of November 17, 2018.This information is current as

Schu4Francisella tularensisImmune Response by

Active Suppression of the Pulmonary

BelisleCatharine M. Bosio, Helle Bielefeldt-Ohmann and John T.

http://www.jimmunol.org/content/178/7/4538doi: 10.4049/jimmunol.178.7.4538

2007; 178:4538-4547; ;J Immunol 

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Active Suppression of the Pulmonary Immune Response byFrancisella tularensis Schu41

Catharine M. Bosio,2 Helle Bielefeldt-Ohmann, and John T. Belisle

Francisella tularensis is an obligate, intracellular bacterium that causes acute, lethal disease following inhalation. As an intracel-lular pathogen F. tularensis must invade cells, replicate, and disseminate while evading host immune responses. The mechanismsby which virulent type A strains of Francisella tularensis accomplish this evasion are not understood. Francisella tularensis hasbeen shown to target multiple cell types in the lung following aerosol infection, including dendritic cells (DC) and macrophages.We demonstrate here that one mechanism used by a virulent type A strain of F. tularensis (Schu4) to evade early detection is bythe induction of overwhelming immunosuppression at the site of infection, the lung. Following infection and replication in multiplepulmonary cell types, Schu4 failed to induce the production of proinflammatory cytokines or increase the expression of MHCIIor CD86 on the surface of resident DC within the first few days of disease. However, Schu4 did induce early and transientproduction of TGF-�, a potent immunosuppressive cytokine. The absence of DC activation following infection could not beattributed to the apoptosis of pulmonary cells, because there were minimal differences in either annexin or cleaved caspase-3staining in infected mice compared with that in uninfected controls. Rather, we demonstrate that Schu4 actively suppressed in vivoresponses to secondary stimuli (LPS), e.g., failure to recruit granulocytes/monocytes and stimulate resident DC. Thus, unlikeattenuated strains of F. tularensis, Schu4 induced broad immunosuppression within the first few days after aerosol infection. Thisdifference may explain the increased virulence of type A strains compared with their more attenuated counterparts. The Journalof Immunology, 2007, 178: 4538–4547.

F rancisella tularensis is a Gram-negative, obligate intra-cellular bacterium that was first identified as the agent ofrabbit fever in the early 1920s (1). There are four sub-

species of F. tularensis, mediasiatica, novicida, holartica (type B),and tularensis (type A). Type A is the most lethal for humans, withas few as 10 inhaled organisms capable of causing acute, lethalpneumonic disease (2, 3). F. tularensis is readily available in theenvironment and has been previously modified to be used as abiological weapon. These features have necessitated its classifica-tion as a category A priority pathogen. Despite identification ofthis bacterium in the early 1920s and subsequent studies on itspathogenesis over the last 80 years, little is understood about manyaspects of immunity to F. tularensis, especially the early eventsfollowing aerosol infection.

The majority of the information describing F. tularensis patho-genesis has been compiled using an attenuated type B strain knownas live vaccine strain (LVS).3 LVS retains dose- and route-related

virulence in mice, although it is relatively attenuated in humans.Using the mouse model, we and others have shown that LVS tar-gets multiple cells, including dendritic cells (DC) and macro-phages for infection and replication (4–6). In the lung, LVS ini-tially targets pulmonary DC for replication. The infection of thesecells results in “phenotypic” activation or increased expression ofMHC class II (MHCII) and CD86 on the surface of DC (6). How-ever, despite this phenotypic activation, DC from LVS-infectedmice do not secrete proinflammatory cytokines, such as TNF-�and IL-12, typically associated with the “functional” maturation ofDC. Furthermore, it has been shown that LVS actively suppressesthe ability of both DC and macrophages to secrete these cytokinesin response to secondary stimuli (6, 7). Given the central role ofTNF-� and IL-12 in the resolution of Francisella infections, thispartial suppression of primary APC is thought to contribute to thevirulence of LVS in mice (8–10). Indeed, in agreement with itsmarked attenuation in humans, LVS stimulates productive proin-flammatory responses following vaccination or in vitro infection ofhuman cells (11, 12).

Other attenuated pathogens have a similar profile of aberrantactivation of DC. For example, intranasal infection of mice withBordetella pertussis results in a similar profile of phenotypic, butnot functional, maturation of DC (13). Virus-like particles that aremorphologically similar to intact virus but lack the componentsrequired for replication and assembly readily activate numerouscells, including NK cells and DC (14, 15). In contrast to these moreattenuated pathogens and preparations, it has been shown thatmore virulent organisms interfere with multiple aspects of host/DCresponses. Both the Ebola virus and Marburg viruses infect andreplicate in DC but suppress the ability of these cells to stimulateT cells and secrete important anti-viral cytokines such as IFN-�(16, 17). It is believed that the targeting and suppression of DCduring the first few hours of infection significantly hobbles the hostresponse, allowing the pathogen to replicate, disseminate, and ul-timately cause death (18).

Department of Microbiology, Immunology and Pathology, Colorado State University,Ft. Collins, CO 80523

Received for publication October 18, 2006. Accepted for publication January17, 2007.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 This work was supported by a grant form the Colorado State University CollegeResearch Council and by Rocky Mountain Regional Center of Excellence NationalInstitutes of Health Grant U01 AI056487-01.2 Address correspondence and reprint requests to Dr. Catharine M. Bosio at the cur-rent address: National Institutes of Health/National Institute of Allergy and InfectiousDiseases/Rocky Mountain Laboratories, 903 South 4th Street, Hamilton, MT 59840.E-mail address: bosioc@niaid.nih.gov3 Abbreviations used in this paper: LVS, live vaccine strain; BAL, bronchoalveolarlavage; BHI, brain-heart infusion; DC, dendritic cell; IHC, immunohistochemistry;MHCII, MHC class II; MLN, mediastinal lymph node.

Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00

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To determine whether virulent type A F. tularensis induces asimilar aberrant activation of host cells as that observed with LVSor executes a broader spectrum of immunosuppression, we exam-ined early events in the pulmonary tissues following low-doseaerosol infection with a representative type A strain of F. tularen-sis, Schu4 (see Table I). In the current study, we demonstrate thatSchu4 replicates within multiple compartments of the pulmonarysystem, including the airways and draining lymph nodes. How-ever, despite this rapid replication and dissemination (in directcontrast to LVS), Schu4 did not induce the phenotypic activationof pulmonary DC, macrophages, or DC in the lymph node drainingthe lung. Schu4 infection also did not induce the secretion of sev-eral proinflammatory cytokines, including TNF-�, IL-12, IL-10,and IL-1�, within 24–48 h of infection. The absence of proin-flammatory cytokines could not be attributed to the loss of cellsdue to apoptosis. Rather, an overwhelming in vivo suppression ofmultiple host responses was observed in Schu4-infected mice. Thisactive suppression included both the failure of resident DC to un-dergo activation and the inability of the host to mobilize effectorcells in response to secondary stimuli (LPS). TGF-�, a potent im-munosuppressive cytokine, was detected at both the site of theinfection and in peripheral tissues within 24 h of infection, beforethe detectable dissemination of the bacterium. The administrationof anti-TGF-� Abs in Schu4-infected mice increased the produc-tion of TNF-� and reduced bacterial loads in the lungs. Theseresults suggest active suppression of host responses by Schu4, in-cluding the elicitation of TGF-� by the bacterium, facilitates itsreplication in the lung. Together, our data may in part explain theenhanced virulence associated with the type A strains of F. tula-rensis and could aid in the identification of novel candidates forvaccine or therapeutic countermeasures.

Materials and MethodsMice

Specific pathogen-free, 6- to 8-wk old female C57BL/6 mice were pur-chased from The Jackson Laboratory. All mice were housed in sterile mi-croisolater cages in the laboratory animal resources facility or in the Bio-hazard Research Building BSL-3 facility at Colorado State University (Ft.Collins, CO) and provided sterile water and food ad libitum. All researchinvolving animals was conducted in accordance with animal care and useguidelines and animal protocols were approved by the Animal Care andUse Committee at Colorado State University.

Bacteria

F. tularensis Schu4 and Yersinia pestis strain MG05 were provided by Drs.J. Peterson and M. Schriefer (Centers for Disease Control, Fort Collins,Colorado). Y. pestis MG05 is a recent virulent isolate obtained from a fatalhuman case of plague (M. Schriefer, unpublished observations). Schu4 wascultured in modified Mueller-Hinton broth at 37°C with constant shakingovernight, aliquoted into 1 ml-samples, frozen at �80°C,and thawed justbefore use as previously described (5). Frozen stocks were titered by enu-merating viable bacteria from serial dilutions plated on modified Mueller-Hinton agar as previously described (5). Y. pestis MG05 was cultured inbrain-heart infusion (BHI) broth at 37°C with constant shaking overnight,aliquoted into 1-ml samples, frozen at �80°C, and thawed just before use.Frozen stocks were titered by enumerating viable bacteria from serial di-lutions plated on BHI agar plates. For both Y. pestis and F. tularensis thenumber of viable bacteria in frozen stock vials varied �5% over a 10-moperiod.

Infection of mice

Mice were infected with Schu4 via a whole body aerosol. Low-dose aero-sol infections were performed as previously described with minor modifi-cations (19). Briefly, mice were placed in a Glas-Col inhalation apparatus.The nebulizer compartment was filled with an 8-ml suspension of F. tu-larensis Schu4 at 106/ml in sterile PBS. The vacuum was set to 35 cubicfeet per hour and the compressor was set to 19 cubic feet per hour. Thesesettings allowed reproducible delivery of �50 CFU to the lungs over a30-min exposure period. Initially, inoculum doses were confirmed by ho-mogenizing the lungs of freshly infected mice and culturing the entirehomogenate as described below. This inoculum routinely results in 100%mortality and a mean time to death of 5 days following infection (data notshown).

In some experiments mice were infected intranasally with Y. pestisMG05 as previously described (43). MG05 was cultured in BHI brothsupplemented with 2 mM CaCl2 broth at 26°C with constant shaking over-night. A 100-�l aliquot was then transferred to fresh BHI broth and grownovernight at 37°C with constant shaking. Bacteria were diluted to achievean inoculum of �1 � 105/ml in sterile PBS immediately before infectionof the mice. The inoculum was titered by enumerating viable bacteria fromserial dilutions plated on BHI agar plates. Mice were anesthetized i.p.with a 200-�l injection of 2.5% Avertin (Sigma-Aldrich). Forty micro-liters (�4 � 104 or 100 LD50) of freshly grown and diluted Y. pestisMG05 was administered to the nares of each mouse. This dose routinelyresults in 100% lethality within 2.5 days of infection. In other experi-ments mice were anesthetized as described above and 5 �g per 40 �lultrapure LPS (Invivogen) was administered to the indicated mice. Micereceiving PBS served as vehicle (untreated) controls.

Delivery of anti-TGF-�

To assess the contribution TGF-� toward the pathogenesis of Schu4 in-fection, mice were treated i.p. with 50 �g per 200 �l either chicken IgY(isotype) or pan-anti-TGF-� Abs (both from R&D Systems) immediatelybefore infection and once more 24 h after infection.

Collection of airway, lung lymph node, and spleen cells

Airway cells were obtained by bronchoalveolar lavage (BAL) as previ-ously described (20). Briefly, mice were euthanized by cervical dislocationand an 18-gauge catheter was immediately inserted into the trachea. Ap-proximately 1.5 ml of ice-cold PBS was injected and then aspirated fromthe lungs. This was repeated three times. BAL cells from each mouse werepooled and then centrifuged at 1200 rpm for 5 min at 4°C. BAL cells werethen resuspended in either complete RPMI or FACS buffer (PBS with 2%FBS and 0.05% sodium azide) before analysis. Lung cells were isolated aspreviously described (21). Briefly, lungs were excised, minced, and incu-bated in PBS supplemented with 5 mg/ml collagenase type 1A, 125 �g/mlDNase, and 250 �g/ml soybean trypsin inhibitor (all from Sigma-Aldrich)

Table I. Comparison of virulence and immunogenicity of LVS and TypeA strains of F. tularensis

LVS Type A

VirulenceHumans Attenuated Highly virulentMice Route and dose

dependentHighly virulent

LD100a

Intraperitoneal �1 (38) �1 (Data notshown)

Intradermal �106 (38) �10 (2)Intranasal/aerosol 500–104 (10, 38, 39) �10 (2)

MTDb 5–7 Days (38, 39) 3–5 Days (2)Target Cellsc

Macrophages Ref. 38 This studyDendritic cells Ref. 6 This studyEpithelial cells Ref. 25 This study

Phenotypic activationAPCHuman Yes (11, 40) UnknownMouse Yes (6) No (This study)

Cytokine Elicitationc

TNF-� No (5–7) No (This studyand Ref. 41)

IL-12p40 No (5–7) No (This study)IL-1� Yes/No (7, 42) No (This study)IL-10 No (5–7) No (This study)TGF-� Yes (6) Yes (This study)

Induce apoptosis Yesd (42) No (This study)

a Among inbred C57BL/6 and BALB/c mice.b Mean time to death.c Within the first 48 h of lethal infection in mice.d Only described following in vitro infection.

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for 30 min at 37°C with 5%CO2. Tissues were triturated using a 5-mlsyringe and an 18-gauge needle. Cells were then pelleted by centrifugationat 1200 rpm for 5 min and RBC were lysed with NHCl4. Cells were thenwashed twice in PBS and resuspended in FACS buffer before analysis. Thedraining lymph nodes (mediastinal lymph node (MLN)) from the lungswere aseptically removed, passed through a 70-�m cell strainer, washedtwice, and resuspended in FACS buffer before analysis. Total live cellsfrom the airways, lungs, and MLN were enumerated using trypan blue anda hemacytometer. For assessment of the ex vivo production of cytokines,the lungs and spleens were excised, weighed, and placed (one per well) in24-well tissue culture plates. Tissues were then uniformly minced and 1 mlof complete RPMI was added to each well. Tissues were incubated at 37°Cand 5% CO2 and supernatants were collected 24 h later and stored at�80°C before analysis for cytokines by ELISA (see below).

Enumeration of bacteria in organs

Lungs, spleens, and MLN were collected and homogenized in sterile PBSusing a stomacher (Tekmar). Bacterial colony counts in each organ weredetermined by plating serial 10-fold dilutions of organ homogenate onmodified Mueller-Hinton agar and incubating the plates at 37°C for 48 h.

Detection of Schu4 by immunohistochemistry (IHC)in lung tissue

Detection of intracellular Schu4 in vivo following infection was accom-plished by performing IHC on lungs at various time points after infection.Briefly, lungs were collected and preserved in 10% buffered formalin. Fol-lowing routine processing and embedding in paraffin, tissues were cut into4-�m sections and mounted on Platinum Line StarFrost slides (MercedesMedical). Tissue sections where deparaffinized with Histoclear (NationalDiagnostics) and rehydrated. The sections were then subjected to Ag re-trieval by incubation for 15 min at 90°C in DakoCytomation Target Re-trieval solution (pH 9.0). The sections were then treated with 3% H2O2 for5 min and blocked with 0.1 M glycine in PBS for 15 min followed byblocking with 1% normal horse and goat serum in TBST with 1% BSA for30 min. The slides were then incubated with a 1/4000 dilution of rabbitanti-Schu4 IgG (a gift from Dr. J. Peterson, Division of Vector-BorneInfectious Diseases/Center for Disease Control) in blocking buffer. Theslides were then washed thoroughly followed by visualization of Ab bind-ing using the Vectastain system (Vector Laboratories) and diaminobenzi-dine as the chromogen. Sections were counterstained with hematoxylin,mounted, and examined on an Olympus BX41 light microscope equippedwith a QColor3 camera and associated QCapture Pro software.

Flow cytometry and analysis of BAL, lung, and MLN cells

Cellular populations in the BAL fluid, lungs, and MLN were assessed byimmunostaining and flow cytometry. Directly conjugated Abs for theseanalyses were purchased from Cedarlane Laboratories, Serotec, BDPharmingen, or eBioscience. The following Abs in various combinationswere used for flow cytometric analysis: anti-CD11c (allophycocyanin orPE; clone N418), anti-CD11b (PE-Cy5 or allophycocyanin-Cy7; clone M1/70), anti-GR-1 (Pey7; clone RB6-8C5), anti-B220 (allophycocyanin-Cy7;clone RA3-6B2), anti-CD8 (FITC or PE; clone 53-6.7), anti-CD4 (allo-phycocyanin; clone RM4-5), anti-CD3 (allophycocyanin-Cy7; clone 145-2C11), anti -I-A/I-E (MHCII, biotin or PE; clone M5/114.15.2), anti-CD86(PE; clone GL1), anti-DEC-205 (biotin; clone NLDC-145), and anti-CD40(Pey5; clone 1C-10). Typical flow cytometric assessment of airway cellsincluded five- to seven-color analysis. Before immunostaining, nonspecificAb binding was blocked by the addition of normal mouse serum and un-labeled anti-FcRIII Ab (clone 24.G2). Staining with directly conjugatedAbs was done in FACS buffer at 4°C followed by washing. In the case ofbiotinylated Abs, the appropriate streptavidin conjugate was added next(eBiosciences or Molecular Probes). After a final wash, the cells were fixedin 1% paraformaldehyde for 72 h at 4°C, washed once, and resuspended inFACS buffer immediately before analysis. Flow cytometry was done usinga Cyan MLE flow cytometer (DakoCytomation). Analysis gates were seton viable unstained airway cells and were designed to include all viablecell populations. Of note, the side scatter and forward scatter settings werereduced from those typically used for the analysis of lymphocytes to in-clude macrophages and DC, which have high forward and side scatterproperties. Approximately 25,000 (BAL samples) and 100,000 (lung)events were analyzed for each sample.

DC were defined as CD11c�DEC-205� cells that expressed variablelevels of other surface determinants, including CD11b, Gr-1, B220, MHCII,CD86, and F4/80, as well as high side and forward scatter properties aspreviously reported (22, 23) Alveolar macrophages were identified asCD11b�F480�CD11c�DEC-205� cells with high forward and side scatter

properties. Monocytes were identified as CD11b�GR-1�/�MHCII�/� cellsthat had forward and side scatter properties more characteristic of lympho-cytes. Granulocytes were identified as GR-1�MHCII�CD11b�/� cells thatdisplayed an elongated side scatter profile typical of granulocytic cells inblood. Isotype control Abs were included when analyses and panels werefirst being performed to assure the specificity of staining but were notroutinely included with each experiment. Data were analyzed using Sum-mit software (DakoCytomation). The percentage of each cell populationwas determined and then total cell numbers for each population were cal-culated from the total number of viable cells collected. Experimental andcontrol groups consisted of 3–5 animals each. SEM and statistical signif-icance between treatment groups were determined by ANOVA followed byTukey-Kramer’s comparison of means.

Assessment of apoptosis

Apoptosis of airway cells and lung cells was assessed using annexin Vstaining and flow cytometry according to the manufacturer’s directions(TACS annexin V kit; R&D Systems). For the detection of apoptosis ofspecific cell types, surface immunostaining was done first followed byincubation with an annexin V reagent. Apoptosis was also monitored viaIHC for cleaved capsase-3 in lung tissues using the SignalStain cleavedcaspase-3 IHC detection kit according to the manufacturer’s instructions(Cell Signaling Technologies). Briefly, lungs were preserved and processedas described above. Deparaffinized tissue sections were subjected to Agunmasking by heating at 90°C for 45 min in 0.01 M sodium citrate buffer.After the slides were cooled, endogenous peroxide was quenched andblocked with the blocking solution provided in the kit. Cleaved caspase-3was detected by first incubating tissues with biotinylated rabbit anti-cleavedcaspase 3 followed by streptavidin-conjugated goat anti-rabbit Abs. BoundAbs were detected with NovaRED substrate (Vector Laboratories).

Pathological analysis of lungs

Lungs were harvested, fixed in 10% buffered formalin, and processed asdescribed above. Tissue sections were stained with H&E and examined onan Olympus BX41 light microscope equipped with a QColor3 camera andassociated QCapture Pro software.

Determination of secreted cytokines

Culture supernatants were assayed for the presence of TNF-�, IL-1�, IL-10, IL-12p40, and TGF-� by ELISA using commercially available kitsaccording to the manufacturer’s instructions (R&D Systems). Sample anal-ysis was accomplished using a Multiskan Ascent ELISA plate reader andAscent software (Thermolab Systems). Each experimental or control groupconsisted of 3–5 animals. SD and the significance of cytokine productionin mice were determined by ANOVA followed by Tukey-Kramer’s com-parison of means.

Statistical analysis

Statistical differences between two groups were determined using an unpairedt test with significance set at p � 0.05. For comparisons between three or moregroups, analysis was done by one-way ANOVA followed by Tukey’s multiplecomparisons test with significance determined at p � 0.05.

ResultsReplication of Schu4 in pulmonary compartments

Virulent and attenuated strains of F. tularensis efficiently replicatein the lungs following pulmonary infection (2). However, the air-ways and draining lymph nodes also represent two important targetorgans for bacterial replication and dissemination following aero-sol infections. The airways also contain a large number of APCthat are essential for initiating immune responses against invadingpathogens following migration to the draining lymph node (23,24). Colonization and replication within these tissues may repre-sent an important aspect of F. tularensis pathogenesis. Therefore,we assessed the ability of Schu4 to colonize and replicate in theairways and the draining lymph node, the MLN, following a low-dose aerosol infection. Schu4 readily colonized the airways andreplicated over time in this pulmonary compartment (Fig. 1). In-terestingly, despite being the site of first contact, bacterial numbersin the airways were significantly lower than those detected in the

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lungs ( p � 0.01) (Fig. 1). In addition to colonization of the air-ways, we also saw rapid dissemination to several peripheral or-gans. Whereas previous reports have suggested that other type Astrains fail to or only poorly disseminate from the lungs until 72 hafter infection, we repeatedly observed 102–103 CFU of Schu4 in

the spleen and, importantly, the MLN as early as 48 h after infec-tion (Fig. 1) (2). Collectively, these data suggest that Schu4 col-onizes and replicates in a variety of pulmonary tissues, includingthe draining lymph nodes. Additionally, dissemination can occur attime points earlier than previously anticipated.

Schu4 does not induce increased expression of MHCIIor CD86 on DC

DC represent a key cellular population that stimulates both in-nate and adaptive immunity. DC participation in and activation ofthe immune response is marked by their ability to rapidly secreteproinflammatory cytokines and increase the expression of surfacereceptors such as CD86 and MHCII that are central to T cell stim-ulation long before other host cells can respond. Many pathogenstarget DC as a mechanism to evade and disable host immunity.Given that Schu4 replicated in multiple pulmonary compartmentsthat have abundant numbers of DC ready to detect pathogens, wenext examined what effect Schu4 had on the phenotypic activationof lung and lymph node DC. We first determined which cell typesSchu4 was targeting following low-dose aerosol infection. Lungswere analyzed by IHC for Schu4 24–72 h after infection. As de-scribed for LVS, Schu4 targets multiple cell types after aerosolinfection including epithelial cells and APC (macrophages andDC) present in both the airways and the interstium of the lung (Fig.2, B–D) (6, 25, 26). Interestingly, by 72 h after infection Schu4was detected as both an intracellular bacterium and cell-free innecrotic lesions (Fig. 2D). Because Schu4 appeared to target manycell types, we next assessed the changes in MHCII and CD86expression on DC and macrophages in the airways and lungs at

FIGURE 1. Schu4 replicates in the airways and draining lymph node ofthe lungs. Mice were infected with a low-dose aerosol of Schu4 (50 CFU)and organs from each mouse (n � 5) were assessed for bacterial loads atthe indicated time points. Twenty-four hours after infection bacteria werereadily detectable in the airways (BAL) and lungs of infected mice. Forty-eight hours after infection bacteria were detected in every organ tested,including the draining lymph node of the lung, MLN. Horizontal bars rep-resent the limit of detection in the MLN or spleen. Error bars representSEM. This data is representative of three experiments of similar design.

FIGURE 2. Schu4 does not induce phe-notypic activation of pulmonary APCs. Atmultiple time points after infection, lungsand airway cells were harvested from mice(n � 3 per group) and analyzed for the pres-ence of Schu4 and/or changes in surface ex-pression of MHCII and CD86. Uninfectedmice (n � 3) served as negative controls.A–D, Lungs were fixed, sectioned, andstained with rabbit anti-Schu4 Ab; represen-tative photomicrographs are shown. Lungsfrom uninfected mice did not appear to non-specifically bind the anti-Schu4 Ab (A).Schu4 targets multiple cell types within thefirst 24 h after infection and was readily de-tected in APC in the airways and interstitiumas well as alveolar epithelial cells (B). Schu4progressed through the interstitium infectingAPC and neutrophils and finally was foundexisting cell-free within necrotic lesions 48and 72 h after infection (C and D, respec-tively). Plates are shown at �20 originalmagnification. E, Despite the infection ofmultiple cell types, no significant changes insurface expression of MHCII or CD86 onairway DC, alveolar macrophages, lung DC,or lung macrophages were detected 48 h af-ter infection compared with uninfected con-trols. Error bars represent SEM. These dataare representative of two experiments ofsimilar design.

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various time points after low-dose aerosol infection. Neither DCnor macrophages changed the expression of MHCII or CD86 dur-ing the first 24 h after infection (data not shown). However, thelack of increased MHCII and CD86 on the surface of APC 24 hafter infection may have been due to the relatively low numbers ofbacteria in these two compartments (Fig. 1). Therefore, we alsoanalyzed cells isolated 48 h following Schu4 infection when highernumbers of bacteria (105–106) were present in the airways andlungs. Once again, we observed no significant differences inMHCII or CD86 expression on either DC or macrophage pop-ulations in Schu4-infected mice compared with uninfected con-trols 48 h after infection (Fig. 2E).

The primary function of DC is to carry Ag to the draining lymphnode for presentation to effector T cells. During this migration DCcan also continue to increase the expression of MHCII and CD86.

Thus, it was possible that activated DC, with an increased surfaceexpression of MHCII and CD86, may have migrated to the MLNfollowing aerosol infection with Schu4. To determine whetherSchu4 infection resulted in the activation and subsequent migra-tion of DC to the MLN, we analyzed cells isolated from the MLN24–72 h after infection. Schu4 did not cause increased numbers ofDC in the MLN at any time during the course of infection (Fig. 3A,and data not shown). Schu4 infection also failed to significantlyincrease expression of MHCII and CD86 on DC in the MLNthroughout infection and, in fact, reduced the expression of MHCIIon DC in the lymph node 48 h after infection ( p � 0.05) (Fig. 3,B and C, and data not shown).

It is possible that the absence of DC activation may have beendue to the inability of Schu4 to infect these cells. However, asshown above, Schu4 is promiscuous in the type of cells it targetsfollowing aerosol infection. Furthermore, we have recently shownthat Schu4 readily infects and replicates in bone marrow-derivedDC and freshly isolated airway APC (C.M. Bosio and S.L. Warner,submitted for publication). Thus, failure to infect these targetsseems unlikely. Together, this data suggests that, unlike attenuatedLVS, Schu4 does not induce the phenotypic activation of residentpulmonary DC, hampering the ability of these cells to activate lym-phocytes following infection (6).

Schu4 does not induce the secretion of proinflammatorycytokines from pulmonary tissues

Pneumonic tularemia infections are often marked by the absenceof early inflammatory responses in the lung despite growing num-bers of bacteria in the lung and airways and the presence of clinicalsymptoms such as fever (27). Proinflammatory cytokines are crit-ical for the development of pulmonary inflammation and their ab-sence can represent a mechanism by which a pathogen can evadehost immune responses. For example, TNF-� and IL-12p40 havebeen shown to be critical for the resolution and survival of Fran-cisella infections (8–10). We have previously shown that the poorproduction of proinflammatory cytokines observed in murine LVSinfection correlates with the dampened pathological changes atearly time points after infection. Thus, we next examined the po-tential for Schu4 to elicit proinflammatory cytokines followingaerosol infection. Airway cells and lungs were removed at theindicated time points after infection and cultured overnight as de-scribed in Materials and Methods. The presence of cytokines in theculture supernatant was then examined by ELISA. In contrast topulmonary infections with a virulent strain of Y. pestis (MG05),Schu4 did not elicit the production of TNF-�, IL-12p40, or IL-10within the first 48 h following aerosol infection (Fig. 4, and data

FIGURE 3. Schu4 does not induce accumulation of phenotypically ac-tivated DC in the lymph node. Forty-eight hours after low-dose aerosolinfection with Schu4 (50 CFU), cells were harvested from the MLN (n �3 mice) and then analyzed for total numbers of DC and changes in MHCIIand CD86 surface expression. Uninfected mice (n � 3) served as negativecontrols. A, Schu4 infection did not increase the number of DC in the MLNcompared with uninfected controls. B and C, Schu4 infection did not increasethe expression of either MHCII (B) or CD86 (C) on DC in the MLN. DC inthe MLN from Schu4-infected mice had significantly less MHCII on theirsurfaces compared with uninfected controls (�, p � 0.05). Error bars representSEM. These data are representative of two experiments of similar design.

FIGURE 4. Schu4 does not elicit production of proinflammatory cytokines in the airways and lungs. Mice (n � 3 per group) were infected intranasallyor by aerosol with Y. pestis MG05 or F. tularensis Schu4, respectively. Uninfected mice served as negative controls (n � 3). Forty-eight hours afterinfection, airway cells and lungs were collected and cultured overnight and supernatants were collected for the analysis of cytokines by ELISA. MG05induced significantly greater concentrations of TNF-� and IL-12p40 compared with uninfected or Schu4-infected mice (�, p � 0.01) from airway or lungcells, respectively. In contrast, Schu4 failed to induce significant changes in either cytokine compared with uninfected controls. Error bars represent SEM.These data are representative of two experiments of similar design.

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not shown). Additionally, although IL-1� has been observed bothin vivo and in vitro following LVS infections, we were unable todetect this cytokine at any time point (24–72 h) after either in vivoor in vitro infection with Schu4 (12, 28) (data not shown). There-fore, despite the exponential replication of Schu4, this organismdoes not elicit the typical proinflammatory responses associatedwith acute pulmonary bacterial infections within the first 48 h ofinfection.

Schu4 does not induce the apoptosis of pulmonary cells

One possible explanation for the absence of proinflammatory cy-tokines and resident APC activation following Schu4 infection isthat the bacterium induced the apoptosis of infected cells, render-ing them unable to produce and/or secrete cytokines and respondto infection. To address this possibility, we next assessed theability of Schu4 to induce apoptosis in pulmonary cells follow-ing in vivo infection. We first examined cellular apoptosis bystaining airway and lung cells with annexin. Airway and lungcells were harvested as described above and analyzed for theirability to bind annexin via flow cytometry. Surprisingly, veryfew annexin-positive DC were observed throughout the courseof infection (Fig. 5A). The lack of annexin staining was notconfined to DC, because even the majority of infiltrating gran-ulocytes remained annexin negative up to 72 h after aerosolinfection (Fig. 5A).

Because the ability to bind annexin is also associated withcells undergoing necrosis, we also assessed apoptosis via in situstaining for cleaved caspase-3. Cleaved caspase-3 is a centralmolecule in apoptosis and its expression is required for theactivation of several components of the apoptotic cascade (29).Lungs from uninfected and Schu4-infected mice were assessedfor the expression of cleaved caspase-3 by the immunohisto-chemical staining of tissue sections sampled 24, 48, and 72 hafter infection. In agreement with the data describing the abilityof cells to bind annexin, very few cells were positive forcleaved caspase-3 throughout 72 h of infection (Fig. 5, F andG). The scarce number of cells positive for cleaved caspase-3were confined to areas of necrosis and appeared to be granulo-cytes or monocytes (Fig. 5, F and G). Cleaved caspase-3-spe-cific staining was confirmed following the preincubation of tis-sues with cleaved caspase-3-blocking peptide (data not shown).Pulmonary endothelial cells are also thought to represent a ma-jor site of Francisella infection and replication. Furthermore, ithas been suggested that it is the infection and destruction ofthese cells that contributes to the dissemination of F. tularensis.However, an examination of the pulmonary endotheliumthroughout Schu4 infection revealed that these cells remain rel-atively intact, undisturbed, and free of apoptosis (Fig. 5D).Therefore, the absence of pulmonary proinflammatory cyto-kines and resident DC activation does not appear to be due tothe loss of cells caused by apoptosis.

Schu4 actively suppresses inflammatory responses in the lung

We and others have previously shown that LVS actively sup-presses the ability of infected cells to respond to secondary stimulias a mechanism of virulence in vitro (6, 7). However, to date theability of Francisella, especially type A strains, to suppress proin-flammatory responses in vivo has not been shown. Given the ab-sence of inflammatory responses in the lungs of Schu4-infectedmice during the first 48 h of infection, we next determined whetherSchu4 merely failed to elicit cellular activation and proinflamma-tory cytokines or could actively inhibit the ability of the host torespond to other inflammatory stimuli. Ultrapure LPS was admin-istered intranasally to mice 24 h after receiving a low-dose aerosol

of Schu4. Twenty-four hours after the inoculation with LPS, air-way cells were harvested and assessed by flow cytometry for theaccumulation of inflammatory cells and the activation of residentAPC. Lungs were also removed, processed, and examined for his-topathological changes. As expected, LPS stimulated significantincreases of CD86 on the surface of airway DC in uninfectedcontrols compared with untreated mice ( p � 0.01) (Fig. 6, Aand B). LPS also induced a marked increase in monocytes in theairways compared with untreated controls ( p � 0.01) (Fig. 6C).In contrast, LPS failed to induce significant changes in CD86expression on airway DC of Schu4-infected mice comparedwith uninfected LPS-treated mice (Fig. 6, A and B). Schu4-infected mice were also refractory to LPS-induced infiltration ofmonocytes into the airways compared with uninfected controls(Fig. 6C).

It was possible that freshly immigrating cells had adhered to theairways of Schu4-infected mice treated with LPS. These cellswould not be readily available in the BAL fluid for analysis. Thus,we also examined the lungs for changes in cellular populations andoverall organ structure. LPS induced marked multifocal margin-ation of monocytes and neutrophils in pulmonary vessels in unin-fected mice (Fig. 6, D and F). Transmigration and marked accu-mulation of these inflammatory cells was also noted in theperivascular stroma with a gradient of infiltration in the alveolarseptae and spaces (Fig. 6F). In marked contrast, Schu4-infectedmice had mild and highly restricted infiltration of monocytes andgranulocytes in the lungs following inoculation with LPS (Fig.6G). Together, these data suggest that Schu4 not only interfereswith the ability of resident DC to respond to further stimulationvia the increased expression of cell surface markers but alsoinhibits the ability of the host to properly mobilize critical cel-lular responses crucial for the resolution of infection at the siteof inoculation.

Schu4 induces local and systemic production of TGF-�

There are many mechanisms by which inflammatory responses aresuppressed and controlled in the host. TGF-� has previously beenassociated with broad immunosuppression and homeostatic pro-cesses in a variety of host tissues including the lung (30–32). Be-cause Schu4 failed to elicit the production of proinflammatory cy-tokines and the activation of pulmonary DC and activelysuppressed responsiveness to LPS, we hypothesized that Schu4may induce the production of immunosuppressive cytokines, in-cluding TGF-�. To determine whether Schu4 could stimulate theproduction of immunosuppressive cytokines airways, lungs andspleens were removed and cultured overnight and the resultingsupernatants were analyzed for TGF-� and IL-10 as describedin Materials and Methods. IL-10 was not detected in any tissueat any time point following Schu4 infection (data not shown).However, 24 h after infection TGF-� levels were significantlygreater in the lungs of Schu4-infected mice compared with un-infected controls ( p � 0.05) (Fig. 7A). Surprisingly, despite theabsence of detectable bacteria at this time point TGF-� was alsoelevated in the spleens of Schu4-infected mice compared withthe uninfected controls (Fig. 1 and 7A). The concentrations ofTGF-� in all tissues tested returned to levels detected in unin-fected controls by 48 h after infection and did not significantlychange throughout the remaining course of infection (data notshown).

We next examined what effect the neutralization of TGF-�would have on the elicitation of proinflammatory cytokines andthe control of Schu4 replication in the lungs 48 h after low-doseaerosol challenge. Mice were treated with anti-pan-TGF-� Abs,isotype control Abs, or PBS (untreated) immediately before

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challenge and 24 h after infection. Forty-eight hours after in-fection (24 h after the last administration of antibodies) micewere analyzed for the production of TNF-� by airway cells and

loads of Schu4 in the lung and spleen. Administration of anti-TGF-� significantly increased the production of TNF-� by air-way cells compared with untreated controls ( p � 0.05) (Fig.

FIGURE 5. Schu4 induces little apoptosis of pulmonary cells following in vivo infection. Mice (n � 3) were infected with a low-dose aerosol of Schu4(50 CFU). Uninfected mice served as negative controls (n � 3). At the indicated time points airway and lung cells or tissues were isolated and assessedfor apoptotic cells. A, Freshly isolated airway and lung cells were assessed via flow cytometry for their ability to bind annexin V. Schu4 did not inducemeasurable changes in annexin V binding by DC 48 or 72 h after infection compared with uninfected controls (0 h time point). Mildly increased annexinstaining was observed in granulocyte populations in the airways and lungs 72 h after infection. B–G, Lung tissues were stained with H&E to evaluate thedevelopment of lesions. Tissues were also evaluated for the expression of cleaved caspase-3 via IHC at the indicated time points after aerosol infection.F and G, Schu4 induced limited expression of cleaved caspase-3 up to 72 h after infection. Cells positive for cleaved caspase-3 were rare and are indicatedby the arrows. C and D, Despite the development of large necrotic lesions in the surrounding tissues, endothelial cells remained intact up to 72 h after Schu4infection as indicated by the black arrows (D, inset). Plates are shown at �20 and insets are at �40 original magnification.

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7B). Treatment with anti-TGF-� also increased the productionof TNF-� compared with mice treated with isotype control;however, this difference was not significant (Fig. 7B).

Mice treated with anti-TGF-� Abs were also assessed for bac-terial loads in the lungs following infection. Forty-eight hours afterinfection, mice treated with anti-TGF-� Abs had significantlyfewer bacteria in the lungs compared with untreated mice ( p �0.05) (Fig. 7C). Administration of anti-TGF-� also reduced

bacterial loads in the lungs compared with isotype-treated mice,but these differences were not significant (Fig. 7C).

These data suggest that the production of TGF-� may contributeto the active suppression observed in the lungs of Schu4-infectedmice. Furthermore, although Schu4 appeared to be localized inthe pulmonary tissues during the first 24 h after aerosolization,pulmonary infection resulted in the systemic production ofTGF-�. Therefore, Schu4 may not only dampen immunity at the

FIGURE 6. Schu4 suppresses the responsiveness of pulmonary cells to secondary stimuli. Mice were infected with a low-dose aerosol of Schu4(50 CFU) (n � 3 per group). Twenty-four hours after infection the mice were anesthetized and given LPS intranasally. Uninfected mice (n � 3 pergroup) treated with or without LPS served as positive and negative controls, respectively. Twenty-four hours after the administration of LPS (48h after infection,) airway cells and lungs were analyzed by flow cytometry (airway) or histopathology (lung) for changes in pulmonary cellpopulations. A and B, Expression of CD86 is significantly lower on DC isolated from Schu4-infected mice regardless of LPS treatment comparedwith uninfected mice treated with LPS (�, p � 0.01). B, Representative histogram of CD86 expression on pulmonary DC following Schu4 challengeand exposure to LPS. LPS induced increased expression of CD86 on DC in uninfected mice (solid thick line) compared with untreated, uninfectedmice (solid thin line). Schu4-infected mice failed to increase the expression of CD86 in response to LPS (filled histogram). As shown above, Schu4infection alone did not induce increased expression of CD86 on airway DC (dashed line). C, Schu4-infected mice also had significantly fewermonocytes in the airways compared with uninfected mice treated with LPS (�, p � 0.01). Error bars represent SEM. D–G, Lungs were fixed,sectioned, and stained with H&E; representative photomicrographs are shown. D and F, Uninfected mice had marked severe inflammation thatconsisted of granulocytes and monocytes in response to LPS compared with untreated, uninfected controls. E and G, Lungs from Schu4-infected micetreated with LPS (G) had mild, contained inflammation compared with uninfected, LPS treated controls (F). Plates are shown at �20 originalmagnification. These data are representative of two experiments of similar design.

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site of infection but may also interfere with protective immuneresponses at peripheral sites before the dissemination of thebacterium.

DiscussionIn this study we demonstrate that, unlike more attenuated strains,virulent type A F. tularensis actively suppresses early proinflam-matory responses in the lung following aerosol infection (Table I).Specifically, F. tularensis Schu4 failed to activate pulmonary DCand macrophages as measured by the changes in key cell surfacereceptors (MHCII and CD86). Schu4 also failed to induce the earlysecretion of several proinflammatory cytokines in the lungs andairways, including TNF-�, IL-12, and IL-1�, which are central toinitiation of effective host immune responses and the control ofFrancisella infections (8–10). The lack of cellular activation couldnot be attributed to the apoptosis of pulmonary cells. In fact, therelative viability and health of multiple pulmonary cell types, in-cluding the endothelial cells, epithelial cells, and monocytes, wereremarkable considering the rapidly increasing bacterial loads.Rather, we found compelling evidence of broad and pervasive ac-tive interference with early (first 24 h) proinflammatory responsesfollowing Schu4 infection. Our evidence also suggests that Schu4

modulates the pulmonary environment, in part via the induction ofTGF-�, to favor anti-inflammatory conditions. Together, thesedata suggest a novel mechanism by which virulent F. tularensisinfects, replicates, and disseminates in the host while evading hostimmune responses.

Many pathogens have evolved strategies to evade host defensesand in many cases can co-opt normal host physiology to gain anadvantage following infection. Once such strategy is the inductionof TGF-�. TGF-� has multiple functions, including maintaininghomeostasis in the host as well as potent anti-inflammatory andantimicrobial activity. For example, the addition of TGF-� toLeishmania-infected macrophages completely abolished the abilityof these cells to kill the infecting parasite (33). The neutralizationof TGF-� in vivo following infection with Leishmania reducedparasite load and increased wound healing compared with un-treated controls (34). In addition to its direct effect on controllingthe replication of pathogens in host cells, investigators have alsoobserved that TGF-� plays an important role in limiting the ex-travasation of granulocytes into various tissues, including the lung.A single administration of DNA encoding TGF-� suppressed therecruitment of eosinophils into the lung following infection withCryptococcus neoformans, the influenza virus, and the respiratorysyncytial virus (30). Interestingly, although this treatment limitedlung pathology, it also significantly increased susceptibility toinfection.

Considering the multifunctionality of TGF-� as a regulatory andsuppressive cytokine and the ability of Schu4 to elicit TGF-�within the first 24 h of infection in both the lung and spleen (Fig.7), it was possible that this cytokine contributed to Schu4 replica-tion in the host. Following the administration of anti-TGF-� Abswe observed decreased bacterial loads in the lungs concomitantwith increased TNF-� in the BAL fluid. However, these differ-ences were not significant compared with isotype-treated controls.Furthermore, there were no differences in the bacterial loads orconcentrations of proinflammatory cytokines in the spleens of anti-TGF-�-treated animals compared with those in controls.

The inability of treatment with anti-TGF-� Abs to significantlyincrease TNF-� while reducing bacterial loads in the lung com-pared with isotype controls may reflect the relatively small con-tribution of this cytokine toward the pathogenesis of pneumonictularemia. However, it is also possible that other molecules capa-ble of modulating the host response work in conjunction withTGF-� to result in the profound inhibition of inflammation ob-served during the first 48 h of Schu4 infections. The interplay ofTGF-� with other immunomodulators such as prostaglandins hasbeen well established. For example, the inhibition of lung NK cellsby alveolar macrophages is through the production of both TGF-�and PGE2 by the macrophages (35). The neutralization of TGF-�and the simultaneous inhibition of prostaglandins significantly in-creased the production of TNF-� and NO while inhibiting repli-cation of M. tuberculosis in the lung (36). More recently Woolardet al. demonstrated that the elicitation of PGE2 by LVS contributesto the successful replication of the bacterium in macrophageswhile suppressing T cell responses in the lung (37). Thus, it ispossible that Schu4 successfully modulates the pulmonary envi-ronment via the induction of TGF-� and PGE2. Studies designedto determine the role of PGE2 toward the pathogenesis of Schu4infections are currently underway in our laboratory.

Many pathogens have developed mechanisms they use to evadehost immunity to ensure their survival and transmission. The datapresented here demonstrate multiple forms of immunosuppressionby which virulent F. tularensis modulates that host response in thelung to its benefit. These mechanisms include the induction ofTGF-�, active inhibition of DC responsiveness, and suppression of

FIGURE 7. Treatment with anti-TGF-� results in limited production ofTNF-� and partial control of Schu4 in the lung. Mice (n � 3) were infectedwith a low-dose aerosol of Schu4 (50 CFU). Uninfected mice (n � 3)served as negative controls. A, Twenty-four hours after infection, airwaycells (BAL) and spleens were removed and cultured overnight and super-natants were collected for analysis of TGF-� by ELISA. Schu4 induced theproduction of significantly greater concentrations of TGF-� by airway cellsand spleens compared with uninfected controls (�, p � 0.05). B and C,Mice were treated with either chicken IgY Ab (isotype) or pan-chickenanti-TGF-� Abs immediately before challenge and 24 h after infection.Forty-eight hours after infection, BAL cells were collected and culturedovernight and the resulting culture supernatants were tested for TNF-�.Additionally, lungs and spleens were collected and assessed for bacterialloads. B, Treatment with anti-TGF-� significantly increased production ofTNF-� following Schu4 infection compared with untreated, but not iso-type, controls (�, p � 0.05). C, Treatment with anti-TGF-� significantlydecreased loads of Schu4 in the lung compared with untreated, but notisotype, controls (�, p � 0.05). There were no significant differences inbacterial loads in the spleens among Schu4-infected mice. Error bars rep-resent SEM. This data are representative of two experiments of similardesign.

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the host’s ability to recruit effector cells crucial for the resolutionof tularemia. These studies also point out important considerations,e.g., the inability of the host to respond to therapeutic stimuli afterinfection, for the development of novel therapeutics intended totreat individuals exposed to aerosolized F. tularensis. Additionalstudies aimed at identifying the specific mechanism and bacterialcomponents responsible for this suppression will be central to thedevelopment of novel vaccines and therapeutics for F. tularensisand other important pulmonary pathogens.

AcknowledgmentsWe thank Dr. Peter Henson for helpful discussions and Shayna Warner andBeth Stallman for excellent technical assistance.

DisclosuresThe authors have no financial conflict of interest.

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