Progress 09/01/02 to 06/30/07
Outputs
OUTPUTS: Bordetella bronchiseptica is a gram-negative respiratory pathogen that is endemic in many animal populations and causes substantial disease in pigs and other animals. B. bronchiseptica is an excellent model for studying host/pathogen interactions involved in bacterial infections. In these studies a murine model of B. bronchiseptica infection was used to explore the role of TLR4, the TTSS, CD11b and the cytokine responses in respiratory immunity. Our findings indicate that the TTSS is a critical virulence factor, and that TLR4, TNF-alpha, TNFR1, and alveolar macrophages limit TTSS associated pathology and bacterial growth in Bordetella bronchiseptica infections. We suggest that in normal mice bacterial LPS binding to TLR4 stimulates alveolar macrophage production of TNF-alpha and protects against TTSS induced pathology. It is also known that more effective vaccines for bacterial respiratory infections are needed. While most vaccines consisting of killed bacteria induce high
serum antibody titers, they do not always confer protection as effective as that induced by infection, particularly against mucosal pathogens. While there are several live attenuated vaccines against this pathogen available for use in a variety of livestock and companion animals, there are few published data on the makeup or efficacy of these vaccines. Here we report the use of a genetically engineered double mutant of B. bronchiseptica, which lacks adenylate cyclase and type III secretion, as a vaccine candidate. This strain is safe at high doses, even for highly immunocompromised animals, and induces immune responses that are protective against highly divergent B. bronchiseptica strains, preventing colonization in the lower respiratory tract and decreasing the bacterial burden in the upper respiratory tract. This novel B. bronchiseptica vaccine candidate induces strong local immunity while eliminating damage caused by the two predominant cytotoxic mechanisms. By an increased
understanding of host pathogen interactions we are able to develop candidate vaccines such as this.
PARTICIPANTS: Mary J. Kennett and Eric Harvill.
TARGET AUDIENCES: Immunologists and Veterinarians
Impacts
This research has helped identify important host immune factors and targeted virulence factors of Bordetella to determine how they interact in the pathogenesis of respiratory disease. We have examined specific host responses to modified or bacterial virulence factors and have identified a candidate vaccine strain. By understanding bacterial virulence factors and how a host can effectively respond, new therapies and better vaccines may be developed.
Publications
- Mann P., E. Goebel, J. Barbarich, M. Pilione, M. Kennett, and E. Harvill. 2007. Use of a genetically defined double mutant strain of Bordetella bronchiseptica lacking adenylate cyclase and type III secretion as a live vaccine. Infect Immun. Jul;75(7):3665-72.
- Loving C. L., M. Kennett, G. M. Lee, V. K. Grippe, and T. J. Merkel. 2007. Murine aerosol challenge model of anthrax. Infect Immun. Jun;75(6):2689-98.
- Tompkins S.M., Y. Lin, G. P. Leser, K. A. Kramer, D. L. Haas, E. W. Howerth, J. Xu, M. J. Kennett, R. K. Durbin, J. E. Durbin, R. Tripp, R. A. Lamb, and B. He. 2007. Recombinant parainfluenza virus 5 (PIV5) expressing the influenza A virus hemagglutinin provides immunity in mice to influenza A virus challenge. Virology. May 25;362(1):139-50
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Progress 01/01/06 to 12/31/06
Outputs
Bordetella bronchiseptica causes respiratory infections in several animal species, and is an excellent model for studying host/pathogen interactions involved in Gram-negative bacterial infections. In this study, a murine model of Bordetella bronchiseptica infection was used to explore the role of CD11b in respiratory immunity. A host cell surface receptor known as CD11b is involved in the generation of a protective immune response against pathogenic organisms. After intranasal inoculation, CD11b-/- mice rapidly succumb to B. bronchiseptica respiratory infection, highlighting the prominent role of CD11b in the generation of a protective immune response in this model. CD11b appears to be required for both the control of bacterial numbers and the regulation of cellular responses in the lungs. An increased accumulation of neutrophils in the lungs of CD11b-/- mice as compared with wild-type mice suggests that CD11b contributes to the regulation of cellular responses to
respiratory infection. This accumulation may be explained by a decrease in apoptosis that is observed in the absence of CD11b following cellular interactions with B. bronchiseptica. Interestingly, this role for CD11b in the regulation of cellular accumulation appears to be critically important for the resolution of damage associated with the type III secretion system (TTSS) of B. bronchiseptica. These data provide new insight into the key role CD11b plays in the resolution of damage in the lower respiratory tract, as well as the B. bronchiseptica virulence determinant that induces it.
Impacts
This research continues to determine the important identify important host immune factors and virulence factors of Bordetella and how they interact in the pathogenesis of respiratory disease. Here we examine specific host responses to identify an effective immune response. By understanding bacterial virulence factors and how a host can effectively respond new therapies and better vaccines may be developed.
Publications
- Marin, H.E., Peraza, M.A., Billin, A.N., Willson, T.M., Ward, J.M., Kennett, M.J., Gonzalez, F.J., and Peters, J.M. 2006. Ligand activation of peroxisome proliferator-activated receptor beta inhibits colon carcinogenesis. Cancer Res. Apr 15; 66(8):4394-4401.
- Pilione, M.R., Agosto, L.M., Kennett, M.J., and Harvill, E.T. 2006. CD11b is required for the resolution of inflammation induced by Bordetella bronchiseptica respiratory infection. Cell Microbiol. May; 8(5):758-68.
- Hollingshead, H.E., Morimura, K., Adachi, M., Kennett, M.J., Billin, A.N., Willson, T.M., Gonzalez, F.J., and Peters J.M. 2006. PPAR beta/delta Protects Against Experimental Colitis in a Ligand-Independent Fashion. Accepted for Publication.
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Progress 01/01/05 to 12/31/05
Outputs
Bordetella bronchiseptica causes respiratory infections in several animal species, and is an excellent model for studying host/pathogen interactions involved in Gram-negative bacterial infections. Although B. bronchiseptica causes chronic asymptomatic infections in normal mice, we have previously demonstrated that C3H/HeJ Toll-like receptor 4 deficient (TLR4-def) mice rapidly develop fatal pneumonia due to an impaired early TNF-alpha response. TLR4 is a mammalian innate immune receptor that detects bacterial lipopolysacharide (LPS) and plays an important role in mediating early production of TNF-alpha and limiting Gram-negative bacterial pneumonia. We examined several specific virulence factor mutant strains of B. bronchiseptica and show that the type three secretion system (TTSS) is required for virulence in TLR4- def and TNF-alpha-def) or TNF alpha depleted mice. The TTSS is encoded by a group of genes found in many Gram-negative bacteria; its proteins form
needle-like complexes allowing bacteria to inject proteins into host cells and subvert them. We further show that TLR4, TNF-alpha, TNF receptor 1 (TNFR1), and alveolar macrophages are required to limit TTSS induced morbidity, and that TNF-alpha mediates protection against TTSS induced pathology. Groups of 4 mice (wild type C3H/HeN, TLR4-def , and TNF-alpha depleted) were intranasally inoculated with wild type B. bronchiseptica (RB50) or a virulence factor mutant (bvg, fha, fim, cya wbm, pagp or bscn) strain. Mice that became ill were removed from the study at specified endpoints and euthanized. The lungs were harvested and prepared for histopathology and quantitative bacterial cultures were performed. Our findings indicate that the TTSS is a critical virulence factor, and that TLR4, TNF-alpha, TNFR1, and alveolar macrophages limit TTSS associated pathology and bacterial growth in Bordetella bronchiseptica infections. We suggest that in normal mice bacterial LPS binding to TLR4
stimulates alveolar macrophage production of TNF-alpha and protects against TTSS induced pathology.
Impacts
This research continues to determine the important virulence factors of Bordetella and how each specifically causes pathological changes in the mammalian host. It also examines the host response to identify an effective immune response. By understanding bacterial virulence factors and how a host can effectively respond, new therapies and better vaccines can be developed.
Publications
- Kirimanjeswara, G. S., Mann, P. B., Pilione, M., Kennett, M.J. and Harvill, E.T. 2005. The Complex Mechanism of Antibody-Mediated Clearance of Bordetella from the Lungs Requires TLR4. J Immunol. Dec 1;175(11):7504-11.
- Kirimanjeswara, G. S., Agosto, L.M., Kennett, M. J., Bjornstad, O. N. and Harvill ET. 2005. Pertussis toxin inhibits neutrophil recruitment to delay antibody-mediated clearance of Bordetella pertussis. J Clin Invest. Dec;115(12):3594-60
- Hays, T., Rusyn, I., Burns, A. M., Kennett, M. J., Ward, J. M., Gonzalez, F. J. and Peters, J. M. 2005. Role of peroxisome proliferator-activated receptor-alpha (PPAR alpha) in bezafibrate-induced hepatocarcinogenesis and cholestasis. Carcinogenesis. Jan;26(1):219-27.
- Foster St. Claire, M. B., Kennett, M. J., Thomas, M. L. and Daly, J.W. 2005. The Husbandry and Care of Dendrobatid Frogs. Contemp Topic Lab Anim Sci. 44 (6) 8-14.
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Progress 01/01/04 to 12/31/04
Outputs
Bordetella bronchiseptica is a Gram-negative respiratory pathogen, which can be used to explore the specific immune responses necessary to eliminate bacteria from the respiratory tract in a murine model. Toll like receptor 4 (TLR4) is the mammalian innate immune receptor that detects bacterial lipopolysacharide (LPS), the major component of the outer membrane of Gram-negative bacteria. Infection models using mice deficient in TLR4 signaling have established an important role for this receptor in limiting Gram-negative infections. We have previously demonstrated that TLR4 deficient mice rapidly develop severe fatal pneumonia after inoculation with the natural mouse pathogen Bordetella bronchiseptica. This model can thus be used to investigate which inflammatory responses are critical for respiratory host defense in the context of natural host pathogen interactions. We hypothesized that microarray technology could be used to identify critically important genes whose
expression is TLR4 dependent and that deficiencies in these gene products would result in increased susceptibility to infection. Wild type control and TLR4 deficient macrophages were exposed to live B. bronchiseptica. RNA was extracted, reverse transcribed, labeled with fluorescent dyes, and hybridized to preprinted gene array chips. Relative gene expression levels were measured using an Axon 4000A scanner and appropriate software. Transcriptional findings were verified at the protein level using ELISA. In vivo infection experiments were then conducted to determine biological relevance. Microarray analysis indicates that although the global expression pattern generated in response to the live bacteria is not dependent on intact TLR4 signaling, the early Tumor Necrosis Factor (TNF-a) mRNA expression is dependent on TLR4. Traditional ELISA based experiments measuring protein levels using samples from in vitro and in vivo infections substantiate this finding. In vivo infection studies
suggest that the early elicited and TLR4 dependent TNFa response is critical to preventing lethal pneumonia in this infection model. Summary: Collectively these results indicate that, during Gram-negative respiratory infection, defective early TNFa responses contributes significantly to the development of fatal pneumonia observed in TLR4 deficient mice. Additionally, the experimental system employed in this study highlights the potential of microarray technology to identify critical genes that may serve as therapeutic targets during bacterial infection.
Impacts
Understanding the mechanisms of immunity important the clearance of a bacterial pathogen from the respiratory tract will help improve vaccines and therapeutic strategies. This study suggest that an early LPS induced proinflammatory response to B. bronchiseptica infection is critical in limiting clinical disease, that inflammation more than bacterial load contributes to clinical disease, and that a diminished early proinflammatory response results in delayed but exaggerated neutrophil response and severe clinical disease. This research will increase our understanding of the immune mechanisms involved in the clearance of B. bronchiseptica from the respiratory tract and will help target the development of new therapies and vaccines that will be able eliminate this infection.
Publications
- Mann, P. A., Kennett, M. J., and Harvill, E. T. 2004. TLR4 is critical to innate and vaccine-mediated protection in a murine model of bordetellosis. J Infect Dis Mar 1;189(5):833-6.
- Pishko, E. J., Kirimanjeswara, G. S., Pilione, M. R., Gopinathan, L., Kennett, M. J., and Harvill, E. T. 2004. Antibody-mediated bacterial clearance from the lower respiratory tract of mice requires complement component C3. Eur J Immunol Jan;34(1):184-93.
- Mann, P.B., Elder, K. D., Kennett, M. J., and Harvill, E. T. 2004. TLR4 dependent early elicited TNF alpha expression is critical for innate host defense against Bordetella bronchiseptica. Infect Immun. Nov; 72(11):6650-8.
- Kennett, M.J. P. B. Mann, K.D. Elder, and E. T. Harvill. 2004. TLR4 Regulates the Early TNFa Response in a Murine Model of Bordetella bronchiseptica Pneumonia. Abstract for 55th National AALAS Meeting, Tampa FL. Contemp Top Lab Anim Sci 43(4):63.
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Progress 01/01/03 to 12/31/03
Outputs
This project addresses ways of eliminating Bordetella bronchiseptica from the respiratory tracts of animals using a mouse infection model. In order to understand defense mechanisms important in bacterial clearance, we used Toll-like Receptor 4 (TLR4) deficient C3H/HeJ mice to explore the early immune mechanisms important in controlling Bordetella bronchiseptica pneumonia. TLR4 is a lipopolysaccharide (LPS) receptor important in triggering signaling cascades that mediate proinflammatory responses, and we have previously shown that TLR4 deficient mice are highly susceptible to B. bronchiseptica infections compared to C3H/HeN (wild type) mice. In this study groups of 3-5 mice were intranasally inoculated with 1 x 105 wild type B. bronchiseptica or an Adenylate Cyclase (AC) and Type III Secretion Apparatus (TTSA) mutant, and sacrificed at 12, 24, and 72 hours post infection. This study showed that lung neutrophil counts (p greater than 0.05), and early inflammatory
cytokine levels (as measured by ELISA, p greater than 0.05) were significantly decreased in TLR4 deficient mouse lung homogenates 12 hours after infection. Contrastingly, lung histology at 72 hours postinfection, when TLR4 deficient mice exhibit clinical signs of bordetellosis, showed marked neutrophilic infiltrates and severe bronchopneumonia. Additionally, infection of TLR4 deficient mice with mutant strains of B. bronchiseptica in which the TTSA and AC toxins are deleted resulted in less severe clinical disease and longer survival, yet the mice did not exhibit decreased bacterial loads. Both TTSA and adenylate cyclase are critical virulence factors that are associated with necrotic cell death and inflammation. Together these results suggest that 1) an early LPS induced proinflammatory response to B. bronchiseptica infection is critical in limiting clinical disease, 2) that inflammation more than bacterial load contributes to clinical disease, and 3) that a diminished early
proinflammatory response results in delayed but exaggerated neutrophil response and severe clinical disease.
Impacts
Understanding the mechanisms of immunity important the clearance of a bacterial pathogen from the respiratory tract will help improve vaccines and therapeutic strategies. This study suggest that 1) an early LPS induced proinflammatory response to B. bronchiseptica infection is critical in limiting clinical disease, 2) that inflammation more than bacterial load contributes to clinical disease, and 3) that a diminished early proinflammatory response results in delayed but exaggerated neutrophil response and severe clinical disease. This research will increase our understanding of the immune mechanisms involved in the clearance of B. bronchiseptica from the respiratory tract and will help target the development of new therapies and vaccines that will be able eliminate this infection.
Publications
- Kennett, M. J. and Harvill, E. T. 2003. The Lipopolysaccharide induced Proinflammatory Response is Critical in Controlling Pneumonia in a Murine Bordetella bronchiseptica Infection Model Contemp. Top. Lab. Anim. Sci. 42:99-100.
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Progress 01/01/02 to 12/31/02
Outputs
We are exploring ways to eliminate `Bordetella bronchiseptica' from the respiratory tracts of animals using a mouse infection model. In order to understand defense mechanisms important in bacterial clearance, we examined differences in the ability of C3H/HeN (wild type) and C3H/HeJ (TLR 4 deficient) mice to clear `B. bronchiseptica' infections. TLR 4 may be important in the immune response to `B. bronchiseptica', as it is a major receptor for lipopolysaccharide and triggers signaling cascades that mediate phagocytic inflammatory cell responses and cytokine expression. The C3H/HeJ mice were colonized by `B. bronchiseptica' at 10-10,000 fold higher levels than their C3H/HeN counterparts. Additionally, bronchoalveolar lavage fluid from C3H/HeJ mice had four fold fewer neutrophils than lavage fluid from the C3H/HeN mice. Lung lesions in C3H/HeJ mice were more severe as compared to C3H/HeN mice. When C3H/HeN mice were injected with immune serum at the time of infection,
they had significantly less CFU in the lungs and trachea as compared to untreated C3H/HeN mice. However, adoptive transfer of antibodies to the TLR 4 deficient C3H/HeJ mice had no effect on the number of CFU in the lungs and trachea. Lung lesions in C3H/HeN mice decreased in severity between days 1 and 3 upon treatment with immune serum, whereas lung lesions increased in severity in treated C3H/HeJ mice. Thus we have shown that these two substrains of C3H mice differ substantially in their ability to clear `B. bronchiseptica', and that TLR 4 is important in controlling bacterial numbers, aids in neutrophil recruitment, helps minimize lung pathology, and is required for antibody-mediated clearance of `B. bronchiseptica' from the lower respiratory tract of mice. This research helps highlights specific immune factors important in response to infection with this respiratory pathogen.
Impacts
Current vaccines and therapies are not able to eliminate many infections from the respiratory tract, and a limited understanding of the mechanisms involved with the clearing of bacteria from the lungs and nasal cavity has prevented therapeutic improvements. This research will increase our understanding of the immune mechanisms involved in the clearance of `B. bronchiseptica' from the respiratory tract and will help target the development of new therapies and vaccines that will be able eliminate this infection.
Publications
- Kennett, M.J., Mann, P. N., Kirimanjeswara, G. S. and Harvill, E. T. 2002. P72 Substrain Differences in the Immune Response to `Bordetella bronchiseptica' Infections in C3H Mice due to TLR4 Mutation. Contemp. Top. Lab. Anim. Sci. 41:103
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