Date of Award

2009-01-01

Degree Name

Doctor of Philosophy

Department

Biological Sciences

Advisor(s)

Kristine M. Garza

Abstract

The incidence of obesity among the distinct world populations has become an ever growing concern. Central to the regulation of obesity is the adipocyte-derived hormone, and in more recent years cytokine, leptin. Leptin plays a central role in the maintenance of food consumption as well as the efficient use of peripheral energy stores. Leptin exerts its physiological role in energy homeostasis by regulating the expression of orexigenic peptides in the central nervous system. However, recent research has implicated leptin in other physiological roles including those of the immune system. As a pleiotropic cytokine, the absence of leptin has been shown to alter macrophage functions especially altering processes directly required for antigen processing. In addition, leptin has also been demonstrated to be critical in T cell proliferation. Having been implicated in these many roles, especially those of the immune system, we sought to determine whether the absence of a functional leptin protein would alter dendritic cell function in mice. Dendritic cells (DCs) are the most potent antigen presenting cells in the immune system and the only reported cells with the capability to activate naïve T cells, or those T cells that have never encountered antigen in the context of a major histocompatability complex. Our original focus was to determine whether dendritic cells that were procured from leptin-deficient mice were different when directly compared to age and sex matched wild type controls. Using flow cytometry, we measured the total detectable number of splenic DCs and their phenotype. Using a molecular approach, we investigated whether leptin-deficiency changed the capability of splenic DCs (sDCs) to acquire and process antigen. Using both approaches, we could detect no significant differences in any of the characteristics of interest. Our efforts shifted from DC phenotype and antigen processing to T cell activation, a property for which DCs are very well known and one that sets them apart from the rest of the cells of the immune system. To investigate the effects of leptin-deficiency on sDC antigen presentation, we used a T cell hybridoma cell line, primary T cells, as well as the in vivo neutralization of M. avium. To our surprise, sDC that were isolated from leptin-deficient mice exhibited a higher capability to activate the ova peptide specific T cell hybridoma cell line. To further corroborate our initial data, sDCs procured from leptin deficient mice also demonstrated an increased ability to activate primary T cells in a mixed lymphocyte reaction. Our in vitro data was then substantiated by our in vivo findings that showed that sDCs from leptin-deficient mice could activate T cells to a higher extent relative to their heterozygous, age, and sex matched controls. It is critical to point out that the increased T cell activation was not due to a decrease in plasmacytoid DCs as they were detected in relatively equal numbers. Having demonstrated that leptin deficiency bestowed DCs with an increased capability to activate T cells, we then wanted to determine the potential mechanism responsible for our results. Leptin potentiates its effects by activating the JAK2/STAT3 pathway and others to a lesser degree. DCs require the activation of the NF-kB transcription factor for their survival as well as their activation and ultimately T cell activation and polarization. Given this, we designed a series of experiments that would allow determining whether the receptor expressed by our DCs was functional. In addition, we wanted to determine whether leptin induced signaling had a negative effect on NF-kB directly. Western blot analysis shows that STAT3 gets activated upon the addition of leptin to DC cultures, a result observed by both western blot analysis and flow cytometry on sDC and BM-DCs. Furthermore, western blot analysis demonstrated that's the STAT3 was functional as SOCS3 expression was detected in a time dependent manner upon leptin treatment. Confocal microscopic analysis showed that the concurrent activation of the NF-kB and STAT3 pathways led to a significant decrease in the nuclear translocation of activated NF-kB. Immunoprecipitation (IP) assays were performed to help determine whether the observed inhibition of NF-kB nuclear translocation was the direct consequence of protein-protein interaction. Upon analysis of leptin-treated DCs, we were able to detect the presence of NF-kB in STAT3 specific IP. Our data therefore implies that in the absence of leptin there are no severe detectable consequences to DC total numbers, phenotype, antigen acquisition, or processing. However, in the absence of a functional leptin protein, DCs demonstrate a superior ability to activate T cells, both cells lines and primary. In addition, this phenomenon is also extended to an in vivo scenario as demonstrated by the effective neutralization of M. avium by sDC from leptin-deficient mice relative to the controls. Upon determining that plasmacytoid DCs (or regulatory DCs) were present in relatively equal numbers we set out to determine any implications that leptin induced signaling might have on DCs. Our experimental data suggests that these observations may be the direct outcome of leptin induced signaling. Our data showed that upon the addition of leptin to DC cultures in the presence of LPS, the detectable levels of NF-kB translocation into the nucleus was significantly decreased. Moreover, IP assays suggest that said decrease is very likely the product of complex formation between STAT3 and NF-kB. Whether the complex being formed consists of only these two transcription factors or involves other still needs to be elucidated. In conclusion, our data demonstrates that leptin-deficiency alone is not enough to disrupt DC numbers phenotype, antigen acquiring properties, or antigen processing abilities. However, in the absence of leptin-induced signaling sDC become highly efficient T cell activators and it is possible that this observation is the direct consequence of STAT3/NF-kB interactions. These data would suggest that in an obese state, an individual would become highly susceptible to microbial infections or pathogenic insults as DCs would encounter an activation signal that would only get screened by the negative/regulatory leptin induced STAT3 signaling more prevalent during this state

Language

en

Provenance

Received from ProQuest

File Size

165 pages

File Format

application/pdf

Rights Holder

Oscar Ramirez

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Microbiology Commons

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