Molecular analysis of LITAF-mediated inflammatory pathways
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TNF-a is a critical cytokine secreted predominantly by macrophages in response to bacterial toxins, inflammatory products and other noxious stimuli. This cytokine plays a significant role in various chronic inflammatory diseases, autoimmune diseases and in endotoxic/septic shock. LITAF is a transcription factor that forms a complex in the cytoplasm with STAT-6B and translocates into the nucleus to bind to the promoter of TNF-a, to participate in the TNF-a gene expression independently of the NF-KB pathway of regulation. It has become essential to elucidate and establish the role of LITAF in inflammatory processes in order to better understand the molecular mechanisms involved in inflammatory processes in hope of designing therapeutic strategies for TNF-a-mediated inflammatory diseases. Based on previous data generated in our laboratory, we hypothesized that (1) LITAF plays a role in the activation of several inflammatory mediators in response to endotoxemia and (2) the absence of LITAF protects animals from the deleterious effects of endotoxemia. Mice genetically deficient of LITAF in monocytes/macrophages (macLITAF-/-) were established in order to aid in the characterization of the functional role of LITAF in vivo. Peritoneal macrophages were collected from each mouse and the animals were screened for LITAF deficiency by PCR and confirmed by Western Blot analysis to determine the expression of LITAF protein and by ELISA to determine the TNF-a level. Characterization of the functional role of LITAF in vivo was elucidated using two inflammatory models: lethal and sub-lethal endotoxemia. LITAF deficient animals better tolerated the lethal endotoxic shock compared to the wild-type animals, when administered with LPS along with D-Galactosamine. Analysis of the profile of inflammatory mediators revealed that the macLITAF-/- mice showed a decreased production of most of the proinflammatory cytokines including TNF-a, as well as all the anti-inflammatory cytokines. Based on the literature, this phenomena can be interpreted that LITAF deficiency causes an immediate reduction in the systemic inflammatory response syndrome (SIRS) which effects the immediate reduction in the compensatory anti-inflammatory response syndrome (CARS), thus protecting the animal from death. In order to determine the systemic effect of LITAF deficiency, the profile of mediators when a sub-lethal dose of LPS was administered, revealed that, there was a delayed peak in the proinflammatory response leading to a similar delay in the anti-inflammatory response. Thus, when inflammation is re-established such as in sub-lethal bacteremia, the anti-inflammatory response follows suit to limit the detrimental effects of a prolonged release of the proinflammatory mediators. To begin to dissect the molecular pathways associated with LITAF-mediated inflammatory response, we tested the role of major serine/threonine kinases in this process. LPS-induced macLITAF-/- exhibited an upregulation of pro-survival kinases Akt, Erkl/2 and RSK compared to wild-type macrophages suggesting that these kinases may be involved in the survival of LITAF deficient animals when exposed to a bacterial challenge. Further studies are in progress to comprehensibly test this hypothesis. Altogether the present data provide new understanding of LITAF mediated inflammatory process and pave the way for further molecular analyses of the kinases involved in this process.
PLEASE NOTE: This work is protected by copyright. Downloading is restricted to the BU community: please click Download and log in with a valid BU account to access. If you are the author of this work and would like to make it publicly available, please contact firstname.lastname@example.org.Thesis (D.Sc.)--Boston University, Henry M. Goldman School of Dental Medicine, 2008 (Dept. of Oral Biology and Periodontology).Includes bibliographical references: leaves 93-104.
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