Salmonella Typhimurium induces NAIP/NLRC4- and NLRP3/ASC-independent, caspase-1/4-dependent inflammasome activation in human intestinal epithelial cells

Abstract

Salmonella enterica serovar Typhimurium is a gram-negative pathogen that causes diseases ranging from gastroenteritis to systemic infection and sepsis. Salmonella uses type III secretion systems (T3SSs) to inject effectors into host cells. While these effectors are necessary for bacterial invasion and intracellular survival, intracellular delivery of T3SS products also enables detection of Salmonella by cytosolic immune sensors. Upon detecting translocated Salmonella ligands, these sensors form multimeric complexes called inflammasomes, which activate caspases that lead to proinflammatory cytokine release and pyroptosis. In particular, the Salmonella T3SS needle, inner rod, and flagellin proteins activate the NAIP/NLRC4 inflammasome in murine intestinal epithelial cells (IECs), which leads to restriction of bacterial replication and extrusion of infected IECs into the intestinal lumen, thereby preventing systemic dissemination of Salmonella . While these processes are studied quite well in mice, the role of the NAIP/NLRC4 inflammasome in human IECs remains unknown. Unexpectedly, we found the NAIP/NLRC4 inflammasome is dispensable for early inflammasome responses to Salmonella in both human intestinal epithelial cell lines and organoids. Additionally, the NLRP3 inflammasome and the adaptor protein ASC are not required for inflammasome activation in Caco-2 cells. Instead, we observed a partial requirement for caspase-1, and a necessity for caspase-4 and GSDMD pore-forming activity in mediating inflammasome responses to Salmonella in Caco-2 cells. These findings suggest that unlike murine IECs, human IECs do not rely on NAIP/NLRC4, and also do not use NLRP3/ASC. Instead, they primarily use caspases-1 and -4 to mediate early inflammasome responses to SPI-1-expressing Salmonella .

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

“…3C, 3D) (37). ASC-positive signal was not observed in HIO epithelial cells, consistent with a recent report that Salmonella-induced epithelial cell death occurs independently of ASC (28), but instead was associated with cells positive for vimentin, a protein expressed by PMNs and mesenchymal cells within the PMN-HIOs. ASC and Vimentin double-positive cells were primarily located within the lumen of PMN-HIOs, suggesting that these cells are PMNs.…”

“…We found that many, but not all luminal epithelial cells were positive for cleaved Caspase-3 (Fig. 2E) suggesting that multiple forms of cell death were occurring in the PMN-HIO likely including Caspase-4/5 mediated shedding of Salmonella- infected epithelial cells as has previously been established in a human enteroid infection model (5, 6) and more recently shown in a Caco-2 model of Salmonella infection (28). Interestingly, PMN-induced cell shedding was not restricted to infected cells, as we observed both infected and uninfected cells in the PMN-HIO lumen (SI Appendix, Fig.…”