Dataset Information

Mice Survival and Plasmatic Cytokine Secretion in a "Two Hit" Model of Sepsis Depend on Intratracheal Pseudomonas Aeruginosa Bacterial Load.

ABSTRACT: Sepsis is characterized by pro- and anti-inflammatory responses following infection. While inflammation is responsible for widespread organ damage, anti-inflammatory mediators lead to immunoparalysis increasing susceptibility to secondary infections (nosocomial pneumonia). We aimed to investigate the impact of bacterial load on survival and cytokine release in a two-hit murine (C57BL/6J) model of CLP followed by P. aeruginosa pneumonia. Plasmatic TNF?, IL-6, IL-10, sTNFr I and II were quantified until 13 days. At D5, splenocytes were processed for immunological assays or mice were intratracheally instilled with Pseudomonas aeruginosa (5.106, 2.107 and 108 CFU) to evaluate survival and cytokines production. TNF?, sTNFrs, IL-6 and IL-10 increased 2h post CLP. TNF? and sTNFrs declined respectively one and two days later. In CLP mice, IL-6 and IL-10 remained high for the whole experiment, as compared to Sham. At D5, for CLP mice, whereas total T cells population (CD3+) decreased, Treg fraction (CD4+/CD25+) increased. In parallel, T cells proliferation and LPS-stimulated splenocytes ability to release TNF? decreased. At D13, survival was 100% after 5.106 CFU, 50% for CLP mice after 2.107 CFU and 0% for CLP and Sham after 108 CFU. After instillation, IL-10 and IL-6 increased and appeared to be dose and time dependent. Pseudomonas was detected in all CLP and Sham's lungs; in spleen and liver only in CLP at 2.107 CFU, and in CLP and Sham at 108 CFU. We demonstrated that post-CLP immunosuppression followed by Pseudomonas aeruginosa lung instillation increases mortality reactivates cytokines secretion and is associated with systemic dissemination in septic mice depending on bacterial load.

SUBMITTER: Restagno D

PROVIDER: S-EPMC5004855 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

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Mice Survival and Plasmatic Cytokine Secretion in a "Two Hit" Model of Sepsis Depend on Intratracheal Pseudomonas Aeruginosa Bacterial Load.

Restagno Damien D Venet Fabienne F Paquet Christian C Freyburger Ludovic L Allaouchiche Bernard B Monneret Guillaume G Bonnet Jeanne-Marie JM Louzier Vanessa V

PloS one 20160830 8

Sepsis is characterized by pro- and anti-inflammatory responses following infection. While inflammation is responsible for widespread organ damage, anti-inflammatory mediators lead to immunoparalysis increasing susceptibility to secondary infections (nosocomial pneumonia). We aimed to investigate the impact of bacterial load on survival and cytokine release in a two-hit murine (C57BL/6J) model of CLP followed by P. aeruginosa pneumonia. Plasmatic TNFα, IL-6, IL-10, sTNFr I and II were quantified ...[more]

PMID: 27574993

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Project description:BACKGROUND: Since the discovery of the human CFTR gene in 1989 various mouse models for cystic fibrosis (CF) have been generated and used as a very suitable and popular tool to approach research on this life-threatening disease. Age related changes regarding the course of disease and susceptibility towards pulmonary infections have been discussed in numerous studies. METHODS: Here, we investigated CftrTgH(neoim)Hgu and Cftrtm1Unc-Tg(FABPCFTR)1Jaw/J CF mice and their non-CF littermates during an acute lung infection with Pseudomonas aeruginosa for age dependent effects of their lung function and immune response.Mice younger than three or older than six months were intratracheally infected with P. aeruginosa TBCF10839. The infection was monitored by lung function of the animals using non-invasive head-out spirometry and the time course of physiological parameters over 192 hours. Quantitative bacteriology and lung histopathology of a subgroup of animals were used as endpoint parameters. RESULTS: Age-dependent changes in lung function and characteristic features for CF like a shallower, faster breathing pattern were observed in both CF mouse models in uninfected state. In contrast infected CF mice did not significantly differ from their non-CF littermates in susceptibility and severity of lung infection in both mouse models and age groups. The transgenic Cftrtm1Unc-Tg(FABPCFTR)1Jaw/J and their non-CF littermates showed a milder course of infection than the CftrTgH(neoim)Hgu CF and their congenic C57Bl/6J non-CF mice suggesting that the genetic background was more important for outcome than Cftr dysfunction. CONCLUSIONS: Previous investigations of the same mouse lines have shown a higher airway susceptibility of older CF mice to intranasally applied P. aeruginosa. The different outcome of intranasal and intratracheal instillation of bacteria implies that infected CF epithelium is impaired during the initial colonization of upper airways, but not in the subsequent response of host defense.

Project description:UnlabelledThe opportunistic pathogen Pseudomonas aeruginosa utilizes an injectisome-type III secretion system (injectisome-T3SS) to elicit cytotoxicity toward epithelial cells and macrophages. Macrophage killing results from the cytotoxic properties of the translocated effector proteins (ExoS, ExoT, ExoU, and ExoY) and inflammasome-mediated induction of pyroptosis. Inflammasome activation can occur following Nlrc4-mediated recognition of cytosolic translocated flagellin (FliC). In the present study, we demonstrate that FliC is a secretion substrate of both the injectisome- and flagellum-associated T3SSs. Molecular analyses indicate that the first 20 amino-terminal residues of FliC are sufficient for secretion by the injectisome-T3SS and that the first 100 residues are sufficient for translocation of FliC into host cells. Although maximal inflammasome activation requires FliC, activation can also occur in the absence of FliC. This prompted us to examine whether other flagellar components might also be translocated into cells to elicit inflammasome activation. Indeed, we find that the flagellar cap (FliD), hook-associated (FlgK and FlgL), hook (FlgE), and rod (FlgE) proteins are secretion substrates of the injectisome-T3SS. None of these proteins, however, result in increased inflammasome activation when they are overexpressed in a fliC mutant and appear to be translocated into host cells. While a role in inflammasome activation has been excluded, these data raise the possibility that flagellar components, which are highly conserved between different bacterial species, trigger other specific host responses from the extracellular milieu or contribute to the pathogenesis of P. aeruginosa.ImportanceThe inflammasome is a host defense mechanism that recognizes invading bacteria and triggers an inflammatory immune response. The opportunistic pathogen P. aeruginosa produces both inflammasome agonists and antagonists. In this study, we demonstrate that overexpression of an agonist suppresses the activity of an antagonist, thereby resulting in inflammasome activation. Since the relative expression levels of agonists and antagonists likely vary between strains, these differences could be important predictors of whether a particular P. aeruginosa strain elicits inflammasome activation.

Dataset Information

Mice Survival and Plasmatic Cytokine Secretion in a "Two Hit" Model of Sepsis Depend on Intratracheal Pseudomonas Aeruginosa Bacterial Load.

Publications

Mice Survival and Plasmatic Cytokine Secretion in a "Two Hit" Model of Sepsis Depend on Intratracheal Pseudomonas Aeruginosa Bacterial Load.

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