Date of Award

2011

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Molecular Biology

Abstract

The differentiation of CD4+ T cells to different effector lineages in response to pathogenic stimuli is the core of the adaptive immune system. One of the effector subsets recently discovered is Thelper 17 (Th17) and it plays a predominant role in autoimmune diseases and inflammatory disorders.

In my thesis, I aimed to study the role of Notch cell surface receptors in Th17 differentiation. Using in vitro Th17 differentiation assays of human naïve CD4+ T cells, I have shown that Notch signaling, particularly Notch1, plays a crucial role in Th17 polarization. By using pharmacological inhibitors and specific knockdown of Notch1, I have shown that Notch1 is necessary for Th17 differentiation. Furthermore, IL-17 promoter assays and chromatin immuno-precipitation (ChIP) experiments revealed that IL-17 and ROR-γt are transcriptional targets of Notch1.

In this study I have also shown that Notch signaling also controls Th1 component of the Th17 differentiation process. By using pharmacological Notch inhibitors and specific Notch1 knockdown in human in vitro Th17 differentiation assays, I have shown that Notch1 signaling regulates IL-17 and TNFα expression but not IL-5 secretion. Notch1 overexpression led to increase in IL-17 and TNFα but not IL-5 secretion in Th17 differentiated cells. Chromatin immuno-precipitations and human T-bet promoter assays support that T-bet and ROR-γt, but not GATA3 are direct targets of Notch1 under Th17 differentiating conditions.

A role of Notch signaling in T cell differentiation is further validated using Experimental Autoimmune Encephalomyelitis (EAE) model in collaboration with Dr Barbara Osborne, University of Massachusetts, Amherst. We observed that Notch signaling inhibition by Gamma Secretase Inhibitors (GSI) in the EAE model ameliorates the symptoms as well as delays the onset of disease. We also observed a decrease in IL-17 and IFNγ cytokine expression levels among restimulated splenocytes and mononuclear cells of the CNS in GSI fed EAE mice as compared to animals fed with control chow.

I propose use of Notch inhibitors (GSI) as a potential therapeutic strategy to treat Multiple Sclerosis due to its ability to regulate pathogenic cytokines such as IL-17 and IFNγ.

Creative Commons License

Creative Commons License
This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 3.0 License.

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