Cryptosporidium impacts epithelial turnover and is resistant to induced death of the host cell.

Infection with the apicomplexan parasite Cryptosporidium is a leading cause of diarrheal disease. Cryptosporidiosis is of particular importance in infants and shows a strong association with malnutrition, both as a risk factor and as a consequence. Cryptosporidium invades and replicates within the small intestine epithelial cells. This is a highly dynamic tissue that is developmentally stratified along the villus axis. New cells emerge from a stem cell niche in the crypt and differentiate into mature epithelial cells while moving toward the villus tip, where they are ultimately shed. Here, we studied the impact of Cryptosporidium infection on this dynamic architecture. Tracing DNA synthesis in pulse-chase experiments in vivo, we quantified the genesis and migration of epithelial cells along the villus. We found proliferation and epithelial migration to be elevated in response to Cryptosporidium infection. Infection also resulted in significant cell loss documented by imaging and molecular assays. Consistent with these observations, single-cell RNA sequencing of infected intestines showed a gain of young and a loss of mature cells. Interestingly, enhanced epithelial cell loss was not a function of enhanced apoptosis of infected cells. To the contrary, Cryptosporidium-infected cells were less likely to be apoptotic than bystanders, and experiments in tissue culture demonstrated that infection provided enhanced resistance to chemically induced apoptosis to the host but not bystander cells. Overall, this study suggests that Cryptosporidium may modulate cell apoptosis and documents pronounced changes in tissue homeostasis due to parasite infection, which may contribute to its long-term impact on the developmental and nutritional state of children. IMPORTANCE: The intestine must balance its roles in digestion and nutrient absorption with the maintenance of an effective barrier to colonization and breach by numerous potential pathogens. An important component of this balance is its constant turnover, which is modulated by a gain of cells due to proliferation and loss due to death or extrusion. Here, we report that Cryptosporidium infection changes the dynamics of this process increasing both gain and loss of enterocytes speeding up the villus elevator. This leads to a much more immature epithelium and a reduction of the number of those cells typically found toward the villus apex best equipped to take up key nutrients including carbohydrates and lipids. These changes in the cellular architecture and physiology of the small intestine may be linked to the profound association between cryptosporidiosis and malnutrition.

Catalytic activity and autoprocessing of murine caspase-11 mediate noncanonical inflammasome assembly in response to cytosolic LPS.

Inflammatory caspases are cysteine protease zymogens whose activation following infection or cellular damage occurs within supramolecular organizing centers (SMOCs) known as inflammasomes. Inflammasomes recruit caspases to undergo proximity-induced autoprocessing into an enzymatically active form that cleaves downstream targets. Binding of bacterial LPS to its cytosolic sensor, caspase-11 (Casp11), promotes Casp11 aggregation within a high-molecular-weight complex known as the noncanonical inflammasome, where it is activated to cleave gasdermin D and induce pyroptosis. However, the cellular correlates of Casp11 oligomerization and whether Casp11 forms an LPS-induced SMOC within cells remain unknown. Expression of fluorescently labeled Casp11 in macrophages revealed that cytosolic LPS induced Casp11 speck formation. Unexpectedly, catalytic activity and autoprocessing were required for Casp11 to form LPS-induced specks in macrophages. Furthermore, both catalytic activity and autoprocessing were required for Casp11 speck formation in an ectopic expression system, and processing of Casp11 via ectopically expressed TEV protease was sufficient to induce Casp11 speck formation. These data reveal a previously undescribed role for Casp11 catalytic activity and autoprocessing in noncanonical inflammasome assembly, and shed new light on the molecular requirements for noncanonical inflammasome assembly in response to cytosolic LPS.

Distinct sequential death complexes regulate pyroptosis and IL-1ß release in response to Yersinia blockade of immune signaling.

Pathogen infection of host cells triggers an inflammatory cell death termed pyroptosis via activation of inflammatory caspases. However, blockade of immune signaling kinases by the Yersinia virulence factor YopJ triggers cell death involving both apoptotic caspase-8 and pyroptotic caspase-1. While caspase-1 is normally activated within inflammasomes, Yersinia-induced caspase-1 activation is independent of known inflammasome components. We report that caspase-8 is an essential initiator, while caspase-1 is an essential amplifier of its own activation through two feed-forward loops involving caspase-1 auto-processing and caspase-1-dependent activation of gasdermin D and NLPR3. Notably, while Yersinia-induced caspase-1 activation and cell death are inflammasome-independent, IL-1ß release requires NLPR3 inflammasome activation. Mechanistically, caspase-8 is rapidly activated within multiple foci throughout the cell, followed by assembly of a canonical inflammasome speck, indicating that caspase-8 and canonical inflammasome complex assemblies are kinetically and spatially distinct. Our findings reveal that functionally interconnected but distinct death complexes mediate pyroptosis and IL-1ß release in response to pathogen blockade of immune signaling.

A TNF-IL-1 circuit controls Yersinia within intestinal pyogranulomas.

Tumor necrosis factor (TNF) is a pleiotropic inflammatory cytokine that mediates antimicrobial defense and granuloma formation in response to infection by numerous pathogens. We previously reported that Yersinia pseudotuberculosis colonizes the intestinal mucosa and induces the recruitment of neutrophils and inflammatory monocytes into organized immune structures termed pyogranulomas (PG) that control Yersinia infection. Inflammatory monocytes are essential for the control and clearance of Yersinia within intestinal PG, but how monocytes mediate Yersinia restriction is poorly understood. Here, we demonstrate that TNF signaling in monocytes is required for bacterial containment following enteric Yersinia infection. We further show that monocyte-intrinsic TNFR1 signaling drives the production of monocyte-derived interleukin-1 (IL-1), which signals through IL-1 receptors on non-hematopoietic cells to enable PG-mediated control of intestinal Yersinia infection. Altogether, our work reveals a monocyte-intrinsic TNF-IL-1 collaborative inflammatory circuit that restricts intestinal Yersinia infection.