Date of Award

January 2017

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biomedical Sciences

First Advisor

Jyotika Sharma

Abstract

Sepsis is one of the oldest and most elusive syndromes in medicine. It is characterized by a systemic inflammatory response leading to acute organ dysfunction. In the United States alone there are over 750,000 cases of sepsis each year, with a mortality rate between 20-50%. Sepsis is the second leading cause of death in the ICU and the tenth leading cause of death overall in the United States. Over half of all ICU resources are consumed in the treatment of sepsis. There are currently no effective therapies available against sepsis, all attempts to develop an effective therapy have failed. Respiratory infections are the leading cause of sepsis, leading to what is called pneumonic sepsis. The Gram-negative bacteria Klebsiella pneumoniae (KPn) is associated with upwards of 20% of all pneumonic sepsis cases. Utilizing a mouse model of pneumonic sepsis induced by intranasal infection of KPn, I studied the role that mammalian C-type lectin receptors (CLRs) play in the development and clearance of pneumonic sepsis. CLRs are primarily expressed on phagocytic immune cells of myeloid origin and are considered pattern recognition receptors that have the ability to shape the immune response by recognizing both pathogen associated molecular patterns and host damage associated molecular patterns in many different pathological conditions. From the pathogen perspective, I studied the effect KPn has on the immune cells and how that may impact the ability of KPn establish the infection.

In my first project, I examined the role of two CLRs, Macrophage Galactose Type Lectin 1 (MGL1) and Clec4e otherwise known as Mincle, in the development and clearance of pneumonic sepsis caused by KPn. This project was built on previous observations made by my advisor Dr. Jyotika Sharma which showed these two CLRs upregulated in the lung during KPn induced pneumonic sepsis. In regards to MGL1, I am the first author on the paper that we recently published in the Journal of Immunology “Macrophage Galactose-Type Lectin-1 Deficiency Is Associated with Increased Neutrophilia and Hyperinflammation in Gram-Negative Pneumonia” (J Immunol. 2016 Apr 1;196(7):3088-96. doi: 10.4049/jimmunol.1501790.). In this paper we showed that MGL1-/- mice were more susceptible to KPn infection, a phenotype that did not correlate with the systemic and local bacterial burden; the ability of macrophages and neutrophils to phagocytose and kill bacteria or neutrophil NETs. We demonstrated that the mechanism underlying the increased mortality of MGL1-/- mice was increased ability of neutrophils to infiltrate the lungs causing their overwhelming accumulation and severe neutrophil-mediated pathology in the lungs in the absence of MGL1. Mechanistic insights into the potential negative regulatory function of MGL1 in neutrophil infiltration, their clearance by efferocytosis as well as the process of granulopoiesis are some of the future directions of this work that are being perused in the laboratory. In the paper I co-authored on Mincle, “Protective Role of Mincle in Bacterial Pneumonia by Regulation of Neutrophil Mediated Phagocytosis and Extracellular Trap Formation” (Infect Dis. (2014) 209 (11): 1837-1846.doi: 10.1093/infdis/jit820), we showed that Mincle mediates two important bacterial clearance mechanisms i.e. bacterial phagocytosis and extracellular trap (NET) formation by neutrophils. As a result, Mincle-/- mice are more susceptible to KPn infection as compared to the Mincle sufficient wild-type mice. This project is being led by Dr. Atul Sharma, a postdoctoral fellow in the lab.

My second project examined the effect of KPn infection on the clearance of neutrophils by a process called efferocytosis. While performing experiments for the role of MGL1 in efferocytosis, I found that in comparison with the uninfected neutrophils, KPn infected cells were engulfed less efficiently by macrophages. As efferocytosis is a receptor mediated process, I discovered that while KPn infection increases the expression of repressive molecules called “Don’t Eat Me” signals on neutrophils, it is the modulation of distribution of a key “Eat Me” signal, phosphatidylserine (PtdSer) and the subsequent delay of apoptosis in neutrophils that is partially involved in KPn mediated inhibition of efferocytosis. Accordingly, KPn infected neutrophils also induce an alternative rout of programmed cell death called necroptosis. It is the combination of the “eat me” signal downregulation and the induction of necroptosis in KPn infected neutrophils that inhibit their efferocytic uptake by macrophages. A part of this data has been communicated for publication. Current work that I have led in the lab on this project involves determining what signaling mechanisms KPn is activating to decrease PtdSer as well as activation of necroptosis to inhibit the efferocytosis of infected PMNs.

The overarching goal of this work was to increase the overall knowledge on the role CLRs play in pneumonic sepsis in terms of neutrophil turnover and how KPn subverts these processes to its own advantage. I believe that this knowledge can be used to identify novel targets for effective therapy of sepsis as well as other inflammatory conditions.

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