Host Intestinal Epithelium Research Articles

Eimeria tenella rhoptry neck protein 2 plays a key role in the process of invading the host intestinal epithelium

The Apicomplexa parasitic phylum rhoptry neck protein 2 (RON2) plays a key role in the process of invading host cells. Eimeria tenella, an intracellular protozoan shares a similar conserved invasion pattern. However, whether E. tenella RON2 participates in the process of invading the host intestinal epithelium is poorly understood. In this study, the sequence of EtRON2 was analyzed and expressed. The expression of the truncated extracellular N-terminal fragment of EtRON2 (403–700 aa, designated EtRON2403–700) with a molecular mass of 38.3 kDa. EtRON2 in the sporozoite protein was detected at 151.4 kDa by rabbit anti-rEtRON2403–700 antibody. Immunofluorescence results showed that EtRON2 was mainly localized to the nucleus and apex of the E. tenella sporozoite. qPCR results showed that the highest expression level of EtRON2 was detected in sporulated oocysts compared with other developmental stages of E. tenella. In vitro invasion inhibition assays showed that the capacity of sporozoites to invade DF-1 cells was significantly inhibited after pretreatment with the rabbit anti-rEtRON2403–700 antibody. Silencing the EtRON2 gene by RNA interference (RNAi) significantly inhibited EtRON2 expression and significantly reduced the invasion of DF-1 cells by sporozoites. In vivo experiments revealed a significant decrease parasite burden and oocyst outputs in chicks after infection with EtRON2 gene-silenced sporozoites by cloacal inoculation. Recombinant EtRON2403–700 (rEtRON2403–700) immunizes chicks effectively against E. tenella infection by inducing humoral immunity and upregulating IFN-γ and CD8+ T lymphocytes. Furthermore, chicks exhibited increased relative weight gain rates, lower cecum lesion scores, and reduced oocyst outputs during the E. tenella challenge. H&E staining showed that the cecum tissue of chicks immunized with rEtRON2403–700 showed relatively mild histopathological changes. In conclusion, the results of this study demonstrated that EtRON2 plays a key role in E. tenella invasion of the host intestinal epithelium and provides a potential target for vaccines against E. tenella infection.

An Adaptable Protocol to Generate a Murine Enteroid-Macrophage Co-Culture System.

Impairment of the intestinal epithelial barrier is frequently seen as collateral damage in various local and systemic inflammatory conditions. The inflammatory process is characterized by reciprocal interactions between the host intestinal epithelium and mucosal innate immune cells, e.g., macrophages. This article provides step-by-step instructions on how to set up a murine enteroid-macrophage co-culture by culturing cellular elements in proximity separated by a porous membrane. Unlike previously published co-culture systems, we have combined enteroids grown from C57BL6j mice with syngeneic bone marrow-derived macrophages to preclude potential allo-reactions between immune cells and epithelium. Transformation of intestinal crypts into proliferative enteroids was achieved by cultivation in Wnt3a-Noggin-R-Spondin-conditioned medium supplemented with ROCK inhibitor Y-27632. The differentiated phenotype was promoted by the use of the Wnt3-deprived EGF-Noggin-R-Spondin medium. The resulting co-culture of primary cells can be employed as a basic model to better understand the reciprocal relationship between intestinal epithelium and macrophages. It can be used for in vitro modelling of mucosal inflammation, mimicked by stimulation of macrophages either while being in co-culture or before being introduced into co-culture, to simulate enterogenic sepsis or systemic conditions affecting the intestinal tract.

Blood-borne extracellular vesicles of bacteria and intestinal cells in patients with psychotic disorders

Background Human cells and bacteria secrete extracellular vesicles (EV) which play a role in intercellular communication. EV from the host intestinal epithelium are involved in the regulation of bacterial gene expression and growth. Bacterial EV (bactEV) produced in the intestine can pass to various tissues where they deliver biomolecules to many kinds of cells, including neurons. Emerging data indicate that gut microbiota is altered in patients with psychotic disorders. We hypothesized that the amount and content of blood-borne EV from intestinal cells and bactEV in psychotic patients would differ from healthy controls. Methods We analyzed for human intestinal proteins by proteomics, for bactEV by metaproteomic analysis, and by measuring the level of lipopolysaccharide (LPS) in blood-borne EV from patients with psychotic disorders (n = 25), tested twice, in the acute phase of psychosis and after improvement, with age- and sex-matched healthy controls (n = 25). Results Patients with psychotic disorders had lower LPS levels in their EV compared to healthy controls (p = .027). Metaproteome analyses confirmed LPS finding and identified Firmicutes and Bacteroidetes as dominating phyla. Total amounts of human intestine proteins in EV isolated from blood was lower in patients compared to controls (p = .02). Conclusions Our results suggest that bactEV and host intestinal EV are decreased in patients with psychosis and that this topic is worthy of further investigation given potential pathophysiological implications. Possible mechanisms involve dysregulation of the gut microbiota by host EV, altered translocation of bactEV to systemic circulation where bactEV can interact with both the brain and the immune system.

Disease-associated gut microbiome and critical metabolomic alterations in patients with colorectal cancer.

Gut microbiota plays a significant role in the colorectal cancer (CRC) process. Ectopic colonization of multiple oral bacteria is reportedly associated with CRC pathogenesis and progression, but the details remain unclear. We enrolled a cohort of 50 CRC patients and 52 healthy controls from an East China population. Taxonomic and functional analysis of the fecal microbiota were performed using 16S rDNA (50 + 52 samples) and shotgun metagenomic sequencing (8 + 6 samples), respectively, with particular attention paid to gut-colonized oral bacteria. The results showed more detected bacterial species but lower species evenness within the samples from CRC patients. To determine the specific bacteria enriched in each group, we analyzed their possible protective, carcinogenic, or opportunistic roles in the CRC process. Among the ectopic oral bacteria, we observed a significant increase in the abundance of Fusobacterium and decreased abundance of Prevotella and Ruminococcus in the CRC group. Main differences in the functional composition of these two groups were related to energy metabolism and biosynthesis, especially the glycolytic pathway. Furthermore, we validated the colonization of Fusobacterium nucleatum subsp. animalis within CRC tissues and studied its impact on the host intestinal epithelium and tumor cells. With high selectivity for cancerous tissues, this subspecies promoted CRC cell proliferation and induced potential DNA damage.

Effects of genes required for exopolysaccharides biosynthesis in Lacticaseibacillus paracasei S-NB on cell surface characteristics and probiotic properties

The cell surface of lactic acid bacteria (LAB) plays an essential role in cell-cell and cell-host interactions. Exopolysaccharides (EPSs) are produced on the cell surface of LAB or in the surrounding medium and are considered to be in favor of the strain- specific probiotic surface characteristics. In this work, the structure features of EPS from Lacticaseibacillus paracasei S-NB were analyzed preliminarily, and the genes involved in EPS biosynthesis of S-NB strain were hypothesized and annotated, and their role in phenotypic characteristics were demonstrated by gene deletion analysis. Four mutant strains with deletion of crucial genes involved in EPS synthesis were analyzed for strain characteristics that are closely related to their ability to interact with the host intestinal epithelium cells, including strain surface characteristics and viability under the gastrointestinal stress conditions (both acid and bile stress). Furthermore, the adherence and immunomodulatory properties of wild-type S-NB and its mutant strains were compared using Caco-2 and RAW 264.7 cell lines, respectively. Taken together, the results indicated the importance of genes associated with EPS biosynthesis in L. paracasei S-NB as a determinant in strain surface characteristics and cell-host interaction, especially for S-NB_2176 (responsible for EPS polymerization) and S-NB_2175 (responsible for CpsD/CapB family tyrosine-protein kinase).

Cloning and Expression of a New Trichinella spiralis Serine Protease and Its Role in Invading Host Intestinal Epithelium

In previous studies, a new Trichinella spiralis serine protease 1.1 (TsSP1.1) was identified in surface proteins of T. spiralis muscle larvae (ML) by proteomics analysis, but its functions in T. spiralis infection are unknown. The aim of this study was to investigate the roles of TsSP1.1 during larval intrusion of gut epithelium. From January 2019 to March 2021, complete TsSP1.1 cDNA sequence was cloned and expressed in Escherichia coli BL21 at the Department of Parasitology, Medical College of Zhengzhou University, Zhengzhou, China. Expression and location of TsSP1.1 in the parasite were investigated using indirect immunofluorescence assay (IIFA) and Western blotting. The in vitro intestinal epithelium cells (IECs) intrusion assay was used to ascertain the roles of TsSP1.1 during larval intrusion of IECs and gut epithelium. TsSP1.1 was a surface and secretory protein, which was expressed at various T. spiralis stages, and principally localized at cuticle, stichosome and embryos of the nematode. rTsSP1.1 accelerated larval intrusion of IECs, whereas anti-rTsSP1.1 antibodies impeded larval intrusion. The acceleration and inhibtion was dose-dependently related to rTsSP1.1 and anti-TsSP1.1 antibodies. Block of the IIL with anti-rTsSP1.1 serum also impeded larval intrusion of gut mucosa. TsSP1.1 participates in T. spiralis intrusion of gut epithelium.

Lycium barbarum polysaccharide modulates gut microbiota to alleviate rheumatoid arthritis in a rat model

Rheumatoid arthritis (RA) seriously impairs the quality of life of sufferers. It has been shown that Lycium barbarum polysaccharide (LBP), a natural active indigestible ingredient with medicinal and edible functions, can effectively relieve RA, however, whether this effect is related to gut microbiota is not known. This study aimed to explore the RA alleviating mechanism of LBP mediated by gut microbiota using a collagen-induced arthritis rat model. The results showed that LBP significantly changed the gut microflora structure accompanied with the RA alleviation. Specifically, a LBP intervention reduced the relative abundance of Lachnospiraceae_NK4A136_group and uncultured_bacterium_f_Ruminococcaceae and significantly increased the abundance of Romboutsia, Lactobacillus, Dubosiella and Faecalibaculum. The mRNA contents of several colonic epithelial genes including Dpep3, Gstm6, Slc27a2, Col11a2, Sycp2, SNORA22, Tnni1, Gpnmb, Mypn and Acsl6, which are potentially associated to RA, were down-regulated due to the DNA hypermethylation, possibly caused by the elevating content of a bacterial metabolite S-adenosyl methionine (SAM). In conclusion, our current study suggests that LBP alleviated RA by reshaping the composition of intestinal microflora which may generate SAM, inducing DNA hypermethylation of RA-related genes in the host intestinal epithelium and subsequently reducing their expression.

Effects of exosomes derived from Trichinella spiralis infective larvae on intestinal epithelial barrier function

Muscle larvae of Trichinella spiralis parasitize the host intestinal epithelium. The mechanisms of exosomes participating in the invasion of T. spiralis muscle larvae are unclear. Hence, the purpose of this study was to explore the effect of exosomes derived from T. spiralis infective larvae (TsExos) on the barrier function of porcine small intestinal epithelial cells (IPEC-J2). First, TsExos were successfully obtained, and their ingestion by epithelial cells was validated. Furthermore, the optimal induction condition was determined by the CCK8 kit, and we found that exposure to 150 μg/mL TsExos for 12/24 h decreased the viability of IPEC-J2 cells by 30%. Based on this outcome, the effects of TsExos on cell biological processes and tight junctions were studied. After coincubation of TsExos and IPEC-J2 cells, the results showed a significant increase in the content of FITC-dextran and in the levels of lactate dehydrogenase (LDH) and reactive oxygen species (ROS). The rate of apoptosis increased by 12.57%, and nuclear pyknosis and nuclear rupture were observed. After the cells were induced by TsExos, the expression of IL-1 was upregulated, but the expression of IL-10, TGF-β, TLR-5, MUC-1 and MUC-2 was downregulated. TsExo induction also led to a decrease in the levels of ZO-1, CLDN-3, and OCLN. In conclusion, TsExos are involved in several cellular biological processes, and they function by disrupting physiological and biochemical processes, hyperactivating innate immunity, and damaging tight junctions.

Probiotics in the management of Giardia duodenalis: an update on potential mechanisms and outcomes.

Giardia duodenalis is a common cause of infection in children and travelers. The most frequent symptom is diarrhea in these patients. G. duodenalis trophozoites use a highly specialized adhesive disc to attach the host intestinal epithelium to induce intestinal damages. Pathological features of the small intestine following giardiasis include villous atrophy; infiltration of granulocytes, lymphocytes, and plasma cells into the lamina propria; and nodular lymphoid hyperplasia. The disturbed intestinal microbiota has been observed in patients with giardiasis. Therefore, a growing body of evidence has emphasized restoring the gut microbiome by probiotics in giardiasis. This study aimed to review the literature to find the pathologic features of giardiasis and its relationship with imbalanced microbiota. Then, benefits of probiotics in giardiasis and their potential molecular mechanisms were discussed. It has been illustrated that using probiotics (e.g., Lactobacillus andSaccharomyces) can reduce the time of gastrointestinal symptoms and repair the damages, particularly in giardiasis. Probiotics' capability in restoring the composition of commensal microbiota may lead to therapeutic outcomes. According to preclinical and clinical studies, probiotics can protect against parasite-induced mucosal damages via increasing the antioxidant capacity, suppressing oxidative products, and regulating the systemic and mucosal immune responses. In addition, they can reduce the proportion of G. duodenalis load by directly targeting the parasite. They can destroy the cellular architecture of parasites and suppress the proliferation and growth of trophozoites via the production of some factors with anti-giardial features. Further researches are required to find suitable probiotics for the prevention and treatment of giardiasis.

Recent Advances in Our Understanding of the Diversity and Roles of Chaperone-Usher Fimbriae in Facilitating Salmonella Host and Tissue Tropism.

Salmonella enterica is one of the most diverse and successful pathogens, representing a species with >2,600 serovars with a variety of adaptations that enable colonization and infection of a wide range of hosts. Fimbriae, thin hair-like projections that cover the surface of Salmonella, are thought to be the primary organelles that mediate Salmonella’s interaction with, and adherence to, the host intestinal epithelium, representing an important step in the infection process. The recent expansion in genome sequencing efforts has enabled the discovery of novel fimbriae, thereby providing new perspectives on fimbrial diversity and distribution among a broad number of serovars. In this review, we provide an updated overview of the evolutionary events that shaped the Salmonella chaperone-usher fimbriome in light of recent phylogenetic studies describing the population structure of Salmonella enterica. Furthermore, we discuss the complexities of the chaperone-usher fimbriae-mediated host-pathogen interactions and the apparent redundant roles of chaperone-usher fimbriae in host and tissue tropism.

Proteomic analysis of hydrolytic proteases in excretory/secretory proteins from Trichinella spiralis intestinal infective larvae using zymography combined with shotgun LC-MS/MS approach

The critical step of Trichinella spiralis infection is that the muscle larvae (ML) are activated to intestinal infective larvae (IIL) which invade the intestinal columnar epithelium to further develop. The IIL excretory/secretory (ES) proteins play an important role in host-parasite interaction. Proteolytic enzymes are able to mediate the tissue invasion, thereby increasing the susceptibility of parasites to their hosts. The aim of the current study was to screen and identify the natural active proteases in T. spiralis IIL ES proteins using Western blot and gel zymography combined with liquid chromatography tandem mass spectrometry (LC-MS/MS). The T. spiralis ML and IIL ES proteins were collected from the in vitro cultures and their enzymatic acitvities were examined by gelatin zymography and azocasein degradation. The protease activities were partially inhibited by PMSF, E-64 and EDTA. Three protein bands (45, 118 and 165 kDa) of T. spiralis IIL ES proteins were identified by shotgun LC-MS/MS because they have hydrolytic activity to gelatin compared to the ML ES proteins. Total of 30 T. spiralis proteins were identified and they are mainly serine proteinases (19), but also metalloproteinases (7) and cysteine proteinases (3). The qPCR results indicated that transcription levels of four T. spiralis protease genes (two serine proteases, a cathepsin B-like cysteine proteinase and a zinc metalloproteinase) at IIL stage were obviously higher than at the ML stage. These proteolytic enzymes are directly exposed to the host intestinal milieu and they may mediate the worm invasion of enteral epithelium and escaping from the host's immune responses. The results provide the new insights into understanding of the interaction of T. spiralis with host and the invasion mechanism.

Expression In Situ Of The Recombinant Human Erythropoetin And Recombinant Insulin

Our aim was to check the suitability of the human intestine for the delivery of the re-combinant proteins in humans. The prospective applications of said technology are huge, since travel in the outer Space to locate new planets for the relocation of the over crowded Earth will face the need for the crew vaccination from the pathological microorganisms, found in these outer Space locations. We invented the technology to treat Diabetes II and his chronic anemia in the adult volunteer as the draft of said new applications of said in vivo technology. The strain of lactobacilli from normal human intestinal microflora was isolated, genetically engineered and then returned back for the adhesion back to the host intestinal epithelium of the volunteer. The volunteer was cured from his Diabetes II and chronic anemia. We discuss the potential applications of said technology for the in vivo expression of the vaccines for the crews of the outer space long range travel missions coming soon.

Nitrate Is Crucial for the Proliferation of Gut Escherichia coli Caused by H9N2 AIV Infection and Effective Regulation by Chinese Herbal Medicine Ageratum-Liquid.

H9N2 avian influenza virus (AIV) infection in chickens is often accompanied by secondary bacterial infection, but the mechanism is unclear. The aim of the present study was to reveal that mechanism and explore non-antibiotic treatment. 16s rRNA sequencing and metabonomics were performed in the intestinal contents of chickens infected with H9N2 AIV or H9N2 AIV and fed with ageratum-liquid (AL) to reveal the metabolite that promote intestinal Escherichia coli (E. coli) proliferation caused by H9N2 AIV, as well as to determine the regulatory effect of AL. It was found that H9N2 AIV infection led E. coli to become the dominant gut microbe and promoted E. coli translocation from the intestinal tract to the visceral tissue through the damaged intestinal barrier. H9N2 AIV infection induces inflammation in the intestinal mucosa and promotes the secretion and release of nitrate from the host intestinal epithelium. In addition, nitrate promoted E. coli proliferation in the inflamed intestinal tract following H9N2 AIV infection. Furthermore, Chinese herbal medicine AL can restore intestinal homeostasis, inhibit the production of nitrate in the intestinal epithelium and effectively prevent the proliferation and translocation of E. coli in the intestines. This is the first report on the mechanism of E. coli secondary infection induced by H9N2 AIV, where herbal medicine AL was shown to have a good preventive effect on the secondary infection.

Intestinal Microbiota Influences DNA Methylome and Susceptibility to Colorectal Cancer.

In a recent publication, Ansari et al. identified gut microbiota as a critical mediator of the intestinal inflammatory response through epigenetic programming of host intestinal epithelium. Exposure to the microbiota induces Ten-Eleven-Translocation (TET)-dependent hypomethylation of genomic elements regulating genes associated with inflammatory response and colorectal cancer. Here, we discuss the impact of such a discovery on the understanding of how the intestinal microbiota may contribute to epigenetic reprogramming and influence the onset of colorectal tumorigenesis. Finally, we examine the prospect of TET inhibition strategies as a therapeutic and/or preventive approach for colorectal cancer in patients afflicted by inflammatory bowel disease.

Binding of elastase-1 and enterocytes facilitates Trichinella spiralis larval intrusion of the host's intestinal epithelium

Elastase-1 is one member of serine protease family, distributes in organisms widely and plays a crucial role in the invasion and development of Trichinella spiralis. In order to identify the binding of T. spiralis elastase-1 (TsEla) with host's intestinal epithelial cells (IECs) and its role in Trichinella larval intrusion, TsEla gene was cloned and expressed in our previous study. The recombinant TsEla (rTsEla) has the enzymatic activity to degrade specific peptide substrate. A specific binding between rTsEla and IECs was detected by Far Western blot and ELISA. In an in vitro invasion assay, rTsEla promoted the larval intrusion, whereas anti-rTsEla serum inhibited the larval penetration. The larval intrusion was also suppressed after the silencing of TsEla by siRNA. Silencing of TsEla gene by siRNA-291 meditated RNA interference suppressed TsEla protein expression, reduced the worm infectivity, development and reproductive capacity. These results indicated that TsEla plays an important role in the T. spiralis intrusion of host's intestinal epithelia, and it could be a prospective vaccine molecular target against T. spiralis infection.

The Role of PI3K/AKT Pathway and NADPH Oxidase 4 in Host ROS Manipulation by Toxoplasma gondii.

Dendritic cell is one of the first innate immune cell to encounter T. gondii after the parasite crosses the host intestinal epithelium. T. gondii requires intact DC as a carrier to infiltrate into host central nervous system (CNS) without being detected or eliminated by host defense system. The mechanism by which T. gondii avoids innate immune defense of host cell, especially in the dendritic cell is unknown. Therefore, we examined the role of host PI3K/AKT signaling pathway activation by T. gondii in dendritic cell. T. gondii infection or T. gondii excretory/secretory antigen (TgESA) treatment to the murine dendritic cell line DC2.4 induced AKT phosphorylation, and treatment of PI3K inhibitors effectively suppressed the T. gondii proliferation but had no effect on infection rate or invasion rate. Furthermore, it is found that T. gondii or TgESA can reduce H2O2-induced intracellular reactive oxygen species (ROS) as well as host endogenous ROS via PI3K/AKT pathway activation. While searching for the main source of the ROS, we found that NADPH oxidase 4 (NOX4) expression was controlled by T. gondii infection or TgESA treatment, which is in correlation with previous observation of the ROS reduction by identical treatments. These findings suggest that the manipulation of the host PI3K/AKT signaling pathway and NOX4 expression is an essential mechanism for the down-regulation of ROS, and therefore, for the survival and the proliferation of T. gondii.

The cysteine protease ATG4B of Trichinella spiralis promotes larval invasion into the intestine of the host

The cysteine proteases of parasites are vital contributors that induce parasite migration to and invasion of host tissue. In this study, we analysed the cysteine protease ATG4B of Trichinella spiralis (TsATG4B) isolated from the soluble proteins of Trichinella spiralis (T. spiralis) adult worms to ascertain its biochemical properties and functions during invasion into the intestine of the host. The 43 kDa recombinant cysteine protease ATG4B protein (rTsATG4B) consists of a conserved peptidase_C54 domain and was expressed in Escherichia coli. Gelatine zymography showed that rTsATG4B could hydrolyse gelatine and that the hydrolytic activity was prevented by the cysteine protease inhibitor E-64 (pH 5.2). Immunofluorescence assays showed that TsATG4B is expressed at different stages and is localized at the cuticles and stichosomes of worms. Far-Western blotting and confocal microscopy revealed that rTsATG4B interacts with intestinal epithelial cells (IECs) and that it was subcellularly localized to the membrane and cytoplasm in IECs. Real‑time quantitative PCR (qPCR) results indicated that the transcription level of the TsATG4B gene was the higher in 6-day-old adult worms (6 days AW) than in any other stage. An in vitro larval invasion assay verified that rTsATG4B promoted larval invasion and that invasion was inhibited when rTsATG4B was pre-incubated with E-64, whereas anti-rTsATG4B serum inhibited larval invasion in a dose-dependent manner. Collectively, these results suggested that the enzymatic activity of TsATG4B significantly influences the hydrolysis process, which is necessary for larval invasion of the host intestinal epithelium.

A115 MODELING CYSTIC FIBROSIS (CF) INTESTINAL DISEASE USING PATIENT DERIVED TISSUES

Abstract Background Cystic Fibrosis is caused by mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene, which encodes for a chloride/bicarbonate anion channel expressed on the apical membrane of most epithelial tissues, such as the lungs, liver, pancreas, small and large intestines, and reproductive tissues. CFTR is responsible for the transport of chloride and bicarbonate ions to maintain tissue surface hydration and pH balance of epithelial tissues. Historically, recurrent lung infections have been the most common cause of mortality in CF patients. With advances in clinical care and therapeutics, the current mean survival age of Canadian patients has increased to 52.3 years. However, this increase in survival has also been associated with an elevated risk of developing gastrointestinal cancers in CF adults. Compared to the general public, CF patients are 10 times more likely to develop cancer. This risk is increased to 25-20 times in patients that have undergone organ transplantations. Although the exact molecular mechanism regarding increased cancer risk in CF remains unclear, chronic intestinal inflammation has been known to contribute to elevated cancer development. Aims CF patients display an increased baseline inflammatory status that is exacerbated with microbiome exposure leading to possible increased risk for inflammation-mediated cancer development. Methods To reduce inter-patient heterogeneity, we have differentiated human intestinal organoids using induced pluripotent stem cells from homozygous F508del CF patients and gene edited isogenic non-CF (Wt-CFTR) controls. We conducted gene expression studies using RT-qPCR to determine baseline differences in gene expression prior to environmental exposures and following exposure to LPS and flagellin. Results We determined the expression levels of stem cell, intestinal epithelial cell, innate immunity genes and differentiation markers and found expression of such genes were not significantly different between 3D CF and gene-edited non-CF organoids. We are currently conducting RNA sequencing to survey expression pattern of all genes to definitively determine possible fundamental changes in the CF intestinal epithelium and determining the effect of LPS and flagellin treatment to determine if there is an altered response to inflammatory stimuli. Conclusions iPSC derived HIOs is a novel, patient based, and renewable model that can be used to dissect the primary intestinal pathologies in CF. Transcriptomic data of CF HIOs at steady state will provide insights to possible developmental defects. Complex interactions between the host intestinal epithelia and the commensal microbiome can also be investigated using this model. Funding Agencies CAG

An Open-Format Enteroid Culture System for Interrogation of Interactions Between Toxoplasma gondii and the Intestinal Epithelium.

When transmitted through the oral route, Toxoplasma gondii first interacts with its host at the small intestinal epithelium. This interaction is crucial to controlling initial invasion and replication, as well as shaping the quality of the systemic immune response. It is therefore an attractive target for the design of novel vaccines and adjuvants. However, due to a lack of tractable infection models, we understand surprisingly little about the molecular pathways that govern this interaction. The in vitro culture of small intestinal epithelium as 3D enteroids shows great promise for modeling the epithelial response to infection. However, the enclosed luminal space makes the application of infectious agents to the apical epithelial surface challenging. Here, we have developed three novel enteroid-based techniques for modeling T. gondii infection. In particular, we have adapted enteroid culture protocols to generate collagen-supported epithelial sheets with an exposed apical surface. These cultures retain epithelial polarization, and the presence of fully differentiated epithelial cell populations. They are susceptible to infection with, and support replication of, T. gondii. Using quantitative label-free mass spectrometry, we show that T. gondii infection of the enteroid epithelium is associated with up-regulation of proteins associated with cholesterol metabolism, extracellular exosomes, intermicrovillar adhesion, and cell junctions. Inhibition of host cholesterol and isoprenoid biosynthesis with Atorvastatin resulted in a reduction in parasite load only at higher doses, indicating that de novo synthesis may support, but is not required for, parasite replication. These novel models therefore offer tractable tools for investigating how interactions between T. gondii and the host intestinal epithelium influence the course of infection.

Human Colonoid Monolayers to Study Interactions Between Pathogens, Commensals, and Host Intestinal Epithelium

Human Colonoid Monolayers to Study Interactions Between Pathogens, Commensals, and Host Intestinal Epithelium