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.

Intestinal cDC1s provide cues required for CD4+ T cell-mediated resistance to Cryptosporidium.

Cryptosporidium is an enteric pathogen and a prominent cause of diarrheal disease worldwide. Control of Cryptosporidium requires CD4+ T cells, but how protective CD4+ T cell responses are generated is poorly understood. Here, Cryptosporidium parasites that express MHCII-restricted model antigens were generated to understand the basis for CD4+ T cell priming and effector function. These studies revealed that parasite-specific CD4+ T cells are primed in the draining mesenteric lymph node but differentiate into Th1 cells in the gut to provide local parasite control. Although type 1 conventional dendritic cells (cDC1s) were dispensable for CD4+ T cell priming, they were required for CD4+ T cell gut homing and were a source of IL-12 at the site of infection that promoted local production of IFN-γ. Thus, cDC1s have distinct roles in shaping CD4+ T cell responses to an enteric infection: first, to promote gut homing from the mesLN, and second, to drive effector responses in the intestine.

Analysis of intestinal epithelial cell responses to Cryptosporidium highlights the temporal effects of IFN-γ on parasite restriction.

The production of IFN-γ is crucial for control of multiple enteric infections, but its impact on intestinal epithelial cells (IEC) is not well understood. Cryptosporidium parasites exclusively infect epithelial cells and the ability of interferons to activate the transcription factor STAT1 in IEC is required for parasite clearance. Here, the use of single cell RNA sequencing to profile IEC during infection revealed an increased proportion of mid-villus enterocytes during infection and induction of IFN-γ-dependent gene signatures that was comparable between uninfected and infected cells. These analyses were complemented by in vivo studies, which demonstrated that IEC expression of the IFN-γ receptor was required for parasite control. Unexpectedly, treatment of Ifng-/- mice with IFN-γ showed the IEC response to this cytokine correlates with a delayed reduction in parasite burden but did not affect parasite development. These data sets provide insight into the impact of IFN-γ on IEC and suggest a model in which IFN-γ signalling to uninfected enterocytes is important for control of Cryptosporidium.

Dendritic cell-mediated responses to secreted Cryptosporidium effectors promote parasite-specific CD8+ T cell responses.

Cryptosporidium causes debilitating diarrheal disease in patients with primary and acquired defects in T cell function. However, it has been a challenge to understand how this infection generates T cell responses and how they mediate parasite control. Here, Cryptosporidium was engineered to express a parasite effector protein (MEDLE-2) that contains the major histocompatibility complex-I restricted SIINFEKL epitope which is recognized by T cell receptor transgenic OT-I(OVA-TCR-I) clusters of differentiation (CD)8+ T cells. These modified parasites induced expansion of endogenous SIINFEKL-specific and OT-I CD8+ T cells that were a source of interferon-gamma (IFN-γ) that could restrict growth of Cryptosporidium. This T cell response was dependent on the translocation of the effector and similar results were observed with another secreted parasite effector (rhoptry protein 1). Although infection and these translocated effector proteins are restricted to intestinal epithelial cells, type 1 conventional dendritic cells were required to generate CD8+ T cell responses to these model antigens. These data sets highlight Cryptosporidium effectors as potential targets of the immune system and suggest that crosstalk between enterocytes and type 1 conventional dendritic cells is crucial for CD8+ T cell responses to Cryptosporidium.