Unravelling the Mechanism of Bactericidal/Permeability-Increasing Protein Expression during Bacterial Pathogenesis
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Anti-microbial proteins (AMP) are the key effector arm of the innate immune system. The prevalence of AMP in single-celled eukaryotes to humans shows its importance during the course of evolution. The first report for the role of the anti-microbial peptide in clearing infection was given by Alexander Fleming in 1990’s through the discovery of Penicillin and Lysozyme. The search for antimicrobial agents in human granulocytes was begun by Ehrlich in 1870’s but the first successful isolation of an antimicrobial agent from rabbit neutrophils was done by Zeya and Spitznagel in 1969. Later work by Peter Elshbach and his group on AMPs in rabbit neutrophils brought to light an AMP that can increase the permeability of the bacterial membrane. This AMP named as Bactericidal/permeability-increasing protein (BPI) was further isolated from human neutrophils. Since then many studies have been carried out to understand the mode of action of BPI, which culminated in understanding the new functional activity of this protein viz opsonisation, LPS neutralization and anti-angiogenic function. Knowing to the role of BPI as an anti-inflammatory agent, multiple studies have tried to use BPI for treating endotoxic shock. Dysregulation of BPI expression is associated with various inflammatory diseases like Crohn’s Disease (CD), Ulcerative colitis (UC) and Infectious enteritis’s. Mutations in BPI are also linked to susceptibility to various infections. Even though there are several studies focusing on the functional aspects of BPI, the regulation of BPI expression is poorly understood. Knowing the clinical importance of dysregulation of BPI, it is vital to understand the regulation of BPI expression during the course of bacterial infection. The Thesis is divided into four chapters. As the main aim of this study is to understand the regulation of BPI expression, in Chapter 1 we introduce the known facts about the protein. A brief overview of the mode of action and regulation of BPI is discussed in this chapter. The subsequent sections describe the diseases associated with Dysregulation of BPI and the use of BPI as a therapeutic agent in various diseases. Towards the end, the objective of the present study is discussed. BPI is primarily known to be expressed in human neutrophils and epithelial cells. Previous studies have shown that among innate immune cells, murine BPI is expressed only in dendritic cells and neutrophils, but not in macrophages. Based on these results, it was presumed that BPI is not expressed in human macrophages. In Chapter 2, we report the presence of BPI in human macrophages. Our studies revealed increased expression of BPI in human macrophages stimulated with various PAMPs (Pathogen-associated molecular patterns) viz., LPS, flagellin as well as during bacterial infection. Further, during the course of an infection, BPI interacted with Gram-negative bacteria, resulting in enhanced phagocytosis and subsequent control of the bacterial replication. However, it was observed that bacteria which can maintain an active replicating niche (Salmonella Typhimurium) avoid the interaction with BPI during later stages of infection. On the other hand Salmonella mutants, which cannot maintain a replicating niche, as well as Shigella flexneri, which quit the endosomal vesicle, showed interaction with BPI. BPI was induced in both M1 and M2 differentiated macrophages suggesting its role in limiting Gram-negative bacteria and parasitic infection. These results propose an active role of BPI in Gram-negative bacterial clearance by human macrophages. This chapter concludes with a discussion on the importance of BPI expression in human but not murine macrophages. The importance of maintaining an active replicating niche by STM to evade interaction with BPI is also discussed. As the first line of defense against invading pathogens, intestinal epithelium produces various antimicrobial proteins (AMP) that help with clearance of pathogen. The precise mechanism of AMP regulation in intestinal epithelium is not clear. Intestinal epithelium being a primary entry point for various pathogens, we tried to understand the regulation of BPI expression in the intestine during the course of bacterial infection. In Chapter 3, we report a direct correlation between intestinal damage and BPI expression. In Caco-2 cells, we see a significant increase in BPI levels upon membrane damage mediated by S.aureus infection and pore-forming toxins (Streptolysin and Listeriolysin). Cells detect changes in potassium levels as a Danger-associated molecular pattern (DAMP) associated with cell damage and induce BPI expression in a p38 dependent manner. These results are further supported by in vivo findings that BPI expression in the murine intestinal epithelium is induced upon infection with bacteria which cause intestinal damage (Salmonella Typhimurium & Shigella flexneri) whereas mutants which don’t cause intestinal damage (STM fliC & STM invC), didn’t induce BPI expression. These findings have a huge impact on our current understanding of AMP response during inflammatory bowel diseases (IBD). Our results suggest that dysregulation of BPI expression might be an effect rather than a cause of IBD. This chapter concludes with a discussion on the importance of potassium efflux associated with membrane damage as an important signal that helps in discriminating the invading pathogen from the pool of gut microflora. Bactericidal/permeability-increasing protein had been shown to possess anti-inflammatory and endotoxin neutralizing activity by interacting with LPS of Gram-negative bacteria. Even though rBPI (recombinant BPI) has cleared phase III clinical trials for treating endotoxemia, the high cost of purified BPI provided by pharmaceutical companies makes it inaccessible or unavailable for the common man. In Chapter 4, we examined the feasibility of using murine BPI (mBPI) expressed on halophilic Archaeal gas vesicle nanoparticles (GVNPs) for the treatment of endotoxemia in high-risk patients, using a murine model of D-galactosamine-induced endotoxic shock. Halobacterium sp. NRC-1 was used to express the N-terminal 199 amino acid residues of mBPI fused to the GVNP GvpC protein, and bound to the surface of the haloarchaeal GVNPs. Our results indicate that delivery of mBPIN-GVNPs increase the survival rate of mice challenged with lethal concentrations of lipopolysaccharide (LPS) and D-galactosamine. Additionally, the mBPIN-GVNP-treated mice displayed reduced symptoms of inflammation including inflammatory anemia, recruitment of neutrophils, liver apoptosis and pro-inflammatory serum cytokine levels. This chapter concludes with a discussion of the advantages of using mBPIN-GVNPs over purified protein in treating endotoxic shock.