Monday, 6 February 2012

Innate immune recognition of Pseudomonas aeruginosa and The role of Pseudomonas Quinolone Signal (PQS) on the interaction of Pseudomonas aeruginosa with lung epithelial cells

As usually guys and because we're right in the middle of those classy reviews of the regulatory affairs course, I am bringing you guys something different. Remember the seminars I do attend now and then, the Bacteriology Forum in the Centre for Biomolecular Sciences in the University of Nottingham. Well, we had two great presentations today and I will be revealing the most interesting facts shared by the investigators responsible for these presentations. Unfortunately, and because I am presently struggling with a serious flu some ideas I was just incapable of listening to adequately, and also the first speaker flashed slides to the speed of light, what actually disturbed my understanding.

To start with, a presentation by Sonali Singh (Lectin Biology Group studying host-pathogen interaction)

Title:     Innate immune recognition of Pseudomonas aeruginosa


     Pseudomonas aeruginosa is an opportunistic pathogen that causes severe respiratory infections in susceptible individuals (e.g. cystic fibrosis patients). The current study aims to identify the conditions required for P. aeruginosa clearance / inhibition and cellular recruitment by one of the major innate immune players in bacterial infections – macrophages (Mf).

    Our results show that under non-opsonic conditions M-CSF-induced human monocyte-derived Mf expressed TNF-a, IL-6, IL-1b, IL-18, IL-8, MCP-1, and MIP-1a in response to viable P. aeruginosa PA01. These Mf eliminated up to 50% of PA01 within 4 hours of infection, but were eventually overwhelmed by the bacteria. Priming Mf prior to infection by the addition of IFN-g or by substitution of M-CSF with GM-CSF did not enhance bacterial clearance or Mf survival significantly, but shifted the cytokine response to a more proinflammatory one. In terms of promoting inflammation, comparison of TNF-a vs. IL-10 ratios revealed an hierarchy where Mf primed with M-CSF < M-CSF + IFN-g ≤ GM-CSF < GM-CSF + IFN-g.


- Cystic fibrosis is the most common inherited disorder in Caucasians (1 in 2000-3500 births);
- 80-97% of cystic fibrosis patients in their sample had been colonised by Pseudomonas aeruginosa;


- Macrophages activation with GM-CSF and IFN-γ enhances pro-inflammatory cytokine profile;
- Reduction in Total PA numbers gets complicated by variation in amount of macrophages-associated  PA;
- They achieved successful purification of minimally activated human neutrophils from whole blood;
- Purified neutrophils produce reactive oxygen species upon stimulation.

Future work: 

- They are trying to answer if macrophages signals affect neutrophils response (in terms of production of reactive oxygen species) to PA01 strain;
- They are also trying to optimise reactive oxygen species assay with X-VIVO15.

In respect to the second presentation of the day conducted by Liu Yi-chia (Chris)

Title:     The role of Pseudomonas Quinolone Signal (PQS) on the interaction of Pseudomonas aeruginosa with lung epithelial cells


     Airway epithelial cells (AECs) serve not only as a physical barrier when challenged by pathogens but also activate immune responses by releasing antimicrobial peptides and recruiting phagocytes to the infection site. P. aeruginosa is thought to be the leading cause of the severe mortality in cystic fibrosis (CF) patients; by exploiting quorum-sensing systems, it can easily adapt to the CF lung.  

     Apart from the AHL-based QS circuit, alkyl quinolone (AQ) molecules can also be detected in CF lungs indicating AQs might involve in the regulation of chronic pulmonary infection, although the mechanism is still unknown.

     To investigate how alkyl-quinolone QS molecules modulate the innate immune system, we utilize P. aeruginosa PA01-L and its isogenic AQ-deficient mutant (pqsA-) to infect polarized airway epithelial cells. Unexpectedly, preliminary data suggest that the AQ-deficient strain might be more invasive than its wild type counterpart. We propose that this increased damaging capacity might facilitate early warning of innate immune cells and promote bacterial clearance. Understanding of the signaling events activated in AECs upon infection may direct the design of better therapeutic interventions for chronic pulmonary infections.


- Epithelial cells are the first line of defence in the lung as they act as physical barrier and have a role as an important contributor to innate mucosal immunity, thus ciliate epithelium lining prevents bacterial colonisation;
- This group is interested in knowing whether Pseudomonas aeruginosa alkyl-quinolone signalling has any effects on the infection of airway epithelial cells;


- They know now that the differentiation and polarisation were achieved in air-liquid interface culture in 3 weeks;
- Calu-3 cells cultured in MEM in non-coated well appears to be physiologically relevant;
- There's no dramatic morphological changes in Calu-3 cells.

Future work:

- Amongst many things they want to analyse PQS molecules in the interaction of PA with Calu-3 cells in the presence and absence of macrophages;

See you tomorrow for the 6th module of the regulatory affairs course review! Bye!

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