Study the modulation of share machinery in macrophage in response to LPS

Abstract

Human body is regularly under attack by various pathogens as it provides optimal environment for growth of pathogens. To defend against these pathogens and their toxic compounds, our immune system has developed an intricate method of recognition and responses. Recognition of antigens by T-cells requires uptake, processing and presentation of pathogen-derived antigens on MHC of APCs. While the pathways of antigen processing and presentation in APCs have been defined, not much is known about the molecular mechanisms and regulation of intracellular traffic involved in antigen processing and presentation pathways in response to pathogens. SNAREs are the proteins that play crucial role in vesicle trafficking in cells and therefore in all immune processes involving exocytosis of inflammatory mediators and uptake and killing of pathogens, also in transport, expression and downregulation of various receptors. So the main aim of our research was to study the modulation of expression of SNARE proteins against LPS challenges. MH-S cell line was choosen as the model for our study as it is a macrophage cell line which is capable of phagocytois and antigen presentation of various pathogens as well as exocytosis of various cytokines produced against the pathogens. We standardize RT-PCR, real-time PCR in term of various parameters and using those standardized condition the modulation of the SNARE proteins was studied in presence of LPS. In order to study the modulation of expression of SNAREs at protein level, western blotting was done. We found that expression of the SNAREs proteins studied was not modulated by LPS treatment pointing to some other mode of regulation of SNARE mediated intracellular traffic in response to LPS challenge in macrophages. Key words: LPS, SNAREs, RT-PCR, Real-time PCR, Western blotting, MH-S cell lines