Author

Monira Obaid

ORCID Identifier(s)

0000-0003-2128-8966

Graduation Semester and Year

2019

Language

English

Document Type

Dissertation

Degree Name

Doctor of Philosophy in Chemistry

Department

Chemistry and Biochemistry

First Advisor

Subhrangsu S Mandal

Abstract

Inflammation is an immune response that protects the human body from infection, illness, or injury. The inflammatory response causes the activation of immune cells, such as leukocyte subsets (i.e., monocytes, macrophages, dendritic cells, neutrophils, eosinophils and others), T cells, and B cells, and induces production of inflammatory mediators such as cytokines, chemokines, and antibodies to fight against injury or infection. Uncontrolled and continued inflammation drives the development of many human diseases, including metabolic diseases, obesity, diabetes, autoimmune disorders, neurological disorder and cancer. Indeed, the signaling process associated with inflammation and immune response is very complex. Our understanding of inflammation and immune response are largely limited to genomic markers and protein-based factors. Even with the huge amount research and therapeutic drugs, many inflammatory diseases still cannot be treated. Therefore, understanding the detailed signaling mechanism associated inflammation and immune response is critical for developing effective therapies. Emerging evidence suggest that noncoding RNAs (ncRNA) play critical roles in inflammation and immune response. As a part of my thesis work, the roles of long noncoding RNAs (lncRNAs) in inflammation and immune response have been investigated. Chapter 1 discusses the functions of known lncRNAs in the regulation of inflammation and immune response. Inflammation is the intrinsic immune response of the body towards invading pathogens. However, uncontrolled and continued inflammation drives the development of many human diseases from autoimmune disorders to cancer. The signaling process associated with inflammation and immune response is a part of immune system which is very complex. Indeed, human immune system constitutes of various immune cells such as neutrophils, eosinophils, monocytes, macrophage, dendritic cells (DCs), mast cells, natural killer (NK) cells, B cells, and T cells, which spread throughout the body and fight against foreign invaders. Emerging evidences suggest that noncoding RNAs (ncRNA) regulate the differentiation, development and function of those immune cells and control the immune response. For instances, lncRNAs (long noncoding RNAs) such as LincRNA-Cox2, THRIL (TNFα and hnRNPL related immunoregulatory lncRNA), PACER (p50-associated COX2 extragenic RNA), and NEAT1 (Nuclear enriched abundant transcript 1) are involved in regulation of inflammatory response of myeloid cells such as macrophage, dendritic cells, and neutrophils. LncRNAs noncoding transcript in CD4+ T cells (NTT), growth-arrest-specific transcript 5 (Gas5), and noncoding repressor of NFAT (NRON) are associated with T cells subset differentiation and function. LncRNA FAS-AS1 plays a crucial role in the B cells development, differentiation, and function. However, understanding the roles of lncRNAs in the regulation of immune response is still limited. In the research, it has been aimed to discover novel lncRNA associated immune response and inflammation and investigate their mechanism of action. In the chapter, functions of various known lncRNAs associated with inflammation and immune response have been summarized. Chapter 2 presents the studies on the functions of a well-known lncRNA HOTAIR in immune response and inflammation. LncRNAs are emerging as major regulators of a variety of cell signaling processes. Many lncRNAs are expressed in immune cells and appear to play critical roles in the regulation of immune response. My study demonstrates that lncRNA HOTAIR expression is induced in immune cells (macrophages) upon treatment with lipopolysaccharide (LPS). Knockdown of HOTAIR reduces NF-B-mediated inflammatory gene and cytokine expression in macrophages. Inhibition of NF-κB resulted in down-regulation of LPS-induced expression of HOTAIR as well as IL-6 and iNOS expression. It has been further demonstrated that HOTAIR regulates activation of NF-κB and its target genes (IL-6 and iNOS) expression via facilitating the degradation of IκBα. HOTAIR knockdown reduces the expression of NF-κB target gene expression via inhibiting the recruitment of NF-κB and associated cofactors at the target gene promoters. Taken together, my finding suggests that HOTAIR is a critical player in NF-κB activation in macrophages suggesting its potential functions in inflammatory and immune response. Chapter 3 presents the studies on the role of lncRNA HOTAIR in the regulation of glucose metabolism in macrophage during inflammatory response. Inflammatory response plays a central role in immune response and the inflammatory response is closely linked with glucose metabolism. My recent study demonstrates that lncRNA HOTAIR plays key roles during inflammation. HOTAIR is upregulated upon inflammation in macrophage and regulates cytokines expression via regulation of NF-B activation. Here it has been further investigated if HOTAIR plays any role in regulation of glucose metabolism in immune cells upon inflammation. HOTAIR in macrophages has been knocked down and measured the glucose uptake potential of macrophages in the absence and presence of LPS (lipopolysaccharide). Interestingly, my study demonstrated that LPS induces the expression of glucose transporter isoform 1 (Glut1) which controls the glucose uptake into macrophages and the lncRNA HOTAIR is required for LPS-induced Glut1 expression. My study demonstrated that HOTAIR induces NF-B activation which in turn increases Glut1 expression in response to LPS. Importantly, it has been found that HOTAIR regulates glucose metabolism in macrophages during LPS-induced inflammation and knockdown of HOTAIR decreases LPS-induced increased glucose uptake. Overall, my study demonstrated that HOTAIR induces Glut1 expression and hence glucose uptake by activating NF-B and regulates metabolic programming in immune cells potentially to meet the energy needs at the priming of immune response to pathogenic infection or other stressors. Chapter 4 presents the studies on the discovery of novel lncRNAs associated with immune response and inflammation. As mentioned earlier that the ncRNAs, especially lncRNAs, are emerging as key players in a variety of cell signaling processes, in health and disease. A large number of lncRNAs have been discovered though most of the studies are linked to cancer. Information on lncRNAs linked to immune response and inflammation, is limited. My recent study has demonstrated that lncRNA HOTAIR plays critical roles in regulation of inflammation and immune response in macrophages. Here, to identify lncRNAs linked to inflammation in an unbiased manner, the whole transcriptome RNAseq analysis in primary BMDM (bone marrow derived macrophages) cells treated with LPS has been performed. A large number of lncRNAs that are potential regulators of inflammatory response have been identified. In particular, a novel lncRNA, termed as LinfRNA1 (Long noncoding inflammation associated RNA 1) whose expression is significantly elevated upon treatment with LPS in macrophage, has been identified. My study demonstrated that LinfRNA1 regulates NF-κB activation and inflammatory response in macrophage. Knockdown of LinfRNA1 (using siRNA specific to LinfRNA1) reduces the level of LPS-induced degradation of IκBα and suppresses the level of LPS-induced accumulation of phospho-p56 (NF-κB), suggesting key roles of LinfRNA1 in NF-κB activation. Knockdown of LinfRNA1 also reduced the LPS-induced expression of NF-B-regulated inflammatory gene and cytokine expression. Furthermore, inhibition of NF-κB resulted in down-regulation of LPS-induced expression of LinfRNA1 as well as IL-6 and iNOS expression. Taken together, my observation demonstrates that LinfRNA1 is a critical and novel regulator of NF-κB activation, inflammation, and immune response. Overall, my research has discovered novel functions of lncRNAs in immune response and inflammation. In particular, my study demonstrated that HOTAIR, which is well-known as repressor lncRNA, plays critical roles in NF-κB activation and inflammation. My study demonstrated that HOTAIR plays critical roles of regulation of glucose transporter and metabolism in macrophage cells especially under inflammatory condition. Finally, my study has discovered novel lncRNAs, called LinfRNA1 associated with inflammation and demonstrates that LinfRNA1 plays critical roles in regulation of NF-κB activation and cytokine expression. My research revealed novel cell signaling pathways involving lncRNA associated inflammation and immune response, and hence may provide novel platform for developing drugs for the treatment of immune and metabolic disorders.

Keywords

Long noncoding RNA, Inflammation, Immune response, Macrophage, Signaling pathway

Disciplines

Chemistry | Physical Sciences and Mathematics

Comments

Degree granted by The University of Texas at Arlington

Included in

Chemistry Commons

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