Salmonella enterica Infection of Murine and Human Enteroid-Derived Monolayers Elicits Differential Activation of Epithelium-Intrinsic Inflammasomes.

Recent studies have determined that inflammasome signaling plays an important role in driving intestinal epithelial cell (IEC) responses to bacterial infections, such as Salmonella enterica serovar Typhimurium. There are two primary inflammasome pathways, canonical (involving caspase-1) and noncanonical (involving caspase-4 and -5 in humans and caspase-11 in mice). Prior studies identified the canonical inflammasome as the major pathway leading to interleukin-18 (IL-18) release and restriction of S Typhimurium replication in the mouse cecum. In contrast, the human C2Bbe1 colorectal carcinoma cell line expresses little caspase-1 but instead utilizes caspase-4 to respond to S Typhimurium infection. Intestinal enteroid culture has enabled long-term propagation of untransformed IECs from multiple species, including mouse and human. Capitalizing on this technology, we used a genetic approach to directly compare the relative importance of different inflammatory caspases in untransformed mouse and human IECs and transformed human IECs upon S Typhimurium infection in vitro We show that caspase-1 is important for restricting intracellular S Typhimurium replication and initiating IL-18 secretion in mouse IECs but is dispensable in human IECs. In contrast, restriction of intracellular S Typhimurium and production of IL-18 are dependent on caspase-4 in both transformed and untransformed human IECs. Notably, cytosolic replication in untransformed cells from both species was less pronounced than in transformed human cells, suggesting that transformation may impact additional pathways that restrict S Typhimurium replication. Taken together, these data highlight the differences between mouse and human IECs and the utility of studying transformed and untransformed cells in parallel.

Intestinal restriction of Salmonella Typhimurium requires caspase-1 and caspase-11 epithelial intrinsic inflammasomes.

We investigated the role of the inflammasome effector caspases-1 and -11 during Salmonella enterica serovar Typhimurium infection of murine intestinal epithelial cells (IECs). Salmonella burdens were significantly greater in the intestines of caspase-1/11 deficient (Casp1/11-/-), Casp1-/- and Casp11-/- mice, as compared to wildtype mice. To determine if this reflected IEC-intrinsic inflammasomes, enteroid monolayers were derived and infected with Salmonella. Casp11-/- and wildtype monolayers responded similarly, whereas Casp1-/- and Casp1/11-/- monolayers carried significantly increased intracellular burdens, concomitant with marked decreases in IEC shedding and death. Pretreatment with IFN-γ to mimic inflammation increased caspase-11 levels and IEC death, and reduced Salmonella burdens in Casp1-/- monolayers, while high intracellular burdens and limited cell shedding persisted in Casp1/11-/- monolayers. Thus caspase-1 regulates inflammasome responses in IECs at baseline, while proinflammatory activation of IECs reveals a compensatory role for caspase-11. These results demonstrate the importance of IEC-intrinsic canonical and non-canonical inflammasomes in host defense against Salmonella.

The Intestinal Epithelium: Central Coordinator of Mucosal Immunity.

The gastrointestinal (GI) tract represents a unique challenge to the mammalian immune system. It must tolerate the presence of the luminal microbiota and thus not respond to their products, but still protect the intestinal mucosa from potentially harmful dietary antigens and invading pathogens. The intestinal epithelium, composed of a single layer of cells, is crucial for preserving gut homeostasis and acts both as a physical barrier and as a coordinating hub for immune defense and crosstalk between bacteria and immune cells. We highlight here recent findings regarding communication between microbes and intestinal epithelial cells (IECs), as well as the immune mechanisms employed by distinct IEC subsets to promote homeostasis, emphasizing the central and active role that these cells play in host enteric defense.

Milk Fat Globule Membrane Supplementation in Formula Modulates the Neonatal Gut Microbiome and Normalizes Intestinal Development.

Breast milk has many beneficial properties and unusual characteristics including a unique fat component, termed milk fat globule membrane (MFGM). While breast milk yields important developmental benefits, there are situations where it is unavailable resulting in a need for formula feeding. Most formulas do not contain MFGM, but derive their lipids from vegetable sources, which differ greatly in size and composition. Here we tested the effects of MFGM supplementation on intestinal development and the microbiome as well as its potential to protect against Clostridium difficile induced colitis. The pup-in-a-cup model was used to deliver either control or MFGM supplemented formula to rats from 5 to 15 days of age; with mother's milk (MM) reared animals used as controls. While CTL formula yielded significant deficits in intestinal development as compared to MM littermates, addition of MFGM to formula restored intestinal growth, Paneth and goblet cell numbers, and tight junction protein patterns to that of MM pups. Moreover, the gut microbiota of MFGM and MM pups displayed greater similarities than CTL, and proved protective against C. difficile toxin induced inflammation. Our study thus demonstrates that addition of MFGM to formula promotes development of the intestinal epithelium and microbiome and protects against inflammation.

Salmonella and the Inflammasome: Battle for Intracellular Dominance.

Inflammasomes are macromolecular cytoplasmic complexes that act as signaling platforms for the activation of inflammatory caspases. Their activation triggers the processing and secretion of the pro-inflammatory cytokines IL-1ß and IL-18, as well as the induction of a specialized form of inflammatory cell death termed pyroptosis. Here, we review the mechanisms of inflammasome activation triggered by the intracellular pathogen Salmonella enterica serovar Typhimurium. We highlight the different inflammasome subfamilies utilized by macrophages, neutrophils, dendritic cells, and intestinal epithelial cells response to a Salmonella infection as well as the Salmonella ligands that trigger each inflammasome's formation. We also discuss the evasion strategies utilized by Salmonella to avoid inflammasome detection. Overall, inflammasomes play a key and multilayered role at distinct stages of host cell defense against Salmonella infection.

Noncanonical inflammasome activation of caspase-4/caspase-11 mediates epithelial defenses against enteric bacterial pathogens.

Inflammasome-mediated host defenses have been extensively studied in innate immune cells. Whether inflammasomes function for innate defense in intestinal epithelial cells, which represent the first line of defense against enteric pathogens, remains unknown. We observed enhanced Salmonella enterica serovar Typhimurium colonization in the intestinal epithelium of caspase-11-deficient mice, but not at systemic sites. In polarized epithelial monolayers, siRNA-mediated depletion of caspase-4, a human ortholog of caspase-11, also led to increased bacterial colonization. Decreased rates of pyroptotic cell death, a host defense mechanism that extrudes S. Typhimurium-infected cells from the polarized epithelium, accounted for increased pathogen burdens. The caspase-4 inflammasome also governs activation of the proinflammatory cytokine, interleukin (IL)-18, in response to intracellular (S. Typhimurium) and extracellular (enteropathogenic Escherichia coli) enteric pathogens, via intracellular LPS sensing. Therefore, an epithelial cell-intrinsic noncanonical inflammasome plays a critical role in antimicrobial defense at the intestinal mucosal surface.

Intestinal epithelium-specific MyD88 signaling impacts host susceptibility to infectious colitis by promoting protective goblet cell and antimicrobial responses.

Intestinal epithelial cells (IECs), including secretory goblet cells, form essential physiochemical barriers that separate luminal bacteria from underlying immune cells in the intestinal mucosa. IECs are common targets for enteric bacterial pathogens, with hosts responding to these microbes through innate toll-like receptors that predominantly signal through the MyD88 adaptor protein. In fact, MyD88 signaling confers protection against several enteric bacterial pathogens, including Salmonella enterica serovar Typhimurium and Citrobacter rodentium. Since IECs are considered innately hyporesponsive, it is unclear whether MyD88 signaling within IECs contributes to this protection. We infected mice lacking MyD88 solely in their IECs (IEC-Myd88(-/-)) with S. Typhimurium. Compared to wild-type (WT) mice, infected IEC-Myd88(-/-) mice suffered accelerated tissue damage, exaggerated barrier disruption, and impaired goblet cell responses (Muc2 and RELMß). Immunostaining revealed S. Typhimurium penetrated the IECs of IEC-Myd88(-/-) mice, unlike in WT mice, where they were sequestered to the lumen. When isolated crypts were assayed for their antimicrobial actions, crypts from IEC-Myd88(-/-) mice were severely impaired in their antimicrobial activity against S. Typhimurium. We also examined whether MyD88 signaling in IECs impacted host defense against C. rodentium, with IEC-Myd88(-/-) mice again suffering exaggerated tissue damage, impaired goblet cell responses, and reduced antimicrobial activity against C. rodentium. These results demonstrate that MyD88 signaling within IECs plays an important protective role at early stages of infection, influencing host susceptibility to infection by controlling the ability of the pathogen to reach and survive at the intestinal mucosal surface.