The innate immune system recognizes and responds to pathogenic organisms. In doing so, this system is responsible for initiating a cytokine response designed to tailor the adaptive immune system to eradicate the offending organism. This process must be tightly regulated as too much activity can lead to inflammatory disease. Because inflammatory diseases are characterized by prolonged innate immune activation and cytokine release, the mechanisms controlling downregulation of the innate immune response are paramount in limiting inflammatory pathology. This grant application aims to study the mechanisms of this downregulation by focusing on the signal transduction mechanisms of NOD2 protein (CARD15 gene) and on NOD2's role in initiating and maintaining the cytokine response. The NOD2 protein is responsible for a number of inflammatory disorders including Blau Syndrome (a familial granulomatosis disease), a subset of Early Onset Sarcoidosis and for 15-20% of genetic Crohn's Disease. NOD2 is activated in response to intracellular exposure to both gram-positive and gram-negative bacteria after which it helps to coordinate NF?B activation and cytokine release through the lysine-63 (K63)-linked polyubiquitination of a novel site (K285) on the IKK scaffolding protein NEMO. We have recently extended this finding to show that the major extracellular innate immune signaling receptors, the Toll-like Receptors (TLRs), also require K285 NEMO ubiquitination to properly signal through NF?B. This work suggests that regulation of the post-translational modifications on the IKK scaffolding protein, NEMO, helps to coordinate cross-talk between intracellular and extracellular innate immune pathways and also helps to regulate the identity, the amount and the duration of cytokines that are released. These findings also suggest that for NF?B signaling, multiple innate immune signaling pathways converge on NEMO and that the post-translational modifications on NEMO serve as a rheostat to control NF?B activity. As such, these post-translational modifications may also be targets for molecules aimed at downregulating the NF?B response activated by NOD2 and other innate immune signaling pathways. The central hypothesis of this grant is that downregulation of NOD2 and TLR-stimulated NF?B activation is paramount in avoiding inflammatory pathology. Failure to properly downregulate the NF?B response and coordinate between alternative (MAP kinase) signaling pathways may underlie the pathophysiology of inflammatory disorders. Study of these pathways of downregulation could lead both to novel insight regarding the pathophysiology of these diseases and to novel druggable target to help treat these diseases. To begin to tackle this important problem, we have generated significant preliminary data. We have identified a novel innate immune-induced phosphorylation site on NEMO that controls NEMO ubiquitination and therefore, controls ultimate NF?B activation. We have also identified a signaling pathway operating through an unexpected MAP3K which inhibits NEMO ubiquitination and shifts innate immune signaling from NF?B activity toward p38 activity.
The Specific Aims of this grant application aim to determine the biochemical mechanisms by which innate immune-induced NF?B activity can be downregulated, to determine the function of MEKK4 in dictating signal specificity downstream of innate immune system activation and to determine the role of these signaling pathways in the pathophysiology of inflammatory disease. Lay Summary As humans, we are constantly exposed to bacteria, fungi and viruses, and we must respond to these pathogens so that we do not become infected. After responding to these pathogens, if our immune systems do not deactivate, we develop inflammatory disorders such as asthma, inflammatory bowel disease, multiple sclerosis and atherosclerosis (heart and vascular disease). Inflammatory diseases such as these are a significant cause of morbidity and mortality across a wide range of populations (infants to elderly). Due to the importance of downregulating the inflammatory response, our bodies have developed sophisticated mechanisms to dampen the inflammatory response. This grant application aims to study the mechanisms that dampen this inflammatory response and the mechanisms by which this dampening is faulty in inflammatory disease. This work aims to help determine the causes of inflammatory disease and aims to identify novel targets for pharmaceutical intervention in these debilitating disorders.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM086550-04
Application #
8204407
Study Section
Special Emphasis Panel (ZRG1-IMM-K (03))
Program Officer
Dunsmore, Sarah
Project Start
2008-12-09
Project End
2013-11-30
Budget Start
2011-12-01
Budget End
2012-11-30
Support Year
4
Fiscal Year
2012
Total Cost
$315,446
Indirect Cost
$114,525
Name
Case Western Reserve University
Department
Pathology
Type
Schools of Medicine
DUNS #
077758407
City
Cleveland
State
OH
Country
United States
Zip Code
44106
Tigno-Aranjuez, Justine T; Benderitter, Pascal; Rombouts, Frederik et al. (2014) In vivo inhibition of RIPK2 kinase alleviates inflammatory disease. J Biol Chem 289:29651-64
Jun, Janice C; Cominelli, Fabio; Abbott, Derek W (2013) RIP2 activity in inflammatory disease and implications for novel therapeutics. J Leukoc Biol 94:927-32
Tigno-Aranjuez, Justine T; Bai, Xiaodong; Abbott, Derek W (2013) A discrete ubiquitin-mediated network regulates the strength of NOD2 signaling. Mol Cell Biol 33:146-58
Jun, Janice C; Kertesy, Sylvia; Jones, Mark B et al. (2013) Innate immune-directed NF-*B signaling requires site-specific NEMO ubiquitination. Cell Rep 4:352-61
Marinis, Jill M; Hutti, Jessica E; Homer, Craig R et al. (2012) I?B kinase ? phosphorylation of TRAF4 downregulates innate immune signaling. Mol Cell Biol 32:2479-89
Marinis, Jill M; Homer, Craig R; McDonald, Christine et al. (2011) A novel motif in the Crohn's disease susceptibility protein, NOD2, allows TRAF4 to down-regulate innate immune responses. J Biol Chem 286:1938-50
Gorfu, Gezahegn; Rivera-Nieves, Jesus; Hoang, Sharon et al. (2010) Beta7 integrin deficiency suppresses B cell homing and attenuates chronic ileitis in SAMP1/YitFc mice. J Immunol 185:5561-8
Liu, Yi C; Gray, Reginald C; Hardy, Gareth A D et al. (2010) CpG-B oligodeoxynucleotides inhibit TLR-dependent and -independent induction of type I IFN in dendritic cells. J Immunol 184:3367-76
Tigno-Aranjuez, Justine T; Asara, John M; Abbott, Derek W (2010) Inhibition of RIP2's tyrosine kinase activity limits NOD2-driven cytokine responses. Genes Dev 24:2666-77
Li, Chaoyang; Yu, Shuiliang; Nakamura, Fumihiko et al. (2009) Binding of pro-prion to filamin A disrupts cytoskeleton and correlates with poor prognosis in pancreatic cancer. J Clin Invest 119:2725-36

Showing the most recent 10 out of 11 publications