Sepsis is a major threat to human health in the US, affecting ~750,000 Americans per year, with an associated mortality rate >28%. Many septic patients develop profound immunosuppression manifested by decreased production of pro-inflammatory cytokines and chemokines. This is highly reminiscent of endotoxin tolerance, a state of re-programming of TLR4 responses after a prior exposure to LPS. Endotoxin tolerance can be used as a model to delineate mechanisms that underlie altered monocyte responses in patients with sepsis. During the period of funding, we have identified new hallmarks of endotoxin tolerance. These include: (i) deficient tyrosine phosphorylation of TLR4 and adapter protein Mal;(ii) suppressed LPS-induced signalosome assembly amongst TLR4, adapter protein TRIF and kinase TBK1;(iii) deficient activation of TBK1 and transcription factor IRF-3;and (iv) increased expression of negative regulators Tollip, IRAK-M, SHIP-1, SOCS-1, SARM, and SIKE. Our preliminary data in THP1 cells and human monocytes show decreased LPS- induced c-Src and Lyn phosphorylation and Lyn-TLR4 interactions;inhibited activation of kinases IRAK4 and TAK-1;impaired K63-linked polyubiquitination of IRAK1 and TRAF-6;suppressed interactions of ubiquitinated IRAK1 with TRAF6 and IKK3;and increased expression of A20, a key deubiquitinating enzyme. Furthermore, we found reduced LPS tolerance induction upon inhibition of protein tyrosine phosphatases or by A20 gene knockdown, indicating a crucial role for altered tyrosine phosphorylation and K63-linked polyubiquitination in tolerance. Based on these data, we hypothesize that LPS tolerance alters post-translational modifications of TLR4, Mal and IRAK kinases and increases levels of negative regulators of TLR signaling, leading to reprogramming of TLR responses through changes in proximal signalosome compositions. This hypothesis will be tested in the following Specific Aims: 1. Identify kinases and phosphatases involved in phosphorylation of TLR4 and Mal and determine the impact of LPS tolerance on their expression and activities;2. Define mechanisms by which LPS tolerance alters signalosome assembly and activation of proximal adapter-kinase modules;and 3. Elucidate molecular basis of interference in TLR4 signaling by negative regulatory molecules associated with LPS tolerance. These studies will determine new mechanisms responsible for tolerance and identify key intermediates affected. They will lay the groundwork for our future research in animal models of sepsis in vivo and translational studies in septic patients, with the goal of facilitating development of new therapeutic strategies to improve treatments for septic patients. These advances would be of key importance for improving public health in septic patients in the U.S.

Public Health Relevance

Sepsis is a major threat to human health worldwide, with an annual incidence of ~750,000 patients and ~215,000 deaths in the U. S. Alterations in signaling by Toll-like receptor (TLR) 4, the main receptor for Gram negative bacteria and lipopolysaccharide, have been associated with infectious diseases and sepsis. Our proposed studies will provide a better understanding of how exposure to bacterial products """"""""re-programs"""""""" functions of TLR4, adapters and kinases at the molecular level. Our research will identify the earliest disturbances in functions of receptor and signaling molecules that are caused by exposures to bacterial products, and will facilitate development of new therapeutic approaches to correct such deficiencies.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Research Project (R01)
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Innate Immunity and Inflammation Study Section (III)
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Minnicozzi, Michael
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University of Maryland Baltimore
Schools of Medicine
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Xiong, Yanbao; Murphy, Michael; Manavalan, Tissa T et al. (2016) Endotoxin Tolerance Inhibits Lyn and c-Src Phosphorylation and Association with Toll-Like Receptor 4 but Increases Expression and Activity of Protein Phosphatases. J Innate Immun 8:171-84
Leifer, Cynthia A; Medvedev, Andrei E (2016) Molecular mechanisms of regulation of Toll-like receptor signaling. J Leukoc Biol 100:927-941
Murphy, Michael; Xiong, Yanbao; Pattabiraman, Goutham et al. (2015) Pellino-1 Positively Regulates Toll-like Receptor (TLR) 2 and TLR4 Signaling and Is Suppressed upon Induction of Endotoxin Tolerance. J Biol Chem 290:19218-32
Medvedev, Andrei E; Murphy, Michael; Zhou, Hao et al. (2015) E3 ubiquitin ligases Pellinos as regulators of pattern recognition receptor signaling and immune responses. Immunol Rev 266:109-22
Murphy, Michael B; Xiong, Yanbao; Pattabiraman, Goutham et al. (2015) Pellino-3 promotes endotoxin tolerance and acts as a negative regulator of TLR2 and TLR4 signaling. J Leukoc Biol 98:963-74
Medvedev, Andrei E (2013) Toll-like receptor polymorphisms, inflammatory and infectious diseases, allergies, and cancer. J Interferon Cytokine Res 33:467-84
Xiong, Yanbao; Pennini, Meghan; Vogel, Stefanie N et al. (2013) IRAK4 kinase activity is not required for induction of endotoxin tolerance but contributes to TLR2-mediated tolerance. J Leukoc Biol 94:291-300
Xiong, Yanbao; Song, Chang; Snyder, Greg A et al. (2012) R753Q polymorphism inhibits Toll-like receptor (TLR) 2 tyrosine phosphorylation, dimerization with TLR6, and recruitment of myeloid differentiation primary response protein 88. J Biol Chem 287:38327-37
Figueroa, Leandra; Xiong, Yanbao; Song, Chang et al. (2012) The Asp299Gly polymorphism alters TLR4 signaling by interfering with recruitment of MyD88 and TRIF. J Immunol 188:4506-15
Xiong, Yanbao; Medvedev, Andrei E (2011) Induction of endotoxin tolerance in vivo inhibits activation of IRAK4 and increases negative regulators IRAK-M, SHIP-1, and A20. J Leukoc Biol 90:1141-8

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