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This dissertation, The Role of Leptin in Regulating Dendritic Cell Maturation and Function by Lai-kwan, Queenie, Lam, 林麗君, was obtained from The University of Hong Kong (Pokfulam, Hong Kong) and is being sold pursuant to Creative Commons: Attribution 3.0 Hong Kong License. The content of this dissertation has not been altered in any way. We have altered the formatting in order to facilitate the ease of printing and reading of the dissertation. All rights not granted by the above license are retained by the author. Abstract: Abstract of thesis entitled The Role of Leptin in Regulating Dendritic Cell Maturation and Function Submitted by Queenie Lai Kwan Lam for the degree of Doctor of Philosophy at The University of Hong Kong in August, 2007 Leptin, a protein hormone produced by the adipocytes, has long been recognized to regulate metabolism, neuroendorine and other physiologic functions. Recent data has established the regulatory function for leptin in immunity, while gene-targeting studies also demonstrate an essential role of leptin in regulating hematopoiesis and lymphopoiesis. Dendritic cells (DC), the most potent antigen- presenting cells (APC) in the immune system, are crucial for the initiation and maintenance of T cell-mediated immune responses. However, it remains unclear whether or not leptin signaling plays a role in DC maturation and function. To study the regulatory role of leptin on DC, I examined the differentiation and function of DC derived from the bone marrow (BM) of leptin-receptor deficient db/db mice. The db/db BM cultures showed a decreased DC yield while specific blockade of leptin with a soluble leptin receptor chimera (OB-R: Fc) in the wild-type (WT) BM cultures inhibited DC generation. Both the db/db BM cultures and the OB- R: Fc-treated WT BM cultures displayed significantly increased apoptosis, which was associated with dysregulated Bcl-2 family gene expressions. The db/db DC also displayed markedly reduced expression of costimulatory molecules and a Th-2 type cytokine profile with a poor capacity in stimulating allogeneic T cell proliferation. Consistently, db/db DC showed downregulated activities of the PI3K/Akt pathway as well as STAT-3 and IκB-α. These data demonstrate a physiological role of leptin signaling in DC maturation and survival. To further elucidate how leptin signaling can affect gene expressions and function in DC, I demonstrated that leptin induced CD40 expression synergistically with LPS. In an Akt-dependent manner, both leptin and LPS activated the downstream transcription factors Nuclear Factor (NF)-κBp65 and Signal Transducer and Activator of Transcription-1α (STAT-1α) in DC. Using pharmacological inhibitors and dominant negative constructs of Akt and IKKα/β/γ as well as small interfering (si)RNA for STAT-1α, I showed that Akt, STAT-1α and NF-κB are important for activated CD40 expression induced by leptin, LPS and their synergistic action. Co-immunoprecipitation (CoIP) analysis revealed that both leptin and LPS promoted the association of Akt with the IKKs and STAT-1α. Blocking studies demonstrated a crucial role for Akt in the translocation of STAT-1α and NF-κBp65 to the nucleus and activation of the CD40 promoter. Finally, analysis with chromatin immunoprecipitation assay (ChIP) confirmed that leptin recruited STAT-1α, NF- κBp65 and RNA polymerase II to the CD40 promoter, and stimulated acetylation of the associated histone 4. To determine the functional significance of leptin signaling in vivo, I first uncovered markedly impaired DC development in the BM and lymph nodes (LN) of db/db mice. Next, administration of leptin into WT mice significantly stimulated DC maturation in the absence or presence of LPS. Moreover, I demonstrated the expression of leptin receptor in BM stromal cells. Leptin treatment could stimulate stromal cell prolife

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