Date of Award

2016

Document Type

Thesis

Degree Name

Master of Science (MS)

Department

Microbiology and Immunology

College

College of Graduate Studies

First Advisor

Xian Kui Zhang

Second Advisor

Tamara Nowling

Third Advisor

Amanda C. LaRue

Fourth Advisor

Bei Liu

Fifth Advisor

Laura Kasman

Sixth Advisor

Natalie Sutkwoski

Abstract

The innate immune system is the primary line of defense to protect the host from pathogen infection. Mammalian cells produce inflammatory cytokines and chemokines in response to innate immune signals and their expression is tightly regulated. IFN-gamma Inducible Protein (IP-10), also known as C-X-C motif chemokine 10 (CXCL10), is an inflammatory chemokine belonging to the CXC chemokine family. CXCL10 is chemotactic for many inflammatory cells, including macrophages, and altered expression of CXCL10 is associated with inflammatory diseases, including lupus nephritis and other autoimmune diseases. The Fli-1 transcription factor is a member of the Ets gene family and regulates the immune response, along with other cellular processes including its role in the pathogenesis of renal injury and systemic lupus erythematosus (SLE). Previous data has shown that Fli-1 heterozygous NZM2410 mice, a murine model of lupus with decreased Fli-1, had significantly decreased infiltration of inflammatory cells including macrophages in kidney. Similarly, in MRL/lpr lupus mice with decreased Fli-1, Decreased T cell infiltrates and CXCL10 levels in the kidney was founded. We hypothesize that Fli-1 is a critical regulator in directly modulating the expression of CXCL10. In this study, Fli-1 protein expression in endothelial cells transfected with Fli-1 specific siRNA was significantly decreased compared to the expression of Fli-1 in cells transfected with control siRNA. Additionally, endothelial cells transfected with Fli-1 specific siRNA produced significantly lower amounts of CXCL10 compared to cells transfected with control siRNA after stimulation by Toll-like receptor (TLR) 4 ligand, lipopolysaccharide (LPS). Chromatin immunoprecipitation (ChIP) assay was performed to show that Fli-1 binds Ets binding sites (GGAA/T) within the mouse CXCL10 promoter. The human CXCL10 gene promoter was used to perform a transient transfection to determine that Fli-1 actively promotes transcription from the CXCL10 promoter. Mutation of the DNA binding domain of Fli-1 demonstrated that Fli-1 activates transcription of CXCL10 in both indirectly and directly ways, likely with the assistance of co-factors or post-transcriptional modifications. Together, the results indicate that Fli-1 is a novel, critical transcription factor in regulating the expression of the pro-inflammatory chemokine CXCL10 and provides a possible mechanism for the protective effect of decreased Fli-1 expression in lupus and other inflammatory autoimmune diseases.

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