Date on Master's Thesis/Doctoral Dissertation

5-2015

Document Type

Doctoral Dissertation

Degree Name

Ph. D.

Department

Microbiology and Immunology

Degree Program

Microbiology and Immunology, PhD

Committee Chair

Jonsson, Colleen B.

Committee Co-Chair (if applicable)

Bodduluri, Haribabu

Committee Member

Kosiewicz, Michele

Committee Member

Lukashevich, Igor

Committee Member

Shirwan, Haval

Committee Member

Suttles, Jill

Subject

H1N1 influenza; Cell culture

Abstract

Influenza A virus (IAV) subtypes and even genotypes within subtypes can show differences in tropism (host, cell type), magnitude of infection, immune response and progression of illness. My dissertation focused on the development and use of two in vitro physiologically-relevant human cell culture models, well-differentiated normal human bronchial epithelial (wdNHBE) cells and human monocyte-derived macrophages (MDM) for the study of early IAV-host interactions. These models have given new insight into early host responses to seasonal H1N1 (BN59) and two pandemic A(H1N1)2009 viruses or H1N1pdm herein. The H1N1pdm are clinical isolates from a fatal (A/KY/180/10) and nonfatal (A/KY/136/09) case. In the wdNHBE model, KY180 showed a significantly higher titer as compared to the other two viruses at 24 hpi (hours post-infection). Interestingly, by microarray analysis, there were no significant differences in the host genome-wide expression intensity profiles of each virus following infection. Soluble cytokine measurements revealed increased apical and basal pro-inflammatory cytokine secretion overtime. A key finding from our data was greater basolateral secretion of cytokines (IL6, CCL5, CCL4 and CCL2) by KY180-infected wdNHBE cells. This finding suggests that the basolateral signals from infected epithelial cells may differ in their potential for recruitment and responses elicited by recruited monocytes/macrophages. In the second model, I used an in vitro model of recruited “resting” MDMs to study virus-host interactions of the clinical H1N1pdm isolates. These viruses replicated in MDM albeit inefficiently. While titers were similar and remained relatively low for all isolates, pro- and anti-inflammatory expression levels differed markedly between KY180 as compared to KY136 and BN59. KY180 had delayed expression at 8 hpi of pro-inflammatory genes (CCL5, TNF, IFN, CXCL10). This apparent delay in response to KY180 depended on the mode of viral entry. For KY180, this occurred primarily through macropinocytosis, mapping to the HA1 gene. In summary, my studies reveal subtle, yet important differences in IAV-host interactions that result in alterations of immune signaling in epithelial and macrophage cell culture models. Continued advancement of the in vitro human cell culture models for the study of IAV is important as they will allow mechanistic insight into the intricate biology of these viruses.

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