Francisella tularensis replicates in macrophages and dendritic cells, but interactions with other cell types have not been well described. F. tularensis LVS invaded and replicated within alveolar epithelial cell lines. Following intranasal inoculation of C57BL/6 mice, Francisella localized to the alveolus and replicated within alveolar type II epithelial cells

Craven, R. R.

Fuller, J. R.

Hall, J. D.

Kawula, T. H.

Pickles, R. J.

Carolina Digital Repository

INFECTION AND IMMUNITY, Feb. 2007, p. 1034–1039 Vol. 75, No. 20019-9567/07/$08.000 doi:10.1128/IAI.01254-06Copyright © 2007, American Society for Microbiology. All Rights Reserved.NOTESFrancisella tularensis Replicates within Alveolar Type II Epithelial CellsIn Vitro and In Vivo following InhalationJoshua D. Hall, Robin R. Craven, James R. Fuller, Raymond J. Pickles, and Thomas H. Kawula*University of North Carolina at Chapel Hill, Chapel Hill, North CarolinaReceived 5 August 2006/Accepted 25 October 2006Francisella tularensis replicates in macrophages and dendritic cells, but interactions with other cell typeshave not been well described. F. tularensis LVS invaded and replicated within alveolar epithelial cell lines.Following intranasal inoculation of C57BL/6 mice, Francisella localized to the alveolus and replicated withinalveolar type II epithelial cells.Francisella tularensis, the etiological agent of tularemia, is azoonotic pathogen with a broad host range, spanning fromprotists to humans. The organism is transmitted to humansthrough handling of contaminated material, insect bites, orinhalation (6, 10). It has been determined that contact with 25or fewer bacteria is sufficient to cause disease (16, 17). Follow-ing contact, the bacteria disseminate to the liver, spleen, andlungs regardless of the initial transmission route (7, 10).The low infectious dose, myriad of transmission routes,broad host range, and severity of disease caused by F. tularensisled a number of nations to develop and stockpile this organismas a biological weapon (9), yet little is known about the basis ofits pathogenesis or virulence. It has been reported that mac-rophages and, potentially, dendritic cells serve as the primaryhost cells for growth of Francisella in humans and other ani-mals (5, 18). This conclusion is supported by the observationsthat F. tularensis and other closely related Francisella speciessurvive and replicate in human and rodent macrophages (1, 4)and that many of the identified attenuating mutations impairintramacrophage growth (2, 11, 12, 14, 15).Clearly, intramacrophage survival is an important virulenceproperty of Francisella. However, apart from one report ofhepatocyte association in infected liver (8), the potential con-tribution of other host cell interactions to F. tularensis viru-lence and pathogenesis remains untested. Given the severity oftularemia acquired by inhalation, we sought to address thispossibility by using in vitro and in vivo models to examinewhether Francisella invades and replicates within epithelialcells of the lung.Francisella tularensis LVS invades and replicates within lungepithelial cells in vitro. To determine whether Francisellacould invade and replicate within lung epithelial cells, in vitroinvasion and replication assays were performed using the hu-man alveolar type II (ATII) epithelial cell-derived cell lineA549 (ATCC), the mouse ATII cell-derived cell line MLE-12(ATCC), and the mouse lung epithelial cell-derived cell lineTc-1 (ATCC). For comparison, the mouse macrophage-likecell line J774A.1 (ATCC) was also used. Intracellular bacteriawere enumerated by a gentamicin protection assay. The celllines were grown to confluence in 24-well plates and inoculatedwith F. tularensis LVS at a multiplicity of infection of 100:1.Four hours after inoculation, the cells were washed with phos-phate-buffered saline (PBS), and medium containing gentami-cin (25 g/ml) was added to kill extracellular bacteria.Intracellular organisms were recovered from all cell lines 6and 24 h postinoculation (Fig. 1A). The number of intracellu-lar organisms recovered 6 h postinoculation and the percent-age of infected cells varied among the lung epithelial cell lines,with the fewest intracellular Francisella organisms recoveredfrom the human ATII cell line, A549. The percentage of in-fected epithelial cells ranged from 0.2% of A549 cells to 17%of Tc-1 cells (Fig. 1A). The number of intracellular bacteriarecovered from each lung epithelial cell line at 6 h was smallerthan the number recovered from J774A.1 cells, as was thepercentage of infected cells, indicating that entry into epithe-lial cells may be a less efficient process than uptake by macro-phages.The number of intracellular bacteria increased by an averageof 100-fold during the following 18 h in each cell line, repre-senting an intracellular doubling time of roughly 3 h (Fig. 1A).Intracellular localization and proliferation were confirmed us-ing fluorescence microscopy and digital image deconvolution(Fig. 1B to E). From 6 to 24 h postinoculation, the relativeincrease of intracellular bacteria within lung epithelial cells wassimilar to that observed within J774A.1 cells, indicating that F.tularensis LVS replicated to the same degree within lung epi-thelial cells as in macrophages.There are many reports of Francisella replicating withinmacrophages in vitro, and here we report its ability to replicatewithin cultured lung epithelial cells. Whereas some work hasbeen done to examine localization of Francisella in the liver(8), little is known about the localization of inhaled Francisellain the lung. Bosio and Dow recently reported that Francisellawas associated primarily with dendritic cells isolated from* Corresponding author. Mailing address: Department of Microbi-ology and Immunology, CB#7290, University of North Carolina,Chapel Hill, NC 27599-7290. Phone: (919) 966-9699. Fax: (919) 962-8103. E-mail: Kawula@med.unc.edu. Published ahead of print on 6 November 2006.1034FIG. 1. F. tularensis LVS invades and replicates within ATII cell lines in vitro. (A) Intracellular bacteria recovered from A549, MLE-12, Tc-1, andJ774A.1 cells 6 and 24 h postinoculation. The percentages above the bars represent percentages of infected cells. (B and C) Fluorescence imaging of A549cells inoculated with GFP-expressing LVS 6 h (B) and 24 h (C) following inoculation. Cell borders were visualized by rhodamine-phalloidin (MolecularProbes) staining (red), and nuclei were visualized with 4,6-diamidino-2-phenylindole (DAPI; blue). (D and E) Fluorescence imaging of J774A.1 cellsinoculated with GFP-expressing LVS (green) 6 h (D) and 24 h (E) postinoculation. Cell borders were visualized using biotinylated lectin from Lensculinaris and streptavidin-conjugated Alexa Fluor 647 (Molecular Probes) (red), and nuclei were stained with DAPI (blue). Intracellular replicationexperiments were carried out in triplicate; error bars represent standard deviations of the means.VOL. 75, 2007 NOTES 1035bronchioalveolar lavage fluid of mice 1 hour after inhalation(5). However, the localization of bacteria in lung tissuethroughout the course of infection has not been addressed.Following inhalation, Francisella localizes to the alveolus.To determine the localization of Francisella in the airwayfollowing inhalation, anesthetized 6- to 8-week-old femaleC57BL/6 mice were inoculated intranasally with 105 CFU ofF. tularensis LVS expressing green fluorescent protein(GFP) suspended in 50 l of PBS. (All mouse work wasperformed according to IACUC-approved protocol.) Immu-nofluorescence analysis was performed on formalin-fixedand paraffin-embedded tissue sections obtained from nasalturbinates, trachea, and lungs harvested 1, 3, and 7 dayspostinoculation. No bacteria were observed in turbinates ortrachea. In the lung, few bacteria associated with the apicalsurface of bronchial epithelial cells, while the majoritycolocalized with alveolar cells (Fig. 2). Fluorescence imagingof sequential vertical planes revealed that most infected alve-olar cells contained multiple bacteria throughout the cyto-plasm, indicative of intracellular replication (data not shown).Bacterial replication within ciliated or nonciliated (Clara)bronchial epithelial cells was not observed. The absolute num-ber of bacteria (Fig. 2D) and the number of infected alveolarcells increased by day 3 (Fig. 2B). By day 7, F. tularensis withinthe lung was widespread but remained exclusively in the alve-olus (Fig. 2C). Throughout the course of infection, the amountof extracellular space observed in the network of alveolar cellsdecreased, causing the alveolar cells to appear more con-densed (Fig. 2).Inhaled Francisella colocalizes to and replicates within ATIIcells in vivo. Given that Francisella invaded and replicatedwithin ATII-derived cell lines in vitro, we probed infectedlung tissue sections with antibodies to the nonsecreted sur-factant protein precursors proSP-B and proSB-C (Chemi-con) to determine whether Francisella localized to and rep-licated within ATII cells in vivo following inhalation.proSP-B is produced by ATII cells and nonciliated bron-chiolar Clara cells (13), whereas proSP-C is produced byATII cells exclusively (3). Three days following inoculation,the majority of alveolar cells associated with F. tularensisalso bound antibodies to the nonsecreted surfactant proteinprecursors proSP-B and proSB-C (Fig. 3A to D). Some cellsassociated with bacteria were not positive for proSP-B orFIG. 2. F. tularensis localizes to the alveolus following inhalation. Mice were intranasally inoculated with 105 CFU of F. tularensis LVSexpressing GFP. One, three, and seven days postinoculation, lungs were harvested and prepared for immunofluorescence analysis. (A to C)Bacterial localization was determined by probing lung sections with a fluorescently labeled antibody to GFP (green). Nuclei were stained withDAPI (blue) to visualize lung cells. Representative images of the alveoli of infected mice 1 (A), 3 (B), and 7 (C) days postinoculation. (D) Bacterialrecovery from lungs 1, 3, and 7 days following intranasal inoculation with 105 CFU LVS. Each bar represents mean recovery from three mice; errorbars represent standard deviations of the means.1036 NOTES INFECT. IMMUN.proSP-C, indicating that Francisella interacts with other celltypes in the lung following inhalation.In addition to the colocalization with proSP proteins, mi-croscopy of sequential vertical planes confirmed that bacteriawere inside infected ATII cells (data not shown). Rabbit anti-body specific for -tubulin (Abcam) did not colocalize with F.tularensis (Fig. 3E), indicating that the colocalization of Fran-cisella with rabbit proSP antibodies was not due to bacteriaFIG. 3. Following inhalation, F. tularensis LVS expressing GFP colocalized with proSP-B and proSP-C, proteins produced by ATII epithelialcells. Bacterial localization was determined with a fluorescently labeled antibody to GFP (green). Nuclei were stained with DAPI (blue). Sectionswere probed with fluorescently labeled antibody to proSP-B (red) (A and B), proSP-C (red) (C and D) to identify ATII cells and -tubulin (red)(E). Representative images are from lung sections 3 days postinoculation.VOL. 75, 2007 NOTES 1037nonspecifically binding rabbit antibodies, nor did the proSPantibodies cross-react with cultured Francisella organisms(data not shown). These collective data demonstrate that F.tularensis replicates within ATII cells following inhalation andthe proportion of ATII cells harboring bacteria increases dur-ing the course of pneumonic tularemia.Bacteria were also observed in probed sections that did notcolocalize with proSP-B and proSP-C (Fig. 3), indicating thatATII cells are not the only alveolar cell type harboring repli-cating Francisella. Macrophages and dendritic cells have beenreported as potential host cells for replicating Francisella or-ganisms (1, 4, 5). Due to difficulties in staining for surfacemacrophage and dendritic cell markers in embedded lung sec-tions, we utilized dispase digestion of infected mouse lungs tostain for surface markers and also to assess our results from theembedded tissue staining.Three days after inoculation with LVS expressing GFP,lungs were infused and incubated with the neutral proteasedispase (BD Biosciences). Digested tissue was washed in PBSand subsequently filtered through 40- and 20-m-pore-sizemesh. Staining with fluorescently labeled antibody specific forthe surface markers F4/80 (eBioscience) and CD11c (eBio-science) was performed prior to fixation with 4% paraformal-dehyde. Staining for the intracellular markers proSP-B andproSP-C occurred following fixation and required the use ofCytoperm (BD Biosciences) to permeabilize the cells. F. tula-rensis LVS was observed within cells expressing F4/80, CD11c,proSP-B, and proSP-C (Fig. 4), indicating that following inha-lation, bacteria survive and replicate within macrophages,dendritic cells, and ATII cells.Here we report that in addition to replicating within mac-rophages and dendritic cells, F. tularensis invades and rep-licates within alveolar type II epithelial cells, indicating thatinteraction with these cells following inhalation may be animportant component of pneumonic tularemia. Future workwill analyze the proportion of various cell types that areinfected in the lung as well as attempt to identify the con-tribution of ATII cell invasion and replication to the diseaseprogression of pneumonic tularemia.We gratefully acknowledge the technical support and invaluablecontributions of Yan Zhao and Kimberly Burns and thank Mats Fors-man for sending us the pKK214GFP plasmid prepublication. We alsothank Jo Rae Wright for sharing her expertise on lung biology andRobert Fulcher for editing assistance and general advisement.This work was supported by a Southeast Regional Center of Excel-lence in Biodefense and Emerging Infections grant (NIH/NIAID U54-AI057157) and by the National Institutes of Health (R21-AI053399).REFERENCES1. Anthony, L. D., R. D. Burke, and F. E. Nano. 1991. Growth of Francisella spp.in rodent macrophages. Infect. Immun. 59:3291–3296.2. Baron, G. S., and F. E. Nano. 1998. MglA and MglB are required for theintramacrophage growth of Francisella novicida. 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Pathog. 34:239–248.8. Conlan, J. W., and R. J. North. 1992. Early pathogenesis of infection in theliver with the facultative intracellular bacteria Listeria monocytogenes, Fran-cisella tularensis, and Salmonella typhimurium involves lysis of infected hepa-tocytes by leukocytes. Infect. Immun. 60:5164–5171.9. Dennis, D. T., T. V. Inglesby, D. A. Henderson, J. G. Bartlett, M. S. Ascher,E. Eitzen, A. D. Fine, A. M. Friedlander, J. Hauer, M. Layton, S. R. Lil-libridge, J. E. McDade, M. T. Osterholm, T. O’Toole, G. Parker, T. M. Perl,P. K. Russell, and K. Tonat. 2001. Tularemia as a biological weapon: medicaland public health management. JAMA 285:2763–2773.FIG. 4. F. tularensis LVS expressing GFP colocalized with cellsexpressing the macrophage marker F4/80, the dendritic cell markerCD11c, and the ATII cell markers proSP-B and proSP-C. Nuclei werestained with DAPI (blue). 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Allelic exchange in Francisella tularensis using PCR products.FEMS Microbiol. Lett. 229:195–202.15. Lauriano, C. M., J. R. Barker, S. S. Yoon, F. E. Nano, B. P. Arulanandam,D. J. Hassett, and K. E. Klose. 2004. MglA regulates transcription of viru-lence factors necessary for Francisella tularensis intraamoebae and intra-macrophage survival. Proc. Natl. Acad. Sci. USA 101:4246–4249.16. Saslaw, S., H. T. Eigelsbach, J. A. Prior, H. E. Wilson, and S. Carhart. 1961.Tularemia vaccine study. II. Respiratory challenge. Arch. Intern. Med. 107:702–714.17. Saslaw, S., H. T. Eigelsbach, H. E. Wilson, J. A. Prior, and S. Carhart. 1961.Tularemia vaccine study. I. Intracutaneous challenge. Arch. Intern. Med.107:689–701.18. Titball, R. W., A. Johansson, and M. Forsman. 2003. Will the enigma ofFrancisella tularensis virulence soon be solved? Trends Microbiol. 11:118–123.Editor: D. L. BurnsVOL. 75, 2007 NOTES 1039

Francisella tularensis Replicates within Alveolar Type II Epithelial Cells In Vitro and In Vivo following Inhalation

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Francisella tularensis Replicates within Alveolar Type II Epithelial Cells In Vitro and In Vivo following Inhalation

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