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Background & Aims: Liver 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. Methods: We 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. Results: LKB1 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. Conclusions: LKB1 in IECs manages BA homeostasis by controlling FGF15/19 production.

Pathobiont expansion, such as that of adherent-invasive Escherichia coli (AIEC), is an emerging factor associated with inflammatory bowel disease. The intestinal epithelial barrier is the first line of defense against these pathogens. Inflammation plays a critical role in altering the epithelial barrier and is a major factor involved in promoting the expansion and pathogenesis of AIEC. AIEC in turn can exacerbate intestinal epithelial barrier dysfunction by targeting multiple elements of the barrier. One critical element of the epithelial barrier is the tight junction. Increasing evidence suggests that AIEC may selectively target protein components of tight junctions, leading to increased barrier permeability. This may represent one mechanism by which AIEC could contribute to the development of inflammatory bowel disease. This review article discusses potential mechanisms by which AIEC can disrupt epithelial tight junction function and intestinal barrier function.

Background and Aims: Gut bacteria-derived short-chain fatty acids (SCFAs) play crucial roles in the maintenance of intestinal homeostasis. However, how SCFAs regulate epithelial turnover and tissue repair remain incompletely understood. In this study, we investigated how the SCFA propionate regulates cell migration to promote epithelial renewal and repair. Methods: Mouse small intestinal epithelial cells (MSIE) and human Caco-2 cells were used to determine the effects of SCFAs on gene expression, proliferation, migration, and cell spreading in vitro. Video microscopy and single cell tracking were used to assess cell migration kinetically. 5-bromo-2’-deoxyuridine (BrdU) and hydroxyurea were used to assess the effects of SCFAs on migration in vivo. Lastly, an acute colitis model using dextran sulfate sodium (DSS) was used to examine the effects of SCFAs in vivo. Results: Using video microscopy and single cell tracking, we found that propionate promoted intestinal epithelial cell migration by enhancing cell spreading and polarization, which led to increases in both cell speed and persistence. This novel function of propionate was dependent on inhibition of class I histone deacetylases (HDAC) and GPR43 and required signal transducer and activator of transcription 3 (STAT3). Furthermore, using 5-bromo-2’-deoxyuridine (BrdU) and hydroxyurea in vivo, we found that propionate enhanced cell migration up the crypt-villus axis under homeostatic conditions, while also protecting against ulcer formation in experimental colitis. Conclusion: Our results demonstrate a mechanism by which propionate stimulates cell migration in an HDAC inhibition, GPR43, and STAT3 dependent manner, and suggest that propionate plays an important role in epithelial migration independent of proliferation.

Background and Aims: Intestinal mucosa undergoes a continual process of proliferation, differentiation, and apoptosis. Disruption of this homeostasis is associated with disorders such as inflammatory bowel disease (IBD). We investigated the role of Sirtuin 2 (SIRT2), a NAD-dependent protein deacetylase, in intestinal epithelial cell (IEC) proliferation and differentiation and the mechanism by which SIRT2 contributes to maintenance of intestinal cell homeostasis. Methods: IECs were collected from SIRT2-deficient mice and patients with IBD. Expression of SIRT2, differentiation markers (mucin2, intestinal alkaline phosphatase, villin, Na,K-ATPase, and lysozyme) and Wnt target genes (EPHB2, AXIN2, and cyclin D1) was determined by western blot, real-time RT-PCR, or immunohistochemical (IHC) staining. IECs were treated with TNF or transfected with siRNA targeting SIRT2. Proliferation was determined by villus height and crypt depth, and Ki67 and cyclin D1 IHC staining. For studies using organoids, intestinal crypts were isolated. Results: Increased SIRT2 expression was localized to the more differentiated region of the intestine. In contrast, SIRT2 deficiency impaired proliferation and differentiation and altered stemness in the small intestinal epithelium ex vivo and in vivo. SIRT2-deficient mice showed decreased intestinal enterocyte and goblet cell differentiation but increased the Paneth cell lineage and increased proliferation of IECs. Moreover, we found that SIRT2 inhibits Wnt/β-catenin signaling, which critically regulates IEC proliferation and differentiation. Consistent with a distinct role for SIRT2 in maintenance of gut homeostasis, intestinal mucosa from IBD patients exhibited decreased SIRT2 expression. Conclusion: We demonstrate that SIRT2, which is decreased in intestinal tissues from IBD patients, regulates Wnt-β-catenin signaling and is important for maintenance of IEC proliferation and differentiation.

Background & Aims: Liver 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., Methods: We generated mice with IEC-specific deletion of LKB1 (LKB1 ΔIEC ) 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., Results: LKB1 deletion resulted in abnormal differentiation of secretory cell lineages. Unexpectedly, BA pool size increased substantially in LKB1 ΔIEC 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 LKB1 ΔIEC 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 LKB1 ΔIEC mice. Moreover, LKB1 ΔIEC mice showed impaired conversion of retinol to retinoic acids in the SI ileum. Subsequently, vitamin A treatment failed to induce FGF15 production. Thus, LKB1 ΔIEC mice fed with a high-fat diet showed improved glucose tolerance and increased energy expenditure., Conclusions: LKB1 in IECs manages BA homeostasis by controlling FGF15/19 production., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Background and Aims: Gut bacteria-derived short-chain fatty acids (SCFAs) play crucial roles in the maintenance of intestinal homeostasis. However, how SCFAs regulate epithelial turnover and tissue repair remain incompletely understood. In this study, we investigated how the SCFA propionate regulates cell migration to promote epithelial renewal and repair., Methods: Mouse small intestinal epithelial cells (MSIE) and human Caco-2 cells were used to determine the effects of SCFAs on gene expression, proliferation, migration, and cell spreading in vitro. Video microscopy and single cell tracking were used to assess cell migration kinetically. 5-bromo-2'-deoxyuridine (BrdU) and hydroxyurea were used to assess the effects of SCFAs on migration in vivo. Lastly, an acute colitis model using dextran sulfate sodium (DSS) was used to examine the effects of SCFAs in vivo., Results: Using video microscopy and single cell tracking, we found that propionate promoted intestinal epithelial cell migration by enhancing cell spreading and polarization, which led to increases in both cell speed and persistence. This novel function of propionate was dependent on inhibition of class I histone deacetylases (HDAC) and GPR43 and required signal transducer and activator of transcription 3 (STAT3). Furthermore, using 5-bromo-2'-deoxyuridine (BrdU) and hydroxyurea in vivo, we found that propionate enhanced cell migration up the crypt-villus axis under homeostatic conditions, while also protecting against ulcer formation in experimental colitis., Conclusion: Our results demonstrate a mechanism by which propionate stimulates cell migration in an HDAC inhibition, GPR43, and STAT3 dependent manner, and suggest that propionate plays an important role in epithelial migration independent of proliferation., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Background and Aims: Intestinal mucosa undergoes a continual process of proliferation, differentiation, and apoptosis. Disruption of this homeostasis is associated with disorders such as inflammatory bowel disease (IBD). We investigated the role of Sirtuin 2 (SIRT2), a NAD-dependent protein deacetylase, in intestinal epithelial cell (IEC) proliferation and differentiation and the mechanism by which SIRT2 contributes to maintenance of intestinal cell homeostasis., Methods: IECs were collected from SIRT2-deficient mice and patients with IBD. Expression of SIRT2, differentiation markers (mucin2, intestinal alkaline phosphatase, villin, Na,K-ATPase, and lysozyme) and Wnt target genes (EPHB2, AXIN2, and cyclin D1) was determined by western blot, real-time RT-PCR, or immunohistochemical (IHC) staining. IECs were treated with TNF or transfected with siRNA targeting SIRT2. Proliferation was determined by villus height and crypt depth, and Ki67 and cyclin D1 IHC staining. For studies using organoids, intestinal crypts were isolated., Results: Increased SIRT2 expression was localized to the more differentiated region of the intestine. In contrast, SIRT2 deficiency impaired proliferation and differentiation and altered stemness in the small intestinal epithelium ex vivo and in vivo. SIRT2-deficient mice showed decreased intestinal enterocyte and goblet cell differentiation but increased the Paneth cell lineage and increased proliferation of IECs. Moreover, we found that SIRT2 inhibits Wnt/β-catenin signaling, which critically regulates IEC proliferation and differentiation. Consistent with a distinct role for SIRT2 in maintenance of gut homeostasis, intestinal mucosa from IBD patients exhibited decreased SIRT2 expression., Conclusion: We demonstrate that SIRT2, which is decreased in intestinal tissues from IBD patients, regulates Wnt-β-catenin signaling and is important for maintenance of IEC proliferation and differentiation., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Background & Aims: Regenerating islet-derived protein (REG3γ), an antimicrobial peptide, typically expressed by intestinal epithelial cells (IEC), plays crucial roles in intestinal homeostasis and controlling gut microbiota. However, the mechanisms that regulate IEC expression of REG3γ are still largely unclear. In this study, we investigated whether and how interleukin (IL) 33, an alarmin produced by IEC in response to injury, regulates REG3γ expression in IEC, thus contributing to intestinal homeostasis., Methods: IEC were isolated from wild-type and IL33 -/- mice to determine expression of REG3γ and other antimicrobial peptides by quantitative real-time polymerase chain reaction and Western blot. IEC cell lines were used for mechanistic studies. 16S rRNA pyrosequencing analysis was used for measuring gut microbiota. Citrobacter rodentium was used for enteric infections., Results: The expression of REG3γ, but not β-defensins, in IECs of IL33 -/- mice was significantly lower than wild-type mice. IL33 treatment induced IEC expression of REG3γ in both mice and human cell lines. Mechanistically, IL33 activated STAT3, mTOR, and ERK1/2 in IEC. Inhibition of these pathways abrogated IL33-induction of REG3γ. IL33 -/- mice demonstrated higher bacteria loads and altered microbiota composition. IL33 did not directly inhibit bacterial growth, but promoted wild-type, not REG3γKO, IECs to kill bacteria in vitro. Consistently, C rodentium infection induced IEC IL33 expression, and IL33 -/- mice demonstrated an impaired bacterial clearance with C rodentium infection., Conclusions: Our study demonstrated that IL33, which is produced by IEC in response to injury and inflammatory stimulation, in turn promotes IEC expression of REG3γ, and controls the gut microbiota of the host., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)