# Enterotoxigenic Escherichia coli–induced intestinal epithelial necroptosis drives lamina propria immune cell pyroptosis and mucosal injury in piglets

## TL;DR

This study shows that intestinal cell death caused by a type of E. coli leads to immune cell damage and gut injury in piglets, and blocking this process can reduce harm.

## Contribution

The study reveals a sequential necroptosis-pyroptosis mechanism in ETEC-induced intestinal injury in piglets.

## Key findings

- ETEC infection triggers necroptosis in jejunal crypt epithelial cells followed by pyroptosis in lamina propria lymphocytes.
- Necroptosis inhibition with Nec-1 reduces inflammation and tissue damage in piglets.
- ETEC-induced cell death correlates with increased inflammatory markers and loss of gut barrier integrity.

## Abstract

Necroptosis is an inflammatory programmed cell death pathway linked to diverse physiological and pathological disorders, yet its role in Enterotoxigenic Escherichia coli (ETEC)−induced intestinal inflammation and mucosal injury remains poorly understood. This study aimed to elucidate the contribution of intestinal epithelial cell necroptosis to the development of intestinal inflammation and injury induced by ETEC infection in piglets. Following ETEC challenge in piglets, key proteins involved in necroptosis, including phosphorylated receptor-interacting protein kinase 3 (p-RIP3) and high-mobility group box 1 (HMGB1), were upregulated in jejunal crypt epithelial cells, which are primarily composed of Paneth cells and stem cells, in a time-dependent manner. In addition, ETEC challenge triggered time−dependent pyroptosis in jejunal lamina propria lymphocytes, a population that includes macrophages, as demonstrated by elevated levels of NLRP3, Caspase−1, GSDMD−N, and Cleaved -IL−1β (p17) proteins in lamina propria lymphocytes. Necroptosis of jejunal crypt epithelial cells occurred prior to pyroptosis of lamina propria lymphocytes, indicating that epithelial cell necroptosis may contribute to the induction of pyroptosis in lamina propria lymphocytes. ETEC challenge induced progressive TNF−α and IL−1β upregulation in plasma, jejunal crypt epithelial cells, and lamina propria lymphocytes of piglets. These changes coincided with intestinal injury and barrier loss, which were indicated by increased plasma i−FABP and decreased jejunal ZO−1 and Occludin. Notably, Nec−1 pretreatment mitigates ETEC−induced intestinal inflammation and tissue damage in piglets by inhibition of crypt epithelial cells necroptosis and the ensuing pyroptosis of lymphocytes. These results indicate that targeting upstream epithelial-cell necroptosis is an important strategy to attenuate inflammation and preserve barrier integrity.