Abstract
Cryptosporidium, a ubiquitous coccidian protozoan parasite that infects the gastrointestinal epithelium and other mucosal surfaces, is an important opportunistic pathogen for immunocompromised individuals and a common cause of diarrhea in young children in the developing countries. One of the pathological hallmarks of intestinal cryptosporidiosis is villous atrophy, which results in a shorter height of intestinal villi. Here, we investigated the effects of Cryptosporidium infection on intestinal epithelial growth, using an ex vivo model of intestinal cryptosporidiosis employing enteroids from mice. We detected infection of enteroids isolated from immunocompetent adult and neonatal mice after ex vivo exposure to Cryptosporidium sporozoites. We observed a significant inhibition of enteroid propagation following infection. Intriguingly, we identified a decreased expression level of intestinal stem cell markers in enteroids following C. parvum infection. We further measured the expression levels of several Wnt antagonists or agonists in infected enteroids, as induction of the Wnt/β‐catenin activation is a key factor for intestinal stem cell function. We detected a markedly increased level of the Dickkopf‐related protein 1 and decreased level of the Wnt family member 5a in enteroids after infection. The low density lipoprotein receptor‐related protein 5, one of the Wnt co‐receptors, is downregulated in the infected enteroids. In addition, increased apoptotic cell death and cell senescence were observed in the infected enteroids. Our results demonstrate a significant inhibitory effect of Cryptosporidium infection on the ex vivo propagation of enteroids from mice, providing additional insights into the impact of Cryptosporidium infection on intestinal epithelial growth.
Highlights
Cryptosporidium, a ubiquitous coccidian protozoan parasite, infects the gastrointestinal epithelium and other mucosal surfaces, causing an asymptomatic or self-limited disease in immunocompetent individuals but a life-threatening diarrheal disease in AIDS patients (Checkley et al 2015)
Since C. parvum infection is mainly localized in the ileum region in the intestine (Farthing 2000), we isolated the crypt/villus units from the small intestine within 2.0 cm above the cecum for adult mice to develop enteroids for ex vivo infection
As previously reported (Mahe et al 2013), these isolated crypt/villus units were cultured in the defined medium in the 3D gel culture dishes, and developed into enteroids lined with a monolayer of cells showing sub-apical membrane staining of actin typical to polarized intestinal epithelium, consistent with results from previous studies (Gao and Kaestner 2010; Mahe et al 2013)
Summary
Cryptosporidium, a ubiquitous coccidian protozoan parasite, infects the gastrointestinal epithelium and other mucosal surfaces, causing an asymptomatic or self-limited disease in immunocompetent individuals but a life-threatening diarrheal disease in AIDS patients (Checkley et al 2015). C. parvum sporozoites attach to the apical membrane of intestinal epithelial cells (mainly villus enterocytes) and form a parasitophorous vacuole in which the organism remains intracellular but extracytoplasmic, limiting a direct infection often only to enterocytes and preventing a direct infection of immune cell types (Chen et al 2002) Due to this “minimally invasive” nature of infection, intestinal epithelium provides the first line of defense and plays a critical role in activating and orchestrating host responses to C. parvum infection (Chen et al 2002). These chemokines/ cytokines of enterocyte origin, as well as epithelial cellderived exosomes, mobilize and activate immune effector cells (e.g., NK cells, macrophages, dendritic cells, CD4,+ and CD8+ lymphocytes) at the site of infection (Chen et al 2002)
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