Signaling In Intestinal Epithelial Cells Research Articles

FGL2 regulates IKK/NF-κB signaling in intestinal epithelial cells and lamina propria dendritic cells to attenuate dextran sulfate sodium-induced colitis

Inflammatory bowel disease (IBD) is an autoimmune disease characterized by an abnormal immune response. Fibrinogen-like protein 2 (FGL2) is known to have immunoregulatory and anti-inflammatory activity. The level of FGL2 is elevated in patients with IBD; however, its comprehensive function in IBD is almost unknown. In our study, we explored the effect of FGL2 on dextran sulfate sodium (DSS)-induced colitis in mice and on NF-κB signaling in intestinal epithelial cells (IECs) and lamina propria dendritic cells (LPDCs). We founded that FGL2−/− mice in the colitis model showed more severe colitis manifestations than WT mice did, including weight loss, disease activity index (DAI), and colon histological scores. FGL2−/− mice treated with DSS produced more proinflammatory cytokines (IL-1β, IL-6, TNF-α) in serum than WT mice did and demonstrated upregulated expression of TNF-α and inflammatory marker enzymes, inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (Cox-2) in the colon tissue. Our data suggested that DSS-treated FGL2−/− mice showed stronger activation of NF-κB signaling, especially in IECs. Next, we demonstrated that recombinant FGL2 (rFGL2) inhibited the production of proinflammatory cytokines and the expression of inflammatory marker enzymes by downregulating the NF-κB signaling in HT-29 cells. Finally, we discovered that LPDCs from the colon of DSS-treated FGL2−/− mice showed significantly upregulated expression of surface maturation co-stimulatory molecules, including CD80, CD86, CD40, and MHC class II molecules compared with that in WT mice. In addition, LPDCs in FGL2−/− treated with DSS exhibited excessive NF-κB activity and the administration of rFGL2 to FGL2−/− mice could rescue the aggravated results of FGL2−/− mice. Taken together, our findings demonstrated that FGL2 might be a target for further therapy of IBD.

Regulation of Ketogenic Enzyme HMGCS2 by Wnt/β-catenin/PPARγ Pathway in Intestinal Cells.

The Wnt/β-catenin pathway plays a crucial role in development and renewal of the intestinal epithelium. Mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase 2 (HMGCS2), a rate-limiting ketogenic enzyme in the synthesis of ketone body β-hydroxybutyrate (βHB), contributes to the regulation of intestinal cell differentiation. Here, we have shown that HMGCS2 is a novel target of Wnt/β-catenin/PPARγ signaling in intestinal epithelial cancer cell lines and normal intestinal organoids. Inhibition of the Wnt/β-catenin pathway resulted in increased protein and mRNA expression of HMGCS2 and βHB production in human colon cancer cell lines LS174T and Caco2. In addition, Wnt inhibition increased expression of PPARγ and its target genes, FABP2 and PLIN2, in these cells. Conversely, activation of Wnt/β-catenin signaling decreased protein and mRNA levels of HMGCS2, βHB production, and expression of PPARγ and its target genes in LS174T and Caco2 cells and mouse intestinal organoids. Moreover, inhibition of PPARγ reduced HMGCS2 expression and βHB production, while activation of PPARγ increased HMGCS2 expression and βHB synthesis. Furthermore, PPARγ bound the promoter of HMGCS2 and this binding was enhanced by β-catenin knockdown. Finally, we showed that HMGCS2 inhibited, while Wnt/β-catenin stimulated, glycolysis, which contributed to regulation of intestinal cell differentiation. Our results identified HMGCS2 as a downstream target of Wnt/β-catenin/PPARγ signaling in intestinal epithelial cells. Moreover, our findings suggest that Wnt/β-catenin/PPARγ signaling regulates intestinal cell differentiation, at least in part, through regulation of ketogenesis.

IFN-I and IL-22 mediate protective effects of intestinal viral infection.

Products derived from bacterial members of the gut microbiota evoke immune signaling pathways from the host that promote immunity and barrier function in the intestine. How immune reactions to enteric viruses support intestinal homeostasis is unknown. Recently, we demonstrated that infection by murine norovirus (MNV) reverses intestinal abnormalities upon depletion of bacteria, indicating that an intestinal animal virus can provide cues to the host that are typically attributed to the microbiota. Here, we elucidate mechanisms by which MNV evokes protective responses from the host. We identify an important role for the viral protein NS1/2 in establishing local replication and a type I interferon (IFN-I) response in the colon. We further show that IFN-I acts on intestinal epithelial cells to increase the proportion of CCR2-dependent macrophages and IL-22 producing innate lymphoid cells (ILCs), which in turn promote pSTAT3 signaling in intestinal epithelial cells and protection from intestinal injury. Additionally, we demonstrate that MNV provides a striking IL-22 dependent protection against early life lethal infection by Citrobacter rodentium. These findings demonstrate novel ways in which a viral member of the microbiota fortifies the intestinal barrier during chemical injury and infectious challenges.

E-101 UPR in coordinated regulation of microbiome and anti-tumor immunity

RNF5 is a ubiquitin ligase implicated in the control of ER stress and ER associated degradation, part of the greater Unfolded Protein Response. RNF5 mutant mice were found to limit tumor growth, of both melanoma and colon cancer models, owing to enhanced anti-tumor immunity. Enhanced TLR signaling resulted in enhanced infiltration of CD4/8+ T cells as well as Dendritic cells to the tumors grown in RNF5 mutant mice. Cross of RNF5 mutant with MYD88 KO or use of neutralizing antibodies against CD4/8+ T cells effectively attenuated anti-tumor immunity and tumor inhibition phenotypes. Coincided with the enhanced anti-tumor immunity was reduced UPR signaling in intestinal epithelial cells of RNF5 mutant mice. Indeed, cultured intestinal epithelial MODE-K cells that were subjected to inhibition of RNF5 expression exhibited reduced UPR signaling, and were able to activate, in co-culture studies, DC and T cells. Surprisingly, co-housing of RNF5 mutant and WT mice resulted in loss of anti-tumor immunity and the loss of tumor growth inhibition, suggesting that microbiota of RNF5 mutant mice may play a key role in activating anti-tumor immunity. Analysis of gut microbiota composition in RNF5 KO mice led to the identification of specific bacterial strains that were able to induce anti-tumor immunity and limit melanoma growth, when administered to germ free mice. The role of altered UPR in the coordinated regulation of microbiome and anti-tumor immunity will be discussed.

Inducibility and role of mTORC1 signaling in intestinal epithelial cells as a result of cell differentiation

Intestinal epithelium undergoes rapid turnover owing to its multiple functions and active metabolism. Stem cells (SE) residing at the base of the crypts give rise to the distinct lineages of specialized intestinal epithelial cells (IEC). In doing so, SE ensure epithelial renewal and maintain intestinal homeostasis. However, SE‐based tissue renewal is not the only means of maintaining intestinal homeostasis. Specialized IEC that become damaged by the repeated physical, chemical or microbial insults attempt to self‐repair by deploying a dedifferentiation program supported by mediators of growth and proliferation such as mechanistic target of rapamycin complex 1 (mTORC1); also contributing in tissue regeneration and repair. In an event of insult, physiological stimuli may reactivate mechanistic target of rapamycin complex 1 (mTORC1) signaling in differentiated IEC thereby, contributing to epithelial dysfunction, return to stemness or tissue damage. We investigated the inducibility of mTORC1 signaling in differentiated vs. undifferentiated Caco‐2 cells in response to growth factor (IGF‐1) or pro‐inflammatory cytokines. To ascertain the role of mTORC1 in the cell response to the mucosal inflammation, Caco‐2 cells were genetically altered resulting in high (Caco‐2shTSC2), low (Caco‐2shRaptor) or baseline (Caco‐2shScramble) mTORC1 activity. Results showed that, mTORC1 activity was several fold lower in differentiated vs. undifferentiated Caco‐2 cells. While IGF‐1 induced mTORC1 signaling in undifferentiated Caco‐2 cells (IGF‐1: +95%, P<0.05), the stimuli failed to do so in differentiated Caco‐2 cells. The cocktail of cytokines (IFNγ, TNFα, IL‐1β, LPS) prominently induced IL‐1β and IL‐8 mRNA abundance in either undifferentiated or differentiated Caco‐2 cells (IL‐1b, range +374–1213%, P<0.05; IL‐8, range +4180–3350%, P<0.05). Despite the overall low mTORC1 activity levels in differentiated Caco‐2 cells, the transepithelial electrical resistance (TEER) was markedly affected by the mTORC1 status of these cells. Caco‐2shRaptor cells developed higher TEER (+180%, P<0.05) than Caco‐2shScramble cells, while Caco‐2shTSC2 cells had a lower TEER (−60%, P<0.05) than Caco‐2shScramble cells. Media transfer studies, wherein differentiated stable Caco‐2 cells received the conditioned media from activated macrophages to model mucosal inflammation, revealed that cellular TEER dropped significantly irrespective of mTORC1 status. However, unlike Caco‐2shRaptor and Caco‐2shScramble cells, in which TEER recovered marginally, Caco‐2shTSC2 cells exhibited 100% TEER recovery within 3 days. In conclusion, the overall inducibility of mTORC1 in response to the tested exogenous stimuli in Caco‐2 was dependent upon the state of differentiation and mTORC1 activity in IEC. Henceforth, mTORC1 activity is downregulated and not readily inducible (by IGF‐1 or the cytokine cocktail) as Caco‐2 cells differentiate in culture. However, development of the highest TEER by Caco‐2shRaptor among the three cell lines; and highest rate of TEER recovery in Caco‐2shTSC2 suggests that mTORC1 may have a crucial role in maintaining barrier function and promoting epithelial recovery in response to inflammation in IEC.Support or Funding InformationUSDA‐NIFAThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Intestinal Epithelial Cells Regulate Gut Eotaxin Responses and Severity of Allergy.

Intestinal epithelial cells (IECs) are known to regulate allergic sensitization. We addressed the role of the intrinsic IKKβ signaling in IECs in the effector phase of allergy following oral allergen challenge and its impact on the severity of responses is poorly. Upon orally sensitization by co-administration of ovalbumin with cholera toxin as adjuvant, wild-type and mice lacking IKKβ in IECs (IKKβΔIEC mice) developed similar levels of serum IgE and allergen-specific secretory IgA in the gut. However, subsequent allergen challenges in the gut promoted allergic lower responses in KKβΔIEC mice. Analysis of cytokines and chemokines in serum and gut tissues after oral allergen challenge revealed impaired eotaxin responses in IKKβΔIEC mice, which correlated with lower frequencies of eosinophils in the gut lamina propria. We also determined that IECs were a major source of eotaxin and that impaired eotaxin production was due to the lack of IKKβ signaling in IECs. Oral administration of CCL11 to IKKβΔIEC mice during oral allergen challenge enhanced allergic responses to levels in wild-type mice, confirming the role of IEC-derived eotaxin as regulator of the effector phase of allergy following allergen challenge. Our results identified targeting IEC-derived eotaxin as potential strategy to limit the severity of allergic responses to food antigens.

Myeloid p38α signaling promotes intestinal IGF‐1 production and inflammation‐associated tumorigenesis

The protein kinase p38α plays a key role in cell homeostasis, and p38α signaling in intestinal epithelial cells protects against colitis‐induced tumorigenesis. However, little is known on the contribution of p38α signaling in intestinal stromal cells. Here, we show that myeloid cell‐specific downregulation of p38α protects mice against inflammation‐associated colon tumorigenesis. The reduced tumorigenesis correlates with impaired detection in the colon of crucial chemokines for immune cell recruitment. We identify insulin‐like growth factor‐1 (IGF‐1) as a novel mediator of the p38α pathway in macrophages. Moreover, using genetic and pharmacological approaches, we confirm the implication of IGF‐1 produced by myeloid cells in colon inflammation and tumorigenesis. We also show a correlation between IGF‐1 pathway activation and the infiltration of myeloid cells with active p38α in colon samples from patients with ulcerative colitis or colon cancer. Altogether, our results uncover an important role for myeloid IGF‐1 downstream of p38α in colitis‐associated tumorigenesis and suggest the interest in evaluating IGF‐1 therapies for inflammation‐associated intestinal diseases, taking into consideration IGF‐1 signaling and immune cell infiltration in patient biopsies.

Composite regulation of ERK activity dynamics underlying tumour-specific traits in the intestine

Acting downstream of many growth factors, extracellular signal-regulated kinase (ERK) plays a pivotal role in regulating cell proliferation and tumorigenesis, where its spatiotemporal dynamics, as well as its strength, determine cellular responses. Here, we uncover the ERK activity dynamics in intestinal epithelial cells (IECs) and their association with tumour characteristics. Intravital imaging identifies two distinct modes of ERK activity, sustained and pulse-like activity, in IECs. The sustained and pulse-like activities depend on ErbB2 and EGFR, respectively. Notably, activation of Wnt signalling, the earliest event in intestinal tumorigenesis, augments EGFR signalling and increases the frequency of ERK activity pulses through controlling the expression of EGFR and its regulators, rendering IECs sensitive to EGFR inhibition. Furthermore, the increased pulse frequency is correlated with increased cell proliferation. Thus, ERK activity dynamics are defined by composite inputs from EGFR and ErbB2 signalling in IECs and their alterations might underlie tumour-specific sensitivity to pharmacological EGFR inhibition.

Walnut phenolic extract inhibits nuclear factor kappaB signaling in intestinal epithelial cells, and ameliorates experimental colitis and colitis-associated colon cancer in mice.

Walnuts (Juglans regia) are known to have anti-cancer and immunomodulatory effects. However, little information is available on the effects of walnut phenolic extract (WPE) on intestinal inflammation and colitis-associated colon cancer. COLO205 cells were pretreated with WPE and then stimulated with tumor necrosis factor (TNF)-α. In the acute colitis model, wild type mice (C57BL/6) were administered 4% dextran sulfate sodium (DSS) for 5 days. In the chronic colitis model, interleukin (IL)-10-/- mice were administered with either the vehicle or WPE (20mg/kg) by oral gavage daily for 2 weeks. In an inflammation-associated tumor model, wild type mice were administered a single intraperitoneal injection of azoxymethane followed by three cycles of 2% DSS for 5 days and 2 weeks of free water consumption. WPE significantly inhibited IL-8 and IL-1α expression in COLO205 cells. WPE attenuated both the TNF-α-induced IκB phosphorylation/degradation and NF-κB DNA binding activity. The administration of oral WPE significantly reduced the severity of colitis in both acute and chronic colitis models, including the IL-10-/- mice. In immunohistochemical staining, WPE attenuated NF-κB signaling in the colons of both colitis models. Finally, WPE also significantly reduced tumor development in a murine model of colitis-associated colon cancer (CAC). WPE ameliorates acute and chronic colitis and CAC in mice, suggesting that WPE may have potentials for the treatment of inflammatory bowel disease.

The role of Gαq/Gα11 signaling in intestinal epithelial cells

Intestinal homeostasis and the coordinated actions of digestion, absorption and excretion are tightly regulated by a number of gastrointestinal hormones. Most of them exert their actions through G-protein-coupled receptors. Recently, we showed that the absence of Gαq/Gα11 signaling impaired the maturation of Paneth cells, induced their differentiation toward goblet cells, and affected the regeneration of the colonic mucosa in an experimental model of colitis. Although an immunohistochemical study showed that Gαq/Gα11 were highly expressed in enterocytes, it seemed that enterocytes were not affected in Int-Gq/G11 double knock-out intestine. Thus, we used an intestinal epithelial cell line to examine the role of signaling through Gαq/Gα11 in enterocytes and manipulated the expression level of Gαq and/or Gα11. The proliferation was inhibited in IEC-6 cells that overexpressed Gαq/Gα11 and enhanced in IEC-6 cells in which Gαq/Gα11 was downregulated. The expression of T-cell factor 1 was increased according to the overexpression of Gαq/Gα11. The expression of Notch1 intracellular cytoplasmic domain was decreased by the overexpression of Gαq/Gα11 and increased by the downregulation of Gαq/Gα11. The relative mRNA expression of Muc2, a goblet cell marker, was elevated in a Gαq/Gα11 knock-down experiment. Our findings suggest that Gαq/Gα11-mediated signaling inhibits proliferation and may support a physiological function, such as absorption or secretion, in terminally differentiated enterocytes.

Combined Mutation of Apc, Kras, and Tgfbr2 Effectively Drives Metastasis of Intestinal Cancer.

Colorectal cancer is driven by the accumulation of driver mutations, but the contributions of specific mutations to different steps in malignant progression are not fully understood. In this study, we generated mouse models harboring different combinations of key colorectal cancer driver mutations (Apc, Kras, Tgfbr2, Trp53, Fbxw7) in intestinal epithelial cells to comprehensively investigate their roles in the development of primary tumors and metastases. ApcΔ716 mutation caused intestinal adenomas and combination with Trp53R270H mutation or Tgfbr2 deletion induced submucosal invasion. The addition of KrasG12D mutation yielded epithelial-mesenchymal transition (EMT)-like morphology and lymph vessel intravasation of the invasive tumors. In contrast, combinations of ApcΔ716 with KrasG12D and Fbxw7 mutation were insufficient for submucosal invasion, but still induced EMT-like histology. Studies using tumor-derived organoids showed that KrasG12D was critical for liver metastasis following splenic transplantation, when this mutation was combined with either ApcΔ716 plus Trp53R270H or Tgfbr2 deletion, with the highest incidence of metastasis displayed by tumors with a ApcΔ716 KrasG12D Tgfbr2-/- genotype. RNA sequencing analysis of tumor organoids defined distinct gene expression profiles characteristic for the respective combinations of driver mutations, with upregulated genes in ApcΔ716 KrasG12D Tgfbr2-/- tumors found to be similarly upregulated in specimens of human metastatic colorectal cancer. Our results show how activation of Wnt and Kras with suppression of TGFβ signaling in intestinal epithelial cells is sufficient for colorectal cancer metastasis, with possible implications for the development of metastasis prevention strategies.Significance: These findings illuminate how key driver mutations in colon cancer cooperate to drive the development of metastatic disease, with potential implications for the development of suitable prevention strategies. Cancer Res; 78(5); 1334-46. ©2017 AACR.

Intestinal epithelial cell-specific RARα depletion results in aberrant epithelial cell homeostasis and underdeveloped immune system.

Retinoic acid (RA), a dietary vitamin A metabolite, is crucial in maintaining intestinal homeostasis. RA acts on intestinal leukocytes to modulate their lineage commitment and function. Although the role of RA has been characterized in immune cells, whether intestinal epithelial cells (IECs) rely on RA signaling to exert their immune-regulatory function has not been examined. Here we demonstrate that lack of retinoic acid receptor alpha (RARα) signaling in IECs results in deregulated epithelial lineage specification, leading to increased numbers of goblet cells and Paneth cells. Mechanistically, lack or RARα resulted in increased KLF4+ goblet cell precursors in the distal bowel, whereas RA treatment inhibited klf4 expression and goblet cell differentiation in zebrafish. These changes in secretory cells are associated with increased reg3g, reduced luminal bacterial detection and an underdeveloped intestinal immune system, as evidenced by an almost complete absence of lymphoid follicles and gut resident mononuclear phagocytes. This underdeveloped intestinal immune system shows a decreased ability to clear infection with Citrobacter rodentium. Collectively, our findings indicate that epithelial cell-intrinsic RARα signaling is critical to the global development of the intestinal immune system.

Intestinal Epithelial Ecto-5'-Nucleotidase (CD73) Regulates Intestinal Colonization and Infection by Nontyphoidal Salmonella.

Ecto-5'-nucleotidase (CD73) is expressed abundantly on the apical surface of intestinal epithelial cells (IECs) and functions as the terminal enzyme in the generation of extracellular adenosine. Previous work demonstrated that adenosine signaling in IECs results in a number of tissue-protective effects during inflammation; however, a rationale for its apical expression has been lacking. We hypothesized that the highly polarized expression of CD73 is indicative of an important role for extracellular adenosine as a mediator of host-microbe interactions. We show that adenosine harbors bacteriostatic activity against Salmonella enterica serovar Typhimurium that is not shared by the related purine metabolite 5'-AMP, inosine, or hypoxanthine. Analysis of Salmonella colonization in IEC-specific CD73 knockout mice (CD73f/fVillinCre ) revealed a nearly 10-fold increase in colonization compared to that in controls. Despite the increased luminal colonization by Salmonella, CD73f/fVillinCre mice were protected against Salmonella colitis and showed reduced Salmonella burdens in viscera, suggesting that adenosine promotes dissemination. The knockdown of CD73 expression in cultured IECs resulted in dramatic defects in intraepithelial localization and replication as well as defective transepithelial translocation by Salmonella In conclusion, we define a novel antimicrobial activity of adenosine in the gastrointestinal tract and unveil an important role for adenosine as a regulator of host-microbe interactions. These findings have broad implications for the development of new therapeutic agents for infectious disease.

G-protein-coupled receptor kinase-2 is a critical regulator of TNFα signaling in colon epithelial cells.

G-protein-coupled receptor kinase-2 (GRK2) belongs to the GRK family of serine/threonine protein kinases critical in the regulation of G-protein-coupled receptors. Apart from this canonical role, GRK2 is also involved in several signaling pathways via distinct intracellular interactomes. In the present study, we examined the role of GRK2 in TNFα signaling in colon epithelial cell-biological processes including wound healing, proliferation, apoptosis, and gene expression. Knockdown of GRK2 in the SW480 human colonic cells significantly enhanced TNFα-induced epithelial cell wound healing without any effect on apoptosis/proliferation. Consistent with wound-healing effects, GRK2 knockdown augmented TNFα-induced matrix metalloproteinases (MMPs) 7 and 9, as well as urokinase plasminogen activator (uPA; factors involved in cell migration and wound healing). To assess the mechanism by which GRK2 affects these physiological processes, we examined the role of GRK2 in TNFα-induced MAPK and NF-κB pathways. Our results demonstrate that while GRK2 knockdown inhibited TNFα-induced IκBα phosphorylation, activation of ERK was significantly enhanced in GRK2 knockdown cells. Our results further demonstrate that GRK2 inhibits TNFα-induced ERK activation by inhibiting generation of reactive oxygen species (ROS). Together, these data suggest that GRK2 plays a critical role in TNFα-induced wound healing by modulating MMP7 and 9 and uPA levels via the ROS-ERK pathway. Consistent with in vitro findings, GRK2 heterozygous mice exhibited enhanced intestinal wound healing. Together, our results identify a novel role for GRK2 in TNFα signaling in intestinal epithelial cells.

Tumour‐associated changes in intestinal epithelial cells cause local accumulation of KLRG1+GATA3+ regulatory T cells in mice

CD4+ Foxp3+ regulatory T (Treg) cells include differentiated populations of effector Treg cells characterized by the expression of specific transcription factors. Tumours, including intestinal malignancies, often present with local accumulation of Treg cells that can prevent tumour clearance, but how tumour progression leads to Treg cell accumulation is incompletely understood. Here using genetically modified mouse models we show that ablation of E-cadherin, a process associated with epithelial to mesenchymal transition and tumour progression, promotes the accumulation of intestinal Treg cells by the specific accumulation of the KLRG1+ GATA3+ Treg subset. Epithelial E-cadherin ablation activates the β-catenin pathway, and we find that increasing β-catenin signals in intestinal epithelial cells also boosts Treg cell frequencies through local accumulation of KLRG1+ GATA3+ Treg cells. Both E-cadherin ablation and increased β-catenin signals resulted in epithelial cells with higher levels of interleukin-33, a cytokine that preferentially expands KLRG1+ GATA3+ Treg cells. Tumours often present reduced E-cadherin expression and increased β-catenin signalling and interleukin-33 production. Accordingly, Treg cell accumulation in intestinal tumours from APCmin/+ mice was exclusively due to the increase in KLRG1+ GATA3+ Treg cells. Our data identify a novel axis through which epithelial cells control local Treg cell subsets, which may be activated during intestinal tumorigenesis.

Curcumin and piperine inhibit mTORC1 signaling in intestinal epithelial cells

Persistent mTORC1 activation is not only linked to metabolic disorders but also cancer and inflammation by inducing transcription and translation of pro‐inflammatory cytokines. Gastrointestinal cancers are frequently associated with chronic inflammation and excessive secretion of IL‐6 family cytokines, which promote tumorigenesis through persistent activation of PI3K/mTORC1 pathway. Curcumin is the major natural phenol in turmeric, Curcuma longa. Curcumin inhibits a number of different molecules that play a role in inflammation such as TNFα, cyclooxygenase‐2, inducible nitric oxide synthase. Curcumin like many other dietary phenolic compounds has poor bioavailability in part due to extensive metabolism in the intestinal mucosa. Piperine, one of the major components of black pepper, has been shown to increase the bioavailability of dietary phenolics including curcumin. The long‐term objective of the study is to determine whether curcumin and piperine have individual and combined effects in the prevention of gut inflammation via inhibition of mTORC1 in Caco‐2 and HT‐29 cells. Results show that curcumin inhibited mTORC1 activity (phosphorylation state of its downstream targets, ribosomal protein S6 kinase β‐1 and ribosomal protein S6) in a dose dependent manner (ribosomal protein S6 phosphorylation: −52% at 2.5 μM, −84% at 20 μM, P<0.05). Piperine inhibited mTORC1 activity in its own right (ribosomal protein S6 kinase β‐1 phosphorylation: −84% at 2.5 μM, −91% at 20 μM, P<0.05). The combination of curcumin + piperine further inhibited the mTORC1 signaling. mTORC1 activity was markedly elevated (+2600%) in undifferentiated vs. differentiated Caco‐2 cells. Since epithelium dedifferentiation precedes the release of pro‐inflammatory cytokines and the neoplastic process, we are investigating the link between mTORC1 and the pro‐inflammatory cascade in these cells, and how curcumin +/− piperine mitigate these events. We conclude that widely available dietary phenolics, curcumin and piperine, have the potential to regulate the mTOR pathway in the intestine. While curcumin and piperine inhibit mTORC1 signaling independently of each other by mechanisms yet to be characterized, piperine may also indirectly potentiate curcumin activity through enhancement of its bioavailability.Support or Funding InformationSupported by USDA‐NIFA.

Flufenamic acid protects against intestinal fluid secretion and barrier leakage in a mouse model of Vibrio cholerae infection through NF-κB inhibition and AMPK activation

Nuclear factor kappa B (NF-κB)-mediated inflammatory responses play crucial roles in the pathogenesis of diarrhea caused by the Vibrio cholerae El Tor variant (EL), which is a major bacterial strain causing recent cholera outbreaks. Flufenamic acid (FFA) has previously been demonstrated to be a potent activator of AMP-activated protein kinase (AMPK), which is a negative regulator of NF-κB signaling. This study aimed to investigate the anti-diarrheal efficacy of FFA in a mouse model of EL infection and to investigate the mechanisms by which FFA activates AMPK in intestinal epithelial cells (IEC). In a mouse closed loop model of EL infection, FFA treatment (20mg/kg) significantly abrogated EL-induced intestinal fluid secretion and barrier disruption. In addition, FFA suppressed NF-κB nuclear translocation and expression of proinflammatory mediators and promoted AMPK phosphorylation in the EL-infected mouse intestine. In T84 cells, FFA induced AMPK activation. Furthermore, FFA promoted tight junction assembly and prevented interferon gamma (IFN-γ)-induced barrier disruption in an AMPK-dependent manner. Biochemical and molecular docking analyses indicated that FFA activates AMPK via a direct stimulation of calcium/calmodulin-dependent protein kinase kinase beta (CaMKKβ) activity. Collectively, our data indicate that FFA represents a class of existing drugs that may be of potential utility in the treatment of cholera caused by EL infection via AMPK-mediated suppression of NF-κB signaling in IEC.

Visualization of Probiotic-Mediated Ca2+ Signaling in Intestinal Epithelial Cells In Vivo.

Probiotics, such as lactic acid bacteria (LAB) and Bacillus subtilis var. natto, have been shown to modulate immune responses. It is important to understand how probiotic bacteria impact intestinal epithelial cells (IECs), because IECs are the first line of defense at the mucosal surface barrier and their activities substantially affect the gut microenvironment and immunity. However, to date, their precise mechanism remains unknown due to a lack of analytical systems available for live animal models. Recently, we generated a conditional Ca2+ biosensor Yellow Cameleon (YC3.60) transgenic mouse line and established 5D (x, y, z, time, and Ca2+) intravital imaging systems of lymphoid tissues including those in Peyer’s patches and bone marrow. In the present study, we further advance our intravital imaging system for intestinal tracts to visualize IEC responses against orally administrated food compounds in real time. Using this system, heat-killed B. subtilis natto, a probiotic TTCC012 strain, is shown to directly induce Ca2+ signaling in IECs in mice housed under specific pathogen-free conditions. In contrast, this activation is not observed in the Lactococcus lactis strain C60; however, when we generate germ-free YC3.60 mice and observe the LAB stimulation of IECs in the absence of gut microbiota, C60 is capable of inducing Ca2+ signaling. This is the first study to successfully visualize the direct effect of probiotics on IECs in live animals. These data strongly suggest that probiotic strains stimulate IECs under physiological conditions and that their activity is affected by the microenvironment of the small intestine, such as commensal bacteria.

Cyanidin-3-O-glucoside inhibits NF-kB signalling in intestinal epithelial cells exposed to TNF-α and exerts protective effects via Nrf2 pathway activation

Chronic intestinal inflammatory disorders, such as Inflammatory Bowel Diseases (IBDs), are characterized by excessive release of proinflammatory mediators, intestinal barrier dysfunction and excessive activation of NF-kB cascade. Previous studies shown that TNF-α plays a central role in intestinal inflammation of IBDs and supported beneficial effects of flavonoids against chronic inflammatory diseases. In this study, we employed an in vitro model of acute intestinal inflammation using intestinal Caco-2 cells exposed to TNF-α. The protective effects of cyanidin-3-glucoside (C3G), an anthocyanin widely distributed in mediterranean diet, were then evaluated. Caco-2 cells exposure to TNF-α activated NF-kB proinflammatory pathway and induced IL6 and COX-2 expression. Cells pretreatment for 24h with C3G (20–40μM) prevented TNF-α-induced changes, and improved intracellular redox status. Our results demonstrated that C3G, also without any kind of stimulus, increased the translocation of the transcription factor Nrf2 into the nucleus so activating antioxidant and detoxifying genes.In conclusion, C3G exhibited protective effects through the inhibition of NF-kB signalling in Caco-2 cells and these beneficial effects appear to be due to its ability to activate cellular protective responses modulated by Nrf2. These data suggest that anthocyanins could contribute, as complementary or preventive approaches, to the management of chronic inflammatory diseases.

Epithelial IL-23R Signaling Licenses Protective IL-22 Responses in Intestinal Inflammation

A plethora of functional and genetic studies have suggested a key role for the IL-23 pathway in chronic intestinal inflammation. Currently, pathogenic actions of IL-23 have been ascribed to specific effects on immune cells. Herein, we unveil a protective role of IL-23R signaling. Mice deficient in IL-23R expression in intestinal epithelial cells (Il23R(ΔIEC)) have reduced Reg3b expression, show a disturbed colonic microflora with an expansion of flagellated bacteria, and succumb to DSS colitis. Surprisingly, Il23R(ΔIEC) mice show impaired mucosal IL-22 induction in response to IL-23. αThy-1 treatment significantly deteriorates colitis in Il23R(ΔIEC) animals, which can be rescued by IL-22 application. Importantly, exogenous Reg3b administration rescues DSS-treated Il23R(ΔIEC) mice by recruiting neutrophils as IL-22-producing cells, thereby restoring mucosal IL-22 levels. The study identifies a critical barrier-protective immune pathway that originates from, and is orchestrated by, IL-23R signaling in intestinal epithelial cells.