Society: AGA

BACKGROUND: Enterochromaffin cells (ECs) are specialized gastrointestinal (GI) epithelial cells that transduce luminal forces and chemicals into release of signal molecules including serotonin (5-HT) to alter GI function including mucosal ion transport. To determine the role of ECs in ion transport, direct activation of ECs is required to localize the downstream effects to ECs versus other cell types.
AIM: Determine whether optogenetic activation of ECs modulates intestinal ion transport.
METHODS: Full-thickness ileum preparations of EC^ReaChR (Tph1^Cre-ERT/+::Rosa26^{LSL-ReaChR-mCitrine/+}) mice treated with tamoxifen (50mg/kg p.o.) four consecutive days prior to tissue harvest were either cryosectioned and immunostained for 5-HT or mounted in 4mL Ussing chambers with Kreb’s solution (NKS). The mucosal side was intermittently stimulated using a fiber optic cable (625-nm LED, Prizmatix) at 2Hz, 40% duty cycle for 20 cycles. Veratridine (30µM), a sodium channel opener, and Substance P (0.3 µM), a neurokinin receptor agonist, were also used to stimulate I_sc. TTX (1µM) was used to block neurotransmission and ondansetron (1µM) and GR113808 (30nM) were used to block serotonin receptors. Responses were measured as change in short-circuit current (ΔI_sc) = I_sc_peak − I_sc_baseline (µA/cm²) with differences detected with Wilcoxson ranked sum tests.
RESULTS: 57% of 5-HT-immunoreactive (ir) cells expressed mCitrine and 96% of mCitrine+ cells were 5-HT-ir illustrating the recombination efficiency and specificity of EC^ReaChR expression. Light stimulation increased I_scin the ileum from EC^ReaChR mice but did not change I_scfrom negative control mice that lacked either Tph1^Cre-ERT or Rosa26^{LSL-ReaChR-mCitrine} transgenes suggesting that light selectively stimulated ReaChR expressed in ECs. Removal of Cl^- from NKS, blocked light-induced I_sc(79 ± 20 µA/cm², NKS, 3.2 ± 0.6 µA/cm², Cl^-free, n=17, P<0.001). While TTX blocked the neurogenic stimuli of veratridine (37 ± 6 µA/cm², veratridine, 3 ± 1 µA/cm², veratridine+TTX, n = 11-23, P<0.001) and Substance P (42 ± 13 µA/cm², Substance P, 9 ± 3 µA/cm² Substance P+TTX, n = 6, P<0.05), TTX reduced but did not fully block light-induced I_sc (73 ± 20 µA/cm² control, 21 ± 5 TTX µA/cm², n=18, P<0.0001). 5-HT receptor antagonists (5HTRa) had no effect on light-induced I_sc(23 ± 6 µA/cm², control, 17 ± 3, 5HTRa, n = 8, P>0.05). However, when TTX and 5HTRa were delivered together, the light induced I_scwas completely blocked (19 ± 5 µA/cm², control, 1.3 ± 0.4 µA/cm², TTX+5HTRa, n = 9, P<0.01).
CONCLUSIONS: Optogenetic stimulation of ECs represents a novel approach to understand mechanisms of EC-initiated ion transport. Optogenetic stimulation of ECs induce mucosal chloride secretion via a combination of neurogenic and paracrine signaling from ECs to secretory epithelial cells.
Supported by NIH AT010875, DK123549, DK129315 and NS118790

Background: Intricate connections exit between dysbiotic microbiome dominated by facultative anaerobes and inflammatory bowel diseases (IBD). Data from Baumler and colleagues support a model where mitochondrial (mito) dysfunction causes disease-associated dysbiosis by increasing oxygen (O2) availability to the microbiome. Here, we posit that a novel compound (AuPhos) restores mito respiration in intestinal epithelial cells (IECs) thereby reducing O2 availability to the microbiome to promote a healthy anaerobic environment (e.g. firmicute-bloom). Methods: The effect of AuPhos on the microbiome was tested in acute (2% DSS-7d) colitis (C57/BL/6) and in germ-free IL10 KO reconstituted with human IBD stool to induce IBD-associated dysbiosis (Hu-IL10 KO; Rousta et al., Nutrients, 2021). Mice were treated with AuPhos (25mg/kg) or vehicle (q3d; n=8/group), and colon and stool samples collected at different time-points. Blinded histological scoring (d32) on Hu-IL10-/- mice was performed and fecal lipocalin-2 (LCN2) levels (d0-d32) determined. Microbial DNA was isolated from stool samples followed by 16S rRNA metagenomic sequencing. Differentially abundant bacterial species and functional potentials of bacterial communities were assessed by Linear Discriminant Analysis (LDA) and PICRUST2, respectively. AuPhos-induced hypoxia in IECs was assessed by Hypoxyprobe-1 staining in sections from pimonidazole HCl-infused DSS-mice. Immunohistochemistry of mito complex I (NDUFB6) was performed on colon sections from Hu-IL10 KO mice. Results: While Hu-IL10 KO mice showed marked histologic inflammation, transmural colitis scores and fecal LCN2 levels in AuPhos-fed Hu-IL10 KO mice significantly decreased (Veh. vs AuPhos; *p<0.05) at d32. Metagenomic (16S) analysis of stool samples from DSS-colitis and Hu-IL10-/- mice showed reduction in relative abundance of (O2 consuming) Proteobacteria, which includes facultatively anaerobic Enterobacteriaceae family, with concomitant increase in obligate anaerobes e.g. Firmicutes including Clostridia (Faecalibacterium prausnitzii, Roseburia sp.), Bifidobacterium, etc. in AuPhos-fed mice. PICRUST2 and LDA revealed that AuPhos decreased bacterial LPS biosynthetic pathway and increased overall fatty acid biosynthesis pathways in AuPhos-fed DSS-colitis mice. Interestingly, hypoxyprobe staining showed AuPhos-induced hypoxia in luminal surface IECs, facilitating obligate anaerobe-promoting environment in the gut. Concomitantly, AuPhos significantly (3-fold; *p<0.05) increased NDUFB6 expression in colonic IECs of Hu-IL10 KO mice compared to vehicle controls. Conclusion: These finding suggest that AuPhos is a “first-in-class” oral therapeutic that corrects microbial dysbiosis in IBD by enhancing mitochondrial respiration and oxygen utilization (i.e. inducing IEC hypoxia) thereby promoting a healthy microbiome dominated by obligate anaerobes.

Backgrounds: Group 3 innate lymphoid cells (ILC3s) recently emerged as important regulators and potential drug targets for IBD. However, the response of ILC3s to environmental stimuli during intestinal inflammation remains elusive. IRE1a-XBP1 is the most conserved pathway of the endoplasmic reticulum stress response that plays an important role in intestinal inflammation. IRE1a mediates the alternative splicing of Xbp1 mRNA to produce a potent transcription factor XBP1s. Our preliminary data suggested that IRE1-XBP1 is induced during the activation of intestinal ILC3s.
Methods: We generated conditional knockout Ire1a^flox/flox,Rorc-Cre (Ire1a^ΔRorc) mice with Ire1a deleted in ILC3s. Intestinal infections were induced by Citrobacter rodentium and Clostridium difficile. In the acute phase of C. rodentium and C. difficile infection, IL-22 secreted by ILC3s, but not adaptive immune cells, is crucial for the host defense. Dextran sodium sulfate (DSS) was given in drinking water to induce colitis. We recruited patients with active Crohn’s disease (CD) and collected intestinal biopsies during routine colonoscopy before starting a new biologic therapy. Mucosal immune cells were isolated from tissue samples for analysis of ILC3s by flow cytometry.
Results: In murine intestinal ILC3s, the activity of IRE1a-XBP1 pathway follows a 24h circadian rhythm which parallels that of clock genes and cytokines including IL-22 and IL-17A. IRE1a-XBP1 in intestinal ILC3s is further activated in response to inflammation/infection in mice and in patients with IBD compared to healthy controls. The IRE1a-XBP1 activation in stimulated ILC3s requires mitochondrial reactive oxygen species. Using the Ire1a^ΔRorc mice, we demonstrate that IRE1a-XBP1 is critical for cytokine expression by ILC3s. Ire1a^ΔRorc mice loss the rhythmic expression of Il-22 and Il-17a by ILC3s in the gut. Additionally, Ire1a^ΔRorc mice are highly vulnerable to C. rodentium and C. difficile infection as well as DSS-induced colitis (Figure 1). The Ire1a^ΔRorc mice exhibit reduced IL-22 and IL-17A in ILC3s and impaired epithelial barrier function with diminished expression of mucins and antimicrobial peptides. The susceptibilities of Ire1a^ΔRorc mice to infections and colitis are rescued by injection of recombinant IL-22. Mechanistically, XBP1s binds to the promoters of Il-22 and Il-17a in activated ILC3s. The frequency of ILC3s expressing XBP1s in ileal mucosa from CD patients positively correlates with response to therapies (Figure 2).
Conclusions: IRE1a-XBP1 pathway controls intestinal ILC3s by regulating the expression of IL-22 and IL-17A. The loss of IRE1a-XBP1 in ILC3s impairs the optimal and rhythmic expression of protective cytokines and renders the mice more susceptible to intestinal infections and colitis. Moreover, IRE1a-XBP1 in mucosal ILC3s may become a useful marker to predict response to IBD therapies.

Background: Metastasis is the primary cause of death in colorectal cancer (CRC) patients. Thyroid hormone receptor interacting protein 6 (TRIP6) functions in recruitment of proteins associated with focal adhesion. We aim to elucidate the role of TRIP6 in driving CRC metastasis and is a therapeutic target to improve chemotherapy efficacy in CRC.
Methods: Clinical significance of TRIP6 was evaluated in our cohorts of primary CRC (cohort I, n=150; cohort II, n=76), paired primary and metastatic CRC samples (n=15), and TCGA database (n=286). Function of TRIP6 was investigated in intestine-specific TRIP6 knockin mice, TRIP6 knockout mice, and experimental metastasis models in nude mice. Interaction of TRIP6 with its downstream effectors was identified by co-immunoprecipitation (co-IP) and mass spectrometry. TRIP6-modulated pathways were identified by RNA-seq. Vesicle-like nanoparticles (VNP)-encapsulated TRIP6-siRNA was constructed to target TRIP6 in vivo.
Results: TRIP6 is significantly up-regulated in CRC compared to adjacent normal tissues in cohort I (P<0.0001), cohort II (P<0.01) and TCGA cohort (P<0.0001). A more pronounced TRIP6 upregulation was demonstrated in liver metastases as compared to their corresponding primary CRC tumors (P<0.001). High TRIP6 expression predicts poor prognosis in CRC patients (P=0.01). Intestine-specific TRIP6 overexpression (Trip6^KIVillin-Cre) in mice accelerated azoxymethane (AOM)-induced CRC formation. TRIP6 promoted submucosal CRC tumor invasion in mice. In contrast, TRIP6 knockout (Trip6^+/-) mice showed dampened tumor formation. Consistent with this, TRIP6 overexpression in DLD1 and SW480 cells significantly promoted epithelial–mesenchymal transition, cell migration and invasion, as well as liver metastases in nude mice. On the other hand, knockdown of TRIP6 in HCT116 and SW1116 cells showed opposite effect. Mechanistically, TRIP6 directly interacted with critical tight junction-associated PDZ domain-containing proteins PARD3 to impair the cell-cell tight junction, activating oncogenic Akt signaling (p-Akt) and inhibiting PTEN. TRIP6-induced pro-metastatic phenotypes and Akt activation was dependent on PARD3, suggesting the functional TRIP6-PARD3-Akt axis in CRC metastasis. Finally, knockdown of TRIP6 in CRC cells increased their sensitivity to oxaliplatin and 5-fluorouacil in vitro, and targeting depletion of TRIP6 by VNP-encapsulated TRIP6-siRNA synergized with chemotherapies of oxaliplatin and 5-fluorouracil to suppress liver metastases of CRC in mice.
Conclusion: TRIP6 promotes CRC metastasis through directly interacting with PARD3 to disrupt the cell-cell tight junction and activating Akt signaling. Targeting of TRIP6 in combination with chemotherapy is a promising strategy for the treatment of metastatic CRC.

Backgrounds: Eosinophilic esophagitis (EoE) is a chronic debilitating disorder characterized by persistent allergic inflammation that leads to diminished epithelial integrity and deranged epithelial proliferation-differentiation gradient. IL-13 induced epithelial disruption, specifically basal cell hyperplasia, is a histologic hallmark of EoE. While it is known that the cytokine milieu disrupts the epithelium, little is known about the mechanisms underlying impaired differentiation. Forkhead box M1 (FOXM1), a transcription factor of the Forkhead box family, is recognized as a major regulator of the cell cycle progression and the mitotic program. FOXM1 has recently been implicated in epithelial differentiation and pathogenesis of allergen-induced inflammatory diseases. However, how the impact of FOXM1 on the esophageal epithelium remains unknown. Herein, we sought to explore the role of FOXM1 in the epithelial disruption in EoE.
Methods: Esophageal FOXM1 expressions were evaluated using patient biopsies by qRT-PCR and immunohistochemistry. To suppress FOXM1, siRNA and FOXM1 inhibitor RCM-1 were used for immortalized non-transformed human esophageal epithelial cells (EPC2-hTERT) and patient-derived organoids (PDOs), respectively. Cultures were stimulated with IL-13 to recapitulate the EoE inflammatory milieu. qRT-PCR, immunoblot, immunofluorescence, and air-liquid interface cultures were performed to examine differentiation differences and barrier integrity in the setting of FOXM1 inhibition. We evaluated proliferation and cell cycle and performed chromatin immunoprecipitation to look for specific targets of FOXM1.
Results: FOXM1 was upregulated in active EoE, compared to non-EoE or inactive EoE patients, and localized to the basal epithelium in biopsies (Figure 1). IL-13 stimulation induced FOXM1 in both EPC2-hTERT cells and PDOs. FOXM1 knockdown and pharmacologic inhibition decreased expression of basal cell markers SOX2 and TP63 and increased expression of differentiation markers involucrin and filaggrin in organoids, while histologically restoring the epithelial differentiation gradient (Figure 2). Air-liquid interface culture showed improved transepithelial resistance in the setting of FOXM1 inhibition. Proliferative ability, S and G2/M phase, and expression of CDK1 and CCNB1 were reduced by FOXM1 knockdown in EPC2-hTERT cells. Further, chromatin immunoprecipitation revealed that FOXM1 bound to transcription start site of CDK1 and CCNB1.
Conclusions: Our results suggest that FOXM1 regulates epithelial homeostasis in esophagus. Inhibition of FOXM1 may promote epithelial differentiation and prevent the disruption via regulation of cell cycle in EoE. Understanding mechanisms of perturbation in epithelial proliferation and differentiation in EoE could lead to the development of novel treatments that promote epithelial healing and restore homeostasis.

Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer-related deaths in the US with a 5-year survival rate of 9%. Current chemotherapeutic strategies are highly aggressive, involving combinations of gemcitabine, Abraxane, and FOLFIRINOX, which are often associated with significant adverse toxicities. Furthermore, despite the aggressive strategies response rates remain poor. As such, it is critical to identify novel targets for therapy to improve patient outcomes. In this regard, we have determined that prolactin (PRL) receptor (PRLR) signaling potentiates PDAC growth, invasive behavior, and stemness. Following systematic knockout (KO) or knockdown (KD) of PRLR by CRISPR/Cas9 or shRNA, respectively, we performed total RNA-sequencing to further delineate the mechanism by which PRLR potentiates these aggressive phenotypes. Ingenuity Pathway Analysis (IPA) was utilized for data analysis, and we identified that glutamine metabolism genes, including GLUD1, GLUD2, GLUL, and SLC1A5 were significantly downregulated in PRLR KD or KO cells compared to controls. We confirmed that PRL treatment increased expression of these glutamine metabolism genes in PRLR competent cells. Moreover, rescue of PRLR in the KO/KD cells also restored expression of these four genes. Given that PDAC is addicted to glutaminolysis, these data suggest that PRLR is the master regulator of glutamine metabolism in PDAC. Interestingly, glutamine has been demonstrated to contribute to cellular invasion and growth. Following these findings, we examined the levels of glutamine metabolites by ELISA in PDAC cells treated with PRL, and in PRLR KO/KD cells. PRL significantly induced production of a-ketoglutarate, citrate, and glutamate, while KO/KD cells displayed significant reduction in these metabolites. We have identified that the anti-psychotic drug penfluridol (Pen) binds to PRLR within the JAK2 binding domain and inhibits PRLR signaling. We also observed that Pen suppresses baseline and PRL-induced expression of the glutamine metabolism genes. Moreover, Pen significantly suppressed migration and invasive potential of PDAC cells. In addition, there was significant reduction in growth of PDAC cancer cells in the syngeneic, orthotopic PDAC models. There was also a reduction in the glutamine response genes in the tumor tissues from the treated animals. Collectively, these data demonstrate that PRL-PRLR signaling regulates glutamine metabolism gene expression, which potentiates aggressive PDAC cell behavior. Inhibition of PRLR signaling, either by genetic or pharmacologic approaches, suppressed glutamine gene expression and metabolite production, which was further associated with the observed decrease in PDAC cell proliferation and migration. Taken together, these data demonstrate the rationale for therapeutic targeting PRLR for PDAC.

David Standing

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PROLACTIN SIGNALING INDUCES GLUTAMINE METABOLISM IN PANCREATIC CANCER

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