A Porcine Ex Vivo Lung Perfusion Model To Investigate Bacterial Pathogenesis

ABSTRACT The use of animal infection models is essential to understand microbial pathogenesis and to develop and test treatments. Insects and two-dimensional (2D) and 3D tissue models are increasingly being used as surrogates for mammalian models. However, there are concerns about whether these models recapitulate the complexity of host-pathogen interactions. In this study, we developed the ex vivo lung perfusion (EVLP) model of infection using porcine lungs to investigate Klebsiella pneumoniae-triggered pneumonia as a model of respiratory infections. The porcine EVLP model recapitulates features of K. pneumoniae-induced pneumonia lung injury. This model is also useful to assess the pathogenic potential of K. pneumoniae, as we observed that the attenuated Klebsiella capsule mutant strain caused less pathological tissue damage with a concomitant decrease in the bacterial burden compared to that in lungs infected with the wild type. The porcine EVLP model allows assessment of inflammatory responses following infection; similar to the case with the mouse pneumonia model, we observed an increase of il-10 in the lungs infected with the wild type and an increase of ifn-γ in lungs infected with the capsule mutant. This model also allows monitoring of phenotypes at the single-cell level. Wild-type K. pneumoniae skews macrophages toward an M2-like state. In vitro experiments probing pig bone marrow-derived macrophages uncovered the role for the M2 transcriptional factor STAT6 and that Klebsiella-induced il-10 expression is controlled by p38 and extracellular signal-regulated kinase (ERK). Klebsiella-induced macrophage polarization is dependent on the capsule. Together, the findings of this study support the utility of the EVLP model using pig lungs as a platform to investigate the infection biology of respiratory pathogens. IMPORTANCE The implementation of infection models that approximate human disease is essential to understand infections and for testing new therapies before they enter into clinical stages. Rodents are used in most preclinical studies, although the differences between mice and humans have fueled the conclusion that murine studies are unreliable predictors of human outcomes. In this study, we have developed a whole-lung porcine model of infection using the ex vivo lung perfusion (EVLP) system established to recondition human lungs for transplant. As a proof of principle, we provide evidence demonstrating that infection of the porcine EVLP with the human pathogen Klebsiella pneumoniae recapitulates the known features of Klebsiella-triggered pneumonia. Moreover, our data revealed that the porcine EVLP model is useful to reveal features of the virulence of K. pneumoniae, including the manipulation of immune cells. Together, the findings of this study support the utility of the EVLP model using pig lungs as a surrogate host for assessing respiratory infections.

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A porcine ex vivo lung perfusion model to investigate bacterial pathogenesis

10.1101/708503 ◽

2019 ◽

Author(s):

Amy Dumigan ◽

Marianne Fitzgerald ◽

Joana Sá Pessoa Graca Santos ◽

Umar Hamid ◽

Cecilia M. O’Kane ◽

...

Keyword(s):

Inflammatory Responses ◽

Respiratory Infections ◽

Ex Vivo ◽

Macrophage Polarization ◽

Lung Perfusion ◽

Respiratory Pathogens ◽

Wild Type ◽

Pathogenic Potential ◽

Ex Vivo Lung Perfusion ◽

Infection Models

ABSTRACTThe use of animal infection models is essential to understand microbial pathogenesis and to develop and test treatments. Insects, and 2D and 3D tissue models are increasingly being used as surrogate for mammalian models. However, there are concerns whether these models recapitulate the complexity of host-pathogen interactions. Here, we developed the ex vivo lung perfusion (EVLP) model of infection using porcine lungs to investigate Klebsiella pneumoniae-triggered pneumonia as model of respiratory infections. The porcine EVLP model recapitulates features of K. pneumoniae-induced pneumonia lung injury. This model is also useful to assess the pathogenic potential of K. pneumoniae as we observed that the attenuated Klebsiella capsule mutant strain caused less pathological tissue damage with a concomitant decrease in the bacterial burden compare to lungs infected with the wild type. The porcine EVLP model allows assessment of inflammatory responses following infection; similar to the mouse pneumonia model, we observed an increase of il-10 in the lungs infected with the wild type and an increase of ifn-γ in lungs infected with the capsule mutant. This model also allows monitoring phenotypes at the single-cell level. Wild-type K. pneumoniae skews macrophages towards an M2-like state. In vitro experiments probing pig bone marrow-derived macrophages uncovered the role of the M2 transcriptional factor STAT6, and that Klebsiella-induced il10 expression is controlled by p38 and ERK. Klebsiella-induced macrophage polarization is dependent on the capsule. Altogether, this study support the utility of the EVLP model using pig lungs as platform to investigate the infection biology of respiratory pathogens.IMPORTANCEThe implementation of infection models that approximate human disease is essential to understand infections and for testing new therapies before they enter into clinical stages. Rodents are used in most of pre-clinical studies, although the differences between mouse and man have fuelled the conclusion that murine studies are unreliable predictors of human outcomes. Here, we have developed a whole lung porcine model of infection using the established ex vivo lung perfusion (EVLP) system established to re-condition human lungs for transplant. As a proof-of-principle, we provide evidence demonstrating that infection of the porcine EVLP with the human pathogen K. pneumoniae recapitulates the known features of Klebsiella-triggered pneumonia. Moreover, our data revealed the porcine EVLP model is useful to reveal features of the virulence of K. pneumoniae including the manipulation of immune cells. Altogether, this study supports the utility of the EVLP model using pig lungs as surrogate host for assessing respiratory infections.

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BspR/BtrA, an Anti-σ Factor, Regulates the Ability ofBordetella bronchisepticaTo Cause Cough in Rats

mSphere ◽

10.1128/msphere.00093-19 ◽

2019 ◽

Vol 4 (2) ◽

Cited By ~ 5

Author(s):

Keiji Nakamura ◽

Noriko Shinoda ◽

Yukihiro Hiramatsu ◽

Shinya Ohnishi ◽

Shigeki Kamitani ◽

...

Keyword(s):

Virulence Factors ◽

Bordetella Pertussis ◽

Rat Model ◽

Whooping Cough ◽

Respiratory Infections ◽

Wild Type ◽

Content Type ◽

Infection Models ◽

Natural Hosts ◽

Σ Factor

ABSTRACTBordetella pertussis,B. parapertussis, andB. bronchisepticacause respiratory infections, many of which are characterized by coughing of the infected hosts. The pathogenesis of the coughing remains to be analyzed, mainly because there were no convenient infection models of small animals that replicate coughing afterBordetellainfection. Here, we present a coughing model of rats infected withB. bronchiseptica. Rats, which are one of natural hosts ofB. bronchiseptica, were readily infected with the organisms and showed frequent coughing.B. pertussisalso caused coughing in rats, which is consistent with previous reports, but the cough response was less apparent than theB. bronchiseptica-induced cough. By using the rat model, we demonstrated that adenylate cyclase toxin, dermonecrotic toxin, and the type III secretion system are not involved in cough production, but BspR/BtrA (different names for the same protein), an anti-σ factor, regulates the production of unknown factor(s) to cause coughing. Rat coughing was observed by inoculation of not only the living bacteria but also the bacterial lysates. Infection withbspR(btrA)-deficient strains caused significantly less frequent coughing than the wild type; however, intranasal inoculation of the lysates from abspR(btrA)-deficient strain caused coughing similarly to the wild type, suggesting that BspR/BtrA regulates the production of the cough factor(s) only when the bacteria colonize host bodies. Moreover, the cough factor(s) was found to be heat labile and produced byB. bronchisepticain the Bvg+phase. We consider that our rat model provides insight into the pathogenesis of cough induced by theBordetellainfection.IMPORTANCEWhooping cough is a contagious respiratory disease caused byBordetella pertussis. This disease is characterized by severe paroxysmal coughing, which becomes a heavy burden for patients and occasionally results in death; however, its pathogenesis remains largely unknown. The major obstacle to analyzingBordetella-induced coughing is the lack of conventional animal models that replicate coughing. AsBordetella pertussisis highly adapted to humans, infection models in experimental animals are not considered to be well established. In the present study, we examined coughing in rats infected withB. bronchiseptica, which shares many virulence factors withB. pertussis. Using this rat model, we demonstrated that some of the major virulence factors ofBordetellaare not involved in cough production, but an anti-σ factor, BspR/BtrA, ofB. bronchisepticaregulates the production of unknown cough-causing bacterial factor(s). Our results provide important clues to understand the mechanism by whichBordetellainduces cough.

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An Assessment of Siderophore Production, Mucoviscosity, and Mouse Infection Models for Defining the Virulence Spectrum of Hypervirulent Klebsiella pneumoniae

mSphere ◽

10.1128/msphere.00045-21 ◽

2021 ◽

Vol 6 (2) ◽

Author(s):

Thomas A. Russo ◽

Ulrike MacDonald ◽

Sidra Hassan ◽

Ellie Camanzo ◽

Francois LeBreton ◽

...

Keyword(s):

Klebsiella Pneumoniae ◽

Clinical Manifestations ◽

Siderophore Production ◽

Mouse Strains ◽

Virulence Plasmid ◽

Pathogenic Potential ◽

Content Type ◽

Infection Models ◽

Cd1 Mice ◽

Virulence Spectrum

ABSTRACT Hypervirulent Klebsiella pneumoniae (hvKp) bacteria are more virulent than classical K. pneumoniae (cKp) with resultant differences in clinical manifestations and management. It is unclear whether all hvKp isolates share a similar pathogenic potential. This report assessed the utility of siderophore production, mucoviscosity, and murine infection for defining the virulence spectrum of hvKp. Three strain cohorts were identified and defined based on the CD1 mouse subcutaneous (SQ) challenge model: (i) fully virulent hvKp strains (fvhvKp), lethal at a challenge inoculum (CI) of ≤103 CFU; (ii) partially virulent hvKp strains (pvhvKp), lethal at a CI of >103 to 107 CFU; (iii) classical K. pneumoniae, not lethal at a CI of 107 CFU. Quantitative siderophore and mucoviscosity assays differentiated fvhvKp and pvhvKp strains from cKp strains but were unable to differentiate between the fvhvKP and pvhvKP strain cohorts. However, SQ challenge of CD1 mice and intraperitoneal (IP) challenge of CD1 and BALB/c mice, but not C57BL/6 mice, were able to discriminate between an fvhvKp and a pvhvKp strain; SQ challenge of CD1 mice may have the greatest sensitivity. cKp was differentiated from hvKp both by SQ challenge of CD1 mice and IP challenge of all three mouse strains. These data identify a means to define the relative virulence of hvKP strains. It remains unclear whether the observed differences of hvKp virulence in mice translates to human infection. However, these data can be used to sort random collections of K. pneumoniae strains into fvhvKp and pvhvKp strain cohorts and assess for differences in clinical manifestations and outcomes. IMPORTANCE The pathogenic potential of hvKp strains is primarily mediated by a large virulence plasmid. The minimal set of genes required for the full expression of the hypervirulent phenotype is undefined. A number of reports describe hvKp strains possessing only a portion of the virulence plasmid; the clinical consequences of this are unclear. Therefore, the goal of this report was to determine whether virulence among hvKp strains varied and, if so, how to best identify the relative virulence of hvKp isolates. Data demonstrate hvKp pathogenic potential varies in CD1 and BALB/c murine infection models. In contrast, measurements of siderophore production and mucoviscosity were unable to discriminate the differences in hvKp isolate virulence observed in mice. This information can be used in future studies to determine the mechanisms responsible for differences between fully virulent hvKp and partially virulent hvKp and whether the differences observed in mice translate to disease in humans.

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