Hannah K. Bayes

Mitochondrial damage contributes to Pseudomonas aeruginosa activation of the inflammasome and is downregulated by autophagy

The nucleotide-binding domain, leucine-rich repeat containing family caspase recruitment domain containing 4 (NLRC4) inflammasome can be activated by pathogenic bacteria via products translocated through the microbial type III secretion apparatus (T3SS). Recent work has shown that activation of the NLRP3 inflammasome is downregulated by autophagy, but the influence of autophagy on NLRC4 activation is unclear. We set out to determine how autophagy might influence this process, using the bacterium Pseudomonas aeruginosa, which activates the NLRC4 inflammasome via its T3SS. Infection resulted in T3SS-dependent mitochondrial damage with increased production of reactive oxygen intermediates and release of mitochondrial DNA. Inhibiting mitochondrial reactive oxygen release or degrading intracellular mitochondrial DNA abrogated NLRC4 inflammasome activation. Moreover, macrophages lacking mitochondria failed to activate NLRC4 following infection. Removal of damaged mitochondria by autophagy significantly attenuated NLRC4 inflammasome activation. Mitochondrial DNA bound specifically to NLRC4 immunoprecipitates and transfection of mitochondrial DNA directly activated the NLRC4 inflammasome; oxidation of the DNA enhanced this effect. Manipulation of autophagy altered the degree of inflammasome activation and inflammation in an in vivo model of P. aeruginosa infection. Our results reveal a novel mechanism contributing to NLRC4 activation by P. aeruginosa via mitochondrial damage and release of mitochondrial DNA triggered by the bacterial T3SS that is downregulated by autophagy.

T helper cell subsets specific for Pseudomonas aeruginosa in healthy individuals and patients with cystic fibrosis.

Background We set out to determine the magnitude of antigen-specific memory T helper cell responses to Pseudomonas aeruginosa in healthy humans and patients with cystic fibrosis. Methods Peripheral blood human memory CD4+ T cells were co-cultured with dendritic cells that had been infected with different strains of Pseudomonas aeruginosa. The T helper response was determined by measuring proliferation, immunoassay of cytokine output, and immunostaining of intracellular cytokines. Results Healthy individuals and patients with cystic fibrosis had robust antigen-specific memory CD4+ T cell responses to Pseudomonas aeruginosa that not only contained a Th1 and Th17 component but also Th22 cells. In contrast to previous descriptions of human Th22 cells, these Pseudomonal-specific Th22 cells lacked the skin homing markers CCR4 or CCR10, although were CCR6+. Healthy individuals and patients with cystic fibrosis had similar levels of Th22 cells, but the patient group had significantly fewer Th17 cells in peripheral blood. Conclusions Th22 cells specific to Pseudomonas aeruginosa are induced in both healthy individuals and patients with cystic fibrosis. Along with Th17 cells, they may play an important role in the pulmonary response to this microbe in patients with cystic fibrosis and other conditions.

Interleukin-17 Is Required for Control of Chronic Lung Infection Caused by Pseudomonas aeruginosa

ABSTRACT Chronic pulmonary infection with Pseudomonas aeruginosa is a feature of cystic fibrosis (CF) and other chronic lung diseases. Cytokines of the interleukin-17 (IL-17) family have been proposed as important in the host response to P. aeruginosa infection through their role in augmenting antibacterial immune responses, although their proinflammatory effect may contribute to lung damage that occurs as a result of chronic infection. We set out to explore the role of IL-17 in the host response to chronic P. aeruginosa infection. We used a murine model of chronic pulmonary infection with CF-related strains of P. aeruginosa. We demonstrate that IL-17 cytokine signaling is essential for mouse survival and prevention of chronic infection at 2 weeks postinoculation using two different P. aeruginosa strains. Following infection, there was a marked expansion of cells within mediastinal lymph nodes, comprised mainly of innate lymphoid cells (ILCs); ∼90% of IL-17-producing (IL-17+) cells had markers consistent with group 3 ILCs. A smaller percentage of IL-17+ cells had markers consistent with a B1 phenotype. In lung homogenates harvested 14 days following infection, there was a significant expansion of IL-17+ cells; about 50% of these were CD3+, split equally between CD4+ Th17 cells and γδ T cells, while the CD3− IL-17+ cells were almost exclusively group 3 ILCs. Further experiments with B cell-deficient mice showed that B cell production of IL-17 or natural antibodies did not provide any defense against chronic P. aeruginosa infection. Thus, IL-17 rather than antibody is a key element in host defense against chronic pulmonary infection with P. aeruginosa.

A murine model of early Pseudomonas aeruginosa lung disease with transition to chronic infection

Pseudomonas aeruginosa (PA) remains an important pathogen in patients with cystic fibrosis (CF) lung disease as well as non-CF bronchiectasis and chronic obstructive airways disease. Initial infections are cleared but chronic infection with mucoid strains ensues in the majority of CF patients and specific interventions to prevent this critical infection transition are lacking. The PA bead model has been widely used to study pulmonary P.aeruginosa infection but has limitations in animal husbandry and in accurately mimicking human disease. We have developed an adapted agar bead murine model using a clinical mucoid strain that demonstrates the key features of transition from transitory to chronic airways infection. Infected animals show very limited acute morbidity and mortality, but undergo infection-related weight loss and neutrophilic inflammation, development of anti-pseudomonal antibodies, variable bacterial clearance, endobronchial infection and microbial adaptation with PA small colony variants. We anticipate this model will allow research into the host and microbial factors governing this critical period in Pseudomonas aeruginosa pulmonary pathogenesis when transition to chronicity is occurring.

IL-22 exacerbates weight loss in a murine model of chronic pulmonary Pseudomonas aeruginosa infection

Background Interleukin (IL)-22 is a critical mediator of mucosal immunity and tissue regeneration, protecting against a number of respiratory pathogens. Whether IL-22 confers protection against chronic Pseudomonas aeruginosa (PA) infection in cystic fibrosis (CF) is unknown. Methods Explanted CF lungs were examined for IL-22 production and immune-localization. A murine model of persistent pulmonary PA infection was used to examine production of IL-22 following infective challenge. The role of IL-22 was examined using IL-22 knockout (KO) animals. Results IL-22 is produced within the adult CF lung and localizes to the airway epithelium. IL-22 is produced by murine pulmonary lymph node cells following lung infection. The absence of IL-22 resulted in no significant difference in acute mortality, bacterial burden, chronic infection rates, histological changes or neutrophilic inflammation in the chronic PA infection model. However, IL-22 KO animals lost less weight following infection. Conclusion IL-22 is produced in the CF lung and in response to PA infection yet is dispensable in protection against chronic pulmonary P. aeruginosa infection in a murine model. However, we identified a novel role for the cytokine in promoting infection-related weight-loss, a significant prognostic factor in the CF population.

IL-17 can be protective or deleterious in murine pneumococcal pneumonia

Streptococcus pneumoniae is the major bacterial cause of community-acquired pneumonia, and the leading agent of childhood pneumonia deaths worldwide. Nasal colonization is an essential step prior to infection. The cytokine IL-17 protects against such colonization and vaccines that enhance IL-17 responses to pneumococcal colonization are being developed. The role of IL-17 in host defence against pneumonia is not known. To address this issue, we have utilized a murine model of pneumococcal pneumonia in which the gene for the IL-17 cytokine family receptor, Il17ra, has been inactivated. Using this model, we show that IL-17 produced predominantly from γδ T cells protects mice against death from the invasive TIGR4 strain (serotype 4) which expresses a relatively thin capsule. However, in pneumonia produced by two heavily encapsulated strains with low invasive potential (serotypes 3 and 6B), IL-17 significantly enhanced mortality. Neutrophil uptake and killing of the serotype 3 strain was significantly impaired compared to the serotype 4 strain and depletion of neutrophils with antibody enhanced survival of mice infected with the highly encapsulated SRL1 strain. These data strongly suggest that IL-17 mediated neutrophil recruitment to the lungs clears infection from the invasive TIGR4 strain but that lung neutrophils exacerbate disease caused by the highly encapsulated pneumococcal strains. Thus, whilst augmenting IL-17 immune responses against pneumococci may decrease nasal colonization, this may worsen outcome during pneumonia caused by some strains.

Review of the British Thoracic Society winter meeting 2012, 5 to 7 December, London, UK.

This is the second annual review of the British Thoracic Society Winter Scientific Meeting held from 7–9 December 2011, which was attended by over 2000 delegates. Although a wide spectrum of respiratory research was presented at the meeting, the content of the review focuses specifically on three key themes: cystic fibrosis, pulmonary vascular disease and thoracic oncology. Advances in clinical and translational respiratory research presented within the major symposia and spoken sessions related to these areas are summarised. Additional sessions recognising topics relevant to the forthcoming 2012 London Olympics are also highlighted.

A review of the British Thoracic Society scientific meeting 1–3 December 2010

This review presents the inaugural report of the British Thoracic Society Winter Scientific Meeting held from 1st–3rd December 2010. Although a wide spectrum of respiratory research was presented at the meeting the content of the review focuses specifically on three key themes of inflammation and lung injury, airways disease and respiratory infection. Advances in both clinical and translational respiratory research presented within the major symposia and spoken sessions related to these themes are summarised.

Genomic inversion drives small colony variant formation and increased virulence in P. aeruginosa

Phenotypic change is a hallmark of bacterial adaptation during chronic infection. In the case of chronic Pseudomonas aeruginosa lung infection in patients with cystic fibrosis, well-characterised phenotypic variants include mucoid and small colony variants (SCVs). It has previously been shown that SCVs can be reproducibly isolated from the murine lung following the establishment of chronic infection with mucoid P. aeruginosa strain NH57388A. Here we show, using a combination of singlemolecule real-time (PacBio) and Illumina sequencing that the genetic switch for conversion to the SCV phenotype is a large genomic inversion through recombination between homologous regions of two rRNA operons. This phenotypic conversion is associated with large-scale transcriptional changes distributed throughout the genome. This global rewiring of the cellular transcriptomic output results in changes to normally differentially regulated genes that modulate resistance to oxidative stress, central metabolism and virulence. These changes are of clinical relevance since the appearance of SCVs during chronic infection is associated with declining lung function.

S105 Innate B1 cells are a novel source of IL-17 in chronic pulmonary Pseudomonas aeruginosa infection

Introduction and Objectives Pseudomonas aeruginosa (PA) is an important respiratory pathogen resulting in damaging neutrophilic responses. The cytokine IL-17 is important in orchestrating such inflammation. Cells producing IL-17, such as Th17 cells, have been shown to be important in host defense in chronic pulmonary PA infection. We set out to determine the origin and role of IL-17 in a model of chronic pulmonary PA infection. Methods Experimental chronic pulmonary infection in mice was produced by intra-tracheal instillation of mucoid PA strains embedded in agar-beads; sterile beads were utilised as controls. Thoracic lymph nodes (LNs), splenocytes and peritoneal B1a cells were restimulated with PA followed by cytokine assay and immunostaining to define responding cell subsets. PA-specific immunoglobulins were measured in sera and culture supernatants. Intrapulmonary B cells were identified via immunohistochemistry. Mice genetically engineered to lack B cells (µMT strain) were utilised to examine the effect on pathogenesis in the absence of B cell responses. Results Chronic PA infection developed in 43% (SD: 25%) of infected animals at 14-days. Following infection, the pulmonary LN B cell compartment expanded, with a large B1 population (B220+ CD19+ CD43+ IgM hi IgD lo and predominantly CD5+ ) that expressed intracellular IL-17A. Infected animals also developed peribronchial B220+ cellular foci. In mediastinal LNs following infection, PA-specific responses were dominated by B220+ CD19+ CD43+ CD23-B5+ cells expressing and producing IL-17A and IL-22 as well as PA-specific IgM but not IgG. This PA-specific B1 response was not seen in the thoracic lymph nodes of sterile-bead treated animals. In splenocytes, there was a pre-existing B cell response to PA with identical features. Peritoneal B1a cells isolated from untreated controls also produced IL-17A, IL-22 and anti-PA IgM following infection, confirming the existence of pre-existing B1 cells that can respond to PA. In µMT animals, chronic colonisation rates, bacterial burden and neutrophilic inflammation did not differ from WT littermates. However, classical PA-specific Th17 responses dominated following infection in µMT animals, suggesting alternative compensatory IL-17 sources acting in the absence of B cells. Conclusions In chronic pulmonary PA infection, innate-like B1 cells migrate to the site of infection and are a novel source of pro-inflammatory IL-17 cytokines.