Autophagy, a process of degradation and recycling of macromolecules and organelles to maintain cellular homeostasis, has also been shown to help eliminate invading pathogens. Conversely, various pathogens including parasites have been shown to modulate/exploit host autophagy facilitating their intracellular infectious cycle. In this regard, Cryptosporidium parvum (CP), a protozoan parasite of small intestine is emerging as a major global health challenge. However, the pathophysiology of cryptosporidiosis is mostly unknown. We have recently demonstrated CP‐induced epithelial barrier disruption via decreasing the expression of specific tight junction (TJ) and adherens junction (AJ) proteins such as occludin, claudin‐4 and E‐cadherin. Therefore, we utilised confluent Caco‐2 cell monolayers as in vitro model of intestinal epithelial cells (IECs) to investigate the potential role of autophagy in the pathophysiology of cryptosporidiosis. Autophagy was assessed by increase in the ratio of LC3II (microtubule associated protein 1 light chain 3) to LC3I protein and decrease in p62/SQSTM1 protein levels. CP treatment of Caco‐2 cells for 24 hr induced autophagy with a maximum effect observed with 0.5 × 106 oocyst/well. CP decreased mTOR (mammalian target of rapamycin, a suppressor of autophagy) phosphorylation, suggesting autophagy induction via mTOR inactivation. Measurement of autophagic flux utilizing the lysosomal inhibitor chloroquine (CQ) showed more pronounced increase in LC3II level in cells co‐treated with CP + CQ as compared to CP or CQ alone, suggesting that CP‐induced increase in LC3II was due to enhanced autophagosome formation rather than impaired lysosomal clearance. CP infection did not alter ATG7, a key autophagy protein. However, the decrease in occludin, claudin‐4 and E‐cadherin by CP was partially blocked following siRNA silencing of ATG7, suggesting the role of autophagy in CP‐induced decrease in these TJ/AJ proteins. Our results provide novel evidence of autophagy induction by CP in host IECs that could alter important host cell processes contributing to the pathophysiology of cryptosporidiosis.

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Inflammatory bowel diseases (IBDs) are chronic inflammatory multifactorial diseases caused by genetic, immune, and environmental factors. A decrease in intestinal serotonin transporter (SERT), which controls the extracellular availability of serotonin (5-HT) has been implicated in IBD. We previously showed that SERT deletion in mice altered gut bacterial community structure. The gut microbiota-derived metabolites are functional intermediaries between the microbiota and host. Here, we investigated the impact of SERT deficiency on gut metabolites under basal conditions and chronic colitis mimicking human IBD. Methods: Global metabolic profiles were analyzed by “Metabolon” (Durham, NC) on fecal samples from wild type littermates (WT) and SERT KO mice given water or chronic DSS (2.5% DSS for 5 weeks, n=7–9/group). Data were analyzed by ANOVA contrasts (difference between groups) and by two-way ANOVA (P<0.05, q<0.01). Results: SERT KO exhibited more severe colitis vs WT as assessed by histological score, myeloperoxidase activity and colon length. There was more pronounced decrease in the mRNA of tight junction proteins (TJs) occludin-1 and ZO-1 in SERT KO DSS vs WT DSS intestine. Metabolic profiling revealed that SERT deficiency alone resulted in extremely low levels of fecal ectoine, a bacterial derived solute that maintains TJ proteins expression. DSS treatment of WT (but not SERT KO) resulted in a significant increase in microbial derived metabolites including phenylalanine, N-acetylphenylalanine, tyrosine derivatives, glutamate, glutamine and benzoate derivatives. An increase in Trimethylamine N-oxide (TMAO), the short chain fatty acids butyrate/isobutyrate, TCA cycle metabolites; and a decrease in several metabolites including spermidine and various primary and secondary bile acids occurred in DSS treated WT and SERT KO to varying degrees; suggesting that these pathways may contribute to the colitis severity in SERT KO mice. Several secondary bile acids, ketone bodies, the metabolites of pterin, riboflavin pathway (FAD and FMN) and fatty acid metabolism pathways were increased in SERT KO (basal and DSS) suggesting genotype related differences in microbial community. We recently showed an impairment of Aryl hydrocarbon receptor (AhR), an IBD susceptible gene, in SERT KO mice, which could be partly due to altered availability of ligands. Indeed, the bacterial derived AhR ligand tryptamine was extremely low in SERT KO (basal and DSS). DSS increased the host derived AhR ligand, kynurenine in WT, but not in SERT KO.Conclusion:These data highlight the impact of serotonergic machinery and SERT inhibition on host physiology and pathophysiology of IBD. The results provide unique insights into gut bacteria derived metabolites and may aid in the development of novel treatment for disorders with altered SERT and 5-HT availability (Supported by CCFA and NIH).

The aryl hydrocarbon receptor (AhR) is an important transcription factor that regulates xenobiotic metabolism and is vital for maintaining immune cell populations, proliferation, and motility in the gastrointestinal (GI) tract. Recent studies have shown that deletion of AhR from mouse intestinal epithelial cells worsens experimental colitis, while activation of AhR by its agonists protects against acuteinflammation. AhR is activated by a variety of ligands including indole‐3‐carbinole (I3C) derived from cruciferous vegetables, such as broccoli and brussels sprouts. Whether AhR ligands derived from the diet impact chronic intestinal inflammation and/or cause gut microbiota alterations has not yet been investigated. Methods C57BL/6 mice (7‐9 wk, M) were fed either standard chow diet or purified diet (depleted of AhR ligands, referred to as ‐I3C diet) or the purified diet supplemented with 200 ppm of I3C (+I3C diet). Along with the diet, the mice were given either water or treated with 5 cycles of 2.5% dextran sulfate sodium (DSS) alternating weekly with water to induce chronic colitis. Total RNA was extracted from mouse colonic mucosal scrapings and amplified using Real Time qRT‐PCR. Myeloperoxidase (MPO) assay and histological analysis was used to assess inflammation. 16S sequencing was performed on DNA extracted from cecal contents for microbiota analysis (Zymo). Results: Depletion of AhR ligands in the diet increased severity of DSS induced colitis as evidenced by: i) a significant decrease in the colon length and an increase in colon weight to length ratio in chronic DSS mice fed the purified ‐I3C diet vs +I3C diet; ii) an increase in myeloperoxidase activity and increased histological scores in chronic DSS mice fed the ‐I3C diet vs DSS mice fed +I3C diet; iii) a significant decrease in mRNA expression of pro‐inflammatory cytokines, TNF‐α, IL‐1β, CCl20, and CXCl2 in the colon of DSS mice fed +I3C diet compared with ‐I3C diet; iv) a significant decrease in tight junction (occludin and ZO‐1) and adherens junction (E‐cadherin) protein levels in chronic DSS mice fed ‐I3C diet and this decrease was attenuated by supplementation of I3C. The different diets modulated gut microbiota under basal and in chronic colitis conditions. ‐I3C diet alone led to the emergence of the pathobiont, Parvibacter caecicola (phylum Actinobacterium) which was not detected in either the chow or +I3C diet. Chronic DSS and ‐I3C diet induced major alterations in phylum Firmicutes (with less abundance of Clostridia and an increase of Erysipeltrichia), and an increase in abundance of Bacteroides thetaoitaomicron (phylumActinobacterium). Interestingly, these changes in microbiota composition in DSS groups were reversed with I3C supplementation to diet. Conclusions Depletion of AhR ligands in the diet worsens colitis and induces dysbiosis. These results highlight the importance of diet in shaping gut microbiota and underscore the significance of potential nutritional strategies to alleviate colitis.

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