Secretory Cell Lineage Research Articles

Impaired activation of succinate-induced type 2 immunity and secretory cell production in the small intestines of Ptk6−/− male mice

Protein tyrosine kinase 6 (PTK6) is an intracellular tyrosine kinase that is distantly related to the SRC family of tyrosine kinases. It is expressed in epithelial linings and regulates regeneration and repair of the intestinal epithelium. Analysis of publicly available datasets showed Ptk6 is upregulated in tuft cells upon activation of type 2 immunity. We found that disruption of Ptk6 influences gene expression involved in intestinal immune responses. Administration of succinate, which mimics infection and activates tuft cells, revealed PTK6-dependent activation of innate immune responses in male but not female mice. In contrast to all wild type and Ptk6−/− female mice, Ptk6−/− male mice do not activate innate immunity or upregulate differentiation of the tuft and goblet secretory cell lineages following succinate treatment. Mechanistically, we found that PTK6 regulates Il25 and Irag2, genes that are required for tuft cell effector functions and activation of type 2 innate immunity, in organoids derived from intestines of male but not female mice. In patients with Crohn’s disease, PTK6 is upregulated in tuft cells in noninflamed regions of intestine. These data highlight roles for PTK6 in contributing to sex differences in intestinal innate immunity and provide new insights into the regulation of IL-25.

Intestinal organoids to model Salmonella infection and its impact on progenitors

In order to survive and replicate, Salmonella has evolved mechanisms to gain access to intestinal epithelial cells of the crypt. However, the impact of Salmonella Typhimurium on stem cells and progenitors, which are responsible for the ability of the intestinal epithelium to renew and protect itself, remains unclear. Given that intestinal organoids growth is sustained by stem cells and progenitors activity, we have used this model to document the effects of Salmonella Typhimurium infection on epithelial proliferation and differentiation, and compared it to an in vivo model of Salmonella infection in mice. Among gut segments, the caecum was preferentially targeted by Salmonella. Analysis of infected crypts and organoids demonstrated increased length and size, respectively. mRNA transcription profiles of infected crypts and organoids pointed to upregulated EGFR-dependent signals, associated with a decrease in secretory cell lineage differentiation. To conclude, we show that organoids are suited to mimic the impact of Salmonella on stem cells and progenitors cells, carrying a great potential to drastically reduce the use of animals for scientific studies on that topic. In both models, the EGFR pathway, crucial to stem cells and progenitors proliferation and differentiation, is dysregulated by Salmonella, suggesting that repeated infections might have consequences on crypt integrity and further oncogenesis.

Effect of air-liquid interface on cultured human intestinal epithelial cells.

The intestinal epithelium is a dynamic barrier that allows the selective exchange of ions, hormones, proteins, and nutrients. To accomplish this, the intestinal epithelium adopts a highly columnar morphology which is partially lost in submerged culturing systems. To achieve this, small intestinal tissue samples were utilized to obtain human intestinal crypts to form enteroids. The Transwell system was subsequently employed to form a monolayer of cells that was cultured in either the submerged condition or the air-liquid Interface (ALI) condition. We found that the human intestinal monolayer under the ALI condition exhibited morphology more similar to the normal intestinal epithelium. F-actin localization and brush border formation were observed apically, and the integrity of the tight junctions was preserved in the ALI condition. Fewer apoptotic cells were observed in the ALI conditions as compared to the submerged conditions. The monolayer of cells expressed a higher level of secretory cell lineage genes in the ALI condition. The ALI condition positively contributes toward a more differentiated phenotype of epithelial cells. It serves as an amplifier that enhances the existing differentiation cue. The ALI system provides a more differentiated platform to study intestinal function compared to submerged conditions.

Intestinal Enteroendocrine Cells: Present and Future Druggable Targets.

Enteroendocrine cells are specialized secretory lineage cells in the small and large intestines that secrete hormones and peptides in response to luminal contents. The various hormones and peptides can act upon neighboring cells and as part of the endocrine system, circulate systemically via immune cells and the enteric nervous system. Locally, enteroendocrine cells have a major role in gastrointestinal motility, nutrient sensing, and glucose metabolism. Targeting the intestinal enteroendocrine cells or mimicking hormone secretion has been an important field of study in obesity and other metabolic diseases. Studies on the importance of these cells in inflammatory and auto-immune diseases have only recently been reported. The rapid global increase in metabolic and inflammatory diseases suggests that increased understanding and novel therapies are needed. This review will focus on the association between enteroendocrine changes and metabolic and inflammatory disease progression and conclude with the future of enteroendocrine cells as potential druggable targets.

Alteration of secretory cell lineages and immune cell migration induced by acute tuft cell ablation in mouse intestine

Background: Intestinal tuft cells have only been recently investigated thoroughly due to their roles in type 2 immunity and chemosensory function. This rare population of intestinal epithelium is implicated in extensively interacting with other cell types and involved in nutrient absorption. Doublecortin-like kinase 1 (DCLK1) is widely used for tuft cell identification in the mouse intestine. However, the effects of tuft cell deletion on mouse intestines have not been fully understood. Here, we investigated the effects of tuft cell deletion on the epithelial cell lineages and overall impact on mouse intestine. Methods: Tuft cell deletion was induced in Dclk1-IRES-GFP-CreERT2/+; Rosa-DTA (Dclk1-DTA) mice by a single dose of tamoxifen injection. Cre-negative DTA/+ mice and DTA-negative Dclk1-CreERT2 mice of littermates received tamoxifen injection at same time as controls. Mouse bodyweight was monitored daily, and intestinal tissues were sampled 2, 4, or 7 days after the tamoxifen injection. Immunostaining of intestinal tissue sections was performed for lysozyme (LYZ), trefoil factor 3 (TFF3), cluster of differentiation 3 (CD3), DCLK1, mast cell protease 1 (MCPT1), Ly6G, and F4/80. Image analysis was conducted using Qupath. Results: Dclk1-DTA mice showed a significant decrease in bodyweight to 11% of their original weight on day 4 after tamoxifen injection. They showed significantly shortened small intestinal length compared to control mice, however, colonic length showed no significant difference. Immunofluorescent staining of induced Dclk1-DTA mouse tissues revealed that DCLK1+ tuft cells were decreased on day 2, almost completely deleted on day 4, and recovered on day 7. Overall brush border structures defined by villin and SGLT1 staining were intact. Correlated with the tuft cell reduction, mislocalized Paneth-like cells, identified with LYZ+ granules, were observed in the middle or upper part of villi with increasing frequency on day 4 and decreasing on day 7. To confirm goblet/Paneth intermediate cells, multiplexed immunofluorescent (MxIF) staining was employed with LYZ and TFF3. Double-positive (LYZ+/TFF3+) cells were significantly increased in the small intestine of tuft cell-depleted mice on day 4. Intraepithelial lymphocytes, mucosal neutrophils, and F4/80+ macrophages/dendritic cells did not show significant changes after the tuft cell deletion. This indicates that there is no inflammatory reactions following tuft cell deletion. Conclusion: Acute ablation of intestinal tuft cell may cause nutrient malabsorption and changes in mucosal defense response in vivo due to the alteration of cell differentiation lineage and the decrease in absorptive area. Our findings support the importance of tuft cell function in nutrient sensing and mucosal defense mechanism. NIH R01DK128190 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Chlorogenic acid improves intestinal morphology by enhancing intestinal stem-cell activity.

Chlorogenic acid (CGA), as one of the most abundant naturally occurring phenolic acids, has been documented to be beneficial for intestinal health. However, the underlying mechanism is still not fully understood. The adult intestinal stem cell is the critical driver of epithelial homeostasis and regeneration. This study hypothesized that CGA exerted intestinal health effects by modulating intestinal stem-cell functions. Lgr5-EGFP mice were treated for 14 days, and intestinal organoids derived from these mice were treated for 3 days, using CGA solution. In comparison with the control group, CGA treatment increased intestinal villous height and crypt depth in mice and augmented the area expansion and the number of budding intestinal organoids. Quantitative polymerase chain reaction (qPCR) analysis revealed that CGA treatment significantly increased the expression of genes coding intestinal stem-cell markers in intestinal tissue and organoids, and upregulated the expression of genes coding secretory cell lineages and enterocytes, although not statistically significantly. Fluorescence-activated cell-sorting analysis further confirmed that CGA augmented the number of stem cells. 5-Ethynyl-2'-deoxyuridine (EdU) incorporation and Ki67 immunostaining results also demonstrated that CGA treatment enhanced intestinal stem-cell proliferation. Altogether, our findings indicate that CGA could activate intestinal stem-cell and epithelial regeneration, which could contribute to the improvement of intestinal morphology or organoid growth of mice. This highlights a promising mechanism for CGA as an excellent candidate for the formulation of dietary supplements and functional foods for intestinal protection. © 2023 Society of Chemical Industry.

Aryl Hydrocarbon Receptor Activation Coordinates Mouse Small Intestinal Epithelial Cell Programming

In the face of mechanical, chemical, microbial, and immunologic pressure, intestinal homeostasis is maintained through balanced cellular turnover, proliferation, differentiation, and self-renewal. Here, we present evidence supporting the role of the aryl hydrocarbon receptor (AHR) in the adaptive reprogramming of small intestinal gene expression, leading to altered proliferation, lineage commitment, and remodeling of the cellular repertoire that comprises the intestinal epithelium to promote intestinal resilience. Ahr gene/protein expression and transcriptional activity exhibit marked proximalHI to distalLO and cryptHI to villiLO gradients. Genetic ablation of Ahr impairs commitment/differentiation of the secretory Paneth and goblet cell lineages and associated mucin production, restricts expression of secretory/enterocyte differentiation markers, and increases crypt-associated proliferation and villi-associated enterocyte luminal exfoliation. Ahr-/- mice display a decrease in intestinal barrier function. Ahr+/+ mice that maintain a diet devoid of AHR ligands intestinally phenocopy Ahr-/- mice. In contrast, Ahr+/+ mice exposed to AHR ligands reverse these phenotypes. Ligand-induced AHR transcriptional activity positively correlates with gene expression (Math1, Klf4, Tff3) associated with differentiation of the goblet cell secretory lineage. Math1 was identified as a direct target gene of AHR, a transcription factor critical to the development of goblet cells. These data suggest that dietary cues, relayed through the transcriptional activity of AHR, can reshape the cellular repertoire of the gastrointestinal tract.

Transcriptional Regulation of Math1 by Aryl Hydrocarbon Receptor: Effect on Math1+ Progenitor Cells in Mouse Small Intestine.

The physiological roles of aryl hydrocarbon receptor (AhR) in the small intestine have been revealed as immunomodulatory and barrier functions. However, its contributions to cell fate regulation are incompletely understood. The Notch-activated signaling cascade is a central component of intestinal cell fate determinations. The lateral inhibitory mechanism governed by Notch directs cell fates toward distinct cell lineages (i.e., absorptive and secretory cell lineages) through its downstream effector, mouse atonal homolog 1 (MATH1). An investigation employing cell lines and intestinal crypt cells revealed that AhR regulates Math1 expression in a xenobiotic response element (XRE)-dependent manner. The AhR-Math1 axis was further addressed using intestinal organoids, where AhR-Math1 and HES1-Math1 axes appeared to coexist within the underlying Math1 transcriptional machinery. When the HES1-Math1 axis was pharmacologically suppressed, β-naphthoflavone-mediated AhR activation increased the number of goblet and Math1+ progenitor cells in the organoids. The same pharmacological dissection of the AhR-Math1 axis was applied invivo, demonstrating an enhanced number of Math1+ progenitor cells in the small intestine following AhR activation. We report here that AhR-Math1 is a direct transcriptional axis with effects on Math1+ progenitor cells in the small intestine, highlighting a novel molecular basis for fine-tuning Notch-mediated cell fate regulation.

H3K27me3 Demethylases Maintain the Transcriptional and Epigenomic Landscape of the Intestinal Epithelium

Although trimethylation of histone H3 lysine 27 (H3K27me3) by polycomb repressive complex 2 is required for intestinal function, the role of the antagonistic process-H3K27me3 demethylation-in the intestine remains unknown. The aim of this study was to determine the contribution of H3K27me3 demethylases to intestinal homeostasis. An inducible mouse model was used to simultaneously ablate the 2 known H3K27me3 demethylases, lysine (K)-specific demethylase 6A (Kdm6a) and lysine (K)-specific demethylase 6B (Kdm6b), from the intestinal epithelium. Mice were analyzed at acute and prolonged time points after Kdm6a/b ablation. Cellular proliferation and differentiation were measured using immunohistochemistry, while RNA sequencing and chromatin immunoprecipitation followed by sequencing for H3K27me3 were used to identify gene expression and chromatin changes after Kdm6a/b loss. Intestinal epithelial renewal was evaluated using a radiation-induced injury model, while Paneth cell homeostasis was measured via immunohistochemistry, immunoblot, and transmission electron microscopy. We did not detect any effect of Kdm6a/b ablation on intestinal cell proliferation or differentiation toward the secretory cell lineages. Acute and prolonged Kdm6a/b loss perturbed expression of gene signatures belonging to multiple cell lineages (adjusted P value < .05), and a set of 72 genes was identified as being down-regulated with an associated increase in H3K27me3 levels after Kdm6a/b ablation (false discovery rate, <0.05). After prolonged Kdm6a/b loss, dysregulation of the Paneth cell gene signature was associated with perturbed matrix metallopeptidase 7 localization (P < .0001) and expression. Although KDM6A/B does not regulate intestinal cell differentiation, both enzymes are required to support the full transcriptomic and epigenomic landscape of the intestinal epithelium and the expression of key Paneth cell genes.

PAX8 as a Potential Target for Ovarian Cancer: What We Know so Far.

The Fallopian tube epithelium harbors the origin cells for the majority of high-grade serous ovarian carcinomas (HGSCs), the most lethal form of gynecologic malignancies. PAX8 belongs to the paired-box gene family of transcription factors and it is a marker of the FTE secretory cell lineage. Its role has been investigated in migration, invasion, proliferation, cell survival, stem cell maintenance, angiogenesis and tumor growth. In this review, we focus on the pro-tumorigenic role of PAX8 in ovarian cancer; in this context, PAX8 possibly continues to exert its transcriptional activity on its physiological targets but may also function on newly available targets after the tumorigenic hits. Acquiring new insights into the different PAX8 mechanism(s) of action in the tumor microenvironment could uncover new viable therapeutic targets and thus improve the current treatment regimen.

Bacillus subtilis programs the differentiation of intestinal secretory lineages to inhibit Salmonella infection.

The role of intestinal microbiota on fate determination of intestinal epithelial cells has not been extensively examined. In this study, we explore the effect of Bacillus subtilis on programmed intestinal epithelial differentiation. We find that B.subtilis stimulates the differentiation of intestinal secretory cells. Moreover, B.subtilis inhibits the Notch pathway to reduce the expression of hairy and enhancer of split 1, thereby shifting intestinal stem cell differentiation toward a secretory cell fate. Moreover, we demonstrate that the programming effect of B.subtilis on intestinal differentiation is Toll-like receptor 2 pathway dependent. B.subtilis is associated with increased numbers of Paneth and goblet cells in the intestine. This results in the production of antimicrobial peptides to protect the intestinal mucosal barrier against Salmonella typhimurium. This study demonstrates that B.subtilis contributes to the differentiation of secretory cells by affecting Notch pathway signaling to maintain the intestinal barrier.

Blocking DCIR mitigates colitis and prevents colorectal tumors by enhancing the GM-CSF-STAT5 pathway.

Dendritic cell immunoreceptor (DCIR; Clec4a2), a member of the C-type lectin receptor family, plays important roles in homeostasis of the immune and bone systems. However, the intestinal role of this molecule is unclear. Here, we show that dextran sodium sulfate (DSS)-induced colitis and azoxymethane-DSS-induced intestinal tumors are reduced in Clec4a2-/- mice independently of intestinal microbiota. STAT5 phosphorylation and expression of Csf2 and tight junction genes are enhanced, while Il17a and Cxcl2 are suppressed in the Clec4a2-/- mouse colon, which exhibits reduced infiltration of neutrophils and myeloid-derived suppressor cells. Granulocyte-macrophage colony-stimulating factor (GM-CSF) administration ameliorates DSS colitis associated with reduced Il17a and enhanced tight junction gene expression, whereas anti-GM-CSF exacerbates symptoms. Furthermore, anti-NA2, a ligand for DCIR, ameliorates colitis and prevents colorectal tumors. These observations indicate that blocking DCIR signaling ameliorates colitis and suppresses colonic tumors, suggesting DCIR as a possible target for the treatment of these diseases.

IL-17RA-signaling in Lgr5+ intestinal stem cells induces expression of transcription factor ATOH1 to promote secretory cell lineage commitment

The Th17 cell-lineage-defining cytokine IL-17A contributes to host defense and inflammatory disease by coordinating multicellular immune responses. The IL-17 receptor (IL-17RA) is expressed by diverse intestinal cell types, and therapies targeting IL-17A induce adverse intestinal events, suggesting additional tissue-specific functions. Here, we used multiple conditional deletion models to identify a role for IL-17A in secretory epithelial cell differentiation in the gut. Paneth, tuft, goblet, and enteroendocrine cell numbers were dependent on IL-17A-mediated induction of the transcription factor ATOH1 in Lgr5+ intestinal epithelial stem cells. Although dispensable at steady state, IL-17RA signaling in ATOH1+ cells was required to regenerate secretory cells following injury. Finally, IL-17A stimulation of human-derived intestinal organoids that were locked into a cystic immature state induced ATOH1 expression and rescued secretory cell differentiation. Our data suggest that the cross talk between immune cells and stem cells regulates secretory cell lineage commitment and the integrity of the mucosa.

LKB1 in Intestinal Epithelial Cells Regulates Bile Acid Metabolism by Modulating FGF15/19 Production.

Background & AimsLiver kinase B1 (LKB1) is a master upstream protein kinase involved in nutrient sensing and glucose and lipid metabolism in many tissues; however, its metabolic role in intestinal epithelial cells (IEC) remains unclear. In this study, we investigated the regulatory role of LKB1 on bile acid (BA) homeostasis.MethodsWe generated mice with IEC-specific deletion of LKB1 (LKB1ΔIEC) and analyzed the characteristics of IEC development and BA level. In vitro assays with small interfering RNA, liquid chromatography/mass spectrometry, metagenomics, and RNA-sequencing were used to elucidate the regulatory mechanisms underlying perturbed BA homeostasis.ResultsLKB1 deletion resulted in abnormal differentiation of secretory cell lineages. Unexpectedly, BA pool size increased substantially in LKB1ΔIEC mice. A significant reduction of the farnesoid X receptor (FXR) target genes, including fibroblast growth factor 15/19 (FGF15/19), known to inhibit BA synthesis, was found in the small intestine (SI) ileum of LKB1ΔIEC mice. We observed that LKB1 depletion reduced FGF15/19 protein level in human IECs in vitro. Additionally, a lower abundance of bile salt hydrolase-producing bacteria and elevated levels of FXR antagonist (ie, T-βMCA) were observed in the SI of LKB1ΔIEC mice. Moreover, LKB1ΔIEC mice showed impaired conversion of retinol to retinoic acids in the SI ileum. Subsequently, vitamin A treatment failed to induce FGF15 production. Thus, LKB1ΔIEC mice fed with a high-fat diet showed improved glucose tolerance and increased energy expenditure.ConclusionsLKB1 in IECs manages BA homeostasis by controlling FGF15/19 production.

Functional MYO5B Loss Disrupts Stem Cell Function and Differentiation of Secretory Cell Lineage

Background and Aims Differentiation of intestinal epithelial cells involves cell division inhibition, cell lineage choice, and brush border elaboration. Functional myosin Vb (MYO5B) loss causes microvillus inclusion disease (MVID) and induces a variety of deficits in enterocyte function. However, the impact of MYO5B loss on differentiated cell lineage choice had not been investigated. Since intestinal progenitor cells predominantly express MYO5B, we hypothesize that MYO5B deficiency alters stem/progenitor cell function and affects proper cell differentiation. Methods Tamoxifen-induced epithelial-specific MYO5B knockout (VilCreERT2; Myo5bflox/flox) mice were used for immunohistochemical analysis of intestinal tissues, organoid generation from crypts, and RNA-sequencing of isolated epithelial cells. We quantified the population of secretory cell lineages in utilizing whole slide imaging and digital image analysis tools. Neonatal pig intestinal organoids, possessing the Navajo mutation MYO5B(P663L), were analyzed as another MVID model that more closely represents human MVID pathology. Results Consistent with our RNA-sequencing data in epithelial cells that demonstrated decreases in Pou2f3, Spib, Dclk1, Chat, and Pgst1, MYO5B loss induced an 80% reduction in DCLK1-positive tuft cells. The frequency of TFF3-producing goblet cells was also significantly decreased. However, the Paneth cell lineage was increased by MYO5B deficiency along with expansion of the progenitor cell zone that express a Notch-dependent stem cell marker, olfactomedin (OLFM)4, and monocarboxylate transporter (MCT)1. No difference was detected in chromogranin-A-expressing enteroendocrine cells. mRNA expression indicated an increase in Notch receptor and decreases in Wnt family ligands in MYO5B deficient intestine, which may explain the alteration of cell lineages by MYO5B loss. We further investigated the effect of lysophosphatidic acid (LPA) receptor activation on epithelial sensory cell differentiation. Intraperitoneal, but not gavaged, LPA significantly increased tuft cell populations both in control and MYO5B knockout mice without alteration of mRNA expression of tuft cell-specific transcription factors. LPA did not affect hyperproliferative crypts in MYO5B deficient mice. In mouse intestinal organoids, the frequency of tuft cells was significantly decreased by 4-hydroxy-tamoxifen-induced MYO5B loss to values corresponding to undifferentiated enteroids. Intestinal organoids that were generated from MYO5B(P663L) pig jejunum had grater organoid forming rate and more proliferative cell population compared to age-matched wild type pig organoids. Differentiation of secretory cell lineages were inhibited in MYO5B(P663L) organoids similarly to the mouse model. Conclusions Functional MYO5B loss disrupts stem cell characteristics and proper differentiation in the small intestine likely through imbalance of Notch/Wnt signaling. Systemic LPA administration specifically activates tuft cell differentiation independent from MYO5B-mediated signaling pathway.

Transcriptomic and proteomic signatures of stemness and differentiation in the colon crypt

Intestinal stem cells are non-quiescent, dividing epithelial cells that rapidly differentiate into progenitor cells of the absorptive and secretory cell lineages. The kinetics of this process is rapid such that the epithelium is replaced weekly. To determine how the transcriptome and proteome keep pace with rapid differentiation, we developed a new cell sorting method to purify mouse colon epithelial cells. Here we show that alternative mRNA splicing and polyadenylation dominate changes in the transcriptome as stem cells differentiate into progenitors. In contrast, as progenitors differentiate into mature cell types, changes in mRNA levels dominate the transcriptome. RNA processing targets regulators of cell cycle, RNA, cell adhesion, SUMOylation, and Wnt and Notch signaling. Additionally, global proteome profiling detected >2,800 proteins and revealed RNA:protein patterns of abundance and correlation. Paired together, these data highlight new potentials for autocrine and feedback regulation and provide new insights into cell state transitions in the crypt.

Regulated IFN signalling preserves the stemness of intestinal stem cells by restricting differentiation into secretory-cell lineages.

Intestinal stem cells (ISCs) are located at the crypt base and fine-tune the balance of their self-renewal and differentiation1,2, but the physiological mechanism involved in regulating that balance remains unknown. Here we describe a transcriptional regulator that preserves the stemness of ISCs by restricting their differentiation into secretory-cell lineages. Interferon regulatory factor 2 (IRF2) negatively regulates interferon signalling3, and mice completely lacking Irf24 or with a selective Irf2 deletion in their intestinal epithelial cells have significantly fewer crypt Lgr5hi ISCs than control mice. Although the integrity of intestinal epithelial cells was unimpaired at steady state in Irf2-deficient mice, regeneration of their intestinal epithelia after 5-fluorouracil-induced damage was severely impaired. Similarly, extended treatment with low-dose poly(I:C) or chronic infection of lymphocytic choriomeningitis virus clone 13 (LCMV C13)5 caused a functional decline of ISCs in wild-type mice. In contrast, massive accumulations of immature Paneth cells were found at the crypt base of Irf2-/- as well as LCMV C13-infected wild-type mice, indicating that excess interferon signalling directs ISCs towards a secretory-cell fate. Collectively, our findings indicate that regulated interferon signalling preserves ISC stemness by restricting secretory-cell differentiation.

Salmonella biases epithelial differentiation in murine enteroids through Wnt and Notch signaling, which may contribute to diarrheal pathogenesis

Salmonella enterica serovar Typhimurium is one of the most burdensome foodborne diarrheal pathogens worldwide. However, despite its prevalence, the mechanism by which Salmonella causes diarrhea is not entirely known. We have shown in mice that Salmonella infection decreases expression of SLC26A3 (Down‐Regulated in Adenoma; DRA), a chloride/bicarbonate exchanger. In order to understand the mechanism of this effect, we employed an enteroid model from murine colon. Enteroid‐derived monolayers (EDM) that recapitulate both the absorptive and secretory cell lineages of the native intestinal epithelium (enterocytes vs. enteroendocrine, goblet, and Paneth cells, respectively) were infected apically with Salmonella and relevant protein expression was studied using qRT‐PCR and western blot. Infection significantly reduced expression of DRA (40%, p = 0.0124) and Hes1 (45%, p = 0.0004) while upregulating expression of ATOH1 (3‐fold, p = 0.0292) and mucin 2 (Muc2) (2‐fold, p = 0.0134). Hes1 is a Notch pathway downstream signaling molecule and thus a precursor to the absorptive epithelial lineage that expresses DRA. ATOH1, on the other hand, is a Wnt pathway downstream signaling molecule and a precursor to secretory lineages, including goblet cells that express Muc2. Infection of EDMs also reduced levels of the Notch intracellular domain. The involvement of Notch was further investigated by inhibiting Notch signaling using a γ‐secretase inhibitor, which reproduced the downregulation in Hes1 and DRA and upregulation in ATOH1 and Muc2 as seen with infection. Because the balance of Wnt and Notch signaling determines relative intestinal epithelial differentiation, our findings suggest that the diarrheal pathogenesis of Salmonella may reflect Notch inhibition and an accompanying shift in epithelial differentiation from absorptive to secretory cell types, a decreased capacity for absorption, and thus the accumulation of diarrheal fluid.

A novel histological index for evaluation of environmental enteric dysfunction identifies geographic-specific features of enteropathy among children with suboptimal growth.

A major limitation to understanding the etiopathogenesis of environmental enteric dysfunction (EED) is the lack of a comprehensive, reproducible histologic framework for characterizing the small bowel lesions. We hypothesized that the development of such a system will identify unique histology features for EED, and that some features might correlate with clinical severity. Duodenal endoscopic biopsies from two cohorts where EED is prevalent (Pakistan, Zambia) and North American children with and without gluten sensitive enteropathy (GSE) were processed for routine hematoxylin & eosin (H&E) staining, and scanned to produce whole slide images (WSIs) which we shared among study pathologists via a secure web browser-based platform. A semi-quantitative scoring index composed of 11 parameters encompassing tissue injury and response patterns commonly observed in routine clinical practice was constructed by three gastrointestinal pathologists, with input from EED experts. The pathologists then read the WSIs using the EED histology index, and inter-observer reliability was assessed. The histology index was further used to identify within- and between-child variations as well as features common across and unique to each cohort, and those that correlated with host phenotype. Eight of the 11 histologic scoring parameters showed useful degrees of variation. The overall concordance across all parameters was 96% weighted agreement, kappa 0.70, and Gwet's AC 0.93. Zambian and Pakistani tissues shared some histologic features with GSE, but most features were distinct, particularly abundance of intraepithelial lymphocytes in the Pakistani cohort, and marked villous destruction and loss of secretory cell lineages in the Zambian cohort. We propose the first EED histology index for interpreting duodenal biopsies. This index should be useful in future clinical and translational studies of this widespread, poorly understood, and highly consequential disorder, which might be caused by multiple contributing processes, in different regions of the world.

Abstract GMM-030: MODELS AND ANALYTIC TECHNIQUES OF MULLERIAN TISSUE-DERIVED ORGANOIDS

Abstract INTRODUCTION: Ovarian cancer is the 5th deadliest cancer found in women and is the deadliest involving the gynecological tract. Most epithelial ovarian cancers have extra-ovarian origins and can be stratified into various histotypes: high and low-grade serous (HGS and LGS), endometrioid (ENOC), clear cell (CCOC), and mucinous – each of which are proposed to have distinct precursor lesions. We present organoids as a useful model to study precursor lesions and the process of tumorigenesis in epithelial ovarian carcinomas. Organoids recapitulate the in vivo growth microenvironment and are useful to study the mechanisms of tumorigenesis from healthy cells. We have previously proposed that ENOC arise from the secretory cell lineage, while CCOC originate from the ciliated cell lineage, and organoids are an ideal model to examine in greater depth the impact of mutation on specific cell populations, such as ciliated cells. METHODS: Surgical fallopian tube and endometrial tissues, removed for non-cancer reasons, were cultured in 2D followed by plating into Matrigel. Matrigel cultures were supplemented with media containing stem/progenitor differentiation factors promoting organoid growth. To study the effect of mutations often found in ovarian cancers on organoid growth and development, gene knockouts were produced using CRISPR lentiviruses on cells prior to Matrigel culture. Lentiviral transductions were optimized for organoid formation and for minimizing invasiveness accrued on cells. CRISPR gRNA constructs were validated by Western Blot and qPCR. Organoids containing knockouts of p53, BRCA1 and BRCA2 were used to model precursor lesions of HGS, whereas ARID1A knockouts and an inducible PIK3CA activating mutations were used to model CCOC. To gain further insight into ciliated cells of the endometrium, organoids were treated with the notch inhibitor-DBZ to drive differentiation of cells towards a ciliated cell lineage. We analyzed organoids by single-cell RNA sequencing (scRNA-seq), immunohistochemistry (IHC), and immunofluorescence staining (IF). Single cells were derived by purifying the organoids from Matrigel followed by a chemical and physical digestion. scRNA-seq was performed utilizing the 10X Genomics Platform and analyzed by in-house bioinformaticians. Bioinformatic analyses included stringent QC to remove low-quality and dead cells, before applying unsupervised learning algorithms like PCA and Gaussian mixture modeling as well as differential expression analysis to understand both how samples relate to each other and cell types discovered within each sample. RESULTS: We successfully recapitulated the histology observed in tissues by growing endometrial and fallopian tube organoids. The notch inhibitor, DBZ forced ciliated cell differentiation, as observed by IHC, IF and scRNA-seq. scRNA-seq clustering of DBZ-treated organoid cultures revealed a possible intermediary state between progenitor and ciliated cells. Initial IHC and IF analyses of CRISPR-mediated organoids reveal successful gene manipulation. CONCLUSIONS: Organoid cultures present as a powerful method for modelling precursor lesions; they can be readily manipulated genetically and with rapid turnaround compared to conventional mouse models. Organoids are also amenable to sequencing at single-cell resolution. The ability to model ovarian cancers with permanent knockouts in human tissue serves as a necessary link between animal models and human therapy. Citation Format: Germain C. Ho, Dawn R. Cochrane, Evan W. Gibbard, Kieran Campbell, Basile Tessier-Cloutier, Kendall Greening, Forouh Kalantari, Genny Trigo-Gonzalez, Yemin Wang, Jessica N. McAlpine, Sohrab P. Shah, David G. Huntsman. MODELS AND ANALYTIC TECHNIQUES OF MULLERIAN TISSUE-DERIVED ORGANOIDS [abstract]. In: Proceedings of the 12th Biennial Ovarian Cancer Research Symposium; Sep 13-15, 2018; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2019;25(22 Suppl):Abstract nr GMM-030.