A short guide to the tight junction.

Tight junctions (TJs) are specialized regions of contact between cells of epithelial and endothelial tissues that form selective semipermeable paracellular barriers that establish and maintain body compartments with different fluid compositions. As such, the formation of TJs represents a critical step in metazoan evolution, allowing the formation of multicompartmental organisms and true, barrier-forming epithelia and endothelia. In the six decades that have passed since the first observations of TJs by transmission electron microscopy, much progress has been made in understanding the structure, function, molecular composition and regulation of TJs. The goal of this Perspective is to highlight the key concepts that have emerged through this research and the future challenges that lie ahead for the field.

Tight junction protein occludin is an internalization factor for SARS-CoV-2 infection and mediates virus cell-to-cell transmission.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spreads efficiently by spike-mediated, direct cell-to-cell transmission. However, the underlying mechanism is poorly understood. Herein, we demonstrate that the tight junction protein occludin (OCLN) is critical to this process. SARS-CoV-2 infection alters OCLN distribution and expression and causes syncytium formation that leads to viral spread. OCLN knockdown fails to alter SARS-CoV-2 binding but significantly lowers internalization, syncytium formation, and transmission. OCLN overexpression also has no effect on virus binding but enhances virus internalization, cell-to-cell transmission, and replication. OCLN directly interacts with the SARS-CoV-2 spike, and the endosomal entry pathway is involved in OCLN-mediated cell-to-cell fusion rather than in the cell surface entry pathway. All SARS-CoV-2 strains tested (prototypic, alpha, beta, gamma, delta, kappa, and omicron) are dependent on OCLN for cell-to-cell transmission, although the extent of syncytium formation differs between strains. We conclude that SARS-CoV-2 utilizes OCLN as an internalization factor for cell-to-cell transmission.

WNK1 collaborates with TGF-ß in endothelial cell junction turnover and angiogenesis.

Angiogenesis is essential for growth of new blood vessels, remodeling existing vessels, and repair of damaged vessels, and these require reorganization of endothelial cell-cell junctions through a partial endothelial-mesenchymal transition. Homozygous disruption of the gene encoding the protein kinase WNK1 results in lethality in mice near embryonic day (E) 12 due to impaired angiogenesis. This angiogenesis defect can be rescued by endothelial-specific expression of an activated form of the WNK1 substrate kinase OSR1. We show that inhibition of WNK1 kinase activity not only prevents sprouting of endothelial cells from aortic slices but also vessel extension in inhibitor-treated embryos ex vivo. Mutations affecting TGF-ß signaling also result in abnormal vascular development beginning by E10 and, ultimately, embryonic lethality. Previously, we demonstrated cross-talk of WNK1 with TGF-ß-regulated SMAD signaling, and OSR1 was identified as a component of the TGF-ß interactome. However, molecular events jointly regulated by TGF-ß and WNK1/OSR1 have not been delineated. Here, we show that inhibition of WNK1 promotes TGF-ß-dependent degradation of the tyrosine kinase receptor AXL, which is involved in TGF-ß-mediated cell migration and angiogenesis. We also show that interaction between OSR1 and occludin, a protein associated with endothelial tight junctions, is an essential step to enable tight junction turnover. Furthermore, we show that these phenomena are WNK1 dependent, and sensitive to TGF-ß. These findings demonstrate intimate connections between WNK1/OSR1 and multiple TGF-ß-sensitive molecules controlling angiogenesis and suggest that WNK1 may modulate many TGF-ß-regulated functions.

Gingipain-carrying outer membrane vesicles from Porphyromonas gingivalis cause barrier dysfunction of Caco-2 cells by releasing gingipain into the cytosol.

Ingestion of Porphyromonas gingivalis, a periodontal pathogen, disrupts the intestinal barrier in mice. However, the involvement of outer membrane vesicles (OMVs) secreted from P. gingivalis in the destruction of the intestinal barrier remains unclear. In this study, we tested the hypothesis that OMVs carrying gingipains, the major cysteine proteases produced by P. gingivalis, affects the intestinal barrier function. OMVs increased the permeability of the Caco-2 cell monolayer, a human intestinal epithelial cell line, accompanied by degradation of the tight junction protein occludin. In contrast, OMVs prepared from mutant strains devoid of gingipains failed to induce intestinal barrier dysfunction or occludin degradation in Caco-2 cells. A close histological examination revealed the intracellular localization of gingipain-carrying OMVs. Gingipain activity was detected in the cytosolic fraction of Caco-2 cells after incubation with OMVs. These results suggest that gingipains were internalized into intestinal cells through OMVs and transported into the cytosol, where they then directly degraded occludin from the cytosolic side. Thus, P. gingivalis OMVs might destroy the intestinal barrier and induce systemic inflammation via OMV itself or intestinal substances leaked into blood vessels, causing various diseases.

Hepatitis C Virus Disrupts Annexin 5-Mediated Occludin Integrity through Downregulation of Protein Kinase Cα (PKCα) and PKCη Expression, Thereby Promoting Viral Propagation.

Annexins (ANXs) comprise a family of calcium- and phospholipid-binding proteins and are implicated in the hepatitis C virus (HCV) life cycle. Here, we demonstrate a novel role of ANX5 in the HCV life cycle. Comparative analysis by quantitative PCR in human hepatoma cells revealed that ANX2, ANX4, and ANX5 were highly expressed among the ANX family proteins. Knockdown of ANX5 mRNA resulted in marked enhancement of HCV RNA replication but had no effect on either HCV translation or assembly. Using the HCV pseudoparticle (HCVpp) system, we observed enhancement of HCVpp infectivity in ANX5 knockdown Huh-7OK1 cells, suggesting that ANX5 is involved in suppression of HCV entry. Additionally, we observed that subcellular localizations of tight-junction proteins, such as claudin 1 (CLDN1) and occludin (OCLN), were disrupted in the ANX5 knockdown cells. It was reported that HCV infection was facilitated by disruption of OCLN distribution and that proper distribution of OCLN was regulated by its phosphorylation. Knockdown of ANX5 resulted in a decrease of OCLN phosphorylation, thereby disrupting OCLN distribution and HCV infection. Further analysis revealed that protein kinase C (PKC) isoforms, including PKCα and PKCη, play important roles in the regulation of ANX5-mediated phosphorylation and distribution of OCLN and in the restriction of HCV infection. HCV infection reduced OCLN phosphorylation through the downregulation of PKCα and PKCη expression. Taken together, these results suggest that ANX5, PKCα, and PKCη contribute to restriction of HCV infection by regulating OCLN integrity. We propose a model that HCV disrupts ANX5-mediated OCLN integrity through downregulation of PKCα and PKCη expression, thereby promoting HCV propagation. IMPORTANCE Host cells have evolved host defense machinery to restrict viral infection. However, viruses have evolved counteracting strategies to achieve their infection. In the present study, we obtained results suggesting that ANX5 and PKC isoforms, including PKCα and PKCη, contribute to suppression of HCV infection by regulating the integrity of OCLN. The disruption of OCLN integrity increased HCV infection. We also found that HCV disrupts ANX5-mediated OCLN integrity through downregulation of PKCα and PKCη expression, thereby promoting viral infection. We propose that HCV disrupts ANX5-mediated OCLN integrity to establish a persistent infection. The disruption of tight-junction assembly may play important roles in the progression of HCV-related liver diseases.

Endothelial BACE1 Impairs Cerebral Small Vessels via Tight Junctions and eNOS.

BACKGROUND: Cerebral small vessel injury, including loss of endothelial tight junctions, endothelial dysfunction, and blood-brain barrier breakdown, is an early and typical pathology for Alzheimer's disease, cerebral amyloid angiopathy, and hypertension-related cerebral small vessel disease. Whether there is a common mechanism contributing to these cerebrovascular alterations remains unclear. Studies have shown an elevation of BACE1 (ß-site amyloid precursor protein cleaving enzyme 1) in cerebral vessels from cerebral amyloid angiopathy or Alzheimer's disease patients, suggesting that vascular BACE1 may involve in cerebral small vessel injury. METHODS: To understand the contribution of vascular BACE1 to cerebrovascular impairments, we combined cellular and molecular techniques, mass spectrometry, immunostaining approaches, and functional testing to elucidate the potential pathological mechanisms. RESULTS: We observe a 3.71-fold increase in BACE1 expression in the cerebral microvessels from patients with hypertension. Importantly, we discover that an endothelial tight junction protein, occludin, is a completely new substrate for endothelial BACE1. BACE1 cleaves occludin with full-length occludin reductions and occludin fragment productions. An excessive cleavage by elevated BACE1 induces membranal accumulation of caveolin-1 and subsequent caveolin-1-mediated endocytosis, resulting in lysosomal degradation of other tight junction proteins. Meanwhile, membranal caveolin-1 increases the binding to eNOS (endothelial nitric oxide synthase), together with raised circulating Aß (ß-amyloid peptides) produced by elevated BACE1, leading to an attenuation of eNOS activity and resultant endothelial dysfunction. Furthermore, the initial endothelial damage provokes chronic reduction of cerebral blood flow, blood-brain barrier leakage, microbleeds, tau hyperphosphorylation, synaptic loss, and cognitive impairment in endothelial-specific BACE1 transgenic mice. Conversely, inhibition of aberrant BACE1 activity ameliorates tight junction loss, endothelial dysfunction, and memory deficits. CONCLUSIONS: Our findings establish a novel and direct relationship between endothelial BACE1 and cerebral small vessel damage, indicating that abnormal elevation of endothelial BACE1 is a new mechanism for cerebral small vessel disease pathogenesis.

Occludin and collagen IV degradation mediated by the T9SS effector SspA contributes to blood-brain barrier damage in ducks during Riemerella anatipestifer infection.

Riemerella anatipestifer infection is characterized by meningitis with neurological symptoms in ducklings and has adversely affected the poultry industry. R. anatipestifer strains can invade the duck brain to cause meningitis and neurological symptoms, but the underlying mechanism remains unknown. In this study, we showed that obvious clinical symptoms, an increase in blood‒brain barrier (BBB) permeability, and the accumulation of inflammatory cytokines occurred after intravenous infection with the Yb2 strain but not the mutant strain Yb2ΔsspA, indicating that Yb2 infection can lead to cerebrovascular dysfunction and that the type IX secretion system (T9SS) effector SspA plays a critical role in this pathological process. In addition, we showed that Yb2 infection led to rapid degradation of occludin (a tight junction protein) and collagen IV (a basement membrane protein), which contributed to endothelial barrier disruption. The interaction between SspA and occludin was confirmed by coimmunoprecipitation. Furthermore, we found that SspA was the main enzyme mediating occludin and collagen IV degradation. These data indicate that R. anatipestifer SspA mediates occludin and collagen IV degradation, which functions in BBB disruption in R. anatipestifer-infected ducks. These findings establish the molecular mechanisms by which R. anatipestifer targets duckling endothelial cell junctions and provide new perspectives for the treatment and prevention of R. anatipestifer infection.

Preventing occludin tight-junction disruption via inhibition of microRNA-193b-5p attenuates viral load and influenza-induced lung injury.

Virus-induced lung injury is associated with loss of pulmonary epithelial-endothelial tight junction integrity. While the alveolar-capillary membrane may be an indirect target of injury, viruses may interact directly and/or indirectly with miRs to augment their replication potential and evade the host antiviral defense system. Here, we expose how the influenza virus (H1N1) capitalizes on host-derived interferon-induced, microRNA (miR)-193b-5p to target occludin and compromise antiviral defenses. Lung biopsies from patients infected with H1N1 revealed increased miR-193b-5p levels, marked reduction in occludin protein, and disruption of the alveolar-capillary barrier. In C57BL/6 mice, the expression of miR-193b-5p increased, and occludin decreased, 5-6 days post-infection with influenza (PR8). Inhibition of miR-193b-5p in primary human bronchial, pulmonary microvascular, and nasal epithelial cells enhanced antiviral responses. miR-193b-deficient mice were resistant to PR8. Knockdown of occludin, both in vitro and in vivo, and overexpression of miR-193b-5p reconstituted susceptibility to viral infection. miR-193b-5p inhibitor mitigated loss of occludin, improved viral clearance, reduced lung edema, and augmented survival in infected mice. Our results elucidate how the innate immune system may be exploited by the influenza virus and how strategies that prevent loss of occludin and preserve tight junction function may limit susceptibility to virus-induced lung injury.

A novel in vitro high-content imaging assay for the prediction of drug-induced lung toxicity.

The development of inhaled drugs for respiratory diseases is frequently impacted by lung pathology in non-clinical safety studies. To enable design of novel candidate drugs with the right safety profile, predictive in vitro lung toxicity assays are required that can be applied during drug discovery for early hazard identification and mitigation. Here, we describe a novel high-content imaging-based screening assay that allows for quantification of the tight junction protein occludin in A549 cells, as a model for lung epithelial barrier integrity. We assessed a set of compounds with a known lung safety profile, defined by clinical safety or non-clinical in vivo toxicology data, and were able to correctly identify 9 of 10 compounds with a respiratory safety risk and 9 of 9 compounds without a respiratory safety risk (90% sensitivity, 100% specificity). The assay was sensitive at relevant compound concentrations to influence medicinal chemistry optimization programs and, with an accessible cell model in a 96-well plate format, short protocol and application of automated imaging analysis algorithms, this assay can be readily integrated in routine discovery safety screening to identify and mitigate respiratory toxicity early during drug discovery. Interestingly, when we applied physiologically-based pharmacokinetic (PBPK) modelling to predict epithelial lining fluid exposures of the respiratory tract after inhalation, we found a robust correlation between in vitro occludin assay data and lung pathology in vivo, suggesting the assay can inform translational risk assessment for inhaled small molecules.

Preclinical Evaluation of Sodium Butyrate's Potential to Reduce Alcohol Consumption: A Dose-Escalation Study in C57BL/6J Mice in Antibiotic-Enhanced Binge-Like Drinking Model.

INTRODUCTION: In our earlier efforts to establish gut-brain axis during alcohol use disorder (AUD), we have demonstrated that supplementation of C57BL/6J male mice with 8 mg/mL sodium butyrate, a major short-chain fatty acid, in drinking water reduced ethanol intake and neuroinflammatory response in antibiotic (ABX)-enhanced voluntary binge-like alcohol consumption model, drinking in the dark (DID). METHODS: To further evaluate the preclinical potential of SB, we have set a dose-escalation study in C57BL/6J male mice to test effects of ad libitum 20 mg/mL SB and 50 mg/mL SB and their combinations with ABX in the DID procedure for 4 weeks. Effects of these SB concentrations on ethanol consumption and bodily parameters were determined for the duration of the treatments. At the end of study, blood, liver, and intestinal tissues were collected to study any potential adverse effects ad to measure blood ethanol concentrations. RESULTS: Increasing SB concentrations in the drinking water caused a loss in the protective effect against ethanol consumption and produced adverse effects on body and liver weights, reduced overall liquid intake. The hypothesis that these effects were due to aversion to SB smell/taste at these high concentrations were further tested in a follow up proof-of-concept study with intragastric gavage administration of SB. The higher gavage dose (320 mg/kg) caused reduction in ethanol consumption without any adverse effects. CONCLUSION: Overall, these findings added more support for the therapeutic potential of SB in management of AUD, given a proper form of administration.

Atorvastatin-induced intracerebral hemorrhage is inhibited by berberine in zebrafish.

Intracerebral hemorrhage (ICH), for which there are currently no effective preventive or treatment methods, has a very high fatality rate. Statins, such as atorvastatin (ATV), are the first-line drugs for regulating blood lipids and treating hyperlipidemia-related cardiovascular diseases. However, ATV-associated ICH has been reported, although its incidence is rare. In this study, we aimed to investigate the protective action and mechanisms of berberine (BBR) against ATV-induced brain hemorrhage. We established an ICH model in zebrafish induced by ATV (2 µM) and demonstrated the effects of BBR (10, 50, and 100 µM) on ICH via protecting the vascular network using hemocyte staining and three transgenic zebrafish. BBR was found to reduce brain inflammation and locomotion injury in ICH-zebrafish. Mechanism research showed that ATV increased the levels of VE-cadherin and occludin proteins but disturbed their localization at the cell membrane by abnormal phosphorylation, which decreased the number of intercellular junctions between vascular endothelial cells (VECs), disrupting the integrity of vascular walls. BBR reversed the effects of ATV by promoting autophagic degradation of phosphorylated VE-cadherin and occludin in ATV-induced VECs examined by co-immunoprecipitation (co-IP). These findings provide crucial insights into understanding the BBR mechanisms involved in the maintenance of vascular integrity and in mitigating adverse reactions to ATV.

Collagen prolyl 4-hydroxylase subunit α member-induced head and neck squamous cell carcinoma aggressiveness is antagonized by LLGL2 via reduced expression of occludin.

There are three isoforms of human collagen prolyl 4-hydroxylases (C-P4Hs), each of which has been reported to play an important role in regulating the progression of a variety of human cancers. By analyzing TGCA datasets on human head and neck squamous cell carcinoma (HNSC), we find that a higher expression of all three C-P4HAs (the α subunit of C-P4Hs) is a superior prognostic indicator than a higher expression of two or a single C-P4HA. Unexpectedly, some patients with higher levels of three C-P4HAs survive longer than patients whose tumors have lower expression of C-P4HAs. Therefore, there may be molecule(s) that can negate the deleterious effects of overexpressing C-P4HAs during cancer progression. By constructing a functional protein interaction network of C-P4HAs and analyzing molecules whose expressions are correlated significantly with that of C-P4HAs, we identify scribble cell polarity complex component 2 (LLGL2) as a factor that antagonizes the effects of overexpressed C-P4HAs on HNSC. Silencing of LLGL2 in the human oral squamous cell line Cal-27 upregulates the expression of occludin and increases cancer cell invasion and migration. In contrast, knocking down C-P4HA alone inhibits cell migration and invasion. Furthermore, simultaneously downregulating three C-P4HAs has more pronounced effects on inhibiting cell migration and invasion. Accordingly, high LLGL2 expression is also a marker indicating improved prognosis in patients with HNSC. These results suggest that the interplay between LLGL2 and C-P4HAs may be targeted to mitigate HNSC tumorigenesis and progression.

Canonical and Non-Canonical Localization of Tight Junction Proteins during Early Murine Cranial Development.

This study investigates the intricate composition and spatial distribution of tight junction complex proteins during early mouse neurulation. The analyses focused on the cranial neural tube, which gives rise to all head structures. Neurulation brings about significant changes in the neuronal and non-neuronal ectoderm at a cellular and tissue level. During this process, precise coordination of both epithelial integrity and epithelial dynamics is essential for accurate tissue morphogenesis. Tight junctions are pivotal for epithelial integrity, yet their complex composition in this context remains poorly understood. Our examination of various tight junction proteins in the forebrain region of mouse embryos revealed distinct patterns in the neuronal and non-neuronal ectoderm, as well as mesoderm-derived mesenchymal cells. While claudin-4 exhibited exclusive expression in the non-neuronal ectoderm, we demonstrated a neuronal ectoderm specific localization for claudin-12 in the developing cranial neural tube. Claudin-5 was uniquely present in mesenchymal cells. Regarding the subcellular localization, canonical tight junction localization in the apical junctions was predominant for most tight junction complex proteins. ZO-1 (zona occludens protein-1), claudin-1, claudin-4, claudin-12, and occludin were detected at the apical junction. However, claudin-1 and occludin also appeared in basolateral domains. Intriguingly, claudin-3 displayed a non-canonical localization, overlapping with a nuclear lamina marker. These findings highlight the diverse tissue and subcellular distribution of tight junction proteins and emphasize the need for their precise regulation during the dynamic processes of forebrain development. The study can thereby contribute to a better understanding of the role of tight junction complex proteins in forebrain development.

The Basic Requirement of Tight Junction Proteins in Blood-Brain Barrier Function and Their Role in Pathologies.

This review addresses the role of tight junction proteins at the blood-brain barrier (BBB). Their expression is described, and their role in physiological and pathological processes at the BBB is discussed. Based on this, new approaches are depicted for paracellular drug delivery and diagnostics in the treatment of cerebral diseases. Recent data provide convincing evidence that, in addition to its impairment in the course of diseases, the BBB could be involved in the aetiology of CNS disorders. Further progress will be expected based on new insights in tight junction protein structure and in their involvement in signalling pathways.

Evaluation of the Antifibrotic Effects of Drugs Commonly Used in Inflammatory Intestinal Diseases on In Vitro Intestinal Cellular Models.

The mechanism underlying intestinal fibrosis, the main complication of inflammatory bowel disease (IBD), is not yet fully understood, and there is no therapy to prevent or reverse fibrosis. We evaluated, in in vitro cellular models, the ability of different classes of drugs currently used in IBD to counteract two pivotal processes of intestinal fibrosis, the differentiation of intestinal fibroblasts to activated myofibroblasts using CCD-18Co cells, and the epithelial-to-mesenchymal transition (EMT) of intestinal epithelial cells using Caco-2 cells (IEC), both being processes induced by transforming growth factor-ß1 (TGF-ß1). The drugs tested included mesalamine, azathioprine, methotrexate, prednisone, methylprednisolone, budesonide, infliximab, and adalimumab. The expression of fibrosis and EMT markers (collagen-I, α-SMA, pSmad2/3, occludin) was assessed by Western blot analysis and by immunofluorescence. Of the drugs used, only prednisone, methylprednisolone, budesonide, and adalimumab were able to antagonize the pro-fibrotic effects induced by TGF-ß1 on CCD-18Co cells, reducing the fibrosis marker expression. Methylprednisolone, budesonide, and adalimumab were also able to significantly counteract the TGF-ß1-induced EMT process on Caco-2 IEC by increasing occludin and decreasing α-SMA expression. This is the first study that evaluates, using in vitro cellular models, the direct antifibrotic effects of drugs currently used in IBD, highlighting which drugs have potential antifibrotic effects.

Biochemical Modulators of Tight Junctions (TJs): Occludin, Claudin-2 and Zonulin as Biomarkers of Intestinal Barrier Leakage in the Diagnosis and Assessment of Inflammatory Bowel Disease Progression.

BACKGROUND: Considering the increasing worldwide prevalence of inflammatory bowel disease (IBD), the early diagnosis of this disease is extremely important. However, non-invasive diagnostic methods remain limited, while invasive techniques are the most commonly used in daily practice. Therefore, there is a serious need to find new non-invasive biomarkers of IBD. METHODS: The serum profiles of occludin, claudin-2, and zonulin were assessed in IBD patients using the ELISA method. The levels of the analyzed biomarkers were measured before and after a year of anti-inflammatory treatment, which was a tumor necrosis factor α (TNF-α) inhibitor (adalimumab) in patients with ulcerative colitis (UC) and conventional therapy in patients with Crohn's disease (CD). RESULTS: In IBD patients, the serum level of occludin (p < 0.001) decreased compared to healthy individuals, while the level of claudin-2 (p < 0.001) increased. Additionally, zonulin (p < 0.01) concentration increased in CD patients compared to the control group. The highest diagnostic ability was presented by occludin measurements with the area under the curve (AUC) of 0.959 (95% CI 0.907-1) in UC and 0.948 (95% CI 0.879-1) in CD. Claudin-2 also demonstrated very good ability in diagnosing UC and CD with AUC values of 0.864 (95% CI 0.776-0.952) and 0.896 (95% CI 0.792-0.999), respectively. The ability of zonulin to diagnose CD was estimated as good with an AUC of 0.74 (95% CI 0.598-0.881). Moreover, a significant correlation was identified between C-reactive protein (CRP), claudin-2 (r = -0.37; p < 0.05), and zonulin (r = -0.44; p < 0.05) in UC patients. Treatment with adalimumab improved the level of occludin, claudin-2, and zonulin in UC patients, while anti-inflammatory conventional therapy decreased the concentration of zonulin in CD. CONCLUSIONS: Occludin and claudin-2 measurements present significant utility in diagnosing both UC and CD, while zonulin assessments may be useful in CD diagnosis. Additionally, claudin-2 and zonulin measurements may be helpful in evaluating the intensity of the inflammatory process. Anti-TNF-α treatment improved the value of occludin, claudin-2, and zonulin, indicating its beneficial effect on the integrity of tight junctions in UC.

Pediatric migraine is characterized by traits of ecological and metabolic dysbiosis and inflammation.

BACKGROUND: Recently, there has been increasing interest in the possible role of the gut microbiota (GM) in the onset of migraine. Our aim was to verify whether bacterial populations associated with intestinal dysbiosis are found in pediatric patients with migraine. We looked for which metabolic pathways, these bacteria were involved and whether they might be associated with gut inflammation and increased intestinal permeability. METHODS: Patients aged between 6 and 17 years were recruited. The GM profiling was performed by the 16S rRNA metataxonomics of faecal samples from 98 patients with migraine and 98 healthy subjects. Alpha and beta diversity analyses and multivariate and univariate analyses were applied to compare the gut microbiota profiles between the two group. To predict functional metabolic pathways, we used phylogenetic analysis of communities. The level of indican in urine was analyzed to investigate the presence of metabolic dysbiosis. To assess gut inflammation, increased intestinal permeability and the mucosal immune activation, we measured the plasmatic levels of lipopolysaccharide, occludin and IgA, respectively. RESULTS: The α-diversity analysis revealed a significant increase of bacterial richness in the migraine group. The ß-diversity analysis showed significant differences between the two groups indicating gut dysbiosis in patients with migraine. Thirty-seven metabolic pathways were increased in the migraine group, which includes changes in tryptophan and phenylalanine metabolism. The presence of metabolic dysbiosis was confirmed by the increased level of indican in urine. Increased levels of plasmatic occludin and IgA indicated the presence of intestinal permeability and mucosal immune activation. The plasmatic LPS levels showed a low intestinal inflammation in patients with migraine. CONCLUSIONS: Pediatric patients with migraine present GM profiles different from healthy subjects, associated with metabolic pathways important in migraine.

Lipopolysaccharide, VE-cadherin, HMGB1, and HIF-1α levels are elevated in the systemic circulation in chronic migraine patients with medication overuse headache: evidence of leaky gut and inflammation.

OBJECTIVE: Medication overuse headache (MOH) was recently shown to be associated with leaky gut in rodents. We aimed to investigate whether chronic migraine (CM) patients with MOH have elevated lipopolysaccharide levels and inflammatory molecules in blood circulation. MATERIALS AND METHODS: The study included women participants (40 CM patients with NSAID overuse headache, 35 episodic migraine (EM) patients, and 20 healthy non-headache sufferers). Migraine duration, monthly migraine headache days, MigSCog, HADS-D, HADS-A, and HIT-6 scores were recorded. Serum samples were collected to measure circulating LPS, LPS binding protein (LBP), tight junction protein occludin, adherens junction protein vascular endothelial cadherin (VE-cadherin), CGRP, HMGB1, HIF-1α, IL-6, and IL-17 levels. RESULTS: Serum LPS, VE-Cadherin, CGRP, HIF-1α, and IL-6 levels were significantly higher in the CM + MOH group compared to the EM group and healthy controls while serum LBP and HMGB1 were higher in the CM + MOH group compared to healthy controls. IL-17 and occludin levels were comparable between the three groups. Serum HMGB1 levels in EM patients were higher compared to the control group. Mig-SCog and HIT-6 scores were higher in the CM + MOH group compared to EM patients. HADS-A and HADS-D scores were significantly higher in the CM + MOH group compared to EM patients and healthy controls, and they were also higher in EM patients compared to healthy subjects. LPS levels were correlated with VE-cadherin and occludin levels. The number of monthly migraine headache days was positively correlated with serum LPS, HIF-1α, VE-cadherin, and IL-6 levels, HADS-A, HADS-D, HIT-6, and MigSCog scores. CONCLUSION: We have evidence for the first time that CM + MOH is associated with elevated serum LPS and LBP levels suggestive of LPS leak into the systemic circulation. Higher levels of nociceptive and/or pro-inflammatory molecules such as HMGB1, HIF-1α, IL-6, and CGRP may play a role in trigeminal sensitization and neurobiology of MOH. Intestinal hyperpermeability and consequent inflammatory response should be considered as a potential contributory factor in patients with MOH.

Berberine decreases S100B generation to regulate gut vascular barrier permeability in mice with burn injury.

Context: Berberine (BBR) can regulate enteric glial cells (EGCs) and the gut vascular barrier (GVB).Objective: To explore whether BBR regulates GVB permeability via the S100B pathway.Materials and methods: GVB hyperpermeability in C57BL/6J mice was induced by burns or S100B enema. BBR (25 or 50 mg/kg/d, 3 d) was gavaged preburn. S100B monoclonal antibody (S100BmAb) was i.v. injected postburn. Mouse intestinal microvascular endothelial cells (MIMECs) were treated with S100B, S100B plus BBR, or Z-IETD-FMK. GVB permeability was assayed by FITC-dextran, S100B by ELISA, caspase-8, ß-catenin, occludin and PV-1 by immunoblot.Results: Burns elevated S100B in serum and in colonic mucosa to a peak (147.00 ± 4.95 ng/mL and 160.30 ± 8.50 ng/mg, respectively) at 36 h postburn, but BBR decreased burns-induced S100B in serum (126.20 ± 6.30 or 90.60 ± 3.78 ng/mL) and in mucosa (125.80 ± 12.40 or 91.20 ± 8.54 ng/mg). Burns raised GVB permeability (serum FITC-dextran 111.40 ± 8.56 pg/mL) at 48 h postburn, but BBR reduced GVB permeability (serum FITC-dextran 89.20 ± 6.98 or 68.60 ± 5.50 ng/mL). S100B enema (1 µM) aggravated burns-raised GVB permeability (142.80 ± 8.07 pg/mL) and PV-1, but the effect of S100B was antagonized by BBR. Z-IETD-FMK (5 µM) increased S100B-induced permeability to FITC-dextran (205.80 ± 9.70 to 263.80 ± 11.04 AUs) while reducing ß-catenin in MIMECs. BBR (5 µM) reduced S100B-induced permeability (104.20 ± 9.65 AUs) and increased caspase-8, ß-catenin and occludin.Discussion and conclusion: BBR decreases burns-induced GVB hyperpermeability via modulating S100B/caspase-8/ß-catenin pathway and may involve EGCs.

Leaky gut and inflammatory biomarkers in a medication overuse headache model in male rats.

Background/aim: Medication overuse is common among chronic migraine patients and nonsteroidal antiinflammatory drugs (NSAIDs) are the most frequently overused drugs. The pathophysiological mechanisms underlying medication overuse headache (MOH) are not completely understood. Intestinal hyperpermeability and leaky gut are reported in patients using NSAIDs. The aim of the study is to investigate the role of leaky gut and inflammation in an MOH model MOH model in male rats. Methods: The study was conducted in male Sprague Dawley rats. There were two experimental groups. The first group was the chronic NSAID group in which the rats received mefenamic acid (n = 8) for four weeks intraperitoneally (ip) and the second group was the vehicle group (n = 8) that received 5% dimethyl sulfoxide+sesame oil (ip) for 4 weeks. We assessed spontaneous pain-like behavior, periorbital mechanical withdrawal thresholds, and anxiety-like behavior using an elevated plus maze test. After behavioral testing, serum levels of occludin and lipopolysaccharide-binding protein (LBP) and brain levels of IL-17, IL-6, and high mobility group box 1 protein (HMGB1) were evaluated with ELISA.Results: Serum LBP and occludin levels and brain IL-17 and HMGB1 levels were significantly elevated in the chronic NSAID group compared to its vehicle (p = 0.006, p = 0.016, p = 0.016 and p = 0.016 respectively) while brain IL-6 levels were comparable (p = 0.67) between the groups. The chronic NSAID group showed pain-like and anxiety-like behavior in behavioral tests. Brain IL-17 level was positively correlated with number of head shakes (r = 0.64, p = 0.045), brain IL-6 level was negatively correlated with periorbital mechanical withdrawal thresholds (r = -0.71, p = 0.049), and serum occludin level was positively correlated with grooming duration (r = 0.73, p = 0.032) in chronic NSAID group. Conclusion: Elevated serum occludin and LBP levels and brain IL-17 and HMGB1 levels indicate a possible role of leaky gut and inflammation in an MOH model in male rats. Additionally, a significant correlation between pain behavior and markers of inflammation and intestinal hyperpermeability, supports the role of inflammation and leaky gut in MOH pathophysiology.