Electronic Thesis and Dissertation Repository

Degree

Master of Science

Program

Pathology

Supervisor

Patrick Luke

Abstract

Immunological Impact of CLI-095 on Dendritic Cell Maturation and Hypoxia-re-oxygenation induced inflammatory injury

Introduction: Ischemia reperfusion injury (IRI) activates innate immunity through the engagement of Toll-Like Receptors (TLRs) by endogenous ligands. TLR4 expressed within the kidney is a potential mediator of innate activation and inflammation. Stimulation of TLR4 induces distinct patterns of gene expression, which not only leads to the activation of innate immunity but also to the development of acquired immunity. CLI-095, a novel synthetic small-molecule, suppresses production of multiple cytokines by inhibiting TLR4 signaling. In this study, we have determined the role of TLR4 in hypoxia and re-oxygenation injury model, which mimics IRI in vitro, and investigated the effect of CLI-095 (a specific TLR4 inhibitor) on TLR4 mediated inflammation and maturation of dendritic cells (DCs).

Hypothesis: TLR4 signaling plays an important role in activation of innate immunity, and that targeting its pathway with CLI-095 will prevent inflammation and subsequent injury caused during hypoxia re-oxygenation.

Method: Bone marrow derived dendritic cells were stimulated by hypoxia re-oxygenation with or without CLI-095. Expression of TLR4, proinflammatory cytokines, and dendritic cells maturation markers were then tested by Flow Cytometry, qRT-PCR, and ELISA.

Results: We have shown that CLI-095 is able to blocks the TLR4 signaling pathway and reduce the expression of pro-inflammatory cytokines (IL6 and TNFα) in response to hypoxia re-oxygenation. In addition, DCs that were pretreated with CLI-095 showed low expression of maturation markers in comparison to cells that were subjected to hypoxia re-oxygenation.

Conclusion: TLR4 is involved in innate immunity activation in response to IRI or hypoxia re-oxygenation and CLI-095 is able to block TLR4 signaling pathway and suppress the activation of the inflammatory response. Therefore, ameliorating TLR4 by new therapies such as CLI-095, which specifically targets TLR4, may have potential implication in reducing IRI in clinical transplantation. Since almost all immune cells including DCs express TLR4.

Keywords:

Kidney, Ischemia Reperfusion Injury, hypoxia re-oxygenation, Toll-like receptors, Innate immunity, CLI-095

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