Modulation of immune recognition by P. aeruginosa through engagement of lectin receptors.

Lead Research Organisation: University of Nottingham
Department Name: School of Life Sciences

Abstract

We have identified a novel interaction between the human disease-causing bacterium Pseudomonas aeruginosa and two carbohydrate (sugar)-binding (lectin) receptors expressed by human immune cells termed DC-SIGN and mannose receptor (MR). The aim of this proposal is to determine how these interactions enable P. aeruginosa to cause life-threatening infections.
Understanding the complex interplay between bacteria and the human host requires characterisation of the interactions that occur between bacteria and receptors expressed by cells of the immune system. These interactions are pivotal in defining the ensuing immune response and, in turn, determine whether the infecting bacterium will be cleared and at what cost. This is important because in some instances the damage inflicted to host tissues by the immune system in order to clear an infection can pose more risk to organ function than the infection itself. Negative regulators of immunity are built into the immune response to ensure that damage to tissues is minimised. In line with this idea we hypothesise that engagement of DC-SIGN and MR by P. aeruginosa could explain the difficulties encountered by the immune system in clearing P. aeruginosa in susceptible individuals once infection is established. These lectin receptors do not directly gear the immune system towards activation. In particular DC-SIGN has been shown to act as an inhibitor and it is plausible to expect that by engaging DC-SIGN, P. aeruginosa is promoting an ineffective immune response that results in persistent chronic bacterial infections and an ongoing inflammation that can damage the host. This scenario has been observed in cystic fibrosis patients.
Our approach is novel in two instances. First it consolidates the concept of carbohydrates being important in the recognition of bacteria by human immune cells. This is a poorly studied area, lectin receptors have been largely neglected when studying the detection of P. aeruginosa infection by the human host. Our current results strongly support an important role for DC-SIGN and, to a lesser extent, MR, in sensing P. aeruginosa infection. Secondly our study includes an investigation of the two major forms of P. aeruginosa growth: in suspension (planktonic) and as biofilms. Planktonic cells are traditionally used to study the ability of bacteria to cause infections and their susceptibility to immune cells and antibiotics. Planktonic cells are considered to model the initial stages of infection (acute). In contrast biofilms consist of bacteria embedded within a 'slime city' composed of extracellular material rich in carbohydrates and make the bacteria highly resistant to immune attack and antibiotic treatment. Biofilms represent a major challenge for clinicians and health practitioners as they are responsible for chronic P. aeruginosa infections Biofilms have been observed, for example in wounds, associated with implanted medical devices and in the airways of cystic fibrosis patients. Our results show that both DC-SIGN and MR recognise P. aeruginosa biofilms while only DC-SIGN binds planktonic P. aeruginosa. Thus, different sets of lectin receptors are involved in the recognition of P. aeruginosa which stresses the importance of studying both planktonic cultures and biofilms with respect to immune recognition. Our experimental approaches will investigate the consequences of DC-SIGN and MR engagement by P. aeruginosa biofilms and planktonic cells as a means to identify a novel Achilles' heel in the infection process of this bacterium that is responsible for life threatening infections in susceptible individuals, particularly individuals with cystic fibrosis, wounds/burns and immunocompromised patients.

Technical Summary

Chronic P. aeruginosa (PA) infections are underpinned by the formation of biofilms. Biofilms consist of an extracellular matrix containing two main carbohydrate polymers, Psl and Pel. Our recent results show that two major lectin receptors expressed by immune cells, DC-SIGN (CD209) and the mannose receptor (MR, CD206), interact with PA biofilms. These findings suggest that biofilms do not just provide an effective barrier against immune attack but could also modulate immune cell activation through engagement of lectin receptors. For MR, biofilm binding is largely mediated through recognition of Psl. DC-SIGN also recognises Psl but in addition binds directly to PA cells independently of the presence of Psl or Pel. Our preliminary results strongly suggest that the common polysaccharide antigen (CPA) LPS, shared by all PA serotypes, is the DC-SIGN ligand in planktonic PA. We hypothesise that engagement of lectin receptors by PA (MR by biofilms and DC-SIGN by planktonic cells and biofilms) provides an advantage to PA by modulating immune cells towards a regulatory phenotype, hence facilitating the establishment of chronic infections.
The overall aim of this proposal is to determine the contribution of MR and DC-SIGN to immunity against PA. In objectives (1) and (2) we will dissect how MR and DC-SIGN engagement by PA biofilms and planktonic cells contributes to human myeloid cell activation. For this we will use (i) PA biofilms deficient in Psl or Pel, (ii) DC-SIGN-binding and non-binding planktonic PA and (iii) human monocyte-derived and dermis-derived MR and DC-SIGN expressing cells. In objective (3) we will employ cystic fibrosis samples (3.1) and huDC-SIGN transgenic mice (3.2) to assess the specific contribution of DC-SIGN to PA pathogenesis. This study will unveil novel mechanisms exploited by PA to modulate immune responses which may ultimately lead to the design of therapies aimed at enhancing the effectiveness of anti-PA immunity.

Planned Impact

The primary objectives of this project are the acquisition of new knowledge, scientific advancement and research capacity building through training. The project has clearly defined tasks and milestones and will lead to new insights into processes driving the establishment of chronic bacterial infections of significance beyond P. aeruginosa (PA). We have discovered that two major carbohydrate-binding (lectin) receptors expressed by cells of the immune system, DC-SIGN (CD209) and the mannose receptor (MR, CD206), interact with PA biofilms (DC-SIGN and MR) and planktonic cells (DC-SIGN) through recognition of surface carbohydrates including the exopolysaccharide Psl and the common polysaccharide antigen (CPA) LPS. Human pathogens associated with biofilm development also include species of Enterococcus faecalis, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, and Enterobacter spp. and we anticipate that exopolysaccharides from biofilms from these ESKAPE organisms could contribute to immune modulation through selected engagement of immune lectin receptors.
PA is responsible for 51,000 healthcare-associated infections in the US each year. In Cystic Fibrosis (CF) nearly 40% of patients are chronically infected by PA in lower airways by the time they reach early adulthood (estimated 9,500 in US and 15,000 in EU). Within the large bronchiectasis population >15% are likely to be chronically infected with PA. A recent evaluation in a US study estimated that the mean annual per-patient costs following initial PA infection increased by an estimated $18,516, with significantly increased hospitalization costs upon entry to an ICU. Innovative strategies for the development of novel therapeutics against PA infection are required to tackle the burden this pathogen poses to the healthcare system.
This research area is at an early developmental stage and further work on the fundamental biology of the interaction of PA and human lectins is required to fully ascertain its therapeutic potential. Nevertheless our work will have short term impact as follows: 1) The previously unappreciated involvement of lectin receptors, in particular DC-SIGN, during PA infection offers a window of opportunity to identify novel therapeutic targets related to this process. In particular, the requirement for the common polysaccharide antigen LPS in PA for DC-SIGN binding (Objective 2) suggests that DC-SIGN binding is a property common to all PA serotypes and agents targeting this process could have general application. Pathways with therapeutic potential include the synthetic pathway(s) leading to the presence of DC-SIGN ligands in planktonic PA; use of DC-SIGN-based chimaeric proteins to target PA for clearance by complement or phagocytes and modulation of DC-SIGN-mediated signalling pathway in immune cells. 2) Assessment of DC-SIGN (and mannose receptor) expression and their link to clinical characteristics in CF patients (Objective 3.1) could provide a novel tool to determine disease progression in this patient group. This approach could be extended to other diseases where changes in monocyte phenotype have been linked to disease outcome. 3) Analysis of PA infection in the hu DC-SIGN transgenic animals (Objective 3.2) will determine the potential for this model to provide a better representation of PA infection process. In addition to its value as a tool to learn about PA pathogenesis, the hu DC-SIGN transgenic mice could be exploited as a novel research platform to test novel anti-microbials targeting PA infection. This model will enable improved testing of bacterial susceptibility to antibiotics, development and testing of therapeutics aimed at blocking biofilm formation and quorum sensing and facilitate research into anti-microbial resistance in a setting more relevant to human disease.
 
Description National Biofilms Innovation Centre
Amount £12,801,513 (GBP)
Funding ID BB/R012415/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 12/2017 
End 11/2022
 
Title Analysis of the effect of purified Psl on human dendritic cells 
Description Immunophenotyping panel for flow cytometric analysis of human DCs and macrophages set up. Markers: MR, DC-SIGN, HLA-DR, CD11b, CD80, CD86, CD14. Also included Zombie NIR (a fixable live/dead stain). Established an assay to determine human DC responses to purified Psl. Preliminary data suggest that there is a decrease in DC-SIGN (and to a lesser extent, MR) surface expression on DCs treated with 50 µg/ml Psl for 6 h. This may indicate internalisation of DC-SIGN due to it binding Psl. Cytokine data from an initial experiment showed that Psl induced upregulation of TNF-alpha, IL-6, IL-10, and IL-1beta by DCs treated with 25 µg/ml Psl for 48 h.7. U937 cells (monocyte-like cell line) with and without DC-SIGN have been obtained, expanded, and DC-SIGN staining on them has been optimised for flow cytometry and fluorescence microscopy. Three new batches of Psl have been prepared. Characterisation and purification of these is on-going. This is a lengthy process due to the numerous steps involved in obtaining sufficiently large quantities of sterile, highly pure Psl, free from protein, lipid, nucleic acid, and LPS contamination. 
Type Of Material Model of mechanisms or symptoms - in vitro 
Year Produced 2017 
Provided To Others? No  
Impact This assay will enable us to determine if the biofilm-associated carbohydrate Psl influences the activation status of immune cells (dendritic cells, in particular) which will inform on the mechanisms enabling establishment of chronic infection by P. aeruginosa. 
 
Title Characteristion of huDC-SIGN-Tg mice 
Description We imported huDC-SIGN-Tg animals from USA (Dr Jordi Ochando's laboratory). Animals were health screened and found clean. They are being genotyped by TransnetYX to determine presence of transgene and presence of human DC-SIGN has been detected in the outer marginal zone of the spleen by immunolabelling. 
Type Of Material Model of mechanisms or symptoms - non-mammalian in vivo 
Year Produced 2018 
Provided To Others? No  
Impact This model will enable us to test if the presence of DC-SIGN influences the outcome of P. aeruginosa infection (Part of Objective 3) 
 
Title Generation of Biofilms in flow chambers 
Description We have developed robust methodology to generate P. aeruginosa biofilms in flow chambers suitable for microscopic examination. This will enable us to investigate the interaction of human immune cells with bacteria biofilms in real time 
Type Of Material Model of mechanisms or symptoms - in vitro 
Year Produced 2018 
Provided To Others? No  
Impact There is little understanding of the consequences of bacterial biofilms on immune cells. Our methodology using a laboratory strain of Pseudomonas aeruginosa (PAO1) will be applicable to clinical isolates and enable the analysis of macrophages, dendritic cell and neutrophil behaviour upon interaction with biofilms. We are able to detect deposition of the polysaccharide Psl, DNA and bacteria within biofilms. 
 
Title Whole blood cultures 
Description Establish culture conditions to analyse monocyte/macrophage phenotype in the context of whole blood. GM-CSF + IL-4 treatment of whole blood for 24 h gave the greatest upregulation of MR and DC-SIGN expression. Immunophenotyping panel set up to allow identification of monocytes (CD45+CD14+ cells) and assess the expression of MR, DC-SIGN, HLA-DR, and CD11b on them. Approval in place for CF pilot study to assess MR and DC-SIGN in whole blood from 10 patients and 10 healthy controls. First patient to be recruited w/c 26th February 2018. 
Type Of Material Model of mechanisms or symptoms - human 
Year Produced 2017 
Provided To Others? No  
Impact This assay will be employed to determine the activation status of monocytes in Cystic Fibrosis patients both in fresh samples and In samples cultured for 24 hours (Part of objective 3). 
 
Description Stall at Festival of Science and Curiosity-17th February 2018 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact We prepared a display to inform the public about phagocytes (macrophages and neutrophils) and how their action was improved by antibodies. This was linked to literature about the benefits of vaccination (based on BSI booklet). We showed a published phagocytosis movie and our own movie showing human neutrophils eliminating bacteria from a biofilm. We also had material to illustrate how antibodies can neutralise pathogens by blocking attachment to cells (neutralisation) and an activity to explain why flu vaccination needs to be done every year.
Year(s) Of Engagement Activity 2018
URL http://www.nottsfosac.co.uk/