Model of host-pathogen Interaction dynamics links In vivo optical imaging and immune responses

Tracking disease progression in vivo is essential for the development of treatments against bacterial infection. Optical imaging has become a central tool for in vivo tracking of bacterial population development and therapeutic response. For a precise understanding of in vivo imaging results in terms of disease mechanisms derived from detailed postmortem observations, however, a link between the two is needed. Here, we develop a model that provides that link for the investigation of Citrobacter rodentium infection, a mouse model for enteropathogenic Escherichia coli (EPEC). We connect in vivo disease progression of C57BL/6 mice infected with bioluminescent bacteria, imaged using optical tomography and X-ray computed tomography, to postmortem measurements of colonic immune cell infiltration. We use the model to explore changes to both the host immune response and the bacteria and to evaluate the response to antibiotic treatment. The developed model serves as a novel tool for the identification and development of new therapeutic interventions

Asthma induced exacerbation of bacterial pneumonia.

Asthma and bacterial pneumonias are major causes of human mortality and morbidity throughout the world. To date many studies have investigated the possibility that bacteria exacerbate asthma but only a handful consider that asthma may cause bacterial infections. Recent clinical evidence suggests that bacterial infections cause serious complications in patients with asthma and that asthmatics show a 2-fold increased risk of invasive pneumococcal disease. This thesis examines the molecular mechanisms causing susceptibility of house dust mite (HDM) exposed lungs to bacterial infection. The main finding of this thesis is that HDM-induced allergic airways disease increases susceptibility to Streptococcus pneumoniae infection. Furthermore, the molecular pathways leading to the production of neutrophil chemoattractants in the lung are compromised and that despite the complexity of anti-bacterial pathways that are disrupted, the re-introduction of a single chemokine to the lungs with allergic airway disease enables clearance of S. pneumoniae that would otherwise prove fatal. However, a reduction in HDM-induced eosinophilia seen in ST2 deficient mice does not restore anti-bacterial immunity. This deficit in anti-bacterial immunity in HDM exposed lungs is associated with a change in resident alveolar macrophages into an alternatively activated phenotype, characterised by high mRNA and protein levels of RELMα, Ym1 and Arg1. These altered alveolar macrophages produce considerably less TNFα in response to Toll-Like Receptor (TLR) stimulation that is not a result of reduced TLR mRNA levels but due to an upregulation of TLR negative regulators particularly A20. A20 targets TRAF-6 that is found upstream of NF-κB activation. To prove a causal link naïve alveolar macrophages were transferred into allergic lungs prior to bacterial infection; such lungs handled the infection better confirming that alveolar macrophages are important in initiating the anti-bacterial response. Overall our findings highlight a change in specific innate immune pathways in the allergic lung that participate in susceptibility to bacterial pneumonia.Open Acces

Bacterial virulence factor inhibits caspase-4/11 activation in intestinal epithelial cells

The human pathogen enteropathogenic Escherichia coli (EPEC), as well as the mouse pathogen Citrobacter rodentium, colonize the gut mucosa via attaching and effacing lesion formation and cause diarrheal diseases. EPEC and C. rodentium type III secretion system (T3SS) effectors repress innate immune responses and infiltration of immune cells. Inflammatory caspases such as caspase-1 and caspase-4/11 are crucial mediators of host defense and inflammation in the gut via their ability to process cytokines such as interleukin (IL)-1β and IL-18. Here we report that the effector NleF binds the catalytic domain of caspase-4 and inhibits its proteolytic activity. Following infection of intestinal epithelial cells (IECs) EPEC inhibited caspase-4 and IL-18 processing in an NleF-dependent manner. Depletion of caspase-4 in IECs prevented the secretion of mature IL-18 in response to infection with EPECΔnleF. NleF-dependent inhibition of caspase-11 in colons of mice prevented IL-18 secretion and neutrophil influx at early stages of C. rodentium infection. Neither wild-type C. rodentium nor C. rodentiumΔnleF triggered neutrophil infiltration or IL-18 secretion in Cas11 or Casp1/11-deficient mice. Thus, IECs have a key role in modulating early innate immune responses in the gut via a caspase-4/11—IL-18 axis, which is targeted by virulence factors encoded by enteric pathogens

Bacterial virulence factor inhibits caspase-4/11 activation in intestinal epithelial cells.

The human pathogen enteropathogenic Escherichia coli (EPEC), as well as the mouse pathogen Citrobacter rodentium, colonize the gut mucosa via attaching and effacing lesion formation and cause diarrheal diseases. EPEC and C. rodentium type III secretion system (T3SS) effectors repress innate immune responses and infiltration of immune cells. Inflammatory caspases such as caspase-1 and caspase-4/11 are crucial mediators of host defense and inflammation in the gut via their ability to process cytokines such as interleukin (IL)-1β and IL-18. Here we report that the effector NleF binds the catalytic domain of caspase-4 and inhibits its proteolytic activity. Following infection of intestinal epithelial cells (IECs) EPEC inhibited caspase-4 and IL-18 processing in an NleF-dependent manner. Depletion of caspase-4 in IECs prevented the secretion of mature IL-18 in response to infection with EPECΔnleF. NleF-dependent inhibition of caspase-11 in colons of mice prevented IL-18 secretion and neutrophil influx at early stages of C. rodentium infection. Neither wild-type C. rodentium nor C. rodentiumΔnleF triggered neutrophil infiltration or IL-18 secretion in Cas11 or Casp1/11-deficient mice. Thus, IECs have a key role in modulating early innate immune responses in the gut via a caspase-4/11-IL-18 axis, which is targeted by virulence factors encoded by enteric pathogens

Model of Host-Pathogen Interaction Dynamics Links In Vivo Optical Imaging and Immune Responses.

Tracking disease progression in vivo is essential for the development of treatments against bacterial infection. Optical imaging has become a central tool for in vivo tracking of bacterial population development and therapeutic response. For a precise understanding of in vivo imaging results in terms of disease mechanisms derived from detailed postmortem observations, however, a link between the two is needed. Here, we develop a model that provides that link for the investigation of Citrobacter rodentium infection, a mouse model for enteropathogenic Escherichia coli (EPEC). We connect in vivo disease progression of C57BL/6 mice infected with bioluminescent bacteria, imaged using optical tomography and X-ray computed tomography, to postmortem measurements of colonic immune cell infiltration. We use the model to explore changes to both the host immune response and the bacteria and to evaluate the response to antibiotic treatment. The developed model serves as a novel tool for the identification and development of new therapeutic interventions

Bacterial superinfection following lung inflammatory disorders.

The lung environment is designed to prevent innate responses to harmless commensal microorganisms and environmental antigens. Features of an intact respiratory epithelium are critical to this process. A damaged or altered lung epithelial surface will therefore remove or alter the suppressive signals delivered to local innate immune cells, and inflammation ensues. Timely resolution of inflammation is important to prevent bystander tissue damage. However, if resolving pathways themselves are prolonged or repeated, they too can cause undesirable consequences, including bacterial superinfections, which we discuss here. </jats:p

Citrobacter rodentium mouse model of bacterial infection.

Infection of mice with Citrobacter rodentium is a robust model to study bacterial pathogenesis, mucosal immunology, the health benefits of probiotics and the role of the microbiota during infection. C. rodentium was first isolated by Barthold from an outbreak of mouse diarrhea in Yale University in 1972 and was 'rediscovered' by Falkow and Schauer in 1993. Since then the use of the model has proliferated, and it is now the gold standard for studying virulence of the closely related human pathogens enteropathogenic and enterohemorrhagic Escherichia coli (EPEC and EHEC, respectively). Here we provide a detailed protocol for various applications of the model, including bacterial growth, site-directed mutagenesis, mouse inoculation (from cultured cells and after cohabitation), monitoring of bacterial colonization, tissue extraction and analysis, immune responses, probiotic treatment and microbiota analysis. The main protocol, from mouse infection to clearance and analysis of tissues and host responses, takes ∼5 weeks to complete

Altered regulation of Toll-like receptor responses impairs antibacterial immunity in the allergic lung

The lung is colonized by commensal bacteria, some of which are associated with asthma exacerbations. Using the intranasal house-dust mite–sensitized mouse model of allergic airway disease, we show an imbalance in novel antibacterial pathways that culminates in a reduction in neutrophil recruitment to the airspaces and leads to bacterial invasion and dissemination. The expression of TREM (Triggering Receptor Expressed on Myeloid cells)-1 that amplifies Toll-like receptor (TLR) signaling and TREM-2 that inhibits this process is reversed. Furthermore, endogenous TLR inhibitors (A20, Tollip, SOCS1, and IRAK-M) and proteins involved in receptor recycling (TRIAD3) are raised. Consequently, the production of neutrophil chemoattractants is reduced. Intranasal administration of either chemokine restores the ability to recruit neutrophils, which prevents bacterial invasion. A background of allergic airway disease therefore exacerbates bacterial infection by altering key antibacterial innate immune pathways that are amenable to therapeutic intervention

Tir Triggers Expression of CXCL1 in Enterocytes and Neutrophil Recruitment during Citrobacter rodentium Infection.

The hallmarks of enteropathogenic Escherichia coli (EPEC) infection are formation of attaching and effacing (A/E) lesions on mucosal surfaces and actin-rich pedestals on cultured cells, both of which are dependent on the type III secretion system effector Tir. Following translocation into cultured cells and clustering by intimin, Tir Y474 is phosphorylated, leading to recruitment of Nck, activation of N-WASP, and actin polymerization via the Arp2/3 complex. A secondary, weak, actin polymerization pathway is triggered via an NPY motif (Y454). Importantly, Y454 and Y474 play no role in A/E lesion formation on mucosal surfaces following infection with the EPEC-like mouse pathogen Citrobacter rodentium. In this study, we investigated the roles of Tir segments located upstream of Y451 and downstream of Y471 in C. rodentium colonization and A/E lesion formation. We also tested the role that Tir residues Y451 and Y471 play in host immune responses to C. rodentium infection. We found that deletion of amino acids 382 to 462 or 478 to 547 had no impact on the ability of Tir to mediate A/E lesion formation, although deletion of amino acids 478 to 547 affected Tir translocation. Examination of enterocytes isolated from infected mice revealed that a C. rodentium strain expressing Tir_Y451A/Y471A recruited significantly fewer neutrophils to the colon and triggered less colonic hyperplasia on day 14 postinfection than the wild-type strain. Consistently, enterocytes isolated from mice infected with C. rodentium expressing Tir_Y451A/Y471A expressed significantly less CXCL1. These result show that Tir-induced actin remodeling plays a direct role in modulation of immune responses to C. rodentium infection