transplant recipients (4, 5). the expression of a large array of immune and inflammatory genes Spread of B. cenocepacia is likely facilitated by patient-to-patient air- (10). Thus, TLRs appear to be directly involved in the fight of borne transmission (6, 7). However, the underlying mechanisms by infections as illustrated by the prominent susceptibility of TLR- and MyD88-deficient mice to numerous pathogens (12). Host defenses can be reduced by the underlying disease but also † *Institut Pasteur, Unite´deDe´fense Inneé et Inflammation, Paris, France; Inserm, by a specific therapy such as chemotherapeutic drugs (13, 14). In U874, Paris, France; ‡Department of Medicine, University of Florida, Gainesville, FL 32610; §Institut Pasteur, Unite´de Recherche et d’Expertise Histotechnologie et Pa- regard to this latter aspect, vinblastine and other vinca alkaloids thologie, Paris, France; ¶University of Orleans and Centre National de la Recherehe cause transient but severe immunosuppression (15). In this study, Scientifique, Orleans, France; and #Microbiologia, Immunologia e Parasitologia, Uni- versidade do Estado do Rio de Janeiro, Brazil to gain insight into the role of TLR signaling in B. cenocepacia- induced pathogenesis in the immunocompromised host, we exam- Received for publication May 8, 2008. Accepted for publication April 28, 2009. ined the responses of vinblastine-treated MyD88- and wild-type 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 mice to infection by this opportunistic bacteria. Strikingly, we ob- with 18 U.S.C. Section 1734 solely to indicate this fact. served that immunosuppressed MyD88-deficient mice had an un- 1 G.V. was financially supported by the “Socie´te´ de Pneumologie de Langue Fran- expected survival advantage. Furthermore, we demonstrated that a çaise” (SPLF). MyD88-dependent, TNF-␣-mediated-inflammatory response in- 2 G.M.deC.V. and V.B. contributed equally to this work. duced by B. cenocepacia triggers fatal pneumonia and sepsis, 3 Address correspondence and reprint requests to Dr. M. Si-Tahar, Unite´De´fense which can be prevented by corticosteroids. Inneé et Inflammation, Inserm U874, Institut Pasteur, 25 rue du Dr. Roux, Paris, France. E-mail address: [email protected] 4 Abbreviations used in this paper: CF, cystic fibrosis; TSA, tryptic soy agar; BAL, Materials and Methods bronchoalveolar lavage; p.i., post-infection; PMN, polymorphonuclear neutrophil; Bacterial strain and growth conditions KC, keratinocyte-derived cytokine. B. cenocepacia of the epidemic ET12 lineage (strain J2315) was provided Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00 by the Pasteur Institute microorganisms depository. Bacteria were grown www.jimmunol.org/cgi/doi/10.4049/jimmunol.0801497 The Journal of Immunology 671 on tryptic soy agar (TSA) at 33°C for 48 h. Single colonies removed from the plate were grown in 5 ml of tryptic soy broth at 33°C with shaking for 16–18 h, corresponding to midlog phase. Bacteria were harvested by centrifugation (3,000 ϫ g for 15 min), resuspended in saline, and optical densities of the suspensions were adjusted to give the desired bacterial concentration; this later was verified by serial dilutions and plating on TSA. Mouse strains TNF␣Ϫ/Ϫ and IL-1RϪ/Ϫ were bred at the animal facility of Centre National de la Recherehe Scientifique. MyD88Ϫ/Ϫ mice were obtained from S. Akira (Osaka University, Osaka, Japan). All mice were backcrossed at least eight times with C57BL/6 to ensure similar genetic backgrounds. Wild- type C57/BL6 mice were supplied by Centre d’Elevage R. Janvier and were used as control animals. Mice were cared for in accordance with Pasteur Institute guidelines in compliance with European animal welfare regulations. Mice were fed normal mouse chow and water ad libitum and were reared and housed under standard conditions with air filtration. Immunosuppressive treatment and infection Chemotherapy-induced granulocytopenia was achieved by the i.v. administration of 5 mg/kg of the antineoplastic drug vinblastine (Cell Pharm) Downloaded from 66 h before infection. Under these conditions, the hematological profile of the mice after vinblastine and during the infection displayed total poly- FIGURE 1. MyD88Ϫ/Ϫ, but not IL-1RϪ/Ϫ, immunosuppressed mice are morphonuclear cell depletion from day 0 (the day of infection) until day 2 Ϫ/Ϫ resistant to B. cenocepacia infection. A, Survival of wild-type and for both wild-type and MyD88 mice. Then, the percentage of polymor- Ϫ Ϫ MyD88 / mice treated with vinblastine and infected by a lethal B. ceno- phonuclear cells was 81.3 and 63 at day 3 post infection (p.i.) and 81 and ϫ 7 57.3 at day 4 p.i., in wild-type and MyD88Ϫ/Ϫ animals, respectively (n ϭ cepacia challenge (4 10 cfu/mouse). Wilcoxon test for comparisons of Kaplan-Meier survival curves indicated a significant increase in the sur- 3 to 5). Of note is that in a distinct investigation, we previously observed http://www.jimmunol.org/ Ϫ Ϫ that under the vinblastine regimen, mouse survival and cytokine production vival of MyD88 / mice (n ϭ 11) compared with that of wild-type animals p Ͻ 0.001). B, Description of wild-type and MyD88Ϫ/Ϫ mice ,ءءء ;were similar to those obtained when neutropenia was induced with the (n ϭ 15 antigranulocyte mAb RB6-8C5 (16). The anti-inflammatory drug cortisone aspects 48 h after B. cenocepacia infection. C, IL-1RϪ/Ϫ immunosup- acetate (10 mg/mouse in saline; Sigma-Aldrich) was administered by the pressed mice are as sensitive as wild-type animals to B. cenocepacia in- i.p. route. Mice were treated 2 days before infection, on the day of infec- fection (n ϭ 6; p Ͼ 0.05). tion, and on day 2 after infection (17). Animals were infected intratrache- ally under general anesthesia achieved with a mixture of ketamine (40 mg/kg) and xylazine (8 mg/kg) administered via the i.m. route. A catheter the Kruskal-Wallis ANOVA test, followed by the Mann-Whitney U test. A (diameter, 0.86 mm) was inserted into the trachea via the oropharynx. Ͻ Proper insertion was verified by checking the formation of mist due to value of p 0.05 was considered statistically significant. expiration on a mirror placed in front of the external end. A 50-␮l B. by guest on September 24, 2021 cenocepacia suspension (4 ϫ 107 cfu/mouse) was placed at the internal end Results of the catheter by introducing a micropipette with a sterile disposable tip Distinct mortality rate of immunocompromised wild-type and for gel loading into the catheter. Mice were then immediately held upright MyD88Ϫ/Ϫ mice infected by B. cenocepacia to facilitate inhalation of bacteria and until normal breathing resumed. This protocol allows highly reproducible infection of the lungs, and 10 times To dissect the role of the TLR adaptor MyD88 in B. cenocepacia more inoculum reaches the lungs via this route than via the intranasal route pathogenesis, we characterized within the immunocompromized (18). Mice were then observed daily for signs of morbidity. Alternatively, host the time-course of major dynamic parameters (i.e., animal mice were killed at different time points by i.p. injection of 300 mg/kg sodium pentobarbital, and 1 ml of heparinized blood was collected by the mortality, pathogen load as well as secretion of critical mediators vena cava. After centrifugation at 300 ϫ g, the resulting plasma was stored. in two distinct compartments, the lung tissues and blood) in in- Airways were washed twice with 1 ml saline, and the bronchoalveolar fected wild-type and MyD88Ϫ/Ϫ animals pretreated with vinblas- lavage (BAL) was collected to further determine cytokine concentrations tine. Fig. 1A shows that all immunocompromized wild-type mice using DuoSet ELISA kits (R&D Systems). Lungs were also washed free of ϫ 7 blood by perfusing the heart and lungs with cold PBS. The lungs were infected with B. cenocepacia at a dose of 4 10 cfu per animal homogenized in 1 ml of cold PBS. One hundred microliters of the homog- died within 7 days. Among the numerous signs of infection, pilo- enates were diluted and plated on TSA plates to determine the number of erection, tachypnea, and anorexia were associated with a loss of cfu of B. cenocepacia. weight (Fig. 1B). Remarkably, we found that MyD88Ϫ/Ϫ-infected Inflammatory protein array mice had a higher survival rate than wild-type infected mice; i.e., ϳ80 and 0%, respectively, at day 7 postinfection (p.i.; n ϭ 11 and A commercial Ab-based protein array designed to detect 32 inflammatory 15, respectively, p Ͻ 0.001; Fig. 1A). Moreover, the aspect of mediators was also used according to the manufacturer’s instructions (Ray- MyD88Ϫ/Ϫ Bio Mouse Cytokine Array II; RayBiotech). Membrane arrays were hy- -infected mice at day 2 p.i. was not different from that bridized with BAL fluids to compare wild-type and MyD88Ϫ/Ϫ mice and observed in noninfected animals (Fig. 1B). These results clearly were always processed simultaneously. show that the absence of MyD88 undeniably protects mice from the lethal effects of B. cenocepacia in immunocompromized Histological analyses individuals. Whole lungs were fixed in 3.7% neutral buffered formaldehyde, embedded As MyD88 is also known as an essential adaptor protein that in- in paraffin, and cut into 5-␮m sections. Sections were stained with H&E for tegrates and transduces intracellular signals generated by IL-1R (19), tissue examination. we checked whether the secured phenotype of MyD88Ϫ/Ϫ mice was Statistics due to a reduced impact of IL-1R-dependent signaling. By comparing the survival rate of IL-1RϪ/Ϫ and wild-type animals infected under Survival of wild-type, MyD88Ϫ/Ϫ, IL-1RϪ/Ϫ, and TNF-␣Ϫ/Ϫ animals was the same conditions as described above, we found that contrary to compared using Kaplan-Meier analysis log-rank test. Inflammatory medi- Ϫ/Ϫ Ϫ/Ϫ ators levels and bacterial counts were expressed as the mean Ϯ SEM. MyD88 mice (Fig. 1A), IL-1R animals were as susceptible as Differences between groups were assessed for statistical significance using wild-type mice to B. cenocepacia fatal pneumonia (Fig. 1C). 672 B. cenocepacia TRIGGERS MyD88-DEPENDENT LETHAL INFLAMMATION

FIGURE 2. Distinct lung inflammatory profile in wild-type and MyD88Ϫ/Ϫ mice immunosuppressed and infected by a lethal B. cenocepacia challenge. Wild-type and MyD88Ϫ/Ϫ mice were treated with vinblastine and infected by 4 ϫ 107 Downloaded from cfu/mouse B. cenocepacia. Different parameters were analyzed during the course of infection. A, BAL fluid levels of KC, IL-6, TNF-␣, MIP-2, G- CSF, and IL-10 in wild-type and MyD88Ϫ/Ϫ mice at 0, 6, 24, 72, and 96 h p.i. ELISA data are the mean Ϯ http://www.jimmunol.org/ SEM values obtained from at least five animals and are representative of three independent experiments. B, BAL fluid levels of inflammatory mediators at 24 h p.i. determined by a protein-array. Signal intensity was normalized to internal positive controls spotted on the membrane and are

expressed as relative units. C, Lung by guest on September 24, 2021 histological features of wild-type and MyD88Ϫ/Ϫ mice at 72 h p.i. H&E stain at a magniﬁcation of ϫ400.

Reduced pulmonary inflammation in B. cenocepacia-infected mentary Fig. S1).5 Next, Fig. 2A shows the time course of the immunocompromized MyD88Ϫ/Ϫ mice expression of inflammatory chemokines and cytokines. In wild- ␣ Immunosuppressed animals were killed at different intervals type-infected mice, KC, TNF- , MIP-2, and G-CSF concentra- postinfection and BAL samples were collected to assess mediators tions peaked early at 6–24 h p.i. and decreased significantly there- content in the airspaces. We first analyzed kinetics of leukocyte after. In regard to IL-6, its secretion was low compared with the influx into the bronchoalveolar spaces of either infected wild-type other mediators and showed a two-wave shape, with the highest or MyD88Ϫ/Ϫ mice. We found that polymorphonuclear neutro- level at 6 h and a sustained expression until day 3 p.i. A very phils (PMN) and mononuclear cells recruitment in wild-type mice modest, delayed, secretion was observed for the anti-inflammatory cytokine IL-10 with a peak at 3 day p.i. Interestingly, the amount was detectable 24 h p.i. and increased at least until 96 h p.i. In Ϫ/Ϫ MyD88Ϫ/Ϫ mice, the kinetics of PMN influx was rather similar. In of the foregoing mediators were all much lower in MyD88 than contrast, mononuclear cells recruitment was impaired when compared with the influx of those cells in wild-type animals (supple- 5 The online version of this article contains supplemental material. The Journal of Immunology 673 in wild-type BALs (Fig. 2A). For instance, keratinocyte-derived cytokine (KC) secretion in MyD88Ϫ/Ϫ animals was ϳ5% of that observed in wild-type mice. Next, BAL fluids were analyzed by an inflammatory protein array to examine whether this distinct profile in wild-type and MyD88Ϫ/Ϫ mice could be confirmed when con- sidering expression at 24 h p.i. of additional mediators. The obtained data (Fig. 2B) not only validated the previous ELISA findings but also revealed a similar reduced expression of MCP-1, MIP-1␣, or sTNFR1␣ in MyD88Ϫ/Ϫ mice relatively to wild-type ones. Finally, histological analysis of the lungs at 72 h p.i. indicated that in wild-type mice, B. cenocepacia induced a severe bronchopneumonia characterized by widespread patchy areas of inflammation that began as a bronchiolitis and alveolitis extending to the alveolar lining epithelium. Numerous PMN and alveolar FIGURE 3. Lung bacterial load of wild-type and MyD88Ϫ/Ϫ mice im- macrophages were recruited at this time point and could be seen munosuppressed and infected by a lethal B. cenocepacia challenge. Wild- Ϫ/Ϫ within the bronchioles and alveolae, mixed with necrotic alveolar type and MyD88 mice were treated with vinblastine and infected by 4 ϫ 107 cfu/mouse B. cenocepacia. Bacterial load in the BAL fluids and epithelial cells and exsudation of RBC (Fig. 2C). By contrast, in lung tissues is expressed as percentage of the initial inoculum, as deter- MyD88Ϫ/Ϫ mice, the lesions were less severe despite the presence mined by serial dilutions and plating on TSA. Downloaded from of few polymorphonuclear cells. The alveolar wall was not dis- rupted, only a few necrotic cells could be observed and no hem- orrhage was apparent (Fig. 2C). Altogether, these data suggest that Reduced systemic inflammation in B. cenocepacia-infected lung lesions induced by B. cenocepacia infection in an immuno- immunocompromized MyD88Ϫ/Ϫ mice compromized host are strongly reduced in the absence of MyD88. It is well established that patients with B. cenocepacia pneumonia are Next, we performed additional experiments to examine B. ceno- likely to develop the so-called “cepacia syndrome”, a multiple organ http://www.jimmunol.org/ cepacia pathogenesis in immunocompetent, nonvinblastin-treated, failure due to a septic shock, associated or not with bacteremia (22). Ϫ/Ϫ ϫ 7 wild-type and MyD88 mice challenged by 4 10 cfu/mouse. In view of this consideration and to better understand the role of We observed no animal death under these experimental conditions MyD88 signaling in the pathogenesis of B. cenocepacia in our im- 8 (even using a higher inoculum, i.e., 10 cfu/mouse; data not munodeficient host model, we assessed blood bacterial content as well shown). Nevertheless, an inflammatory response was observed in as the expression of circulating inflammatory cytokines in infected the BALs of infected immunocompetent wild-type animals al- wild-type and MyD88Ϫ/Ϫ mice. Whatever the time point at which though its intensity and/or the duration were low. Thus, except for blood samples were collected (6 or 24 h p.i.), we could not recover IL-6, which was increased (by 55%), the level of all other studied growing B. cenocepacia in either group (data not shown). Next, we mediators were reduced in the BALs of immunocompetent wild- used a multiplex cytokine assay (assessing IFN-␥, KC, TNF-␣, IL-1␣, by guest on September 24, 2021 type mice in comparison with vinblastin-treated wild-type animals IL-5, IL-10, IL-17, and IL-12p40), as well as specific ELISA to de- at 24 h postinfection, i.e., 100% for KC, ϳ75% for TNF-␣, 100% termine whether inflammatory mediators could be present in plasma for MIP2␣, and ϳ48% for G-CSF. Moreover, this immune re- of both murine groups and whether their relative amount could be sponse was strongly reduced in the BALs of immunocompetent affected by MyD88 deficiency. Among all searched molecules, only MyD88Ϫ/Ϫ mice. As a single example, TNF-␣ content at 6 h p.i. IL-6 and KC were significantly detected in plasma harvested at 6 and Ϫ Ϫ in the BALs of immunocompetent wild-type and MyD88Ϫ/Ϫ was 24 h p.i. in wild-type and MyD88 / mice. Their concentration was 2447 Ϯ 216 pg/ml and 554 Ϯ 68 pg/ml, respectively (n ϭ 4). further precisely quantified by ELISA. Fig. 4 shows that in wild-type

Paradoxical increased lung B. cenocepacia load in immunocompromized MyD88Ϫ/Ϫ mice The previous results indicated that a potent inflammatory reaction occurs in the lungs of wild-type mice after B. cenocepacia infection and that this process is critically reduced in MyD88Ϫ/Ϫ animals. Inflammatory signaling pathways during microbial infection have been interpreted in some cases as a protective response of the host, whereas in other cases pathogens can use these pathways to enhance their replication (20, 21). Thus, to investigate whether MyD88-dependent host response might regulate the amount of B. cenocepacia, immunosuppressed wild-type and MyD88Ϫ/Ϫ mice were killed 6, 24, and 72 h after infection and bacterial numbers both in BAL fluids and in lung tissues were determined. Fig. 3 shows a similar elevated bacterial load at 6 h p.i in lung tissues from both wild-type and MyD88Ϫ/Ϫ mice. Paradoxically, while FIGURE 4. Distinct systemic inflammatory profile in wild-type and MyD88Ϫ/Ϫ B. cenocepa- lungs of wild-type animals had very significantly cleared B. ceno- mice immunosuppressed and infected by a lethal cia challenge. Wild-type and MyD88Ϫ/Ϫ mice were treated with vinblas- cepacia as early as 24 h p.i., bacteria persisted at that time in tine and infected by 4 ϫ 107 cfu/mouse B. cenocepacia. Blood levels of KC Ϫ/Ϫ ϳ MyD88 mice with a load 10 times higher than in wild-type and IL-6 were quantified at 0, 6 and 24 h p.i. Histograms are the mean Ϯ animals. However, for both groups of mice, lungs were almost SEM values obtained from at least five animals and are representative of .(p Ͻ 0.001 ,ءءء p Ͻ 0.01 and ,ءء) cleared from the bacterial infection by 72 h p.i. three independent experiments 674 B. cenocepacia TRIGGERS MyD88-DEPENDENT LETHAL INFLAMMATION Downloaded from

FIGURE 5. Corticosteroid treatment and absence of TNF-␣ protect immunosuppressed mice from a B. cenocepacia lethal challenge. Wild-type mice were treated with vinblastine alone or with a combination of vinblastine plus cortisone acetate and further infected by 4 ϫ 107 cfu/mouse B. cenocepacia. http://www.jimmunol.org/ A, Wilcoxon test for comparisons of Kaplan-Meier survival curves indicated a significant increase in the survival of vinblastine/cortisone acetate treated p Ͻ 0.001). B, BAL fluid levels of KC, IL-6, TNF-␣, MIP-2, G-CSF, and ,ءءء ;mice (n ϭ 10) compared with those treated with vinblastine alone (n ϭ 6 IL-10 in wild-type mice at 0, 24, and 48 h p.i. Data are the mean Ϯ SEM values obtained from five animals and are representative of three independent experiments. C, Survival of wild-type and TNF-␣Ϫ/Ϫ mice treated with vinblastine and infected as described above. Wilcoxon test for comparisons of Kaplan-Meier survival curves indicated a significant increase in the survival of TNF-␣Ϫ/Ϫ mice (n ϭ 7) compared with that of wild-type animals (n ϭ 10; .(p Ͻ 0.001 ,ءءء mice, a peak of IL-6 was observed at 6 h p.i. followed by a rapid the anti-inflammatory cytokine IL-10 was moderately, but not sig- decrease within 24 h toward the basal values measured in noninfected nificantly, increased ( p Ͼ 0.05). In regard to leukocyte recruitment by guest on September 24, 2021 animals. In contrast, the systemic expression of KC was sustained into the bronchoalveolar space of infected wild-type mice pre- with a 2-fold higher level at 24 h than at 6 h p.i. In regard to treated with vinblastine alone or with the combination of vinblas- MyD88Ϫ/Ϫ animals, concentrations of both KC and IL-6 were sig- tine and corticosteroids, the supplementary Fig. S2 indicates that nificantly reduced compared with wild-type samples, especially at 6 h leukocyte influx differed only moderately between the two groups p.i. ( p Ͻ 0.01 and p Ͻ 0.001, respectively). of animals. More importantly, using a specific gene-deficient mouse Corticosteroids rescue immunocompromised wild-type mice model, we demonstrated a critical role of TNF-␣ in mediating from B. cenocepacia pneumonia B. cenocepacia pathogenesis. Indeed, when wild-type and TNF- Ϫ Ϫ In view of the apparent deleterious MyD88-dependent acute ␣ / mice were challenged by 4 ϫ 107 cfu/animal, the absence inflammation triggered by B. cenocepacia in the immunocom- of TNF-␣ conferred a striking advantage (0 and 100% of animal Ϫ Ϫ promised host, we speculated that administration of an anti- death in TNF-␣ / and wild-type, respectively, even at day inflammatory regimen such as corticosteroids (23) would limit the 10 p.i.; Fig. 5C). life-threatening consequences of this bacterial infection. To verify such hypothesis, cortisone acetate was administered i.p. to vinblas- Discussion tine-treated wild-type mice (n ϭ 10). A second group of vinblas- Combating nosocomial disease associated to B. cenocepacia is still tine-treated mice received saline under the same experimental con- a major clinical challenge despite considerable advances in epide- ditions and served as controls (n ϭ 6). Mice were then observed miology, taxonomy, as well as in identification of virulence factors for 6 days to measure their survival. Fig. 5A indicates a significant (24, 25). Therefore, it is of importance to gain insight into the protective effect of corticosteroids. Indeed, while Ͼ80% of vin- immune response to B. cenocepacia infection to potentially iden- blastine plus saline-treated mice died by day 6 p.i., all animals tify new therapeutic strategies. Our knowledge of how the immune receiving vinblastine plus cortisone acetate survived. In a separate system recognizes pathogens has increased exponentially in recent set of experiments, mice were infected and treated exactly as years due to the discovery of the TLR family. Recently, we dem- above, and BAL fluids were collected at different time points p.i. onstrated using in vitro approaches that TLR5 and MyD88, but to ascertain whether the administration of cortisone acetate was neither TLR2 nor TLR4, play a central role in the response of effective against the acute pulmonary inflammation triggered by B. bronchial epithelial cells to B. cenocepacia (26). These findings cenocepacia. Consistent with the survival data, corticosteroid- implied that flagellin, the agonist of TLR5 was the key B. ceno- treated immunocompromised and infected mice had a contained cepacia proinflammatory molecule. Our results were consistent inflammatory response as illustrated by the highly significant de- with an in vivo study showing that flagellin-induced signaling was crease of KC, IL-6, TNF-␣, MIP-2, and G-CSF concentrations required for B. cenocepacia virulence in a mouse lung infection compared with saline-treated mice (Fig. 5B). Interestingly, only model (27). More generally, several clinical reports confirmed the The Journal of Immunology 675 contribution of TLRs to the pathophysiology of infectious dis- Meanwhile, our study suggests that corticosteroids are a prom- eases, and polymorphisms in TLR genes are associated with pre- ising therapeutic approach to specifically dampen inflammation disposition to severe infections (28–30). during B. cenocepacia pneumonia. This possibility is supported by In view of this major information, we anticipated at the start of previous works showing that corticosteroids spare or even enhance our investigation that MyD88 expression would be a prerequisite numerous lung innate immune responses while they concomitantly for effective host defense in vivo against B. cenocepacia. Con- suppress inflammation (44). Incidentally, the present work sheds a versely, its absence would render MyD88Ϫ/Ϫ mice more suscep- new light on the limited number of clinical case reports that indi- tible to B. cenocepacia pneumonia. Surprisingly, our study reveals cated improvement of critically sick CF and non-CF patients with that mice deficient in MyD88, even after immunosuppression by B. cepacia syndrome, after use of corticosteroids (22, 45). vinblastine, have an unexpected advantage. Thus, in comparison with wild-type mice, we found in MyD88Ϫ/Ϫ animals a clearly Acknowledgments reduced level of inflammatory mediators both in lung airspaces We are grateful to Micheline Lagranderie (Laboratoire Immunothe´rapie, and in blood, and, most important, a paradoxical extended sur- Institut Pasteur) for the multiplex cytokine assay and for helpful vival. Based on these findings and in view of our results describing discussions. the rescuing effect of corticosteroids in the pathogenesis of B. cenocepacia infection, we discuss below the fact that the enhanced Disclosures Ϫ Ϫ resistance of B. cenocepacia-infected MyD88 / might be due to The authors have no financial conflict of interest. a lower MyD88-mediated release of inflammatory mediators, especially of TNF-␣. References Downloaded from The abrogated response to TLR ligands by MyD88 knock-out 1. Davies, J. C., and B. K. Rubin. 2007. Emerging and unusual gram-negative in- mice has provided an invaluable tool for analyzing the critical role fections in cystic fibrosis. Semin. Respir. Crit. Care Med. 28: 312–321. 2. Lipuma, J. J. 2005. Update on the Burkholderia cepacia complex. Curr. Opin. of MyD88-dependent signaling in host defense against infection Pulm. Med. 11: 528–533. (12). Multiple studies have demonstrated that MyD88 deficiency 3. Mahenthiralingam, E., T. A. Urban, and J. B. Goldberg. 2005. The multifarious, renders animals highly susceptible to microbial infection. As a few multireplicon Burkholderia cepacia complex. Nat. Rev. 3: 144–156. 4. Pegues, C. F., D. A. Pegues, D. S. Ford, P. L. Hibberd, L. A. Carson, C. M. Raine, examples, one can mention the protective in vivo role of MyD88 and D. C. Hooper. 1996. Burkholderia cepacia respiratory tract acquisition: ep- http://www.jimmunol.org/ in infections by the Gram-positive or negative bacteria Pseudomo- idemiology and molecular characterization of a large nosocomial outbreak. Epi- nas aeruginosa, Staphylococcus aureus, Chlamydia pneumoniae, demiol. Infect. 116: 309–317. 5. Siddiqui, A. H., M. E. Mulligan, E. Mahenthiralingam, J. Hebden, J. Brewrink, or Mycobacterium tuberculosis, the fungus Candida albicans, the S. Qaiyumi, J. A. Johnson, and J. J. LiPuma. 2001. An episodic outbreak of parasite Toxoplasma gondii, and the herpes simplex virus-2 (18, genetically related Burkholderia cepacia among non-cystic fibrosis patients at a Ϫ/Ϫ university hospital. Infect. Control Hosp. Epidemiol. 22: 419–422. 31–35). Accordingly, our current demonstration that MyD88 6. Bressler, A. M., K. S. Kaye, J. J. LiPuma, B. D. Alexander, C. M. Moore, mice are protected from the lethal effects of B. cenocepacia revisits L. B. Reller, and C. W. Woods. 2007. Risk factors for Burkholderia cepacia noticeably the paradigm. Moreover, our findings are especially re- complex bacteremia among intensive care unit patients without cystic fibrosis: a case-control study. Infect. Control Hosp. Epidemiol. 28: 951–958. markable because, in our experimental model, animals were im- 7. Yamagishi, Y., J. Fujita, K. Takigawa, K. Negayama, T. Nakazawa, and munocompromised while all other previous studies have tested the J. Takahara. 1993. Clinical features of Pseudomonas cepacia pneumonia in an by guest on September 24, 2021 role of MyD88 in immunocompetent hosts. epidemic among immunocompromised patients. Chest 103: 1706–1709. 8. Kawai, T., and S. Akira. 2007. TLR signaling. Semin. Immunol. 19: 24–32. There is growing evidence that the mediators that have a central 9. Miyake, K. 2007. Innate immune sensing of pathogens and danger signals by cell role in the resolution of bacterial infections, are the same that surface Toll-like receptors. Semin. Immunol. 19: 3–10. causes many clinical signs related to these diseases. Thus, cyto- 10. O’Neill, L. A., and A. G. Bowie. 2007. The family of five: TIR-domain-contain- ing adaptors in Toll-like receptor signaling. Nat. Rev. Immunol. 7: 353–364. kines trigger a vigorous localized inflammatory response that is 11. Trinchieri, G., and A. Sher. 2007. Cooperation of Toll-like receptor signals in required for elimination of the particular pathogen. However, the innate immune defense. Nat. Rev. Immunol. 7: 179–190. 12. Takeuchi, O., and S. Akira. 2002. Genetic approaches to the study of Toll-like magnitude of inflammatory cytokine expression must be tightly receptor function. Microbes Infect. 4: 887–895. regulated and compartmentalized to prevent excessive tissue injury 13. Donowitz, G. R., D. G. Maki, C. J. Crnich, P. G. Pappas, and K. V. Rolston. 2001. (36–39). We show in the present study that B. cenocepacia infec- Infections in the neutropenic patient: new views of an old problem. Hematology 113–139. tion leads to the synthesis of major inflammatory cytokines and 14. Hubel, K., K. Hegener, R. Schnell, G. Mansmann, F. Oberhauser, P. Staib, chemokines in lung tissues. More importantly, our study estab- V. Diehl, and A. Engert. 1999. Suppressed neutrophil function as a risk factor for lishes that MyD88 plays a major role in their production, the lack severe infection after cytotoxic chemotherapy in patients with acute nonlympho- cytic leukemia. Annals Hematol. 78: 73–77. of this signaling molecule resulting in a significant decrease of KC, 15. Noble, R. L. 1990. The discovery of the vinca alkaloids: chemotherapeutic agents IL-6, TNF-␣, MIP-2, and G-CSF synthesis. Interestingly, elevated against cancer. Biochem. Cell Biol. 68: 1344–1351. 16. Balloy, V., J. M. Sallenave, B. Crestani, M. Dehoux, and M. Chignard. 2003. cytokine levels have been implicated in both systemic and local Neutrophil DNA contributes to the antielastase barrier during acute lung inflam- inflammation and may play a role in “cepacia syndrome” (40). mation. Am. J. Respir. Cell Mol. Biol. 28: 746–753. How lung inflammation triggers multiorgan dysfunction is unclear 17. Balloy, V., M. Huerre, J. P. Latge, and M. Chignard. 2005. Differences in patterns of infection and inflammation for corticosteroid treatment and chemotherapy in but neural and humoral mechanisms may be involved (41). In that experimental invasive pulmonary aspergillosis. Infect. Immun. 73: 494–503. regard, our study is the first to reveal the involvement of the proin- 18. Ramphal, R., V. Balloy, M. Huerre, M. Si-Tahar, and M. Chignard. 2005. TLRs flammatory cytokine TNF-␣ in the development of fatal pneumo- 2 and 4 are not involved in hypersusceptibility to acute Pseudomonas aeruginosa lung infections. J. Immunol. 175: 3927–3934. nia induced by B. cenocepacia. Moreover, a clear conclusion from 19. Li, C., J. Zienkiewicz, and J. Hawiger. 2005. Interactive sites in the MyD88 our work is that a reduction in MyD88-mediated inflammation Toll/interleukin (IL) 1 receptor domain responsible for coupling to the IL1␤ reduces the clinical manifestations of B. cenocepacia-induced signaling pathway. J. Biol. Chem. 280: 26152–26159. 20. Backert, S., and W. Konig. 2005. Interplay of bacterial toxins with host defence: pneumonia. It is thus reasonable to hypothesize that targeting molecular mechanisms of immunomodulatory signalling. Int. J. Med. Microbiol. MyD88 and/or TNF-␣ signaling pathway in B. cenocepacia-in- 295: 519–530. 21. Knodler, L. A., J. Celli, and B. B. Finlay. 2001. Pathogenic trickery: deception of fected immunocompromised patients has great therapeutic poten- host cell processes. Nat. Rev. Mol. Cell. Biol. 2: 578–588. tial, although significant complexities must be considered. Indeed, 22. Kazachkov, M., J. Lager, J. LiPuma, and P. M. Barker. 2001. Survival following MyD88 and TNF-␣ fulfils important functions in innate immunity, Burkholderia cepacia sepsis in a patient with cystic fibrosis treated with corticosteroids. Pediatric Pulmonol. 32: 338–340. cell death, and is intimately related to adaptive immunity, endo- 23. Skerrett, S. J., and D. R. Park. 2001. Anti-inflammatory treatment of acute and crine, circulatory, and nervous systems (42, 43). chronic pneumonia. Semin. Respir. Infect. 16: 76–84. 676 B. cenocepacia TRIGGERS MyD88-DEPENDENT LETHAL INFLAMMATION

24. Chiarini, L., A. Bevivino, C. Dalmastri, S. Tabacchioni, and P. Visca. 2006. 34. Tengvall, S., and A. M. Harandi. 2008. Importance of myeloid differentiation Burkholderia cepacia complex species: health hazards and biotechnological po- factor 88 in innate and acquired immune protection against genital herpes infec- tential. Trends Microbiol. 14: 277–286. tion in mice. J. Reprod. Immunol. 78: 49–57. 25. Valvano, M. A. 2006. Infections by Burkholderia spp.: the psychodramatic life of 35. Villamon, E., D. Gozalbo, P. Roig, C. Murciano, J. E. O’Connor, D. Fradelizi, an opportunistic pathogen. Future Microbiol. 1: 145–149. and M. L. Gil. 2004. Myeloid differentiation factor 88 (MyD88) is required for murine resistance to Candida albicans and is critically involved in Candida- 26. Ventura, G., R. Le Goffic, V. Balloy, M. C. Plotkowski, M. Chignard, and induced production of cytokines. Eur. Cytokine Netw. 15: 263–271. M. Si-Tahar. 2008. TLR 5, but neither TLR2 nor TLR4, is involved in lung 36. Deng, J. C., and T. J. Standiford. 2005. The systemic response to lung infection. epithelial cell response to Burkholderia cenocepacia. FEMS Immunol. Med. Mi- Clin. Chest Med. 26: 1–9. crobiol. 54: 37–44. 37. Hippenstiel, S., B. Opitz, B. Schmeck, and N. Suttorp. 2006. Lung epithelium as 27. Urban, T. A., A. Griffith, A. M. Torok, M. E. Smolkin, J. L. Burns, and a sentinel and effector system in pneumonia: molecular mechanisms of pathogen J. B. Goldberg. 2004. Contribution of Burkholderia cenocepacia flagella to in- recognition and signal transduction. Respir. Res. 7: 97. fectivity and inflammation. Infect. Immun. 72: 5126–5134. 38. Khan, S. A., P. Everest, S. Servos, N. Foxwell, U. Zahringer, H. Brade, 28. Cook, D. N., D. S. Pisetsky, and D. A. Schwartz. 2004. Toll-like receptors in the E. T. Rietschel, G. Dougan, I. G. Charles, and D. J. Maskell. 1998. A lethal role pathogenesis of human disease. Nat. Immunol. 5: 975–979. for lipid A in Salmonella infections. Mol. Microbiol. 29: 571–579. 29. Texereau, J., J. D. Chiche, W. Taylor, G. Choukroun, B. Comba, and J. P. Mira. 39. Si-Tahar, M., L. Touqui, and M. Chignard. 2009. Innate immunity and inflam- 2005. The importance of Toll-like receptor 2 polymorphisms in severe infections. mation: two facets of the same anti-infectious reaction. Clin. Exp. Immunol. 156: Clin. Infect. Dis. 41 Suppl 7: S408–S415. 194–198. 30. Uematsu, S., and S. Akira. 2006. The role of Toll-like receptors in immune 40. Govan, J. R. 2003. The Burkholderia cepacia complex and cytokine induction: an disorders. Expert Opin. Biol. Therapy 6: 203–214. inflammatory tale. Pediatric. Rese. 54: 294–296. 41. Munford, R. S., and K. J. Tracey. 2002. Is severe sepsis a neuroendocrine dis- 31. Fremond, C. M., V. Yeremeev, D. M. Nicolle, M. Jacobs, V. F. Quesniaux, and ease? Mol. Med. 8: 437–442. B. Ryffel. 2004. Fatal Mycobacterium tuberculosis infection despite adaptive 42. Brown, K. L., C. Cosseau, J. L. Gardy, and R. E. Hancock. 2007. Complexities immune response in the absence of MyD88. J. Clin. Invest. 114: 1790–1799. of targeting innate immunity to treat infection. Trends Immunol. 28: 260–266. 32. Naiki, Y., K. S. Michelsen, N. W. Schroder, R. Alsabeh, A. Slepenkin, W. Zhang, 43. Liebermann, D. A., and B. Hoffman. 2002. Myeloid differentiation (MyD) pri- S. Chen, B. Wei, Y. Bulut, M. H. Wong, E. M. Peterson, and M. Arditi. 2005. mary response genes in hematopoiesis. Oncogene 21: 3391–3402. Downloaded from MyD88 is pivotal for the early inflammatory response and subsequent bacterial 44. Schleimer, R. P. 2004. Glucocorticoids suppress inflammation but spare innate clearance and survival in a mouse model of Chlamydia pneumoniae pneumonia. immune responses in airway epithelium. Proc. Am. Thor. Soc. 1: 222–230. J. Biol. Chem. 280: 29242–29249. 45. Okano, M., M. Yamada, M. Ohtsu, N. Kawamura, Y. Sakiyama, K. Aoi, 33. Scanga, C. A., J. Aliberti, D. Jankovic, F. Tilloy, S. Bennouna, E. Y. Denkers, S. Gandoh, M. Fujita, and K. Kobayashi. 1999. Successful treatment with meth- R. Medzhitov, and A. Sher. 2002. Cutting edge: MyD88 is required for resistance ylprednisolone pulse therapy for a life-threatening pulmonary insufficiency in a to Toxoplasma gondii infection and regulates parasite-induced IL-12 production patient with chronic granulomatous disease following pulmonary invasive as- by dendritic cells. J. Immunol. 168: 5997–6001. pergillosis and Burkholderia cepacia infection. Respiration 66: 551–554. http://www.jimmunol.org/ by guest on September 24, 2021 SUPPLEMENTARY MATERIAL

Lack of MyD88 protects the immunodeficient host against fatal lung inflammation triggered by the opportunistic bacteria Burkholderia cenocepacia

Grasiella M. de C. Ventura1,2*, Viviane Balloy1,2*, Reuben Ramphal3, Huot Khun4, Michel

Huerre4, Bernhard Ryffel5, Maria-Cristina M. Plotkowski 6, Michel Chignard1,2 and Mustapha

Si-Tahar1,2‡.

Fig. S1. Polymorphonuclear (PMN) and mononuclear cells recruitment into the bronchoalveolar space of wild-type and MyD88-/- immunosuppressed mice, infected by a lethal B. cenocepacia challenge. Wild-type and MyD88-/- mice were treated with vinblastine and infected by 4x107 cfu/mouse of B. cenocepacia. Leukocyte amount recovered in the BAL fluids is the mean ± SEM obtained from 4 animals. Data are expressed as the number of cells/ml.

Fig. S2. PMN and mononuclear cells recruitment into the bronchoalveolar space of wild- type mice pretreated with vinblastine alone or in combination with cortisone acetate, infected by a lethal B. cenocepacia challenge. Wild-type mice were treated with the different molecules and infected by 4x107 cfu/mouse of B. cenocepacia. Leukocyte amount recovered in the BAL fluids is the mean ± SEM obtained from at least 4 animals. Data are expressed as the number of cells/ml.