Obesity-induced chronic inflammation plays a key role in the pathogenesis of insulin resistance and the metabolic syndrome. Proinflammatory cytokines can cause insulin resistance in adipose tissue, skeletal muscle and liver by inhibiting insulin signaling transduction. Interleukin-1 receptor-associated kinase-1 (IRAK-1) is a serine/threonine kinase functioning in Toll-like Receptor signaling pathways, and plays an important role in inflammation and immune response. In our studies, we demonstrated that IRAK-1 is involved with the negative regulation of PI3K-Akt dependent signaling pathway induced by insulin and TLR 2&4 agonists. Out data also indicate that IRAK-1 can interact with IRS-1 protein both in vivo and in vitro. The binding sites for the IRAK1-IRS1 biochemical interaction are IRS-1's PH domain and IRAK-1's proline-rich LWPPPP motif. Our studies also indicate that IRAK-1 is involved with the negative regulation of glycogen synthesis through inhibiting PI3K-Akt signaling pathway and thus releasing GSK3β's inhibitory effect on glycogen synthase. Moreover, our studies also suggest that IRAK-1 is involved in the activation of transcription factors CREB and ATF-1 by stimulating CREB-Ser133 and ATF-1 phosphorylation. CREB transcription factor family induces genes involved in cellular metabolism, gene transcription, cell cycle regulation, cell survival, as well as growth factor and cytokine genes. That may partially explain our finding that IRAK-1 may be also involved with cell proliferation and survival pathway. / Master of Science

Interleukin-1 receptor associated kinase -1 is a protein kinase pivotal in mediating signals for innate immune responses. Here, I report that IRAK-1 also regulates cell-mediated immune responses. NFATc2 (nuclear factor of activated T cells) was found to be associated with IRAK-1 in T cells in vitro and its activity was elevated in the absence of IRAK-1. In addition, IRAK-1-/- mice had increased naturally occurring regulatory T cells and inducible regulatory T cells as well as Th1 responses as compared to WT mice. The findings suggest that activated T cells might employ IRAK-1 to mediate the regulation of acquired immunity. Therefore, IRAK-1 may participate in direct signaling cross talk between the innate and the acquired immunity. / Master of Science

We examined the role of Tollip in the hypertrophic response of cardiomyocytes. C57BL/6 mice were subjected to transverse aortic constriction (TAC) for 2 weeks and age-matched sham surgical operated mice served as control. TAC significantly reduced the association of Tollip with IRAK-1 by 66.4 percent and increased NF-kappaB binding activity by 86.5 percent and the levels of phosphop38 by 114.6 percent in the myocardium compared with sham control, respectively. In vitro experiments showed that IL-1beta stimulation also significantly reduced the association of Tollip with IRAK-1 and increased NFkappaB binding activity in neonatal cardiomyocytes. Tollip overexpression by transfection of cardiac myocytes significantly attenuated the IL-1beta-induced hypertrophic response of cardiac myocytes as evidenced by reduced cell size (16.4 percent) and decreased ANP expression (33.3 percent). Overexpression of Tollip also reduced NFkappaB binding activity by 30.7 percent and phospho-p38 by 47.1 percent, respectively. The results suggest that Tollip could be a negative regulator during the development of cardiac hypertrophy. The negative regulation of cardiac hypertrophy by Tollip may involve downregulation of the MyD88-dependent NF-kappaB activation pathway.

Oxidative stress plays a vital role in the pathogenesis of many chronic and acute inflammatory diseases. Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are two key mediators that are known to induce cellular and tissue oxidative stress. The generation of ROS and RNS is mediated by innate immune signaling processes. Lipopolysaccharide (LPS), a major inflammatory signal, is known to be a potent inducer of ROS/RNS. Thus, strategies that may block LPS-mediated generation of free radicals may hold promise in treating various inflammatory disease processes. However, the molecular mechanisms underlying LPS-mediated ROS/RNS production are not fully defined. Interleukin-1 Receptor associated kinase (IRAK-1), an intracellular kinase downstream of Toll-like Receptor 4 (TLR4) has been shown to contribute to the inflammatory cascade associated with LPS-TLR4 signaling pathway. However, its role in ROS production has not been defined. Therefore, we tested the hypothesis that IRAK-1 plays an important role in regulating ROS/RNS production. Both in vitro and in vivo studies were conducted to investigate the role of IRAK-1 in modulating free radicals as well as oxidative stress. In vitro studies demonstrate that IRAK-1 is a critical molecule involved in the induction of ROS/RNS. IRAK-1 deletion ablated free radical production following LPS challenge in a variety of cell types including macrophages, fibroblasts and microglia. Mechanistically, we observed that IRAK-1 is required for optimal expression and activity of NADPH oxidase subunits and iNOS. IRAK-1 deletion reduced LPS-triggered p47phox membrane translocation, suppressed NOX-1 expression and protein levels as well as hampered Rac1 activation. On the other hand, IRAK-1 deletion sustained antioxidative enzyme activity and levels in IRAK-1-/- macrophages and fibroblasts. In terms of the in vivo physiological consequences, IRAK-1-/- mice exhibited attenuated lipid peroxidation in vital organs, attenuated histopathological lesions in liver and kidney, and reduced endotoxemia-associated mortality. Taken together, IRAK-1 may, at least in part, serve as an important therapeutic target in the treatment of various inflammatory disease processes. / Ph. D.

Generation of reactive oxygen species (ROS) by mammalian host cells is a double-edged sword. ROS are clearly beneficial in directly killing pathogens and as a signaling molecule to alert macrophages and neutrophils to the site of infection. However, ROS are also capable of damaging host cells by destroying DNA, oxidizing proteins and lipids, inactivating enzymes, and eliciting apoptosis. Therefore the balance of ROS generation and clearance is essential for homeostasis. Although multiple mechanisms can contribute to the generation of ROS, NADPH oxidase (Nox) is a primary producer. In terms of clearance, several ROS scavenging enzymes are induced by Nrf2, a sensor of excessive ROS. The mechanisms behind the skewing of this balance toward prolonged accumulation of ROS under chronic inflammatory conditions are not well understood. Lipopolysaccharide (LPS), a major component of the Gram-negative bacteria cell wall, is specifically recognized by Toll-like receptor 4 (TLR4). LPS triggers robust activation of Nox and ROS production through TLR4, while also activating Nrf2 and ROS clearance. Intracellular pathways regulating ROS generation and clearance mediated by TLR4 are not well defined. Since interleukin-1 receptor associated kinase 1 (IRAK-1) is a key downstream component of TLR4, we test the hypothesis that IRAK-1 may play a critical role in maintaining the balance of LPS triggered ROS generation and clearance. Using wild type and IRAK-1 deficient murine embryonic fibroblasts, we tested the dynamic induction of Nox1 (a key NADPH oxidase) and Nrf2 by varying dosages of LPS. Our data confirm that high dose LPS (as seen in acute bacterial infection) induced both Nox1 and Nrf2. The generation of Nox1 is IRAK-1 dependent. Low dose LPS (as seen in chronic metabolic endotoxemia) fails to induce Nrf2 and induces mild and prolonged expression of Nox1. Cells pre-challenged with low dose LPS are primed for more robust expression of ROS following a second LPS challenge. The conclusions and implications generated by this study are that chronic low dose endotoxemia (prevalent in adverse health conditions) may skew the balance of ROS generation and clearance to favor prolonged ROS accumulation, and that IRAK-1 represents a potential therapeutic target to treat chronic inflammatory diseases. / Ph. D.