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

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.

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.

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.