# GSDMD-dependent IL-1 signaling in intestinal inflammation

> **NIH NIH P01** · CASE WESTERN RESERVE UNIVERSITY · 2020 · $541,671

## Abstract

Abstract:
About 1.3 million people suffer from IBD (chronic inflammation of the intestine) in the United States. The etiology
of IBD remains elusive and preventive measures or a cure are not available. Inflammasomes and derived
cytokines IL-1β are being intensely investigated as the signaling hub that is dys-regulated in inflammatory bowel
disease (IBD). IL-1β is synthesized as inactive pro-forms with no secretory signal sequence. Recent studies
have found that a lipid binding protein, Gasdermin D (GSDMD), is required for release of IL-1β in response to
caspase-1/11 inflammasome activation. This breakthrough led to a rapid growth of literature that focuses on the
pore forming and associated pyroptotic activity of GSDMD in myeloid cells. Interestingly, we discovered a novel
nonpyrototic role of GSDMD in guiding the release of IL-1β containing vesicles from intestinal epithelia cells and
T cells in response to Caspase 8 (Casp8) but not casp1 activation. Through unbiased proteomic analysis, we
identified a set of novel GSDMD-interacting proteins in intestinal epithelial cells (IECs), including NEDD4 (an E3
ligase) and the Hsp90 co-chaperone CDC37. Ablation of GSDMD or NEDD4 abolished LPS and ATP-induced
IL-1β production from IECs. Strikingly, LPS+ATP stimulation led to the polyubiquitination of pro-IL-1β, which was
secreted and processed into mature IL-1β along with a complex containing full-length GSDMD, Hsp90/CDC37,
NEDD4, Atg7, Casp8 but not Casp1. In vitro ubiquitination assay demonstrated that NEDD4, known to interact
with LC3 and promote cargo loading into secretory vesicles, catalyzed the polyubiquitination of pro-IL-1β. Indeed,
while GSDMD was associated with LC3+ vesicles; GSDMD-dependent release of extracellular vesicles (< 200
nm) were detected by electron microscopy, which contains the GSDMD/NEDD4/IL-1β complex. Moreover,
inactivating mutation in the Asp276 of GSDMD, which abolishes its pore-forming and pyroptotic activity, did not
impact the GSDMD-guided IL-1β secretion from IECs. In TH17 cells, a T helper cell subset highly relevant to
intestinal inflammation, this GSDMD-guided secretion of polyubiquitinated pro-IL-1β complex (GSDMD, Hsp90,
Casp8 and NEDD4) was also readily detected in response to ATP stimulation and TCR activation. Collectively,
the data revealed a novel nonpyroptotic role for GSDMD in IL-1β release from non-myeloid cells. The pathogenic
role of the GSDMD-guided IL-1β release was investigated in two mouse models of intestinal inflammation. Firstly,
while polyubiquitinated pro-IL-1β, mIL-1β and the GSDMD/NEDD4-secretary complex were induced in a
GSDMD-dependent manner in the intestinal explants in response to dextran sodium sulfate (DSS)-induce colitis,
GSDMD-deficiency attenuated the intestinal inflammation. Secondly, GSDMD or IL-1β deficiency in naïve T cells
reduced their ability to elicit intestinal inflammation. Based on these findings, we hypothesize that the GSDMD
mediates distinct pathways (guided secretion and...

## Key facts

- **NIH application ID:** 10024455
- **Project number:** 1P01AI141350-01A1
- **Recipient organization:** CASE WESTERN RESERVE UNIVERSITY
- **Principal Investigator:** Xiaoxia Li
- **Activity code:** P01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $541,671
- **Award type:** 1
- **Project period:** 2020-07-24 → 2025-06-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10024455

## Citation

> US National Institutes of Health, RePORTER application 10024455, GSDMD-dependent IL-1 signaling in intestinal inflammation (1P01AI141350-01A1). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/10024455. Licensed CC0.

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