Date of Award

8-15-2013

Document Type

Thesis

Degree Name

Doctor of Philosophy (PhD)

First Advisor

Kevin Tracey

Abstract

High mobility group protein 1(HMGB1), released from activated immune cells or damaged cells, is a mediator of inflammation during sterile and infectious injury,  and  importantly  contributes  to  the  pathogenesis  of  a  number  of inflammatory diseases. As HMGB1 contains two nuclear location sequence (NLS) sites, most HMGB1 is localized in the nucleus in quiescent cells. Efficient HMGB1 release during inflammation requires the acetylation of HMGB1 at the NLS sites and subsequent HMGB1 accumulation in the cytoplasm. Further, unlike other classic cytokines which harbor a leader peptide that facilitates their secretion through the endoplasmic reticulum - Golgi exocytotic route, HMGB1 lacks a leader peptide and is released by activated immune cells via pyroptosis, a proinflammatory programmed cells death, which is mediated by the inflammasome. However, the signaling transduction pathways that control HMGB1 cytoplamsic accumulation and inflammasome activation remain largely unknown. As the mechanistic studies of HMGB1 release by activated immune cells have important clinical implications, the aims of my thesis focus on the identification of the key pathways which critically regulate the HMGB1 cytoplamsic accumulation or the inflammasome activation in activated immune cells.

In Aim 1, we  discovered  an  important  role of  double-stranded  RNA dependent protein kinase (PKR) in inflammasome activation. PKR inactivation by genetic deletion or pharmacological inhibition severely impaired inflammasome activation in response to double-stranded RNA, ATP, monosodium urate, adjuvant aluminum, rotenone, live E. coli, anthrax lethal toxin, DNA transfection, and S. Typhimurium infection. PKR deficiency significantly inhibited the secretion of IL-1beta, IL-18 and HMGB1 in E. coli-induced peritonitis. PKR physically interacts with multiple inflammasome components, including NLRP3, NLRP1, NLRC4, AIM2, and broadly regulates inflammasome activation. PKR autophosphorylation in  a  cell  free  system  with  recombinant  NLRP3,  ASC  and  pro-casapse-1 reconstitutes inflammasome activity.

In Aim 2, we show that the JAK/STAT1 pathway is essential for HMGB1 cytoplasmic accumulation in macrophages. Pharmacological inhibition of the JAK/STAT1  pathway  blocked  LPS-induced  HMGB1  nuclear  translocation. Conversely, activation of the JAK/STAT1 pathway by type 1 interferon induced robust HMGB1 nucleus to cytoplasm translocation. Mass spectrometric analysis unequivocally revealed that that the pharmacological inhibition of the JAK/STAT1 pathway or genetic deletion of STAT1 abrogated LPS- or type 1 interferon-induced HMGB1 acetylation within the NLS sites.

In  Aim  3,  we  show  that  cholinergic  neuronal  signals  attenuate inflammasome  activation  by  preventing  mitochondrial  DNA  release  into cytoplasm. Cholinergic receptor agonists or vagus nerve stimulation significantly inhibited the inflammasome activation and mitochondrial perturbation, whereas genetic deletion of alpha 7 nicotinic acetylcholine receptor (alpha 7 nAchR) significantly  enhanced  the  inflammasome  activation  and  mitochondrial perturbation. We also found that extracellular acetylcholine rapidly influxes into macrophage cytoplasm upon ATP stimulation in an alpha 7 nAchR-independent manner. Importantly, acetylcholine significantly attenuated calcium or hydrogen oxide-induced mitochondrial damage and the subsequent mitochondrial DNA release in purified mitochondria.

In  conclusion, this  study revealed  an  important role of PKR in the inflammasome activation and an essential role of the JAK/STAT1 pathway in HMGB1 acetylation and cytoplasmic accumulation, suggesting novel potential drug target molecules to treat HMGB1-dependent diseases. In addition, this study unraveled  a  novel  mechanism  of  how  cholinergic  neural  signals  inhibit inflammation,  in  which  acetylcholine  translocates  into  t h e  cytoplasm  of immune cells and inhibits inflammasome activation by preserving mitochondrial membrane integrity.

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