# Novel sterile inflammatory pathways in alcoholic hepatitis

> **NIH VA I01** · VETERANS ADMIN PALO ALTO HEALTH CARE SYS · 2020 · —

## Abstract

Alcoholic hepatitis (AH) affects disproportionally high numbers of veterans, and has a mortality rate that has not
changed substantially over the last decade. As AH pathogenesis has distinct features and is characterized by
very severe hepatocyte injury, sterile inflammation and neutrophil recruitment; our goal is to identify molecular
targets that have significant roles in these processes. We found that dysregulation of the Src homology 2 domain
containing (Shc) collagen-related proteins play important roles in calreticulin (CTR) exposure, and immunogenic
cell death during AH, exacerbating sterile inflammatory signalling cascades. To study the regulatory role of Shc
proteins in AH, we demonstrated that Shc was induced in patients with AH, and inhibition of hepatocyte Shc in
an animal model with AH resulted in significantly attenuated inflammation and oxidative stress. In hepatocytes
Shc played a role in CTR translocation and exposure, a critical DAMP that determines immunogenicity of cell
death elicited by alcohol. Furthermore in neutrophils Shc/NOX2-dependent signals induced neutrophil
extracellular trap formation thereby further augmenting inflammatory injury in AH. Based on these, we
hypothesize that activation of Shc signals leads to immunogenic cell death and pro-oxidant sterile
inflammatory pathways in AH. We propose three SPECIFIC AIMS to address the key areas generated by the
main hypothesis:
Our first aim is to determine the molecular mechanism by which Shc proteins are involved in redox signaling
and calreticulin (CTR) exposure as DAMP in hepatocytes during alcoholic hepatitis.
a) We propose to study the mechanism of redox-mediated p52Shc activation. To evaluate the mechanistic
aspects of p52Sh signals, deletion mutants will be generated by site-directed mutagenesis to delineate the key
phosphorylation sites. Stress signaling will be evaluated in conjunction with ROS production, and CTR
translocation. b) We will define the signals eliciting CTR exit from the ER using both biochemical approaches
and the novel real-time, live-cell confocal fluorescence microscopy. In our second aim we will focus on the role
of neutrophil extracellular trap (NET) formation in alcoholic hepatitis. a) We propose studies that evaluate the
key role of Shc/NOX2 in NET formation, and interrogate downstream signaling cascades. We discovered that
p52Shc directly binds to the p47phox NOX2 subunit and directly plays a role in enzyme activation. The
interaction will be assessed by biolayer interferometry assays and lucigenin/Amplex red assays for ROS
production. We will study the mechanistic aspects of p47phox mobilization in correlation to p52Shc binding,
activation of the NOX2 complex in lipid rafts and correlate to NET formation (by FRET microscopy, and by
dynamic live cell imaging). b) As persistence of NETs can fuel further inflammation, we will investigate the
mechanism of defective NET phagocytosis in AH. Our third aim is to study the in vivo effects of Shc s...

## Key facts

- **NIH application ID:** 9890961
- **Project number:** 2I01BX002418-06A2
- **Recipient organization:** VETERANS ADMIN PALO ALTO HEALTH CARE SYS
- **Principal Investigator:** Natalie J. Torok
- **Activity code:** I01 (R01, R21, SBIR, etc.)
- **Funding institute:** VA
- **Fiscal year:** 2020
- **Award amount:** —
- **Award type:** 2
- **Project period:** 2014-07-01 → 2023-12-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9890961

## Citation

> US National Institutes of Health, RePORTER application 9890961, Novel sterile inflammatory pathways in alcoholic hepatitis (2I01BX002418-06A2). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/9890961. Licensed CC0.

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