Document Type

Thesis - University Access Only

Award Date

2012

Degree Name

Master of Science (MS)

Department / School

Biology and Microbiology

First Advisor

Radhey Shyam Kaushik

Abstract

Microbial infection initiates complex interactions between the pathogen and the host. Several immune cells of the host interact with pathogenic infectious agents through several immune-surveillance mechanisms and pathogen-a< ;sociated virulence factors. There are different groups of cells that protect the host from such infectious agents. One such group of antimicrobial warriors is macrophages. Macrophages play an important role in mediating innate immune responses to various pathogens. These cells also initiate specific defense mechanisms in adaptive immunity. Another group of cells that play a vital role in the pathogenesis of several enteric diseases is intestinal epithelial cells. Epithelial-pathogenic interactions induce epithelial innate immune responses to various enteric pathogens. Both epithelial and macrophages express various pathogen-recognition receptors (PRRs) such as toll-like receptors (TLRs) that recognize pathogen-associated molecular pattems (PAMPs) and induce signals to activate genes responsible for host defense. Some TLRs recognize viral ligands. These include TLR3 which recognizes viral dsRNA and TLR7 and 8 which recognize viral single stranded RNA. RIG-1 and MDA-5 proteins present in the cytoplasm can recognize dsRNA of viruses. Swine influenza vims (SIV) has been shown to infect both lung epithelial cells and alveolar macrophages. SIV causes significant morbidity in pig population and is an economically important disease of swine industry. Recently novel HlNl SIV caused severe respiratory disease in humans and World Health Organization (WHO) declared human novel HlNl SIV infections as pandemic in 2009. As there is no universal vaccine available for influenza viruses, it is important to study the innate immune responses to this vims for devising appropriate antiviral therapies. Recently two primary porcine macrophage cell lines (Cdelta2+ and Cdelta2-) had been described, and we obtained these cells lines in our lab. The aims of this project were to characterize porcine intestinal epithelial cells IPEC-J2 and porcine macrophage Cdelta2+ and Cdelta2- cell lines for the presence of virus specific TLRs, RIG-1 and MDA-5 expression, study the innate responses of these cells to viral ligands, determine the infectivity of macrophage cell lines to porcine influenza A and human influenza B strains, and study the innate responses of Cdelta2+ cells to SIV infection. Real time Reverse Transcriptase Polymerase Chain Reaction (real time RT-PCR) was employed to examine the expression of various PRRs, cytokines and chemokine genes in IPEC-J2, Cdelta2+ and Cdelta2- cells. These cell lines expressed PRRs and immune genes necessary for viral ligand recognition and innate responses. Then innate immune responses of both IPEC-J2 and Cdelta2+ cells to various viral ligands were determined at transcriptional level. These cells showed significant transcriptional changes for various TLRs and cytokines genes upon stimulation with Imiquimod. Poly IC and Poly IC with Lyovec. In the IPEC-J2 cells, TLR-2, TLR-6, RIG-I. MDA-5, NOD-1, IFN a, IL-la, -12p40, -6, -8, -15, -18, TNF-a, MCP-1 and beta-defensin-2 were up regulated at 3 hours following stimulation. TLR-4, IL-7 and beta-defensin-1 were down regulated Vl at 3 hours. At 24 hours. TLR-8, NOD-1 and beta-defensin-1 were up regulated. TLR-L - 5, RIG-1, NOD-2, IL-1 p, -12p35, -15, MCP-1 and beta-defensin-2 were down regulated at 24 hours. The innate immune responses to these viral ligands were quite different in the Cdelta2+ cell line. Anti-viral TLRs such as TLR-3, -7, -8, -9 as well as RIG-1, NOD- 1, IFN-a, -B, IL-la, -6, -12, -8. TNF-a and beta-detensin-1 and -2 were up regulated at 3 hours post stimulation. TLR-2, -4, -6. -9, MDA-5, NOD-2, IL-7, -15 and MIF were down regulated at 3 hours. At 24 hours, TLR-3, -8, -9, RIG-1, MDA-5, NOD-1, IL-la, - 12p40, -7, TNF-a, MCP-1, beta-defensin-1 and 2 were up regulated. NOD-2, IFN-a, -P, IL-IP.-6, -8, MIF and beta-defensin-1 were down regulated at 24 hours. The mRNA transcriptional levels induced by these viral ligands indicated that both IPEC-J2 and Cdelta2+ cells were able to mount innate immune responses against the viral ligands. As a first step to determine the infectivity of the Cdelta2+ and Cdelta2-cells to SIV, we checked the binding ofMaackia amurensis lectin-II (MAL-II) and Sambucus nigra lectin (SNA) for both cell lines. Both cell lines- Cdelta2+ and Cdelta2- showed 71.5±4.99% and 52±14.7% binding to SNA lectin and 22.75±14.14% and 27±19.01% binding to MAL-II lectin respectively. The next step was to check if Cdelta2+ and Cdelta2- cells were susceptible to SIV and if they do, then detennine if these viruses can replicate in these cells. The cytopathic effects of SIV on both cell types were observed following SIV infection. Immunoflouresc,ence assays were further used to confitm the infection of these cells with porcine Influenza A and human influenza B viruses. Flow cytometric analysis was used to measure the percentage of Maldin Darby canine kidney (MOCK) epithelial cells, Cdelta2+ and Cdelta2- cells that were infected by the porcine Influenza A and human influenza B strains. Hemagglutination assays were used to determine the replication rates of the Influenza A and B strains in these cells. The replication and infection rates of the Cdelta2+ and Cdelta2- cells were comparable to the MOCK cell line. Both Cdelta2+ and Cdelta2- had a large number of cells that were infected with HlNl Influenza A (37.94±12.35% and 11.82±4.49%) and Influenza B virus (34.52±8.33% and 13.67±4.43%) respectively. Although the number of cells infected wa5 less for Influenza B, both cell lines had higher replication rates with Influenza B as judged by hemagglutination titers. After detennining that these cells were susceptible to influenza infection and allowed viral replication, the Cdelta2+ cells were infected with porcine Influenza A and human influenza B strains for 6 and 24 hrs. The innate immune responses to porcine influenza A HlNl and human Influenza B vims were determined using real time RT-PCR, and the data were analyzed using Student's t -test. Both Influnza A and B viruses induced significant changes in various PRRs and cytokines. Further work could be conducted to determine if the various TL Rs and cytokines are significantly modulated at the protein levels by using immunohistochemistry and Enzyme -linked Immunosorbent Assay (ELISA). Concluding remarks: Both IPEC-J2 and Cdelta2+ cells express PRRs that can initiate innate and inflammatory responses against viral ligands. The Immunofluorescence assays~ flow cytometric analysis, hemagglutination assays and innate immune response studies following influenza infection clearly demonstrated that the Cdelta2+ cells may be a useful tool for studying the influenza virus pathogenesis and innate immune responses in macrophages of swine origin.

Library of Congress Subject Headings

Swine -- Virus diseases
Swine influenza
Immune response
Macrophages

Publisher

South Dakota State University

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