Leprosy is an ancient infectious scourge, caused by a microscopic organism called Mycobacterium leprae. This organism can live for years inside human cells, and has developed various, but poorly understood, mechanisms for escaping human immune defenses. In the absence of antibiotic treatment, a large proportion of leprosy patients suffer lifelong debilitating disease where the mycobacteria are able to reproduce to very high numbers. In these patients, defenses against other infections appear to be normal, so the immune system has a specific “blind spot” with regard to Mycobacterium leprae only. If we were able to understand how leprosy avoids immunity, we might be able to learn new ways to specifically shut down the immune response where it is unwanted, as in autoimmune diseases such as multiple sclerosis and diabetes.
We think that an important way in which these mycobacteria either avoid recognition by the immune system, or, if recognized, trick the system into making an ineffectual response, is by modifying the actions of a key set of immune cells called dendritic cells. Dendritic cells act as immune gatekeepers, strategically located in those parts of the body that interface with the environment. In a properly functioning immune response, upon contact with infectious organisms, these cells respond by moving into areas where other immune cells, called T cells, are concentrated, and generating various signals which “instruct” the T cells to seek, recognize, and destroy cells infected with the offending organism.
To test the idea that in leprosy patients, there is a defect in the function of dendritic cells in response to Mycobacterium leprae compared to other triggers of dendritic cell activity, we first developed a method for rapidly measuring dendritic cell function in the blood of patients, where dendritic cells are present in very low concentrations. Our technique involved the direct testing of dendritic cell function in a drop of blood taken from a patient, with a minimum of manipulation, which we think most closely reflects the way these cells might function in the body. Having developed this approach, we tested the blood of 11 leprosy patients and 11 controls, and observed no differences in the function of these cells. We did, however, note that the degree to which dendritic cells from both patients and controls were “excited” by Mycobacterium leprae was modest compared to other stimuli, including other types of mycobacteria. This led us to conclude that there was no obvious intrinsic defect in the dendritic cell response in leprosy patients, but that the mycobacterium itself might have ways of subverting the function of normal dendritic cells. We next tested another idea, that Mycobacterium leprae is able to subvert dendritic cell function by inserting some of its chemicals into the surface of dendritic cells in such a way that the T cell instructing properties of the dendritic cell are changed. We demonstrated that a chemical from the mycobacterium is indeed inserted into the dendritic cell surface, in such a location that it would be strategically positioned to affect an interacting T cell. Furthermore, we found that this chemical activates immune proteins that circulate in the blood that may further deviate the responses of T cells.
These findings represent a novel mechanism of immune escape by an infectious organism and suggest new approaches to developing therapies that could be used to influence immune function.
Mycobacterium leprae (M. leprae), the cause of leprosy, is an intracellular bacterium with a striking ability to elude effective Th1 immunity in patients who have otherwise normal immune function. To test whether this defect results from a specific failure of dendritic cell (DC) activation in leprosy patients, we developed a rapid flow cytometric method that enabled us to examine the function of both plasmacytoid and myeloid DCs in the peripheral blood of 11 leprosy patients and 11 demographically matched controls. We found no difference in the expression of the maturation markers, CD80, CD83, CD86 and CCR7, nor in the expression of the cytokines, tumor necrosis factor-α and interferon-α, following stimulation with lysates of M. leprae, M. tuberculosis, and generic ligands for TLR 2, 4, 7, 8 and 9. Thus, no intrinsic defect in DC function was observed in leprosy patients. However, we did note very modest responses of DCs to M. leprae lysates compared to M. tuberculosis lysates and other stimuli. Since M. leprae is known to contain multiple TLR2 ligands, we hypothesized that the organism is able to suppress the response of DCs to TLR signaling. In further experiments, we showed that a complex glycolipid called phenolic glycolipid-1 (PGL-1), which comprises up to 2% of the mass of M. leprae, is inserted into the lipid rafts of human dendritic cells infected with M. leprae. Since TLRs are co-located in lipid rafts, PGL-1 is strategically inserted to potentially interfere with the function of these receptors. We next demonstrated that M. leprae-infected, but not M. bovis-infected DCs are able to activate complement present in the serum of both leprosy patients, and to a lesser extent, healthy controls. This process was calcium and not magnesium-dependent, consistent with complement activation via either the classical or mannose-binding lectin pathway. This activation results in the colocalization of C3 and PGL-1 in the lipid rafts of the dendritic cell plasma membrane. This finding is consistent with previously published work demonstrating complement activation by PGL-1. The presence of C3 in lipid rafts of M. leprae-infected DCs suggests a mechanism by which DCs may subvert acquired immunity by engaging the complement receptor CD46 during T cell priming, a process that is known to stimulate potentially immunosuppressive IL-10 secreting regulatory T cells.
We conclude that M. leprae has the potential to subvert T cell responses by multiple mechanisms that include complement-dependent stimulation of regulatory T cells, as well as the physical disruption of TLR signaling by insertion of PGL-1 into lipid rafts.