<i>Ex Vivo</i> and <i>In Vivo</i> Mice Models to Study <i>Blastocystis</i> spp. Adhesion, Colonization and Pathology: Closer to Proving Koch's Postulates

<div><p><i>Blastocystis</i> spp. are widely prevalent extra cellular, non-motile anerobic protists that inhabit the gastrointestinal tract. Although <i>Blastocystis</i> spp. have been associated with gastrointestinal symptoms, irritable bowel syndrome and urticaria, their clinical significance has remained controversial. We established an <i>ex vivo</i> mouse explant model to characterize adhesion in the context of tissue architecture and presence of the mucin layer. Using confocal microscopy with tissue whole mounts and two axenic isolates of <i>Blastocystis</i> spp., subtype 7 with notable differences in adhesion to intestinal epithelial cells (IEC), isolate B (ST7-B) and isolate H (more adhesive, ST7-H), we showed that adhesion is both isolate dependent and tissue trophic. The more adhesive isolate, ST7-H was found to bind preferentially to the colon tissue than caecum and terminal ileum. Both isolates were also found to have mucinolytic effects. We then adapted a DSS colitis mouse model as a susceptible model to study colonization and acute infection by intra-caecal inoculation of trophic <i>Blastocystis</i> spp.cells. We found that the more adhesive isolate ST7-H was also a better colonizer with more mice shedding parasites and for a longer duration than ST7-B. Adhesion and colonization was also associated with increased virulence as ST7-H infected mice showed greater tissue damage than ST7-B. Both the <i>ex vivo</i> and <i>in vivo</i> models used in this study showed that <i>Blastocystis</i> spp. remain luminal and predominantly associated with mucin. This was further confirmed using colonic loop experiments. We were also successfully able to re-infect a second batch of mice with ST7-H isolates obtained from fecal cultures and demonstrated similar histopathological findings and tissue damage thereby coming closer to proving Koch’s postulates for this parasite.</p></div

Fecal and intestinal cultures using Jones media from (A) normal mice, (B) DSS-treated mice and (C) Parasites recovered in culture were used to re-infect DSS-treated mice.

<p>Fecal and intestinal cultures using Jones media from (A) normal mice, (B) DSS-treated mice and (C) Parasites recovered in culture were used to re-infect DSS-treated mice.</p

<i>Blastocystis</i> spp. remains luminal and binds to mucin <i>in vivo</i>.

<p>Representative histology of colon loops from DSS treated C57BL/6 mice (starved overnight to minimize intestinal contents) inoculated with 5x10⁷<i>Blastocystis</i> spp. ST7-H or ST7-B or saline (controls) for 1 hour <i>in vivo</i>, gently washed and preserved in methacarn. <i>Blastocystis spp</i>. were found only in the lumen and mucus layer for both isolates. Black arrows indicate luminal and mucin bound parasites and the white arrow in the control tissue shows well-preserved PAS stained inner mucin layer.</p

The MUC13 cell-surface mucin protects against intestinal inflammation by inhibiting epithelial cell apoptosis

Background and Aims: The MUC13 transmembrane mucin is highly and constitutively expressed in the small and large intestine. Although MUC13 polymorphisms have been associated with human inflammatory bowel diseases and susceptibility to Escherichia coli infection in pigs, the biological functions of MUC13 are unknown. This study aimed to explore whether MUC13 modulates intestinal inflammation. Methods: Muc13 mice were generated, phenotyped and challenged with the colitis-inducing agent, dextran sodium sulphate (DSS). Colitis was assessed by clinical symptoms and intestinal histopathology. Intestinal epithelial cell apoptosis and proliferation, macrophage infiltration and cytokine production were also quantified. Apoptosis of human LS513 intestinal epithelial cells in response to apoptotic agents, including DSS, was also measured, following knockdown of MUC13 with siRNA. Results: Muc13 mice were viable, fertile and developed normally, with no spontaneous intestinal pathology except mild focal neutrophilic inflammation in the small and large intestines of old mice. In response to DSS challenge, Muc13 mice developed more severe acute colitis, as reflected by increased weight loss, rectal bleeding, diarrhoea and histological colitis scores compared with wild-type mice. Increased numbers of F4/80 macrophages in inflamed mucosa of Muc13 mice were accompanied by increased expression of intestinal IL-1β and TNFα mRNA. Muc13 mice had significantly increased intestinal epithelial cell apoptosis within 3 days of DSS exposure. LS513 cells were more susceptible to DSS, actinomycin-D, ultraviolet irradiation and TRAIL-induced apoptosis when MUC13 was knocked down by siRNA. Conclusions: These novel findings indicate a protective role for Muc13 in the colonic epithelium by inhibiting toxin-induced apoptosis and have important implications for intestinal infections, inflammatory diseases and the development of intestinal cancer