Epithelial Function Research Articles

The artificial sweetener neotame negatively regulates the intestinal epithelium directly through T1R3-signaling and indirectly through pathogenic changes to model gut bacteria.

Recent studies have indicated considerable health risks associated with the consumption of artificial sweeteners. Neotame is a relatively new sweetener in the global market however there is still limited data on the impact of neotame on the intestinal epithelium or the commensal microbiota. In the present study, we use a model of the intestinal epithelium (Caco-2) and microbiota (Escherichia coli and Enterococcus faecalis) to investigate how physiologically-relevant exposure of neotame impacts intestinal epithelial cell function, gut bacterial metabolism and pathogenicity, and gut epithelium-microbiota interactions. Our findings show that neotame causes intestinal epithelial cell apoptosis and death with siRNA knockdown of T1R3 expression significantly attenuating the neotame-induced loss to cell viability. Similarly, neotame exposure results in barrier disruption with enhanced monolayer leak and reduced claudin-3 cell surface expression through a T1R3-dependent pathway. Using the gut bacteria models, E. coli and E. faecalis, neotame significantly increased biofilm formation and metabolites of E. coli, but not E. faecalis, reduced Caco-2 cell viability. In co-culture studies, neotame exposure increased adhesion capacity of E. coli and E. faecalis onto Caco-2 cells and invasion capacity of E. coli. Neotame-induced biofilm formation, E.coli-specific Caco-2 cell death, adhesion and invasion was identified to be meditated through a taste-dependent pathway. Our study identifies novel pathogenic effects of neotame on the intestinal epithelium or bacteria alone, and in co-cultures to mimic the gut microbiome. These findings demonstrate the need to better understand food additives common in the global market and the molecular mechanisms underlying potential negative health impacts.

MTOR signaling pathway regulation HIF-1 α effects on LPS induced intestinal mucosal epithelial model damage

BackgroundSepsis-induced small-intestinal injury is associated with increased morbidity and mortality. Our previous study and other papers have shown that HIF-1α has a protective effect on intestinal mucosal injury in septic rats. The purpose of this study is to further verify the protective effect of HIF-1α on intestinal mucosa and its molecular mechanism in vitro experiments.MethodsCaco-2 cells were selected and experiment was divided into 2 parts. Part I: HIF-1α activator and inhibitor were used to treat lipopolysacchrides (LPS)-stimulated Caco-2 cells respectively, to explore the effect of HIF-1α on LPS induced Caco-2 cell epithelial model; Part II: mTOR activator or inhibitor combined with or without HIF-1α activator, inhibitor to treat LPS-stimulated Caco-2 cells respectively, and then the molecular mechanism of HIF-1α reducing LPS induced Caco-2 cell epithelial model damage was detected.ResultsThe results showed that HIF-1α activator decreased the permeability and up regulated tight junction (TJ) expression, while HIF-1α inhibitor had the opposite effect with the HIF-1α activator. mTOR activation increased, while mTOR inhibition decreased HIF-1α protein and expression of its downstream target molecules, which can be attenuated by HIF-1α activator or inhibitor.ConclusionThis study once again confirmed that HIF-1α alleviates LPS-induced mucosal epithelial model damage through P70S6K signalling pathway. It is of great value to explore whether HIF-2α plays crucial roles in the regulation of mucosal epithelial model functions in the future.

CAPN5 attenuates cigarette smoke extract-induced apoptosis and inflammation in BEAS-2B cells.

Apoptosis and chronic inflammation are the main phenotypes in chronic obstructive pulmonary disease (COPD) pathogenesis. Cigarette smoke exposure is the leading risk factor for COPD, which causes aberrant airway epithelial structure and function. As a non-classical calpain, the molecular function of calpain5 (CAPN5) in COPD remains unclear. This study investigated the role of CAPN5 in mediating cigarette smoke extract (CSE)-induced apoptosis and inflammation. Immunohistochemistry (IHC) and Western blotting (WB) were performed to detect the location and expression of CAPN5. In vitro, BEAS-2B cells were transfected with CAPN5 siRNA or CAPN5 plasmid, followed by phosphate-buffered saline (PBS) or cigarette smoke extract (CSE) treatment. The protein expression levels of CAPN5, NF-κB p65, p-p65, IκBα, p-IκBα and apoptosis proteins (BCL-2, BAX) were measured by WB. Flow cytometry (FCM) was performed to analyze the cell apoptosis index. CAPN5 was mainly expressed in the airway epithelium and significantly decreased in the COPD-smoker and emphysema-mouse groups. Silencing CAPN5 significantly decreased the protein expression of BCL-2, IκBα, and increased p-p65 and BAX protein expression. Additionally, an increased apoptosis index was detected after silencing CAPN5. Moreover, overexpression of CAPN5 partly inhibited IκBα degradation and p65 activation, and reduced CSE-induced inflammation and apoptosis. These combined results indicate that CAPN5 could protect against CSE-induced apoptosis and inflammation, which may provide a potential therapeutic target for smoking-related COPD.

Revisiting airway epithelial dysfunction and mechanisms in chronic obstructive pulmonary disease: Role of mitochondrial damage.

Chronic exposure to environmental hazards causes airway epithelial dysfunction, primarily impaired physical barriers, immune dysfunction, and repair or regeneration. Impairment of airway epithelial function subsequently leads to exaggerated airway inflammation and remodeling, the main features of chronic obstructive pulmonary disease (COPD). Mitochondrial damage has been identified as one of the mechanisms of airway abnormalities in COPD, which is closely related to airway inflammation and airflow limitation. In this review, we evaluate updated evidence for airway epithelial mitochondrial damage in COPD and focus on the role of mitochondrial damage in airway epithelial dysfunction. In addition, the possible mechanism of airway epithelial dysfunction mediated by mitochondrial damage is discussed in detail, and recent strategies related to airway epithelial-targeted mitochondrial therapy are summarized. Results have shown that dysregulation of mitochondrial quality and oxidative stress may lead to airway epithelial dysfunction in COPD. This may result from mitochondrial damage as a central organelle mediating abnormalities in cellular metabolism. Mitochondrial damage mediates pro-cellular senescence effects due to mitochondrial reactive oxygen species, which effectively exacerbate different types of programmed cell death, participate in lipid metabolism abnormalities, and ultimately promote airway epithelial dysfunction and trigger COPD airway abnormalities. These can be prevented by targeting mitochondrial damage factors and mitochondrial transfer. Thus, because mitochondrial damage is involved in COPD progression as a central factor of homeostatic imbalance in airway epithelial cells, it may be a novel target for therapeutic intervention to restore airway epithelial integrity and function in COPD.

Differentiation of intestinal stem cells toward goblet cells under systemic iron overload stress are associated with inhibition of Notch signaling pathway and ferroptosis

Iron overload can lead to oxidative stress and intestinal damage and happens frequently during blood transfusions and iron supplementation. However, how iron overload influences intestinal mucosa remains unknown. Here, the aim of current study was to investigate the effects of iron overload on the proliferation and differentiation of intestinal stem cells (ISCs). An iron overload mouse model was established by intraperitoneal injection of 120 mg/kg body weight iron dextran once a fortnight for a duration of 12 weeks, and an iron overload enteroid model was produced by treatment with 3 mM or 10 mM of ferric ammonium citrate for 24 h. We found that iron overload caused damage to intestinal morphology with a 64 % reduction in villus height/crypt depth ratio, and microvilli injury in the duodenum. Iron overload mediated epithelial function by inhibiting the expression of nutrient transporters and enhancing the expression of secretory factors in the duodenum. Meanwhile, iron overload inhibited the proliferation of ISCs and regulated their differentiation into secretory mature cells, such as goblet cells, through inhibiting Notch signaling pathway both in mice and enteroid. Furthermore, iron overload caused oxidative stress and ferroptosis in intestinal epithelial cells. In addition, ferroptosis could also inhibit Notch signaling pathway, and affected the proliferation and differentiation of ISCs. These findings reveal the regulatory role of iron overload on the proliferation and differentiation of ISCs, providing a new insight into the internal mechanism of iron overload affecting intestinal health, and offering important theoretical basis for the scientific application of iron nutrition regulation.

Translational Aspects in Metaplastic Breast Carcinoma.

Breast cancer is the most common cancer among women. Metaplastic breast carcinoma (MpBC) is a rare, heterogeneous group of invasive breast carcinomas, which are classified as predominantly triple-negative breast carcinomas (TNBCs; HR-negative/HER2-negative). Histologically, MpBC is classified into six subtypes. Two of these are considered low-grade and the others are high-grade. MpBCs seem to be more aggressive, less responsive to neoadjuvant chemotherapy, and have higher rates of chemoresistance than other TNBCs. MpBCs have a lower survival rate than expected for TNBCs. MpBC treatment represents a challenge, leading to a thorough exploration of the tumor immune microenvironment, which has recently opened the possibility of new therapeutic strategies. The epithelial-mesenchymal transition in MpBC is characterized by the loss of intercellular adhesion, downregulation of epithelial markers, underexpression of genes with biological epithelial functions, upregulation of mesenchymal markers, overexpression of genes with biological mesenchymal functions, acquisition of fibroblast-like (spindle) morphology, cytoskeleton reorganization, increased motility, invasiveness, and metastatic capabilities. This article reviews and summarizes the current knowledge and translational aspects of MpBC.

Abstract PL03-02: From microniches to single cells: Understanding the impact of intratumoral microbes on spatial and cellular heterogeneity in cancer

Abstract The tumor microenvironment (TME) is a dynamic network that includes a variety of non-malignant cells such as endothelial cells, fibroblasts, and immune cells, which play a crucial role in tumor behavior. Advances in molecular analysis have recently identified the presence of intratumoral microbiota in several solid cancer types. However, the specific ways these microorganisms interact with human cells within the TME and contribute to disease progression are not yet fully understood. Our recent research provides new insights into how bacterial components of the intratumoral microbiota contribute to tumor heterogeneity, particularly in head and neck and colorectal cancers. To investigate these interactions, we have adapted and applied emerging technologies in spatial transcriptomics, spatial proteomics, and single-cell sequencing (INVADEseq). Our spatial analyses show that the intratumoral microbiota are not distributed randomly within human tumors; instead, they localize to distinct microniches that promote immune and epithelial cell functions supportive of cancer progression. These bacterial-colonized microniches are characterized by myeloid cell infiltration, upregulation of MAPKs and immune checkpoint proteins, and reduced T-cell levels. Furthermore, INVADEseq single-cell RNA sequencing has enabled us to identify the dominant host cell-associated bacteria, the human cell types they infect, and their impact on the transcriptional programs of infected cells from patient tumor specimens. In oral squamous cell carcinoma, Fusobacterium and Treponema species have been identified as the dominant cell-associated bacterial taxa, primarily infecting subsets of macrophages and aneuploid cancer epithelial cells and upregulating pathways associated with cancer progression. Our findings offer insights into the complex interplay between intratumoral microbiota and human components of the TME, emphasizing their role in supporting intratumoral heterogeneity and influencing cancer progression. Understanding these dynamics holds significant promise for the development of targeted therapeutic strategies aimed at disrupting bacterial-human spatial and cellular interactions within the TME. Citation Format: Susan Bullman. From microniches to single cells: Understanding the impact of intratumoral microbes on spatial and cellular heterogeneity in cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr PL03-02.

Abstract LB417: Leveraging advanced human lung models to explore mechanisms underlying T-DXd-associated interstitial lung disease (ILD)

Abstract Trastuzumab deruxtecan (T-DXd) is a HER2-directed antibody-drug conjugate approved for several indications, including HER2-positive unresectable or metastatic breast cancer that progressed on two or more prior therapies, HER2-positive locally advanced or metastatic gastric or gastroesophageal junction adenocarcinoma who have received a prior trastuzumab-based regimen and HER2-mutant unresectable or metastatic non-small cell lung cancer (USPI-ENH-C9-1122-r006). While the safety profile of T-DXd is manageable, ILD is an important identified risk and considered as an Adverse Event (AE) of special interest (Powell et al ESMO Open 2022). The therapeutic options for patients that develop ILD are limited to steroid medications, including prednisone and methylprednisolone. Hence, a greater understanding of T-DXd-associated ILD pathophysiology has the potential to guide alternative interventions to prevent ILD progression and improve patient care. Here, we used advanced human lung models to explore if T-DXd can affect pulmonary epithelial cell homeostasis and function. Transcriptome and gene set enrichment analysis of bronchial epithelial cells cultured under air-liquid interface (ALI) conditions revealed that genes and pathways associated with senescence, inflammation, and barrier integrity were dysregulated upon treatment with T-DXd. These transcriptomic findings linked to senescence and inflammation were confirmed in follow-up ALI experiments where T-DXd was shown to activate the p53 pathway, and upregulate pro-senescence, -inflammatory and -fibrotic mediators. Similarly, T-DXd activated senescence- and inflammation-associated markers in a microphysiological alveolar lung-on-chip system, thus indicating that T-DXd has similar effects in alveoli epithelia. In addition, T-DXd induced epithelial injury of ALI cells, which was reflected by loss of barrier function and release of epithelial damage markers. These changes were also accompanied by upregulation of mesenchymal cell markers, suggesting cells transition to a partial epithelial-to-mesenchymal phenotype. Whereas the payload (deruxtecan; DXd) and IgG control antibody-DXd conjugate (IgG-DXd) could recapitulate effects measured with T-DXd on lung epithelial cells, trastuzumab did not have any effect, suggesting limited contribution of HER2 targeting to T-DXd-induced epithelial changes in the experimental models used. Altogether, these data demonstrate that DXd can drive epithelial mechanisms and pathways associated with pulmonary fibrosis, inflammation, and damage, which are dysregulated events observed in ILD. Ongoing studies aim to evaluate the translation of these discoveries to the clinic and to support the development of novel therapeutic strategies to manage or prevent ILD. Citation Format: Ingrid Lua, Lin Jia, Jeff Chen, Kevin Contrepois, Mary McFarlane, Anton I. Rosenbaum, Peter Newham, Sonia Terrillon. Leveraging advanced human lung models to explore mechanisms underlying T-DXd-associated interstitial lung disease (ILD) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB417.

MURAMYL DIPEPTIDE CAUSES MITOCHONDRIAL DYSFUNCTION AND INTESTINAL INFLAMMATORY CYTOKINE RESPONSES IN RATS.

Introduction: Intestinal flora and the translocation of its products, such as muramyl dipeptide (MDP), are common causes of sepsis. MDP is a common activator of the intracellular pattern recognition receptor NOD2, and MDP translocation can cause inflammatory damage to the small intestine and systemic inflammatory responses in rats. Therefore, this study investigated the effects of MDP on the intestinal mucosa and distant organs during sepsis and the role of the NOD2/AMPK/LC3 pathway in MDP-induced mitochondrial dysfunction in the intestinal epithelium. Methods: Fifty male Sprague Dawley rats were randomly divided into five treatment groups: lipopolysaccharide (LPS) only, 1.5 and 15 mg/kg MDP+LPS, and 1.5 and 15 mg/kg MDP+short-peptide enteral nutrition (SPEN)+LPS. The total caloric intake was the same per group. The rats were euthanized 24 h after establishing the model, and peripheral blood and small intestinal mucosal and lung tissues were collected. Results: Compared to the LPS group, both MDP+LPS groups had aggravated inflammatory damage to the intestinal mucosal and lung tissues, increased IL-6 and MDP production, increased NOD2 expression, decreased AMPK and LC3 expression, increased mitochondrial reactive oxygen species production, and decreased mitochondrial membrane potential. Compared to the MDP+LPS groups, the MDP+SPEN+LPS groups had decreased IL-6 and MDP production, increased AMPK and LC3 protein expression, and protected mitochondrial and organ functions. Conclusions: MDP translocation reduced mitochondrial autophagy by regulating the NOD2/AMPK/LC3 pathway, causing mitochondrial dysfunction. SPEN protected against MDP-induced impairment of intestinal epithelial mitochondrial function during sepsis.

Approaching Thrombospondin-1 as a Potential Target for Mesenchymal Stromal Cells to Support Liver Regeneration after Partial Hepatectomy in Mouse and Humans.

Extended liver resection carries the risk of post-surgery liver failure involving thrombospondin-1-mediated aggravation of hepatic epithelial plasticity and function. Mesenchymal stromal cells (MSCs), by interfering with thrombospondin-1 (THBS1), counteract hepatic dysfunction, though the mechanisms involved remain unknown. Herein, two-thirds partial hepatectomy in mice increased hepatic THBS1, downstream transforming growth factor-β3, and perturbation of liver tissue homeostasis. All these events were ameliorated by hepatic transfusion of human bone marrow-derived MSCs. Treatment attenuated platelet and macrophage recruitment to the liver, both major sources of THBS1. By mitigating THBS1, MSCs muted surgery-induced tissue deterioration and dysfunction, and thus supported post-hepatectomy regeneration. After liver surgery, patients displayed increased tissue THBS1, which is associated with functional impairment and may indicate a higher risk of post-surgery complications. Since liver dysfunction involving THBS1 improves with MSC treatment in various animal models, it seems feasible to also modulate THBS1 in humans to impede post-surgery acute liver failure.

Novel air-liquid interface culture model to investigate stiffness-dependent behaviors of alveolar epithelial cells

Pulmonary alveoli are functional units in gas exchange in the lung, and their dysfunctions in lung diseases such as interstitial pneumonia are accompanied by fibrotic changes in structure, elevating the stiffness of extracellular matrix components. The present study aimed to test the hypothesis that such changes in alveoli stiffness induce functional alteration of epithelial cell functions, exacerbating lung diseases. For this, we have developed a novel method of culturing alveolar epithelial cells on polyacrylamide gel with different elastic modulus at an air-liquid interface. It was demonstrated that A549 cells on soft gels, mimicking the modulus of a healthy lung, upregulated mRNA expression and protein synthesis of surfactant protein C (SFTPC). By contrast, the cells on stiff gels, mimicking the modulus of the fibrotic lung, exhibited upregulation of SFTPC gene expression but not at the protein level. Cell morphology, as well as cell nucleus volume, were also different between the two types of gels.

Dupilumab prevents nasal epithelial function alteration by IL-4 in vitro: Evidence for its efficacy.

Chronic rhinosinusitis with nasal polyp (CRSwNP) is a typical type 2 inflammation involving interleukin (IL)-4 and IL-13. Dupilumab is a fully human monoclonal antibody targeting IL-4 receptor α subunit, thereby blocking signaling by both cytokines. Our hypothesis was that IL-4 and IL-13, by inducing a severe epithelial dysregulation, are involved in CRSwNP pathogenesis. This study aimed to evaluate the in vitro direct effect of IL-4, IL-13, and dupilumab on nasal epithelial functions. Nasal polyps and control mucosa from 28 patients, as well as human nasal epithelial cells (HNEC) from 35 patients with CRSwNP were used. Three major epithelial functions were investigated: the epithelial barrier function (characterized by transepithelial electrical resistance measurements and tight junction protein expression), the ciliary motion (characterized by the ciliary beating efficiency index), and wound healing (characterized by the wound repair rate) under various stimulations (IL-4, IL-13, and dupilumab). The main outcome was a significant change in epithelial functions following exposure to IL-4, IL-13, and dupilumab for 48h in the basal media. IL-4 (1, 10, and 100ng/mL) but not IL-13 induced a significant decrease in occludin and zonula-occludens protein expression, ciliary beating efficiency, and wound repair rate in HNEC. Dupilumab (0.04mg/mL) had no effect on HNEC and specifically restored all epithelial functions altered when cells were exposed to a 48-h IL-4 stimulation. Dupilumab, in vitro, restored epithelial integrity by counteracting the effect of IL-4 on the epithelial barrier (increased epithelial permeability, decreased ciliary beating efficiency, and decreased wound repair rate).

Cell division cycle 42 attenuates high glucose-treated renal tubular epithelial cell apoptosis, fibrosis, and inflammation, but activates the PAK1/AKT pathway.

Cell division cycle 42 (CDC42) modulates metabolism, inflammation, and fibrosis to engage in the pathology of diabetic complications. This study intended to further investigate the influence of CDC42 on viability, apoptosis, inflammation, epithelial-mesenchymal transition, and fibrosis in high glucose (HG)-treated renal tubular epithelial cells. HK-2 cells were exposed to HG medium (30mM) to establish the diabetic nephropathy (DN) cellular model, then the cells were transfected with scramble overexpression control (oeNC) or CDC42 overexpression (oeCDC42) vectors. Both the level of CDC42 mRNA and protein were decreased in HG-treated HK-2 cells in a dose- and time-dependent manner. Then HG-treated HK-2 cells were proposed for the following experiments. It was found that CDC42 increased CCK-8 detected viability and EdU positive cells. On the contrary, CDC42 reduced cell apoptosis, which was reflected by decreased TUNEL positive rate, increased BCL2, and reduced BAX. Interestingly, CDC42 inhibited fibrosis, which was reflected by increased E-Cadherin, as well as decreased Vimentin, TGF-β1, Collagen1, and α-SMA. Apart from these, CDC42 also attenuated proinflammatory cytokine production, including TNF-α, IL-1β, and IL-6. Moreover, CDC42 activated the PAK1/AKT pathway, which was reflected by increased p-PAK1 and p-AKT. However, CDC42 did not affect p-ERK. CDC42 may retard DN progression via its regulation of renal tubular epithelial cell functions, which may be due to its stimulation of the PAK1/AKT pathway.

CPAP may promote an endothelial inflammatory milieu in sleep apnoea after coronary revascularization

Continuous positive airway pressure (CPAP) has failed to reduce cardiovascular risk in obstructive sleep apnoea (OSA) in randomized trials. CPAP increases angiopoietin-2, a lung distension-responsive endothelial proinflammatory marker associated with increased cardiovascular risk. We investigated whether CPAP has unanticipated proinflammatory effects in patients with OSA and cardiovascular disease. Patients with OSA (apnoea-hypopnea index [AHI] ≥15 events/h without excessive sleepiness) in the Randomized Intervention with CPAP in Coronary Artery Disease and OSA study were randomized to CPAP or usual care following coronary revascularization. Changes in plasma levels of biomarkers of endothelial (angiopoietin-2, Tie-2, E-selectin, vascular endothelial growth factor [VEGF-A]) and lung epithelial (soluble receptor of advanced glycation end-products [sRAGE]) function from baseline to 12-month follow-up were compared across groups and associations with cardiovascular morbidity and mortality assessed. Patients with OSA (n=189; 84% men; age 66±8 years, BMI 28±3.5kg/m2, AHI 41±23 events/h) and 91 patients without OSA participated. Angiopoietin-2 remained elevated whereas VEGF-A declined significantly over 12 months in the CPAP group (n=91). In contrast, angiopoietin-2 significantly declined whereas VEGF-A remained elevated in the usual care (n=98) and OSA-free groups. The changes in angiopoietin-2 and VEGF-A were significantly different between CPAP and usual care, whereas Tie-2, sRAGE and E-selectin were similar. Greater 12-month levels of angiopoietin-2 were associated with greater mortality. Greater CPAP levels were associated with worse cardiovascular outcomes. Greater CPAP levels increase proinflammatory, lung distension-responsive angiopoietin-2 and reduce cardioprotective angiogenic factor VEGF-A compared to usual care, which may counteract the expected cardiovascular benefits of treating OSA. National Institutes of Health/National Heart, Lung, and Blood Institute; Swedish Research Council; Swedish Heart-Lung Foundation; ResMed Foundation.

The Aryl Hydrocarbon Receptor Regulates Epidermal Differentiation through Transient Activation of TFAP2A

The aryl hydrocarbon receptor (AHR) is an evolutionary conserved environmental sensor identified as indispensable regulator of epithelial homeostasis and barrier organ function. Molecular signaling cascade and target genes upon AHR activation and their contribution to cell and tissue function are however not fully understood. Multi-omics analyses using human skin keratinocytes revealed that, upon ligand activation, AHR binds open chromatin to induce expression of transcription factors (TFs), e.g., Transcription Factor AP-2α (TFAP2A), as a swift response to environmental stimuli. The terminal differentiation program including upregulation of barrier genes, filaggrin and keratins, was mediated by TFAP2A as a secondary response to AHR activation. The role of AHR-TFAP2A axis in controlling keratinocyte terminal differentiation for proper barrier formation was further confirmed using CRISPR/Cas9 in human epidermal equivalents. Overall, the study provides additional insights into the molecular mechanism behind AHR-mediated barrier function and identifies potential targets for the treatment of skin barrier diseases.

Allopurinol Disrupts Purine Metabolism to Increase Damage in Experimental Colitis.

Inflammatory bowel disease (IBD) is marked by a state of chronic energy deficiency that limits gut tissue wound healing. This energy shortfall is partially due to microbiota dysbiosis, resulting in the loss of microbiota-derived metabolites, which the epithelium relies on for energy procurement. The role of microbiota-sourced purines, such as hypoxanthine, as substrates salvaged by the colonic epithelium for nucleotide biogenesis and energy balance, has recently been appreciated for homeostasis and wound healing. Allopurinol, a synthetic hypoxanthine isomer commonly prescribed to treat excess uric acid in the blood, inhibits the degradation of hypoxanthine by xanthine oxidase, but also inhibits purine salvage. Although the use of allopurinol is common, studies regarding how allopurinol influences the gastrointestinal tract during colitis are largely nonexistent. In this work, a series of in vitro and in vivo experiments were performed to dissect the relationship between allopurinol, allopurinol metabolites, and colonic epithelial metabolism and function in health and during disease. Of particular significance, the in vivo investigation identified that a therapeutically relevant allopurinol dose shifts adenylate and creatine metabolism, leading to AMPK dysregulation and disrupted proliferation to attenuate wound healing and increased tissue damage in murine experimental colitis. Collectively, these findings underscore the importance of purine salvage on cellular metabolism and gut health in the context of IBD and provide insight regarding the use of allopurinol in patients with IBD.

Somatic variant profiling of a thymoma in Good syndrome

Good syndrome (GS) is a combined immunodeficiency that is associated with thymomas. The cause of the reduction in B-cells in patients with GS may be multifactorial and may include dysregulated T-cell responses. It has been proposed that tumorigenesis in a normal thymus alters thymic epithelial cell function, which leads to attenuated elimination of T-cells autoreactive to B-cells. Although the comprehensive genetic analysis of thymoma has been performed and reported in many articles, the comprehensive genetic analysis specified for GS-related thymoma has not been reported. Herein, we report comprehensive genetic analysis of a thymoma taken from a patient with GS. Oncogenesis-associated genes that may contribute to thymoma development were detected. Additionally, alteration of VCAM1, which is required in the interaction between T-cells and thymic epithelial cells, was observed. Aberrantly expressed VCAM1 in thymic epithelial cells may decrease the efficacy of negative of selection autoreactive T-cells and contribute to autoimmunity to B-cells.

In-Ovo Glutamine Administration Enhances Intestinal Development and Functions in Broiler Chickens: Insights from Enteroid Models

BackgroundEarly life events play significant roles in tissue development and animal health in their later life. Early nutrition, through in-ovo delivery, has shown beneficial effects on improving intestinal health in broiler chickens. However, the underlying mechanism is not fully investigated. A recently developed enteroid culture technique allows investigations on intestinal epithelial functions that are close to physiologic conditions. ObjectivesIn this study, we evaluated the short- and long-term effects of in-ovo administration of glutamine (Gln) on intestinal epithelial development and functions by using intestinal enteroid culture and tissue electrophysiologic analysis. MethodsA hundred eggs of commercial Cobb500 broilers were in-ovo injected with 0.2 mL of either phosphate-buffered saline (PBS) or 3% Gln at embryonic day 18 (E18). Chicks were killed on the day of hatch, and at 3- and 14-d posthatch. Enteroids were generated from the small intestine. After 4 d of culture, enteroids were harvested for 5-ethynyl-2′-deoxyuridine proliferation, fluorescein isothiocyanate-4 kDa dextran permeability, and glucose absorption assays. At day 3 (d3) and day 14 (d14), intestinal barrier and nutrient transport functions were measured by the Ussing chamber. The gene expression of epithelial cell markers, nutrient transporters, and tight-junction proteins were analyzed in both intestinal tissues and enteroids. ResultsIn comparison with the PBS control group, in-ovo Gln increased intestinal villus morphology, epithelial cell proliferation, and differentiation, and altered epithelial cell population toward increased number of enteroendocrine and goblet cells while decreasing Paneth cells. Enteroids gene expression of nutrient transporters (B0AT1, SGLT1, and EAAT3), tight junction (ZO2), glucose absorption, and barrier functions were enhanced on the day of hatch. Long-term increases of intestinal di-peptide and alanine transport were observed at day 14 posthatch. ConclusionsTogether our results suggested that the in-ovo injection of Gln stimulated intestinal epithelium proliferation and programmed the epithelial cell differentiation toward absorptive cells.

HDAC6-selective inhibitor CAY10603 ameliorates cigarette smoke-induced small airway remodeling by regulating epithelial barrier dysfunction and reversing

BackgroundSmall airway remodelling is a vital characteristic of chronic obstructive pulmonary disease (COPD), which is mainly caused by epithelial barrier dysfunction and epithelial-mesenchymal transition (EMT). Recent studies have indicated that histone deacetylase 6 (HDAC6) plays an important role in the dysregulation of epithelial function. In this study, we investigated the therapeutic effects and underlying mechanisms of an inhibitor with high selectivity for HDAC6 in COPD.MethodsCigarette smoke (CS) exposure was used to establish a CS-induced COPD mouse model. CAY10603 at doses of 2.5 and 10 mg/kg was injected intraperitoneally on alternate days. The protective effects of CAY10603 against CS-induced emphysema, epithelial barrier function and small airway remodeling were evaluated using hematoxylin and eosin (H&E) staining, Masson’s trichrome staining, immunohistochemical staining, and western blot. The human lung bronchial epithelial cell line (HBE) was used to elucidate the underlying molecular mechanism of action of CAY10603.ResultsHDAC6 levels in the lung homogenates of CS-exposed mice were higher than that those in control mice. Compared to the CS group, the mean linear intercept (MLI) of the CAY10603 treatment group decreased and the mean alveolar number (MAN)increased. Collagen deposition was reduced in groups treated with CAY10603. The expression of α-SMA was markedly upregulated in the CS group, which was reversed by CAY10603 treatment. Conversely, E-cadherin expression in the CS group was further downregulated, which was reversed by CAY10603 treatment. CAY10603 affects the tight junction protein expression of ZO-1 and occludin. ZO-1 and occludin expression were markedly downregulated in the CS group. After CAY10603treatment, the protein expression level of ZO-1 and occludin increased significantly. In HBE cells, Cigarette smoke extract (CSE) increased HDAC6 levels. CAY10603 significantly attenuated the release of TGF-β1 induced by CSE. CAY10603 significantly increased the E-cadherin levels in TGF-β1 treated HBE cells, while concurrently attenuated α-SMA expression. This effect was achieved through the suppression of Smad2 and Smad3 phosphorylation. CAY10603 also inhibited TGF-β1 induced cell migration.ConclusionsThese findings suggested that CAY10603 inhibited CS induced small airway remodelling by regulating epithelial barrier dysfunction and reversing EMT via the TGF-β1/Smad2/3 signalling pathway.

Breaking Down Barriers: Epithelial Contributors to Monogenic IBD Pathogenesis.

Monogenic causes of inflammatory bowel diseases (IBD) are increasingly being discovered. To date, much attention has been placed in those resulting from inborn errors of immunity. Therapeutic efforts have been largely focused on offering personalized immune modulation or curative bone marrow transplant for patients with IBD and underlying immune disorders. To date, less emphasis has been placed on monogenic causes of IBD that pertain to impairment of the intestinal epithelial barrier. Here, we provide a comprehensive review of monogenic causes of IBD that result in impaired intestinal epithelial barrier that are categorized into 6 important functions: (1) epithelial cell organization, (2) epithelial cell intrinsic functions, (3) epithelial cell apoptosis and necroptosis, (4) complement activation, (5) epithelial cell signaling, and (6) control of RNA degradation products. We illustrate how impairment of any of these categories can result in IBD. This work reviews the current understanding of the genes involved in maintaining the intestinal barrier, the inheritance patterns that result in dysfunction, features of IBD resulting from these disorders, and pertinent translational work in this field.