Avoidance of the immune response is an important aspect of the biology of many pathogens, and a wide variety of mechanisms have been defined. For Gram-positive bacteria, secretion of proteins belonging to the family of cholesterol dependent cytolysins (CDC) may lead to lysis of some cell types at high concentrations, but at lower concentrations may also sensitize cells such as macrophages to apoptosis-inducing stimuli by engagement of TLR4. This was recently reported for anthrolysin O, a CDC produced by B. anthracis, and for a number of other CDC family members. Stimulation of TLR however could be considered a double edged sword, since this might also stimulate the immune response, particularly when involving potent ARC such as dendritic cells (DC). We have investigated the effects of three CDC proteins, anthrolysin O, tetanolysin O and streptolysin O, and observed that human DC rapidly respond to treatment with non-toxic levels of these proteins by fluxing calcium and then secreting or shedding microvesicles (MV) that have on their surface class II MHC proteins. At the same time, DC lose the ability to up-regulate class II MHC in response to IPS. Based on these results, we hypothesize that CDC act to impair antigen presentation by DC by inducing secretion of MV containing class II MHC and other proteins important to antigen presenting function precisely at the time of pathogen encounter. Furthermore, we suggest that released MV may themselves have immunosuppressive properties, based on similarities to vesicles called exosomes, and that CDC toxins might be disseminated throughout the body and taken up by phagocytes, in this way impacting antigen presentation more widely than at the site of infection. We propose to further characterize these effects of a representative CDC protein, TLO, on the function of human monocyte-derived DC and to investigate the impact of TLO-induced MV on immune reponses in the following aims: 1. how TLO affects class II localization in moDC will be determined; 2. the protein contents of TLO-induced MV will be characterized; 3. the mechanism of MV production and uptake by cells will be probed; 4. the effect of TLO-induced and constitutively produced MV on CD4 and CDS T cell function will be determined. Lay statement; Understanding how toxins from bacteria affect the immune response could help devise new treatments for tetanus, streptococcal disease, and anthrax. ? ? ?
