Date of Award

Winter 2020

Project Type

Dissertation

Program or Major

Biochemistry

Degree Name

Doctor of Philosophy

First Advisor

Sherine SE Elsawa

Second Advisor

Sherine SE Elsawa

Third Advisor

Linda LY Yasui

Abstract

Inflammation is an important and healthy mechanism that our body uses for wound-healing and fighting off infections by utilizing molecular pathways of immune and non-immune cells. However, when inflammation is persistent or chronic it can have severe consequences that can lead to auto-immune diseases or cancer. Pathogen-associated molecular patterns (PAMPs), such as Lipopolysaccharide (LPS), present on gramnegative bacteria, or double-stranded RNA (dsRNA), present in RNA-viruses, were shown to be potent inducers of inflammation. These PAMPs are known ligands of Toll like receptor 4 (TLR4), activated by LPS and Toll-like receptor 3 (TLR3), activated by dsRNA which are carried on the outside and inside of immune cells. Interaction of PAMPs with their receptors lead to production of cytokines such as Interleukin 6 (IL6), Tumor necrosis factor α (TNFα) or C-C Motif Chemokine Ligand 2 (CCL2), which bind to other receptors on immune and non-immune cells which leading to an amplification of the inflammatory signal inside the host. Besides TLR signaling, studies have found other important signaling events involved in inflammation and chronic inflammation. One of those pathways is the Hedgehog (HH) signaling pathway. At the center of HH signaling are Patched1 (PTCH1) and Smoothened (SMO) receptor system but also GLI transcription factor family members GLI1, GLI2 and GLI3. Whilethe role of GLI1 and GLI2 was described very frequently in inflammation and inflammation related diseases such as cancer, the role of GLI3 was barely studied. This dissertation focuses on the role of GLI3 in inflammation.Upon activation of TLR4 signaling by LPS, either Myeloid Differentiation Primary Response Protein (MYD88) or TIR-domain-containing adapter-inducing interferon-β (TRIF) dependent signaling can occur. Downstream of MYD88, Nuclear Factor-κB (NFκB) and Mitogen-Activated Protein Kinase (MAPK) pathways are activated while downstream of TRIF, Interferon Regulatory Factor 3 (IRF3) pathway in addition to NFκB and MAPK signaling are initiated. These transcription factors bind to promoter regions of cytokine related genes and initiate cytokine expression and secretion. The TRIF cascade also applies to TLR3 signaling, which is the only adaptor molecule involved in this signaling and which can be initiated by Poly (I:C), a dsRNA homolog. We found that upon stimulation of monocytes with LPS, GLI3 expression is upregulated and that this upregulation is highly dependent on the TLR-TRIF-cascade. Additionally, we found that IRF3 is the transcription factor which regulates GLI3 expression levels downstream of TRIF by directly binding to its promoter region. The role of IRF3 regulation of GLI3 expression was further confirmed by treatment of monocytes with Poly(I:C) which robustly induced GLI3 levels and significantly increased binding of IRF3 to the GLI3 promoter. This strongly supports a role for GLI3 in inflammation. To further investigate the role of GLI3 in inflammation we utilized the CreloxP system to generate conditional GLI3 knock-out mice, lacking GLI3 expression in macrophages termed as M-GLI3-/- . We isolated peritoneal macrophages from wild-type (WT) mice and M-GLI3-/- mice and treated those with LPS and found a significant difference in expression of inflammation related genes by RNA-seq analysis. Furthermore, we identified GLI3 asa new candidate in M2 macrophage polarization, an anti-inflammatory macrophage subset highly involved in promoting tumor growth. Finally, we identified GLI3 as a regulator of inflammatory response since isolated macrophages from M-GLI3-/- mice secreted significantly less proinflammatory cytokines IL6, CCL2 and TNFα than macrophages from WT mice. Therefore, our studies identified TLR signaling as novel molecular mechanism in regulating GLI3 expression and GLI3 as regulator of inflammation.

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