GASTROINTESTINAL DISEASE-RELATED SINGLE-NUCLEOTIDE VARIANTS (SNV_S) COLOCALIZE WITH <i>CIS</i>-REGULATORY ELEMENTS (CRE_S) OF THE ELECTRICAL SLOW WAVE (SW)-ASSOCIATED GENE <i>SLC4A4</i> IN A HUMAN MODEL OF PACEMAKER INTERSTITIAL CELLS OF CAJAL (ICC)

Background & Aim:
Phosphatase and tensin homolog (PTEN) is a lipid phosphatase, while its protein phosphatase activity is being suggested. Since the classical role of PTEN as a tumor suppressor is unclear in the gut, we hypothesize PTEN’s alternative function in regulating gut homeostasis.

Methods:
We examined human colonic mucosal biopsies of pediatric Ulcerative Colitis (pUC) patients and control subjects to evaluate PTEN mRNA levels. We generated intestinal epithelial cell (IEC)-Pten-knockout (KO) (Pten-dIEC/dIEC) mice using the Villin-Cre system. We tested in vivo gut permeability with FITC-Dextran, intestinal epithelial cell junction, and autophagy using transmission electron microscopy and fluorescence confocal microscopy. We knocked down (KD) endogenous PTEN gene in human colonic epithelial HT-29 and DLD-1 cells to perform immunoblotting and immunoprecipitation. We used fluorescence in situ hybridization and 16S rRNA gene sequencing. We generated Pten-dIEC/dIEC;Myd88-KO, Pten-dIEC/dIEC;Tlr5-KO, and Pten-dIEC/dIEC;Tlr4-KO mice to study the pathological link of IEC-Pten-KO.

Results:
PTEN mRNA level is 70% reduced (P<0.01, Mann-Whitney U test) in colonic mucosa tissues of pUC (n=9) compared to controls (n=19). IEC-Pten-KO mice exhibit disrupted intestinal epithelial cell junctions and increased in vivo gut permeability. These mice have reduced levels of cell-junction proteins (ZO-1, JAM-C, Claudin-1) in IECs. PTEN-KD decreases cell-junction protein levels, but increases SNAIL/SLUG transcription repressor proteins in HT-29 and DLD-1 cells. PTEN-KD promotes the phosphorylation of Ubiquitin protein and increases the ubiquitination of SNAIL/SLUG in IECs. Loss of PTEN initiates the autophagy pathway in IECs. But, it lowers YKT6 levels to inhibit the fusion between autophagosome and lysosome in IECs. Thereby, PTEN deficiency inhibits the protein degradation machinery of autophagy. We further observed that IEC-Pten-KO mice have increased Bacteroides, but decreased Akkermansia muciniphila in the feces. IEC-Pten-KO mice have increased gut bacterial dissemination to the colon submucosa. Accordingly, Pten-dIEC/dIEC;Myd88-KO, Pten-dIEC/dIEC;Tlr5-KO mice develop massive inflammation in extra-intestinal organs, including the liver and kidney. Pten-dIEC/dIEC;Tlr4-KO mice exhibit mild inflammation in the liver where the abundance of Bacteroides is markedly increased.

Conclusion:
PTEN deficiency promotes the ubiquitination of SNAIL/SLUG transcription repressor protein, while blocking the autophagy flux by inhibiting the autolysosome formation in IECs. Thus, ubiquitinated SNAIL/SLUG cannot be degraded in PTEN-deficient IECs, resulting in augmented SNAIL/SLUG that suppresses the expression of cell-junction proteins and then disrupts the intestinal epithelium. Consequently, PTEN deficiency in IECs increases the gut permeability and risk of gut microbe-induced disorders.

Background and Aims: The Aryl Hydrocarbon Receptor (AhR) plays important functions in intestinal stem cell differentiation, intestinal homeostasis, and immune regulation in the gut, however, its role in regulating intestinal immune tolerance remains poorly understood. In this study, we assessed the role of AhR knockout on the intestinal paracellular tight junction (TJ) barrier, and how it affects the factors influencing intestinal anergy and immune tolerance.
Methods: Ahr^fl/fl, and AhR^Vil-Cre mice were maintained in a specific pathogen-free area until injected with tamoxifen to delete the AhR gene, and then moved to the normal housing. The AhR deletion was maintained by weekly tamoxifen injections for 3 weeks. We performed physiological assessments of the gut epithelial barrier and used flow cytometry, and qRT-PCR analysis to determine the effect of epithelia-specific AhR^-/- on the gut immune system.
Results: Conditional gut epithelium-specific knockout of AhR in AhR^Vil-Cre mice significantly increased the colonic transepithelial resistance (TER) and reduced the paracellular flux of small molecule, urea, and macromolecule inulin, compared to AhR^fl/fl mice. Assessment of transcript levels in the mouse colonic tissues, after 3 weeks of AhR deletion, showed that AhR knockout did not alter the baseline levels of TNF-α, IFN-γ, IL-4, and TGF-β, however, the transcript levels of IL-6, IL-1β, and Il-17A showed marked elevation. Inflammatory stimuli in the form of intraperitoneal LPS administration further amplified the increase in IL-6, IL-1β, and IL-17A mRNA levels in AhR^Vil-Cre mice. Knocking out AhR in the gut epithelial cells also reduced the colonic transcript levels of IL-10, a key anti-inflammatory cytokine, and Programmed death-ligand 1 (PD-L1) and cytotoxic T-lymphocyte-associated protein 4 (CTLA4), the two key proteins, involved in limiting the pro-inflammatory responses in the gut mucosa. Flow cytometry analysis using control Ahr^fl/fl and AhR^Vil-Cre mouse colons subjected to the same tamoxifen administration cycles and LPS challenge showed an increased abundance of IL-6^hi inflammatory M1 macrophages and IL-17A^hi RoRγt⁺ CD4+ Th17 cells in the AhR^Vil-Cre colonic lamina propria under baseline conditions which were further increased by the administration of LPS.
Conclusion: Our data show that deletion of the AhR gene in the gut epithelial cells reduces the epithelial paracellular TJ permeability. This causes the down-regulation of immunological-anergy-promoting proteins and increases the presence of circulating M1 polarized macrophages and Th17 cells in the mice colon in the event of an inflammatory challenge. Our study also highlights the role of paracellular TJ permeability in intestinal immune homeostasis.

Background: Microvillus inclusion disease (MVID) is a congenital disorder characterized by severe diarrhea that presents within the first few days of life. MVID is caused by inactivating mutations in myosin Vb (MYO5B), a motor protein. Mice lacking MYO5B in their intestinal epithelium (VilCre^ERT2; MYO5B^fl/fl) have demonstrated the importance of MYO5B in enterocyte brush border formation and epithelial cell differentiation. Additionally, the proliferative zone of the intestinal crypt of MYO5B-deficient mice is elongated compared to controls. This study aims to better understand the differentiation deficits and crypt-specific effects caused by MYO5B loss utilizing the progenitor cell-specific Cre driver under Lrig1.

Methods: Tamoxifen-inducible Lrig1-Cre^ERT2;R26R-YFP and Myo5b^fl/fl mice were crossbred to generate Lrig1-Cre^ERT2;R26R-YFP;MYO5B^fl/fl (Lrig1^ΔMYO5B) mice. Eight to 10-week-old Lrig1^ΔMYO5B mice and control littermates received 2 mg tamoxifen intraperitoneally at day 0. Cells derived from Cre-induced cells also express YFP (Fig. 1A). GI tissues were collected 2.5, 3, 4, and 5 days after tamoxifen administration for histological characterization.

Results: Lrig1^ΔMYO5B mice began experiencing significant weight loss 4 days after tamoxifen injection and lost 20% of their starting body weight on day 5 (Fig. 1B). At day 5, the Lrig1^ΔMYO5B mouse colon was devoid of solid feces and their small intestine contained clear luminal contents, together suggesting a watery diarrheal phenotype (Fig. 1C). Enterocytes of Lrig1^ΔMYO5B mice on day 5 contained abnormal cytoplasmic accumulation of PAS staining (Fig. 1D). Like the differentiation deficits seen in VilCre^ERT2; MYO5B^fl/fl mice, fewer goblet cells were apparent in intestinal sections from day 3 and 5 Lrig1^ΔMYO5B mice compared to controls. Crypts were elongated starting at day 3. Similarly, the PCNA-expressing epithelial region was expanded in Lrig1^ΔMYO5B mice, even reaching into the villi at day 5 (Fig. 2A). The time course of the PCNA⁺ region expansion corresponded to the wave of MYO5B-loss beginning at the base of crypts, indicating that MYO5B loss directly altered progenitor cell proliferation and differentiation. MYO5B expression was diminished in the crypts at day 3 and nearly absent from the epithelium by day 5. Sodium/glucose cotransporter 1 (SGLT1), which is present along the apical surface of mature enterocytes, was progressively diminished and mis-localized away from the apical surface after tamoxifen administration (Fig. 2B).
Conclusion: We established a novel mouse model that demonstrates progressive MYO5B loss in the intestinal epithelium, starting at the base of the crypt and reaching villi tips over the course of 5 days following Cre induction. This Lrig1^ΔMYO5B strain will allow for lineage tracing studies to better understand the effect of MYO5B loss on intestinal epithelial differentiation.

Background:. TGF-β induces multiple transitions in hepatic stellate cells (HSC) including mitochondrial biogenesis, and transcriptional changes. However, the connection between these mitochondrial signals and fibrogenic activation of HSC are not clearly established. In the current study we have demonstrated a novel pathway involving TGF-β induced HSC activation via mitochondrial DNA release and activation of the cGAS-STING-IRF3 pathway. Methods: Hepatic stellate cells were isolated from C57BL6 mice and LX2 cells (human HSC line) and were subjected to 5ng/ml TGF-β for 16h and were assayed for transdiffertation. Inhibitors such as VBIT4 10μM (VDAC inhibitor), STING inhibitor (IFM-4490 at 20μM) were used. A mouse model of high fat/fibrogenic diet (Choline deficient – L-amino acid sufficient) for 6 weeks with/without STING antagonist 5mg/kg. Results: TGF-β stimulus in HSC resulted in phospho-STING (S366) localization to Golgi (p<0.001) and phospho-IRF3 nuclear translocation and expression of α-smooth muscle actin. Knockdown of STING in LX2 cells significantly suppressed the above changes. Live cell imaging of LX2 cells expressing TFAM-eGFP and TOM20-mPlum with super resolution microscopy showed DNA release following 8h of stimulation with TGF-β. We have termed the cGAS binding to DNA on TOM20 structures as “mtDNA cap”. Similar mitochondrial membrane protrusions were also observed in electron micrographs of TGFβ stimulated LX2. Depletion of mtDNA in LX2 abrogated TGF-β induced HSC activation. Blocking Ca²⁺ flux in LX2 mitochondria resulted in suppression of TGFβ induced activation. Immunofluorescence and proximity ligation assay confirmed that mtDNA is released via VDAC3 upon TGF-β stimulus. Furthermore, VBIT4 significantly suppressed the ECM gene signature upon TGFβ stimulus further providing evidence for VDAC3 mediated mtDNA release. Pretreatment of LX2 with a STING inhibitor significantly suppressed TGF-β induced IRF3 nuclear translocation as well as ACTA2 transcription. Moreover, RNA sequence analysis of LX2 cells subjected to TGF-β stimulus with or without STING inhibitor shows significant suppression of gene signatures involved in ECM production and HSC differentiation. Mice treated with STING antagonist and subjected to high fat – CDAA diet were significantly protected from steatosis induced hepatic fibrosis (n=8; P<0.001). Conclusion: TGF-β signaling in HSC results in mtDNA release via VDCA3 onto the outer mitochondrial membrane. Cytosolic cGAS binds this mtDNA to form a “mtDNA-cap” structure which activates the STING-IRF3 axis. IRF3 further mediates transactivation of a panel of genes leading to HSC differentiation and fibrosis progression. These findings provide novel insights into fibrogenic activation of HSCs and potential therapeutic targets for hepatic fibrosis.

Introduction: Chronic pancreatitis (CP) is a fibro-inflammatory disease of pancreas with unknown pathophysiology. Hedgehog (Hh) pathway is a developmental signaling pathway with key roles during embryogenesis. Its aberrant activation in adults has been implicated in several fibrotic diseases. Our previous work suggest that pharmacological inhibition of Hh signaling improves CP in multiple animal models. Here, we investigate the mechanism by which Hh signaling influences the outcome of CP.
Methods: L-Arginine CP was induced in C57BL/6 mice by repeated administration of L-Arginine (4.5 g/kg × 2 hourly, ip, per week ×4). Mice were euthanized and pancreas was harvested and analyzed by single cell RNA sequencing (scRNAseq) and RNA Fluorescent In situ Hybridization (RNAFish) to identify the cellular compartment of Hh activation. Thereafter, we generated Col1A2-CreER; Smo^FL/FL strain of mice to facilitate tamoxifen driven, Pancreatic Stellate Cell (PSC)-specific deletion of Smo, which is a critical mediator of Hh signaling. L-Arginine model of CP was induced in these mice followed by their randomized into 2 groups – The tamoxifen (75mg/kg) and the Vehicle (corn oil) group (Figure 1). Mice were euthanized and tissue specimens were collected. Pancreatic atrophy, acinar cell loss, inflammatory cell infiltration and fibrosis were evaluated. Additionally, PSCs harvested from Col1A2-CreER; Smo^FL/FL mice and cultured with or without 4-hydroxytamoxifen (4-HT) were used to evaluate the role of Hh signaling in PSC-macrophage crosstalk.
Results: scRNAseq and RNAFish of CP pancreas in C57BL/6 mice revealed PSC specific activation of Hh signaling, as indicated by Gli1 expression (Hh pathway target gene). Based on this finding, we used Col1A2-CreER; Smo^FL/FL for our further experiments. PSC-specific deletion of Smo mediated by tamoxifen in Col1A2-CreER; Smo^FL/FL mice resulted in significant improvement in the parameters of CP as evidenced by increased pancreas-to-mouse weight ratio, decreased inflammatory infiltrate, reduced deposition of collagen as well as downregulation of pro-fibrotic genes such as α-SMA, fibronectin and vimentin compared to the vehicle (corn oil) group (Figure 2). Ex vivo, deletion of Smo in PSCs by 4-HT treatment resulted in their decreased activation as well as reduced secretion of IL4 and IL13, thereby downregulating M2 polarization of macrophages in PSC-macrophage co-culture experiment.
Conclusion: Activation of Hh signaling leads to fibrosis in pancreas and plays an important role in the pathogenesis of CP. PSC specific deletion of Hh signaling reduces the disease severity by re-programming PSCs and consequently preventing M2 polarization of macrophages.

Background & Aims: Previously, we reported selective expression of the electrogenic Na⁺/HCO3⁻ cotransporter SLC4A4 (NBCe1-B) in pacemaker ICC and described its role in SW amplitude and frequency control. Here, we investigated the potential role of SLC4A4 in GI diseases by colocalizing GI-relevant noncoding SNVs from a phenome-wide association study (PheWAS) with cREs for the SLC4A4 gene identified by epigenomic analysis in a human ICC model. Methods: We developed GIST882 cells, which originated from transformed gastric ICC, as a pacemaker ICC model expressing SLC4A4 verified by RNA-sequencing, immunofluorescence, and immunoblotting and by detecting coordinated electrical SWs and Ca²⁺ signals by imaging fluorescence from DiBAC₄(3) and RGECO, respectively. Epigenomic profiling was performed by chromatin immunoprecipitation-sequencing (ChIP-seq) targeting the promoter-associated mark H3K4me3, the enhancer-associated mark H3K4me1, and the active cRE-associated mark H3K27ac and by analyzing open chromatin and transcription factor (TF) footprints by ATAC-seq. cRE interactions were identified by chromosome conformation capture-sequencing (HiC). PheWAS screening of genome-wide significant (P≤5e-8) SNVs was performed in the ~1.16 Mb topologically associating domain (TAD) containing SLC4A4. The SNV-associated traits detected in the UK Biobank were filtered for GI relevance. Results: In GIST882 cells, SWs and Ca⁺ oscillations occurred at 1.17±0.36 min^-1 (mean±SD; n=27). ChIP-seq and ATAC-seq identified 2 SLC4A4-intronic and 3 intergenic enhancer clusters with direct coupling to the SLC4A4 promoter. These cREs were missing from GIST-T1 cells lacking SLC4A4 expression. Occupied sequences within differentially open sites included motifs for the ICC/GIST TF FOXF1, TFs regulating organogenesis, and members of the MYC/MAX/MAD network. PheWAS traits associated with the SLC4A4 promoter included cholelithiasis, flatulence, and gastrocote use. The 5’ intergenic and SLC4A4-intronic enhancers were associated with gastritis, cholelithiasis, cholangitis, cholangiocarcinoma, peritoneal metastasis, senna use, ulcerative colitis, diverticulitis with perforation and abscess, GI hemorrhage, anus, appendix, and rectosigmoid neoplasm. The 3’ enhancer cluster, which contained many gastric expression quantitative trait loci for SLC4A4 in linkage disequilibrium, was associated with peritoneal metastasis, diverticulitis, duodenum, intestinal perforation, anal cancer, and cholelithiasis. 3 cholelithiasis-related SNVs mapped into TF footprints. Conclusions: Combined PheWAS and epigenomic analysis revealed an association of several GI disease-relevant SNVs with active cREs of the SW-regulating gene SLC4A4 and identified candidate TFs that may contribute to GI pathogenesis. We also established GIST882 cells as a pacemaker ICC model. Grants: NIH R01 DK57061, R01 DK58185, P30 DK84567.