Understanding phagocytosis, one of the most common cellular degradative phenomenon, has significant implications in dentistry. P. gingivalis, a gram negative anaerobic bacillus, is a red complex periodontal pathogen due to its high association with severe periodontal disease. Macrophages, a critical component of the innate immune system, are likely some of the first responders to this bacterium. Understanding the interplay between macrophages and P. gingivalis can have significant impact on our understanding of the pathogenesis of periodontal disease and may impact future pharmaceutical interventions.
Macrophages degrade extracellular cargo through phagocytosis. In phagocytosis, specialized cells engulf apoptotic cells, pathogens or other debris internalizing the cargo in vesicles called phagosomes. Through fusion with lysosomes and other endosomes, phagosomes mature into degradative phagolysosomes. Evidence suggests that melanoregulin (MREG), a small highly charged cargo sorting protein, regulates intracellular trafficking and lysosomal intermediates.
The Boesze-Battaglia lab in the Biochemistry Department at University of Pennsylvania – School of Dental Medicine is investigating P. gingivalis’s journey upon phagocytosis by macrophages and how changes in MREG expression may influence this pathway. We specifically determine whether P. gingivalis can alter endosomal trafficking by effecting MREG expression. Evidence suggests that P. gingivalis escapes immediate degradation through colocalization with MREG and LC3II positive autophagosomes in murine macrophages. This could allow P. gingivalis to survive in nutrient-rich intracellular niches and may be a virulence factor.
The aim of this research project is to reduce MREG expression in human derived monocytes (THP1 cells) and use confocal microscopy to understand co-localization of P. gingivalis with other autophagosome markers. This study can be split into two parts: (1) knockdown of MREG expression; (2) perform colocalization studies with THP-1 transfected with P. gingivalis with reduced or normal levels of MREG expression.
Using lipofectamine 2000, I have been attempting to knockdown MREG expression in THP-1 Cells. The overall protocol involves differentiating THP-1 monocytes into macrophages, and then incubating cells in different concentrations of both the lipid vector and RNAi for different amounts of time. After treating the cells for 24Hr, 48Hr or 72Hr, protein from these cells is isolated and analyzed on a western blot to determine the success or failure of the specific treatment conditions.
So far lipofectamine’s lipid vector has proven cytotoxic for differentiated THP-1 cells. Cell death likely increases post incubation with the lipid reaching 90-100% by 72Hr. Although no stable knockdown protocol exists yet for MREG in THP-1 cells, my next steps are to continue varying the conditions until a stable repeatable protocol can be received providing sufficient MREG knockdown to proceed.
~Jaskaran Saggu, Pennsylvania ’16