# Alternative pathways of gasdermin function and IL-1beta secretion in granulocytic leukocytes

> **NIH NIH P01** · CASE WESTERN RESERVE UNIVERSITY · 2022 · $410,012

## Abstract

Abstract
This project aims to define novel signaling mechanisms for the regulation of innate immune responses by
Gasdermins D and E (GSDMD/E) in granulocytic leukocytes that include neutrophils, mast cells, and
eosinophils. Physiological roles for GSDMD in both pyroptosis and IL-1β release during inflammasome
signaling have been extensively characterized in macrophages and other mononuclear myeloid leukocytes.
This involves cleavage of GSDMD by caspase-1 to generate N-GSDMD fragments which oligomerize in the
plasma membrane (PM) to form pores that mediate IL-1β efflux and pyroptosis. Notably, the granulocytic
myeloid leukocytes are also important sources of IL-1β during multiple innate immune responses, but few
studies have examined roles for GSDMD or other GSDM-family proteins in these cells. Our preliminary data
shows that caspase-1-generated N-GSDMD in neutrophils does not localize to the PM to form pores or drive
pyroptosis but is required for IL-1β secretion. This absence of PM pores reflects alternative subcellular
trafficking whereby N-GSDMD associates with azurophilic secretory granules (AG) and LC3+ autophagosomes.
Analyses using ATG7-deficient neutrophils indicate that IL-1β is secreted from neutrophils via an autophagy
machinery-assisted mechanism. These findings reveal fundamental differences in GSDM biology, including
high expression of GSDME, between granulocytes and macrophages that will shape granulocyte roles in
innate immunity. We hypothesize that the abundant secretory granules which define neutrophils, mast
cells and eosinophils underlie granulocyte-specific mechanisms for non-canonical production of
bioactive IL-1β and for regulated cell death via alternative pathways of GSDM family processing and
subcellular trafficking. In Aim 1, we will define signaling hierarchies based on activation of inflammatory
caspases, granule-derived serine proteases, apoptotic caspases, GSDMD, and GSDME that facilitate
alternative paths of bioactive IL-1β production by neutrophils, mast cells, and eosinophils. Studies will include
analyses of GSDMD/E trafficking to secretory granules versus the plasma membrane and GSDMD's ability to
act as a chaperone for autophagy protein-assisted secretion of proIL-1β that can be extracellularly cleaved by
serine proteases. In Aim 2, we will define GSDMD/E signaling networks that regulate granulocyte death
pathways which are induced by triggers of extracellular DNA trap release/ETosis, apoptosis, and progression
to granulocyte senescence. The project will draw on the strengths of the other three projects to facilitate our
functional studies. The results will define novel mechanisms for how GSDMD and GSDME mediate
granulocyte-specific signaling responses which play physiological roles during acute infection but contribute to
tissue dysfunction in humans with common autoimmune, allergic, or chronic inflammatory diseases.

## Key facts

- **NIH application ID:** 10441355
- **Project number:** 5P01AI141350-03
- **Recipient organization:** CASE WESTERN RESERVE UNIVERSITY
- **Principal Investigator:** GEORGE R DUBYAK
- **Activity code:** P01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $410,012
- **Award type:** 5
- **Project period:** 2020-07-24 → 2025-06-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10441355

## Citation

> US National Institutes of Health, RePORTER application 10441355, Alternative pathways of gasdermin function and IL-1beta secretion in granulocytic leukocytes (5P01AI141350-03). Retrieved via AI Analytics 2026-05-25 from https://api.ai-analytics.org/grant/nih/10441355. Licensed CC0.

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