Human neutrophils direct epithelial cell extrusion to enhance intestinal epithelial host defense during Salmonella infection

Abstract

Infection of the human gut by Salmonella enterica Typhimurium (STM) results in a localized inflammatory disease that is not mimicked in murine infections. To determine mechanisms by which neutrophils, as early responders to bacterial challenge, direct inflammatory programming of human intestinal epithelium, we established a multi-component human intestinal organoid (HIO) model of STM infection. HIOs were micro-injected with STM and then seeded with primary human polymorphonuclear leukocytes (PMN-HIOs), specifically neutrophils and analyzed for bacterial growth and host cell survival. Surprisingly, PMNs did not affect luminal colonization of Salmonella, but their presence reduced intraepithelial bacterial burden. Adding PMNs to infected HIOs resulted in substantial accumulation of shed intestinal epithelial cells that could be blocked by Caspase-1 or Caspase-3 inhibition. Cleaved Caspase-3 was present in epithelial cells, but expression of the inflammasome adaptor, ASC, was only detected in PMNs. Caspase inhibition also increased bacterial burden in the epithelium of the PMN-HIO, suggesting PMNs enhance activation of cell death pathways in human intestinal epithelial cells as a protective response to infection. These data support a critical function for neutrophils beyond their antimicrobial role whereby they amplify cell death and extrusion of epithelial cells from the Salmonella-infected intestinal monolayer.Significance statementNeutrophils are early responders to Salmonella intestinal infection, but how they influence infection progression and outcome is unknown. Here we use a co-culture model of human intestinal organoids and human primary neutrophils to study the contribution of human neutrophils to Salmonella infection of the intestinal epithelium. We found that neutrophils markedly enhanced epithelial defenses, including enhancing cell extrusion to reduce intraepithelial burden of Salmonella and association with the epithelium, rather than directly killing Salmonella in the HIO lumen. These findings reveal a novel role for neutrophils in the gut beyond killing invading pathogens and illuminate how neutrophils can reprogram cells in the gut environment to enhance antimicrobial defenses.

Neutrophils prime unique transcriptional responses in intestinal organoids during infection with nontyphoidal Salmonella enterica serovars

“…3A), which were all highly upregulated during infection in both HIOs and PMN-HIOs. Since PMNs represent approximately less than 5% of the cells in the PMN-HIO model (22), the contribution of PMN transcripts in the overall bulk-RNA-seq is likely minimal. However, analysis of culture supernatants via ELISA revealed that all these mediators were present at significantly higher levels in infected PMN-HIOs compared to HIOs (Fig.…”

“…PMNs are potent drivers of inflammation, so we reasoned that PMNs would likely modulate the intestinal host response to Salmonella infection. Using the PMN-HIO model, we previously characterized the degree of PMN recruitment and effect on bacterial colonization during Salmonella infection (22). Here, to probe the contribution of PMNs to the global intestinal transcriptional responses during infection, HIOs and PMN-HIOs were microinjected with SE, STM or PBS and harvested at 8h post-infection (8hpi) for bulk RNA-sequencing (RNA-seq).…”