# Extracellular Lipopolysaccharide Triggers the Release of Unconjugated Interferon-Stimulated Gene 15 (ISG15) Protein from Macrophages via Type-I Interferon/Caspase-4/Gasdermin-D Pathway

## TL;DR

This study reveals how macrophages release ISG15 protein without dying, using a pathway involving interferon, caspase-4, and Gasdermin D.

## Contribution

The paper identifies a new non-lytic pathway for ISG15 release via the IFN-Casp4-GSDMD axis.

## Key findings

- Extracellular LPS triggers ISG15 release through GSDMD pores.
- Type-I interferon activates caspase-4 to cleave GSDMD for ISG15 release.
- ISG15 release occurs without lytic cell death.

## Abstract

Interferon-stimulated gene 15 (ISG15) is an interferon-induced ubiquitin-like protein that plays an important role in antiviral defense and inflammatory responses, primarily through the process of ISGylation, whereby ISG15 is covalently conjugated to target proteins. Beyond its intracellular functions, a portion of free unconjugated ISG15 is also released into the extracellular environment during infections and diseases such as cancer. Extracellular ISG15 is known to regulate immune cell activity and cytokine production. Despite its immune-modulatory role, how ISG15 is released from cells has remained unclear. In this study, we have identified a non-lytic mechanism by which human macrophages release ISG15. Using lipopolysaccharide (LPS) as a stimulus, we show that extracellular LPS triggers unconjugated ISG15 release by utilizing plasma membrane-localized Gasdermin D (GSDMD) pores. Mechanistically, LPS via the autocrine/paracrine action of type-I interferon (IFN) activates caspase-4 (Casp4) to cleave the N-terminal domain of GSDMD for the formation of cell surface GSDMD pores that permit the extracellular release of unconjugated ISG15 in the absence of lytic cell death. Together, our studies have identified the IFN-Casp4-GSDMD axis as a previously unrecognized non-classical pathway for unconjugated ISG15 release from cells.