Maintenance Of Epithelial Barrier Research Articles

Exploring Microbial Metabolite Receptors in Inflammatory Bowel Disease: An In Silico Analysis of Their Potential Role in Inflammation and Fibrosis.

Metabolites produced by dysbiotic intestinal microbiota can influence disease pathophysiology by participating in ligand-receptor interactions. Our aim was to investigate the differential expression of metabolite receptor (MR) genes between inflammatory bowel disease (IBD), healthy individuals (HIs), and disease controls in order to identify possible interactions with inflammatory and fibrotic pathways in the intestine. RNA-sequencing datasets containing 643 Crohn's disease (CD) patients, 467 ulcerative colitis (UC) patients and 295 HIs, and 4 Campylobacter jejuni-infected individuals were retrieved from the Sequence Read Archive, and differential expression was performed using the RaNA-seq online platform. The identified differentially expressed MR genes were used for correlation analysis with up- and downregulated genes in IBD, as well as functional enrichment analysis using a R based pipeline. Overall, 15 MR genes exhibited dysregulated expression in IBD. In inflamed CD, the hydroxycarboxylic acid receptors 2 and 3 (HCAR2, HCAR3) were upregulated and were associated with the recruitment of innate immune cells, while, in the non-inflamed CD ileum, the cannabinoid receptor 1 (CNR1) and the sphingosine-1-phospate receptor 4 (S1PR4) were downregulated and were involved in the regulation of B-cell activation. In inflamed UC, the upregulated receptors HCAR2 and HCAR3 were more closely associated with the process of TH-17 cell differentiation, while the pregnane X receptor (NR1I2) and the transient receptor potential vanilloid 1 (TRPV1) were downregulated and were involved in epithelial barrier maintenance. Our results elucidate the landscape of metabolite receptor expression in IBD, highlighting associations with disease-related functions that could guide the development of new targeted therapies.

Nicotinic acetylcholine receptor signaling maintains epithelial barrier integrity.

Disruption of epithelial barriers is a common disease manifestation in chronic degenerative diseases of the airways, lung, and intestine. Extensive human genetic studies have identified risk loci in such diseases, including in chronic obstructive pulmonary disease (COPD) and inflammatory bowel diseases. The genes associated with these loci have not fully been determined, and functional characterization of such genes requires extensive studies in model organisms. Here, we report the results of a screen in Drosophila melanogaster that allowed for rapid identification, validation, and prioritization of COPD risk genes that were selected based on risk loci identified in human genome-wide association studies (GWAS). Using intestinal barrier dysfunction in flies as a readout, our results validate the impact of candidate gene perturbations on epithelial barrier function in 56% of the cases, resulting in a prioritized target gene list. We further report the functional characterization in flies of one family of these genes, encoding for nicotinic acetylcholine receptor (nAchR) subunits. We find that nAchR signaling in enterocytes of the fly gut promotes epithelial barrier function and epithelial homeostasis by regulating the production of the peritrophic matrix. Our findings identify COPD-associated genes critical for epithelial barrier maintenance, and provide insight into the role of epithelial nAchR signaling for homeostasis.

Caveolin-1: A Review of Intracellular Functions, Tissue-Specific Roles, and Epithelial Tight Junction Regulation.

Caveolin-1 (Cav1) is a vital protein for many cellular processes and is involved in both the positive and negative regulation of these processes. Cav1 exists in multiple cellular compartments depending on its role. Of particular interest is its contribution to the formation of plasma membrane invaginations called caveolae and its involvement in cytoskeletal interactions, endocytosis, and cholesterol trafficking. Cav1 participates in stem cell differentiation as well as proliferation and cell death pathways, which is implicated in tumor growth and metastasis. Additionally, Cav1 has tissue-specific functions that are adapted to the requirements of the cells within those tissues. Its role has been described in adipose, lung, pancreatic, and vascular tissue and in epithelial barrier maintenance. In both the intestinal and the blood brain barriers, Cav1 has significant interactions with junctional complexes that manage barrier integrity. Tight junctions have a close relationship with Cav1 and this relationship affects both their level of expression and their location within the cell. The ubiquitous nature of Cav1 both within the cell and within specific tissues is what makes the protein important for ongoing research as it can assist in further understanding pathophysiologic processes and can potentially be a target for therapies.

Potential Biomarkers for Noninfectious Scleritis Identified by Serum and Tear Fluid Proteomics

Scleritis is an extremely painful and potentially blinding inflammation of the sclera with unknown pathogenesis and unpredictable course. To gain insight in its disease process and identify biomarker candidates, we performed extensive proteomics in serum and tear fluid. Prospective multicenter cohort study. A total of 121 patients with noninfectious scleritis (of which 39 active cases), 30 healthy controls, and 23 disease controls (uveitis and rheumatoid arthritis) were enrolled in the Netherlands from 2020 to2022. Serum, tear fluid of both eyes, and clinical data were gathered. The level of 368 inflammatory proteins was measured using proximity extension assays. Results were validated in an independent cohort of 15 patients with scleritis, and using addressable laser bead immunoassay, or enzyme-linked immunoassays. In addition, we studied an extended panel of matrix metalloproteinases in tear fluid of necrotizing scleritis with addressable laser bead immunoassay. Statistically significant differences in the level of inflammatory proteins between patients with scleritis and control groups. Proteomics revealed 18 significantly upregulated or downregulated serum proteins in active scleritis cases compared with all control groups in both the discovery cohort and the validation cohort. The most upregulated protein was nuclear migration protein nudC (NudC; P= 0.0032), a protein involved in neurogenesis. The other significant hits included proteins involved in T-cell activation, apoptosis, epithelial barrier maintenance, and angiogenesis. Our tear fluid analysis showed matrix metalloproteinase 9 (MMP9) to be upregulated in the tear fluid of patients with scleral necrosis. The results of our proteomics analysis suggest a role for neurogenesis, T-cell activation, disruption of epithelial barrier, and angiogenesis in the pathogenesis of scleritis, and highlight MMP9 and NudC as biomarkers with potential clinical relevance. The authors have no proprietary or commercial interest in any materials discussed in this article.

Mannose-Decorated Co-Polymer Facilitates Controlled Release of Butyrate to Accelerate Chronic Wound Healing.

Butyrate is a key bacterial metabolite that plays an important and complex role in modulation of immunity and maintenance of epithelial barriers. Its translation to clinic is limited by poor bioavailability, pungent smell, and the need for high doses, and effective delivery strategies have yet to realize clinical potential. Here, a novel polymeric delivery platform for tunable and sustainable release of butyrate consisting of a methacrylamide backbone with butyryl ester or phenyl ester side chains as well as mannosyl side chains, which is also applicable to other therapeutically relevant metabolites is reported. This platform's utility in the treatment of non-healing diabetic wounds is explored. This butyrate-containing material modulated immune cell activation in vitro and induced striking changes in the milieu of soluble cytokine and chemokine signals present within the diabetic wound microenvironment in vivo. This novel therapy shows efficacy in the treatment of non-healing wounds through the modulation of the soluble signals present within the wound, and importantly accommodates the critical temporal regulation associated with the wound healing process. Currently, the few therapies to address non-healing wounds demonstrate limited efficacy. This novel platform is positioned to address this large unmet clinical need and improve the closure of otherwise non-healing wounds.

YES-associated protein-regulated Smad7 worsen epithelial barrier injury of chronic sinusitis with nasal polyps.

Chronic rhinosinusitis with nasal polyps (CRSwNP) was potentially due to the epithelial barrier injury. YES-associated protein (YAP) is a multifunctional transcriptional factor and plays versatile roles in the regulation and maintenance of epithelial barrier in different organs and tissues. The purpose of this study is to elucidate possible effect and mechanism of YAP on the epithelial barrier of CRSwNP. Patients were divided into CRSwNP group (n = 12) and control group (n = 9). The location of YAP, PDZ-binding transcriptional co-activator (TAZ), and Smad7 were estimated by immunohistochemistry and immunofluorescence. Meanwhile, the expression of YAP, TAZ, Zona occludens-1 (ZO-1), E-cadherin, and transforming growth factor-beta1 (TGF-β1) were detected by Western blot. After primary human nasal epithelial cells were treated with YAP inhibitor, the expression level of YAP, TAZ, ZO-1, E-cadherin, TGF-β1, and Smad7 were measured by Western blot. Compared with the control group, the protein levels of YAP, TAZ, and Smad7 were significantly upregulated, while TGF-β1, ZO-1, and E-cadherin were downregulated in CRSwNP. YAP and Smad7 demonstrated lower levels, while the expression of ZO-1, E-cadherin, and TGF-β1 rose slightly after YAP inhibitor treatment in primary nasal epithelial cells. Higher level of YAP may lead to CRSwNP epithelial barrier injury via the TGF-β1 signaling pathway, and the inhibition of YAP can partially reverse epithelial barrier function.

Investigation of murine host sex as a biological variable in epithelial barrier function and muscle contractility in human intestinal organoids.

Intestinal failure (IF) occurs when intestinal surface area or function is not sufficient to support digestion and nutrient absorption. Human intestinal organoid (HIO)-derived tissue-engineered intestine is a potential cure for IF. Research to date has demonstrated successful HIO transplantation (tHIO) into mice with significant in vivo maturation. An area lacking in the literature is exploration of murine host sex as a biological variable (SABV) in tHIO function. In this study, we investigate murine host SABV in tHIO epithelial barrier function and muscle contractility. HIOs were generated in vitro and transplanted into nonobese diabetic, severe combined immunodeficiency gamma chain deficient male and female mice. tHIOs were harvested after 8-12 weeks in vivo. Reverse transcriptase polymerase chain reaction and immunohistochemistry were conducted to compare tight junctions and contractility-related markers in tHIOs. An Ussing chamber and contractility apparatus were used to evaluate tHIO epithelial barrier and muscle contractile function, respectively. The expression and morphology of tight junction and contractility-related markers from tHIOs in male and female murine hosts is not significantly different. Epithelial barrier function as measured by transepithelial resistance, short circuit current, and fluorescein isothiocyanate-dextran permeability is no different in tHIOs from male and female hosts, although these results may be limited by HIO epithelial immaturity and a short flux time. Muscle contractility as measured by total contractile activity, amplitude, frequency, and tension is not significantly different in tHIOs from male and female hosts. The data suggest that murine host sex may not be a significant biological variable influencing tHIO function, specifically epithelial barrier maintenance and muscle contractility, though limitations exist in our model.

Crosstalk between epithelium, myeloid and innate lymphoid cells during gut homeostasis and disease.

The gut epithelium not only provides a physical barrier to separate a noxious outside from a sterile inside but also allows for highly regulated interactions between bacteria and their products, and components of the immune system. Homeostatic maintenance of an intact epithelial barrier is paramount to health, requiring an intricately regulated and highly adaptive response of various cells of the immune system. Prolonged homeostatic imbalance can result in chronic inflammation, tumorigenesis and inefficient antitumor immune control. Here we provide an update on the role of innate lymphoid cells, macrophages and dendritic cells, which collectively play a critical role in epithelial barrier maintenance and provide an important linkage between the classical innate and adaptive arm of the immune system. These interactions modify the capacity of the gut epithelium to undergo continuous renewal, safeguard against tumor formation and provide feedback to the gut microbiome, which acts as a seminal contributor to cellular homeostasis of the gut.

Effects of TAMP family on the tight junction strand network and barrier function in epithelial cells.

Occludin, tricellulin, and marvelD3 belong to the tight junction (TJ)-associated MARVEL protein family. Occludin and tricellulin jointly contribute to TJ strand branching point formation and epithelial barrier maintenance. However, whether marvelD3 has the same function remains unclear. Furthermore, the roles of the carboxy-terminal cytoplasmic tail, which is conserved in occludin and tricellulin, on the regulation of TJ strand morphology have not yet been explored in epithelial cells. We established tricellulin/occludin/marveld3 triple-gene knockout (tKO) MDCK II cells and evaluated the roles of marvelD3 in the TJ strand structure and barrier function using MDCK II cells and a mathematical model. The complexity of TJ strand networks and paracellular barrier did not change in tKO cells compared to that in tricellulin/occludin double-gene knockout (dKO) cells. Exogenous marvelD3 expression in dKO cells did not increase the complexity of TJ strand networks and epithelial barrier tightness. The expression of the carboxy-terminal truncation mutant of tricellulin restored the barrier function in the dKO cells, whereas occludin lacking the carboxy-terminal cytoplasmic tail was not expressed on the plasma membrane. These data suggest that marvelD3 does not affect the morphology of TJ strands and barrier function in MDCK II cells and that the carboxy-terminal cytoplasmic tail of tricellulin is dispensable for barrier improvement.

Genome-wide siRNA screening reveals several host receptors for the binding of human gut commensal Bifidobacterium bifidum

Bifidobacterium spp. are abundant gut commensals, especially in breast-fed infants. Bifidobacteria are associated with many health-promoting effects including maintenance of epithelial barrier and integrity as well as immunomodulation. However, the protective mechanisms of bifidobacteria on intestinal epithelium at molecular level are poorly understood. In this study, we developed a high-throughput in vitro screening assay to explore binding receptors of intestinal epithelial cells for Bifidobacterium bifidum. Short interfering RNAs (siRNA) were used to silence expression of each gene in the Caco-2 cell line one by one. The screen yielded four cell surface proteins, SERPINB3, LGICZ1, PKD1 and PAQR6, which were identified as potential receptors as the siRNA knock-down of their expression decreased adhesion of B. bifidum to the cell line repeatedly during the three rounds of siRNA screening. Furthermore, blocking of these host cell proteins by specific antibodies decreased the binding of B. bifidum significantly to Caco-2 and HT29 cell lines. All these molecules are located on the surface of epithelial cells and three out of four, SERPINB3, PKD1 and PAQR6, are involved in the regulation of cellular processes related to proliferation, differentiation and apoptosis as well as inflammation and immunity. Our results provide leads to the first steps in the mechanistic cascade of B. bifidum-host interactions leading to regulatory effects in the epithelium and may partly explain how this commensal bacterium is able to promote intestinal homeostasis.

PI3Kγ is a novel regulator of TNFα signaling in the human colon cell line HT29/B6

Phosphoinositide 3-kinases (PI3Ks) are a family of enzymes phosphorylating phospholipids in the membrane, thereby, promoting the PI3K/AKT signaling cascade. PI3Ks are involved in a variety of fundamental cellular functions, including tumor necrosis factor α (TNFα)-induced tight junction (TJ) impairment-a hallmark of inflammatory bowel diseases. Most of the studies analyzing the role of class I PI3K signaling in epithelial barrier maintenance did not decipher which of the isoforms are responsible for the observed effects. By using wild-type and PI3Kγ-deficient HT-29/B6 cells, we characterized the functional role of PI3Kγ in these cells under inflammatory conditions. Measurement of the transepithelial electrical resistance and the paracellular flux of macromolecules revealed that monolayers of PI3Kγ-deficient cells, compared with wild-type cells, were protected against TNFα-induced barrier dysfunction. This effect was independent of any PI3K activity because treatment with a pan-PI3K inhibitor did not alter this observation. By immunostaining, we found correlative changes in the distribution of the TJ marker ZO-1. Furthermore, the absence of PI3Kγ reduced the basal level of the pore-forming TJ protein claudin-2. Our study suggests a novel noncanonical, kinase-independent scaffolding function of PI3Kγ in TNFα-induced barrier dysfunction.

You shall not pass! Protective role of autophagic machinery in response to plasma membrane damage triggered by Candida albicans invasion

ABSTRACT Candida albicans (C. albicans) is an opportunistic pathogen causing infections ranging from superficial to life-threatening dissemination, in which C. albicans is able to translocate through the gut barrier into deeper organs. In its filamentous form (hyphae), C. albicans can invade epithelial cells by two mechanisms: epithelial cell-driven endocytosis and C. albicans-driven active penetration of host cell plasma membrane (PM). Autophagic machinery is known to be involved in the epithelial barrier maintenance, especially the intestinal barrier that is continuously challenged by exposure to the gut microbiota or to xenobiotics. The protective role of autophagy during C. albicans infection has been investigated in myeloid cells, however, far less was known regarding its role during infection of epithelial cells. Here, we demonstrated that key proteins of the autophagic machinery and vesicles presenting features of autophagosomes are recruited at C. albicans invasion sites. These events are associated with host PM damage caused by the active penetration of C. albicans. We showed that ATG5 and ATG16L1 proteins contribute to PM repair mediated by lysosomal membrane exocytosis and participate in protection of epithelial cells’ integrity against C. albicans-induced cell death. Our findings extend the knowledge on emerging roles of the autophagic machinery in stress-related membrane dynamics.

Presence of innate lymphoid cells in allogeneic hematopoietic grafts correlates with reduced graft-versus-host disease

BackgroundAllogeneic hematopoietic cell transplantation (HCT) can be devastating when graft-versus-host disease (GvHD) develops. GvHD is characterized by mucosal inflammation due to breaching of epithelial barriers. Innate lymphoid cells (ILCs) are immune modulatory cells that are important in the maintenance of epithelial barriers, via their production of interleukin (IL)-22 and their T cell suppressive properties. After chemo- and radiotherapy, ILCs are depleted, and recovery after remission-induction therapy and after allogeneic HCT is slow and incomplete in a significant number of patients, which is associated with an increased risk to develop acute GvHD. ObjectiveTo investigate whether the presence of mature ILCs within G-CSF–mobilized HCT grafts is correlated with the development of acute GvHD after allogeneic HCT. Study DesignWe analyzed ILCs in a cohort of 36 patients who received allogeneic HCT for a hematologic malignancy, by flow-cytometric immune-phenotyping of prospectively collected, cryopreserved peripheral blood mononuclear cells (PBMCs) and donor-derived HCT grafts collected for the same patients. Biased analysis, with ILCs defined as CD3−lineage−CD45+CD127+CD161+ lymphocytes, was performed using FlowJo version 10 software. Unbiased analysis was done using FlowSOM, which uses a self-organizing map (SOM) with a minimal spanning tree (MST) to define and visualize different clusters present in the samples. ResultsRemission-induction therapy significantly depleted ILCs from the blood, and patients who had a relatively low percentage of ILCs before allogeneic HCT were significantly more prone to develop acute GvHD, confirming previous findings in a separate cohort. Allogeneic HCT grafts, which were all obtained from the blood of G-CSF–mobilized healthy donors, contained ILCs at a frequency very similar to the peripheral blood of healthy individuals. The ILC subset composition was also comparable to that of the blood of healthy individuals, with the exception of NKp44+ ILC3s, which were significantly more abundant in HCT grafts. The relative ILC content of the graft tended to correlate with ILC reconstitution after allogeneic HCT, suggesting that peripheral expansion of transplanted mature ILCs may contribute to early ILC reconstitution after allogeneic HCT. Patients who received a relatively ILC-poor HCT graft had a significantly increased risk to develop acute GvHD, compared with patients who received relatively ILC-rich allogeneic HCT grafts. Unbiased phenotypic analysis with the FlowSOM algorithm confirmed that allogeneic HCT grafts of patients who developed acute GvHD contained a lower frequency of ILCs that clustered in NKp44+ ILC3 signature groups. ConclusionThe presence of ILCs in allogeneic HCT grafts is associated with a reduced risk to develop acute GvHD. These data suggest that enhancement of ILC reconstitution of ILC3s in particular, for example via adoptive transfer of ILCs, may prevent acute GvHD and has the potential to improve outcome of allogeneic HCT recipients.

Zinc Inhibits Cadherin 1 Expression Induced by 1α,25-Dihydroxyvitamin D3 in Colon Cancer Cells.

Zinc is a mineral that is essential for biological molecules, such as transcription factors, and is involved in the maintenance of intestinal homeostasis. Vitamin D signaling is mediated by vitamin D receptor (VDR) activated by 1α,25-dihydroxyvitamin D3 [1,25(OH)2D3] and is also important in intestinal functions, such as calcium absorption and epithelial barrier maintenance. However, the crosstalk between vitamin D signaling and zinc signaling in intestinal cells remains poorly understood. Colon cancer SW480 and HCT116 cells were treated with zinc chloride (ZnCl2) with/without 1,25(OH)2D3 Expression of zinc-inducible genes [metallothionein 1A (MT1A) and MT2A] and VDR target genes [cytochrome P450 family 24 subfamily A member 1 (CYP24A1), transient receptor potential cation channel subfamily V member 6 (TRPV6) and cadherin 1 (CDH1)] was examined. Treatment of cells with ZnCl2 effectively induced MT1A and MT2A mRNA expression, and interestingly suppressed mRNA expression of CDH1, which was induced by 1,25(OH)2D3 in both cell lines. ZnCl2 also reduced the CDH1 protein level in HCT116 cells. Zinc signaling suppresses VDR-induced expression of CDH1.

Intestinal microbiota-derived tryptophan metabolites are predictive of Ah receptor activity

ABSTRACT Commensal microbiota-dependent tryptophan catabolism within the gastrointestinal tract is known to exert profound effects upon host physiology, including the maintenance of epithelial barrier and immune function. A number of abundant microbiota-derived tryptophan metabolites exhibit activation potential for the aryl hydrocarbon receptor (AHR). Gene expression facilitated by AHR activation through the presence of dietary or microbiota-generated metabolites can influence gastrointestinal homeostasis and confer protection from intestinal challenges. Utilizing untargeted mass spectrometry-based metabolomics profiling, combined with AHR activity screening assays, we identify four previously unrecognized tryptophan metabolites, present in mouse cecal contents and human stool, with the capacity to activate AHR. Using GC/MS and LC/MS platforms, quantification of these novel AHR activators, along with previously established AHR-activating tryptophan metabolites, was achieved, providing a relative order of abundance. Using physiologically relevant concentrations and quantitative gene expression analyses, the relative efficacy of these tryptophan metabolites with regard to mouse or human AHR activation potential is examined. These data reveal indole, 2-oxindole, indole-3-acetic acid and kynurenic acid as the dominant AHR activators in mouse cecal contents and human stool from participants on a controlled diet. Here we provide the first documentation of the relative abundance and AHR activation potential of a panel of microbiota-derived tryptophan metabolites. Furthermore, these data reveal the human AHR to be more sensitive, at physiologically relevant concentrations, to tryptophan metabolite activation than mouse AHR. Additionally, correlation analyses indicate a relationship linking major tryptophan metabolite abundance with AHR activity, suggesting these cecal/fecal metabolites represent biomarkers of intestinal AHR activity.

Cold-inducible RNA-binding protein maintains intestinal barrier during deep hypothermic circulatory arrest.

The intestinal injury during deep hypothermic circulatory arrest (DHCA) is harmful to clinical outcomes. Cold-inducible RNA-binding protein (CIRBP) plays a protective role in hypothermia. The aim of this study was to explore the effects of CIRBP on intestinal barrier during DHCA. Sprague-Dawley (wild type, n = 13) and knockout of Cirbp (Cirbp-/-, n = 8) rats were used in the model of DHCA. The histomorphology of the epithelial barrier was evaluated by haematoxylin-eosin, Chiu's scores, Gram's stain and Ki67. The function of the intestinal barrier was evaluated by serum intestinal fatty acid-binding protein, diamine oxidase and d-lactate. The structure of the epithelial barrier, phosphocreatine-creatine kinase system and adenosine triphosphate were assessed in the intestine. The expression of CIRBP significantly increased in the intestine during DHCA. Cirbp-/- rats showed obvious destruction of intestinal barrier after DHCA. Chiu's scores, intestinal fatty acid-binding protein, diamine oxidase and d-lactate significantly increased in the Cirbp-/- group. Ki67 showed that cell proliferation decreased in the Cirbp-/- rats. In the Cirbp-/- group, zonula occludens-1, E-cadherin and occludin levels were significantly decreased, and these proteins either disappeared or redistributed in the monolayer. Besides, Cirbp-/- resulted in decreased levels of creatine kinase B, glycine amidinotransferase, adenosine triphosphate and creatine contents in the intestine, affecting energy metabolism and balance, which is associated with the maintenance of epithelial barrier during acute injury. CIRBP is related to the maintenance of the intestinal epithelial barrier during DHCA, which is expected to be a new target for the prevention of intestinal injury.

Model Systems to Investigate NOX-Dependent Cell Migration and Invasiveness.

There is mounting evidence indicating that reactive oxygen species (ROS) play a crucial role in cell migration and invasion. Our previous studies have demonstrated the NADPH oxidase (NOX) family of enzymes are a source of ROS in different cell types undergoing migration. Several NOX enzymes are induced or activated in processes including wound repair and maintenance of epithelial barriers, as well as in promoting metastatic cell migration and invasiveness. This chapter outlines three different in vitro assays used to examine how NOX enzymes are involved in cell motility: scratch-wound repair, Matrigel invasion, and migration from confluent cell monolayer boundaries created by cell culture inserts. The three methods provide a range of experimental approaches for delineating roles of NOX enzymes in cell migration through manipulation of the expression or activities of the endogenous or overexpressed oxidases.

Functional roles of Grainyhead-like transcription factors in renal development and disease.

Proper renal function relies on the tightly regulated development of nephrons and collecting ducts. This process, known as tubulogenesis, involves dynamic cellular and molecular changes that instruct cells to form highly organized tubes of epithelial cells which compartmentalize the renal interstitium and tubular lumen via assembly of a selective barrier. The integrity and diversity of the various renal epithelia is achieved via formation of intercellular protein complexes along the apical-basal axis of the epithelial cells. In recent years, the evolutionarily conserved family of Grainyhead-like (GRHL) transcription factors which encompasses three mammalian family members (Grainyhead-like 1, 2, 3) has emerged as a group of critical regulators for organ development, epithelial differentiation, and barrier formation. Evidence from transgenic animal models supports the presence of Grainyhead-like-dependent transcriptional mechanisms that promote formation and maintenance of epithelial barriers in the kidney. In this review, we highlight different Grhl-dependent mechanisms that modulate epithelial differentiation in the kidney. Additionally, we discuss how disruptions in these mechanisms result in impaired renal function later in life.

The cerebral cavernous malformation disease causing gene KRIT1 participates in intestinal epithelial barrier maintenance and regulation.

Epithelial barrier maintenance and regulation requires an intact perijunctional actomyosin ring underneath the cell-cell junctions. By searching for known factors affecting the actin cytoskeleton, we identified Krev interaction trapped protein 1 (KRIT1) as a major regulator for epithelial barrier function through multiple mechanisms. KRIT1 is expressed in both small intestinal and colonic epithelium, and KRIT1 knockdown in differentiated Caco-2 intestinal epithelium decreases epithelial barrier function and increases cation selectivity. KRIT1 knockdown abolished Rho-associated protein kinase-induced and myosin II motor inhibitor-induced barrier loss by limiting both small and large molecule permeability but did not affect myosin light chain kinase-induced increases in epithelial barrier function. These data suggest that KRIT1 participates in Rho-associated protein kinase- and myosin II motor-dependent (but not myosin light chain kinase-dependent) epithelial barrier regulation. KRIT1 knockdown exacerbated low-dose TNF-induced barrier loss, along with increased cleaved caspase-3 production. Both events are blocked by pan-caspase inhibition, indicating that KRIT1 regulates TNF-induced barrier loss through limiting epithelial apoptosis. These data indicate that KRIT1 controls epithelial barrier maintenance and regulation through multiple pathways, suggesting that KRIT1 mutation in cerebral cavernous malformation disease may alter epithelial function and affect human health.-Wang, Y., Li, Y., Zou, J., Polster, S. P., Lightle, R., Moore, T., Dimaano, M., He, T.-C., Weber, C. R., Awad, I. A., Shen, L. The cerebral cavernous malformation disease causing gene KRIT1 participates in intestinal epithelial barrier maintenance and regulation.

Anti-TNF Therapy Induces CD4+ T-Cell Production of IL-22 and Promotes Epithelial Repairs in Patients With Crohn’s Disease

Anti-tumor necrosis factor (TNF) therapy appears to be effective in the treatment of Crohn's disease (CD), a chronic inflammatory disease of the gastrointestinal tract. However, the mechanisms involved are not completely understood. Fifty-seven active CD patients were enrolled, and cytokine profiles in colonic biopsies of patients with active CD receiving anti-TNF monoclonal antibody (mAb) (infliximab [IFX]) treatment were determined using quantitative real-time polymerase chain reaction (qRT-PCR). Colonic biopsies of active CD patients and healthy donors were cultured with IFX in vitro, and cytokine profiles were measured by qRT-PCR. Peripheral blood (PB)-CD4+ T cells were stimulated with anti-CD3 and anti-CD28 mAbs in the presence of human immunoglobin (HIg), IFX, recombinant human TNF-α converting enzyme (rhTACE), and aryl hydrocarbon receptor (AhR) inhibitor (CH-223191), respectively, to determine interleukin (IL)-22 expression by CD4+ T cells. Caco2 cells were also utilized to study their potential role in modulating epithelial cell barrier repairs in vitro. IFX therapy markedly upregulated IL-22 mRNA expression in the gut mucosa of CD patients. In vitro treatment with IFX greatly promoted CD CD4+ T cells to express IL-22, which was inhibited by rhTACE, indicating that reverse signaling through binding to membrane-bound TNF mediates anti-TNF-induced IL-22 expression of CD CD4+ T cells. However, blockade of AhR markedly inhibited anti-TNF-induced IL-22+CD4+ T (Th22) cell differentiation in CD patients. Moreover, treatment with IL-22 induced intestinal epithelial cell expression of tight junction proteins (eg, claudin1 and ZO-1) and facilitated transepithelial resistance, indicating that IL-22 protects intestinal mucosa from inflammation via maintenance of epithelial barrier integrity. Our results uncover a novel mechanism whereby anti-TNF therapy upregulates IL-22 production in CD patients through promoting Th22 cell differentiation and contributes to intestinal epithelial barrier repairs.