Document Type

Thesis - Open Access

Award Date

2022

Degree Name

Master of Science (MS)

Department / School

Biology and Microbiology

First Advisor

Radhey Kaushik

Abstract

In response to a pathogenic attack, the host produces a series of defense mechanisms through various intracellular signaling pathways. The byproduct of these signaling pathways helps tackle the invading pathogen and protects the body from getting into a diseased state. This system is called the immune system. The immune system can be divided into two branches namely the innate immune system and adaptive immune system. The groups of immune cells that provide protection regardless of the pathogen specificity constitute the innate immune system. The system that acts according to the pathogen specificity is called the adaptive immune response. The production of antibodies by B cells is a prime example of adaptive immune responses. Macrophages, neutrophils, and monocytes are a few examples of innate immune cells. Besides them, mucosal epithelial cells of the intestinal and respiratory systems are crucial in generating innate immune responses. Invading pathogens and their recognition by the host is pivotal in preventing the subsequent infection and diseases. Epithelial cells express various Pathogen Recognition Receptors (PRRs). These PRRs recognize pathogen-associated molecular patterns (PAMPs) or damage-associated molecular patterns (DAMPs). The binding of PAMPs and/or DAMPs to epithelial cells initiates intracellular signaling pathways that lead to the generation of innate immune responses through the regulation of gene expression. Porcine respiratory epithelial cells and their expression of PRRs render them vital not only for the regulation of innate immune responses but also to study respiratory disease pathogenesis. Ironically, only a handful of studies can be found on these cells and the limited number of studies have hindered our understanding of the role of porcine respiratory cells in innate immunity. In this study, we have characterized previously established porcine primary respiratory epithelial cells from nasal turbinate and trachea followed by their immortalization using hTERT and SV40 large T-antigen. We also studied their innate immune responses to various bacterial and viral ligands. Both the primary and immortalized cells showed typical epithelial cobblestone morphology with a heavy expression of cytokeratin indicating epithelial origin. Cells did not change their morphological characteristics even after immortalization. Immortalization was confirmed by immunofluorescence assay for SV40 and immunocytochemistry for hTERT. However, they did look more granulated than the primary cells. Growth curve analysis showed a faster growth rate of immortalized cells in comparison to the primary cells of both nasal and tracheal origin. Finally, we stimulated the primary cells with various bacterial and viral ligands. Upon stimulation, porcine primary respiratory cells mounted innate immune responses through modulation of the expression of various PRRs and the production of cytokines/chemokines. Modulation of gene expression on mRNA level was measured using ΔΔct method. The research findings may be vital in studying the role of respiratory epithelial cells in the pathogenesis of various respiratory diseases and innate immune responses in pigs.

Number of Pages

176

Publisher

South Dakota State University

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Rights Statement

In Copyright