The use of crd-fc proteins as artificial opsonins to enhance bacterial killing

The inflammatory response associated with bacterial infection does not only affect the well-being of an animal, but also decreases production parameters in food-producing animals. Treatment of bacterial infections with new antibiotics becomes more and more problematic due to the increase in multi-drug resistant bacteria. The current project-proposal aims to use cells of the innate immune system, polymorph-nucleated neutrophils (PMNs) and dendritic cells (DC) in conjunction with recombinant carbohydrate recognition domains (CRD) of C-type lectin receptors (CLR) as 'artificial' opsonins to enhance bacterial phagocytosis. The project would therefore combine the potential development of a new treatment-strategy, which is of interest for the pharmaceutical partner, as well as increase our understanding of the interaction of PMN and DC in the bovine (ruminant) system. PMNs are well known for their ability in innate immunity to instantly kill pathogens when they invade tissues. However, evidence indicates that PMNs can also directly play a role in adaptive immunity by directly instructing DC and T cells. Upon inflammation, PMNs can travel from the site of infection to the nearest lymph node, where they undergo apoptosis and are taken up by DCs. As a consequence, DCs can present PMN-derived antigens to T cells. In addition, PMNs have been demonstrated to acquire antigen-presenting functions themselves and can directly transfer antigens to DCs. Interestingly, both, PMNs and DC express a variety of receptors for antigen-uptake on their surface, including receptors for the recognition of the Fc-part of antibodies (CD16, CD32, CD64, respectively). These receptors are critical surface receptors for facilitating phagocytic movement of antibody-opsonized particles, and ingestion through pathways affecting cytoskeletal reorganization. In addition to these, DC (and to a certain degree neutrophils) express CLRs that are involved in the recognition and capture of many glycoproteins of pathogens. These CLRs serve as antigen receptors allowing internalization and antigen presentation, but also function as adhesion and/or signalling molecules. The expression of CLRs is very sensitive to maturation stimuli, leading to down-regulation as DCs mature. CLRs such as DC-SIGN and Dectin-1 recognize high-mannose-containing structures expressed on bacteria such as mycobacteria and streptococci. It was recently demonstrated that glycan modification of antigen can strongly enhance MHC class I responses and the induction of antigen-specific cytotoxic T-lymphocytes, indicating that glycosylated antigen targets CLRs to enhance antigen-specific T-cell responses. Moreover, these CLRs induce signalling processes in DCs and specific cytokine responses in combination with Toll-like receptor triggering. This implies that specific CLR-targeted antigens can regulate T-cell polarization. We have recently described the presence of Dectin-1 and DC-SIGN in the bovine system, and have shown that these bind several bacteria. Our hypothesis is that soluble chimeric proteins consisting of the CRD of a CLR and the Fc-part of an IgG molecule will enhance phagocytosis of bacteria by PMNs, leading to stimulation of DC and T cells. To test the hypothesis, the student will assess the potential of CRDs as 'artificial' opsonines by: 1) Cloning, sequencing and expressing CRD domains of murine/human/bovine dectin-1/DC-SIGN as Fc-tagged proteins 2) Assessing the functionality of CRD-Fc proteins by assessing their ability to opsonise bacteria and to induce increased phagozytosis in different cell-types 3) Assessing effects of CRD-Fc protein opsonised bacteria on PMN function 4) Assessing the ability of PMNs to transfer phagocytised bacteria to DC and to mount a subsequent CD4 or CD8 response