Conjugated Nanoparticles for Solid Tumor Theranostics: Unraveling the Interplay of Known and Unknown Factors.

Cancer diagnoses have been increasing worldwide, and solid tumors are among the leading contributors to patient mortality, creating an enormous burden on the global healthcare system. Cancer is responsible for around 10.3 million deaths worldwide. Solid tumors are one of the most prevalent cancers observed in recent times. On the other hand, early diagnosis is a significant challenge that could save a person's life. Treatment with existing methods has pitfalls that limit the successful elimination of the disorder. Though nanoparticle-based imaging and therapeutics have shown a significant impact in healthcare, current methodologies for solid tumor treatment are insufficient. There are multiple complications associated with the diagnosis and management of solid tumors as well. Recently, surface-conjugated nanoparticles such as lipid nanoparticles, metallic nanoparticles, and quantum dots have shown positive results in solid tumor diagnostics and therapeutics in preclinical models. Other nanotheranostic material platforms such as plasmonic theranostics, magnetotheranostics, hybrid nanotheranostics, and graphene theranostics have also been explored. These nanoparticle theranostics ensure the appropriate targeting of tumors along with selective delivery of cargos (both imaging and therapeutic probes) without affecting the surrounding healthy tissues. Though they have multiple applications, nanoparticles still possess numerous limitations that need to be addressed in order to be fully utilized in the clinic. In this review, we outline the importance of materials and design strategies used to engineer nanoparticles in the treatment and diagnosis of solid tumors and how effectively each method overcomes the drawbacks of the current techniques. We also highlight the gaps in each material platform and how design considerations can address their limitations in future research directions.

Paneth cell dysfunction in radiation injury and radio-mitigation by human α-defensin 5.

Introduction: The mechanism underlying radiation-induced gut microbiota dysbiosis is undefined. This study examined the effect of radiation on the intestinal Paneth cell α-defensin expression and its impact on microbiota composition and mucosal tissue injury and evaluated the radio-mitigative effect of human α-defensin 5 (HD5). Methods: Adult mice were subjected to total body irradiation, and Paneth cell α-defensin expression was evaluated by measuring α-defensin mRNA by RT-PCR and α-defensin peptide levels by mass spectrometry. Vascular-to-luminal flux of FITC-inulin was measured to evaluate intestinal mucosal permeability and endotoxemia by measuring plasma lipopolysaccharide. HD5 was administered in a liquid diet 24 hours before or after irradiation. Gut microbiota was analyzed by 16S rRNA sequencing. Intestinal epithelial junctions were analyzed by immunofluorescence confocal microscopy and mucosal inflammatory response by cytokine expression. Systemic inflammation was evaluated by measuring plasma cytokine levels. Results: Ionizing radiation reduced the Paneth cell α-defensin expression and depleted α-defensin peptides in the intestinal lumen. α-Defensin down-regulation was associated with the time-dependent alteration of gut microbiota composition, increased gut permeability, and endotoxemia. Administration of human α-defensin 5 (HD5) in the diet 24 hours before irradiation (prophylactic) significantly blocked radiation-induced gut microbiota dysbiosis, disruption of intestinal epithelial tight junction and adherens junction, mucosal barrier dysfunction, and mucosal inflammatory response. HD5, administered 24 hours after irradiation (treatment), reversed radiation-induced microbiota dysbiosis, tight junction and adherens junction disruption, and barrier dysfunction. Furthermore, HD5 treatment also prevents and reverses radiation-induced endotoxemia and systemic inflammation. Conclusion: These data demonstrate that radiation induces Paneth cell dysfunction in the intestine, and HD5 feeding prevents and mitigates radiation-induced intestinal mucosal injury, endotoxemia, and systemic inflammation.

TRPV6 deficiency attenuates stress and corticosterone-mediated exacerbation of alcohol-induced gut barrier dysfunction and systemic inflammation.

Introduction: Chronic stress is co-morbid with alcohol use disorder that feedback on one another, thus impeding recovery from both disorders. Stress and the stress hormone corticosterone aggravate alcohol-induced intestinal permeability and liver damage. However, the mechanisms involved in compounding tissue injury by stress/corticosterone and alcohol are poorly defined. Here we explored the involvement of the TRPV6 channel in stress (or corticosterone) 3and alcohol-induced intestinal epithelial permeability, microbiota dysbiosis, and systemic inflammation. Methods: Chronic alcohol feeding was performed on adult wild-type and Trpv6-/- mice with or without corticosterone treatment or chronic restraint stress (CRS). The barrier function was determined by evaluating inulin permeability in vivo and assessing tight junction (TJ) and adherens junction (AJ) integrity by immunofluorescence microscopy. The gut microbiota composition was evaluated by 16S rRNA sequencing and metagenomic analyses. Systemic responses were assessed by evaluating endotoxemia, systemic inflammation, and liver damage. Results: Corticosterone and CRS disrupted TJ and AJ, increased intestinal mucosal permeability, and caused endotoxemia, systemic inflammation, and liver damage in wild-type but not Trpv6-/- mice. Corticosterone and CRS synergistically potentiated the alcohol-induced breakdown of intestinal epithelial junctions, mucosal barrier impairment, endotoxemia, systemic inflammation, and liver damage in wild-type but not Trpv6-/- mice. TRPV6 deficiency also blocked the effects of CRS and CRS-mediated potentiation of alcohol-induced dysbiosis of gut microbiota. Conclusions: These findings indicate an essential role of TRPV6 in stress, corticosterone, and alcohol-induced intestinal permeability, microbiota dysbiosis, endotoxemia, systemic inflammation, and liver injury. This study identifies TRPV6 as a potential therapeutic target for developing treatment strategies for stress and alcohol-associated comorbidity.

TRPV6 channel mediates alcohol-induced gut barrier dysfunction and systemic response.

Intestinal epithelial tight junction disruption is a primary contributing factor in alcohol-associated endotoxemia, systemic inflammation, and multiple organ damage. Ethanol and acetaldehyde disrupt tight junctions by elevating intracellular Ca2+. Here we identify TRPV6, a Ca2+-permeable channel, as responsible for alcohol-induced elevation of intracellular Ca2+, intestinal barrier dysfunction, and systemic inflammation. Ethanol and acetaldehyde elicit TRPV6 ionic currents in Caco-2 cells. Studies in Caco-2 cell monolayers and mouse intestinal organoids show that TRPV6 deficiency or inhibition attenuates ethanol- and acetaldehyde-induced Ca2+ influx, tight junction disruption, and barrier dysfunction. Moreover, Trpv6-/- mice are resistant to alcohol-induced intestinal barrier dysfunction. Photoaffinity labeling of 3-azibutanol identifies a histidine as a potential alcohol-binding site in TRPV6. The substitution of this histidine, and a nearby arginine, reduces ethanol-activated currents. Our findings reveal that TRPV6 is required for alcohol-induced gut barrier dysfunction and inflammation. Molecules that decrease TRPV6 function have the potential to attenuate alcohol-associated tissue injury.

Prevention and mitigation of alcohol-induced neuroinflammation by Lactobacillus plantarum by an EGF receptor-dependent mechanism.

Neuroinflammation is implicated in the pathogenesis of alcohol use disorders. We investigated the role of Gut-Brain interactions in alcohol-induced neuroinflammation by probiotic-mediated manipulation of intestinal dysbiosis in mice. Chronic ethanol feeding induced dysbiosis, as evidenced by an increase in Firmicutes/Bacteroidetes ratio and depletion of Lactobacillus species in the colon. Ethanol increased the levels of IL-1ß, IL-6, and TNFα in plasma and the mRNA for IL-1ß, IL-6, TNFα, and MCP1 genes in the cerebral cortex and hippocampus. Ethanol feeding increased inulin flux from the circulation into different brain regions, accompanied by the increase in TLR4 mRNA levels in the cerebral cortex and hippocampus. The immunofluorescence confocal microscopy showed that ethanol elevates the expression of microglial activation marker TMEM119 in the cerebral cortex. Feeding L. plantarum suppressed the ethanol-induced dysbiosis to some extent, as evidenced by attenuation of ethanol effects on Firmicutes/Bacteroidetes ratio and abundance of Lactobacillus spp. L. plantarum blocked ethanol-induced elevation of plasma cytokines, inulin permeability to the brain, mRNA for TLR4, IL-1ß, IL-6, TNFα, and MCP1 in brain regions, and the expression of TMEM119 in the cerebral cortex. The L. plantarum effect was absent in mice that express a dominant-negative EGFR, suggesting that the EGFR receptor plays an essential role in the protective effect of L. plantarum against ethanol-induced neuroinflammation. L. plantarum, when administered after chronic ethanol-induced injury, rescued the ethanol-induced systemic inflammation and neuroinflammation. This study demonstrates that L. plantarum in the gut prevents and mitigates ethanol-induced neuroinflammation by an EGFR-dependent mechanism.

Central role of intestinal epithelial glucocorticoid receptor in alcohol- and corticosterone-induced gut permeability and systemic response.

Corticosterone, the stress hormone, exacerbates alcohol-associated tissue injury, but the mechanism involved is unknown. We examined the role of the glucocorticoid receptor (GR) in corticosterone-mediated potentiation of alcohol-induced gut barrier dysfunction and systemic response. Hepatocyte-specific GR-deficient (GRΔHC ) and intestinal epithelial-specific GR-deficient (GRΔIEC ) mice were fed ethanol, combined with corticosterone treatment. Intestinal epithelial tight junction integrity, mucosal barrier function, microbiota dysbiosis, endotoxemia, systemic inflammation, liver damage, and neuroinflammation were assessed. Corticosterone potentiated ethanol-induced epithelial tight junction disruption, mucosal permeability, and inflammatory response in GRΔHC mouse colon; these effects of ethanol and corticosterone were absent in GRΔIEC mice. Gut microbiota compositions in ethanol-fed GRΔHC and GRΔIEC mice were similar to each other. However, corticosterone treatment in ethanol-fed mice shifted the microbiota composition to distinctly different directions in GRΔHC and GRΔIEC mice. Ethanol and corticosterone synergistically elevated the abundance of Enterobacteriaceae and Escherichia coli and reduced the abundance of Lactobacillus in GRΔHC mice but not in GRΔIEC mice. In GRΔHC mice, corticosterone potentiated ethanol-induced endotoxemia and systemic inflammation, but these effects were absent in GRΔIEC mice. Interestingly, ethanol-induced liver damage and its potentiation by corticosterone were observed in GRΔHC mice but not in GRΔIEC mice. GRΔIEC mice were also resistant to ethanol- and corticosterone-induced inflammatory response in the hypothalamus. These data indicate that the intestinal epithelial GR plays a central role in alcohol- and corticosterone-induced gut barrier dysfunction, microbiota dysbiosis, endotoxemia, systemic inflammation, liver damage, and neuroinflammation. This study identifies a novel target for potential therapeutic for alcohol-associated tissue injury.

Chronic stress and corticosterone exacerbate alcohol-induced tissue injury in the gut-liver-brain axis.

Alcohol use disorders are associated with altered stress responses, but the impact of stress or stress hormones on alcohol-associated tissue injury remain unknown. We evaluated the effects of chronic restraint stress on alcohol-induced gut barrier dysfunction and liver damage in mice. To determine whether corticosterone is the stress hormone associated with the stress-induced effects, we evaluated the effect of chronic corticosterone treatment on alcoholic tissue injury at the Gut-Liver-Brain (GLB) axis. Chronic restraint stress synergized alcohol-induced epithelial tight junction disruption and mucosal barrier dysfunction in the mouse intestine. These effects of stress on the gut were reproduced by corticosterone treatment. Corticosterone synergized alcohol-induced expression of inflammatory cytokines and chemokines in the colonic mucosa, and it potentiated the alcohol-induced endotoxemia and systemic inflammation. Corticosterone also potentiated alcohol-induced liver damage and neuroinflammation. Metagenomic analyses of 16S RNA from fecal samples indicated that corticosterone modulates alcohol-induced changes in the diversity and abundance of gut microbiota. In Caco-2 cell monolayers, corticosterone dose-dependently potentiated ethanol and acetaldehyde-induced tight junction disruption and barrier dysfunction. These data indicate that chronic stress and corticosterone exacerbate alcohol-induced mucosal barrier dysfunction, endotoxemia, and systemic alcohol responses. Corticosterone-mediated promotion of alcohol-induced intestinal epithelial barrier dysfunction and modulation of gut microbiota may play a crucial role in the mechanism of stress-induced promotion of alcohol-associated tissue injury at the GLB axis.

LPAR2 receptor activation attenuates radiation-induced disruption of apical junctional complexes and mucosal barrier dysfunction in mouse colon.

The tight junction (TJ) and barrier function of colonic epithelium is highly sensitive to ionizing radiation. We evaluated the effect of lysophosphatidic acid (LPA) and its analog, Radioprotein-1, on γ-radiation-induced colonic epithelial barrier dysfunction using Caco-2 and m-ICC12 cell monolayers in vitro and mice in vivo. Mice were subjected to either total body irradiation (TBI) or partial body irradiation (PBI-BM5). Intestinal barrier function was assessed by analyzing immunofluorescence localization of TJ proteins, mucosal inulin permeability, and plasma lipopolysaccharide (LPS) levels. Oxidative stress was analyzed by measuring protein thiol oxidation and antioxidant mRNA. In Caco-2 and m-ICC12 cell monolayers, LPA attenuated radiation-induced redistribution of TJ proteins, which was blocked by a Rho-kinase inhibitor. In mice, TBI and PBI-BM5 disrupted colonic epithelial tight junction and adherens junction, increased mucosal permeability, and elevated plasma LPS; TJ disruption by TBI was more severe in Lpar2-/- mice compared to wild-type mice. RP1, administered before or after irradiation, alleviated TBI and PBI-BM5-induced TJ disruption, barrier dysfunction, and endotoxemia accompanied by protein thiol oxidation and downregulation of antioxidant gene expression, cofilin activation, and remodeling of the actin cytoskeleton. These data demonstrate that LPAR2 receptor activation prevents and mitigates γ-irradiation-induced colonic mucosal barrier dysfunction and endotoxemia.

Summary of the 2018 Alcohol and Immunology Research Interest Group (AIRIG) meeting.

On January 26, 2018, the 23rd annual Alcohol and Immunology Research Interest Group (AIRIG) meeting was held at the University of Colorado Denver, Anschutz Medical Campus, Aurora, Colorado. The meeting consisted of plenary sessions with oral presentations and a poster presentation session. There were four plenary sessions that covered a wide range of topics relating to alcohol use: Alcohol and Liver Disease; Alcohol, Inflammation and Immune Response; Alcohol and Organ Injury; Heath Consequences and Alcohol Drinking. The meeting provided a forum for the presentation and discussion of novel research findings regarding alcohol use and immunology.

EGF receptor plays a role in the mechanism of glutamine-mediated prevention of alcohol-induced gut barrier dysfunction and liver injury.

Recent study indicated that glutamine prevents alcoholic tissue injury in mouse gut and liver. Here we investigated the potential role of Epidermal Growth Factor Receptor (EGFR) in glutamine-mediated prevention of ethanol-induced colonic barrier dysfunction, endotoxemia and liver damage. Wild-type and EGFR*Tg transgenic (expressing dominant negative EGFR) mice were fed 1-6% ethanol in Lieber-DeCarli diet. Gut permeability was measured by vascular-to-luminal flux of FITC-inulin, and junctional integrity assessed by confocal microscopy. Liver injury was evaluated by plasma transaminases, histopathology and triglyceride analyses. Glutamine effect on acetaldehyde-induced tight junction disruption was investigated in Caco-2 cell monolayers. Doxycycline-induced expression of EGFR* blocked glutamine-mediated prevention of ethanol-induced disruption of colonic epithelial tight junction, mucosal permeability and endotoxemia. Ethanol activated cofilin and disrupted actin cytoskeleton, which was blocked by glutamine in an EGFR-dependent mechanism. Ethanol down-regulated antioxidant gene expression and up-regulated cytokine and chemokine gene expression, which were blocked by glutamine in wild-type mice in the presence or absence of doxycycline, but not in EGFR*Tg mice in the presence of doxycycline. Histopathology, plasma transaminases, triglyceride and expression of chemokine and antioxidant genes indicated ethanol-induced liver damage, which were blocked by glutamine in an EGFR-dependent mechanism. Src kinase activity and extracellular ligand binding domain of EGFR are required for glutamine-mediated protection of barrier function in Caco-2 cell monolayers. Glutamine released metalloproteinases into the medium, and metalloproteinase inhibitors blocked glutamine-mediated protection of barrier function. Results demonstrate that EGFR plays an important role in glutamine-mediated prevention of alcoholic gut permeability, endotoxemia and liver damage.

Human Defensin-5 Blocks Ethanol and Colitis-Induced Dysbiosis, Tight Junction Disruption and Inflammation in Mouse Intestine.

Alcohol consumption has been shown to cause dysbiosis, but the mechanism involved in it is unknown. Recurrent colitis is known to induce expression of α-defensins in the colon, but the effect of alcohol consumption on it is not known. We investigated the effect of ethanol on α-defensin expression in the small intestine and colitis-induced expression in colon in mice. Furthermore, we evaluated the effect of human defensin-5 (HD5) on ethanol and colitis-induced gut barrier dysfunction and mucosal damage. Recurrent colitis was induced by feeding dextran sulfate sodium (DSS), 3 cycles of 5-days each with 15 days intervals, followed by 30-days remission. Ethanol was fed during the intervals and recovery in a liquid diet with or without HD5. Expression of α-defensins, tight junction (TJ) integrity and cytokine/chemokine expression were analyzed. Chronic ethanol feeding reduced α-defensin expression in the small intestine and colitis-induced defensin expression in the colon. HD5 attenuated the growth of enterotoxigenic Bacteriodes fragilis and E. coli, but had no effect on non-toxigenic Bacteriodes fragilis or probiotics, the Lactobacilli. Ethanol and colitis elevated Enterobacteriaceae, Firmicutes and Firmicutes to Bacteriodetes ratio in colonic mucosa. HD5 feeding attenuated ethanol and colitis-induced dysbiosis, disruption of intestinal epithelial TJ, mucosal inflammation, expression of pro-inflammatory cytokines and chemokines in the small intestine and colon, and endotoxemia. These results demonstrate that ethanol suppresses intestinal α-defensin expression, leading to dysbiosis, barrier dysfunction, inflammation and endotoxemia. HD5 feeding attenuates intestinal injury caused by ethanol and colitis, indicating that defensin expression is a potential target for treatment of alcoholic tissue injury and colitis.

Deletion of TLR-4 attenuates fetal alcohol exposure-induced gene expression and social interaction deficits.

Fetal alcohol spectrum disorders (FASD) are associated with social interaction behavior and gastrointestinal (GI) abnormalities. These abnormal behaviors and GI abnormalities overlap with autism spectrum disorder (ASD). We investigated the effect of fetal alcohol exposure (FAE) on social interaction deficits (hallmark of autism) in mice. Evidence indicates that exogenous lipopolysaccharide (LPS) administration during gestation induces autism-like behavior in the offspring. LPS regulates the expression of genes underlying differentiation, immune function, myelination, and synaptogenesis in fetal brain by the LPS receptor, TLR-4-dependent mechanism. In this study, we evaluated the role of TLR-4 in FAE-induced social behavior deficit. WT and TLR4-/- pregnant mice were fed Lieber-DeCarli liquid diet with or without ethanol. The control group was pair-fed with an isocaloric diet. Social behavior was tested in the adult offspring at postnatal day 60. Frontal cortex mRNA expression of autistic candidate genes (Ube3a, Gabrb3, Mecp2) and inflammatory cytokine genes (IL-1ß, IL-6, TNF-α) were measured by RT-qPCR. Adult male offspring of ethanol-fed WT dams showed low birth weight compared to offspring of pair-fed WT dams. However, their body weights at adulthood were greater compared to the body weights of offspring of pair-fed WT dams. There were no body weight differences in offspring of TLR4-/- dams. Social interaction deficit was observed only in male offspring of ethanol-fed WT dams, but it was not observed in both male and female offspring of ethanol-fed TLR4-/- dams. Expressions of autism candidate genes, Gabrb3 and Ube3a, were elevated, while that of the Mecp2 gene was suppressed in the frontal cortex of male, but not female, offspring of ethanol-fed WT mice. The expressions of inflammatory cytokine genes, IL-1ß, IL-6, and TNF-α, were also significantly increased in the frontal cortex of male, but not female, offspring of ethanol-fed dams. The changes in the expression of autistic and cytokine genes were unaffected in the offspring of ethanol-fed TLR4-/- dams. These data also indicate that TLR4 mediates FAE-induced changes in social interactions and gene expression in brain, suggesting that ethanol-induced LPS absorption from the maternal gut may be involved in gene expression changes in the fetal brain.

Prevention and treatment of secretory diarrhea by the lysophosphatidic acid analog Rx100.

IMPACT STATEMENT: A critical barrier in treating diarrheal disease is easy-to-use effective treatments. Rx100 is a first in class, novel small molecule that has shown efficacy after both subcutaneous and oral administration in a mouse cholera-toxin- and Citrobacter rodentium infection-induced diarrhea models. Our findings indicate that Rx100 a metabolically stable analog of the lipid mediator lysophosphatidic acid blocks activation of CFTR-mediated secretion responsible for fluid discharge in secretory diarrhea. Rx100 represents a new treatment modality which does not directly block CFTR but attenuates its activation by bacterial toxins. Our results provide proof-of-principle that Rx100 can be developed for use as an effective oral or injectable easy-to-use drug for secretory diarrhea which could significantly improve care by eliminating the need for severely ill patients to regularly consume large quantities of oral rehydration therapies and offering options for pediatric patients.

Lactobacillus plantarum prevents and mitigates alcohol-induced disruption of colonic epithelial tight junctions, endotoxemia, and liver damage by an EGF receptor-dependent mechanism.

Pathogenesis of alcohol-related diseases such as alcoholic hepatitis involves gut barrier dysfunction, endotoxemia, and toxin-mediated cellular injury. Here we show that Lactobacillus plantarum not only blocks but also mitigates ethanol (EtOH)-induced gut and liver damage in mice. L. plantarum blocks EtOH-induced protein thiol oxidation, and down-regulation of antioxidant gene expression in colon L. plantarum also blocks EtOH-induced expression of TNF-α, IL-1ß, IL-6, monocyte chemotactic protein 1 ( MCP1), C-X-C motif chemokine ligand ( CXCL)1, and CXCL2 genes in colon. Epidermal growth factor receptor (EGFR) signaling mediates the L. plantarum-mediated protection of tight junctions (TJs) and barrier function from acetaldehyde, the EtOH metabolite, in Caco-2 cell monolayers. In mice, doxycycline-mediated expression of dominant negative EGFR blocks L. plantarum-mediated prevention of EtOH-induced TJ disruption, mucosal barrier dysfunction, oxidative stress, and inflammatory response in colon. L. plantarum blocks EtOH-induced endotoxemia as well as EtOH-induced pathologic lesions, triglyceride deposition, oxidative stress, and inflammatory responses in the liver by an EGFR-dependent mechanism. L. plantarum treatment after injury accelerated recovery from EtOH-induced TJ, barrier dysfunction, oxidative stress, and inflammatory response in colon, endotoxemia, and liver damage. Results demonstrate that L. plantarum has both preventive and therapeutic values in treatment of alcohol-induced tissue injury, particularly in alcoholic hepatitis.-Shukla, P. K., Meena, A. S., Manda, B., Gomes-Solecki, M., Dietrich, P., Dragatsis, I., Rao, R. Lactobacillus plantarum prevents and mitigates alcohol-induced disruption of colonic epithelial tight junctions, endotoxemia, and liver damage by an EGF receptor-dependent mechanism.

Phosphorylation hotspot in the C-terminal domain of occludin regulates the dynamics of epithelial junctional complexes.

The apical junctional complex (AJC), which includes tight junctions (TJs) and adherens junctions (AJs), determines the epithelial polarity, cell-cell adhesion and permeability barrier. An intriguing characteristic of a TJ is the dynamic nature of its multiprotein complex. Occludin is the most mobile TJ protein, but its significance in TJ dynamics is poorly understood. On the basis of phosphorylation sites, we distinguished a sequence in the C-terminal domain of occludin as a regulatory motif (ORM). Deletion of ORM and expression of a deletion mutant of occludin in renal and intestinal epithelia reduced the mobility of occludin at the TJs. ORM deletion attenuated Ca2+ depletion, osmotic stress and hydrogen peroxide-induced disruption of TJs, AJs and the cytoskeleton. The double point mutations T403A/T404A, but not T403D/T404D, in occludin mimicked the effects of ORM deletion on occludin mobility and AJC disruption by Ca2+ depletion. Both Y398A/Y402A and Y398D/Y402D double point mutations partially blocked AJC disruption. Expression of a deletion mutant of occludin attenuated collective cell migration in the renal and intestinal epithelia. Overall, this study reveals the role of ORM and its phosphorylation in occludin mobility, AJC dynamics and epithelial cell migration.

Calcium-mediated oxidative stress: a common mechanism in tight junction disruption by different types of cellular stress.

The role of reactive oxygen species (ROS) in osmotic stress, dextran sulfate sodium (DSS) and cyclic stretch-induced tight junction (TJ) disruption was investigated in Caco-2 cell monolayers in vitro and restraint stress-induced barrier dysfunction in mouse colon in vivo Live cell imaging showed that osmotic stress, cyclic stretch and DSS triggered rapid production of ROS in Caco-2 cell monolayers, which was blocked by depletion of intracellular Ca2+ by 1,2-bis-(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid. Knockdown of CaV1.3 or TRPV6 channels blocked osmotic stress and DSS-induced ROS production and attenuated TJ disruption and barrier dysfunction. N-Acetyl l-cysteine (NAC) and l-NG-Nitroarginine methyl ester (l-NAME) blocked stress-induced TJ disruption and barrier dysfunction. NAC and l-NAME also blocked stress-induced activation of c-Jun N-terminal kinase (JNK) and c-Src. ROS was colocalized with the mitochondrial marker in stressed cells. Cyclosporin A blocked osmotic stress and DSS-induced ROS production, barrier dysfunction, TJ disruption and JNK activation. Mitochondria-targeted Mito-TEMPO blocked osmotic stress and DSS-induced barrier dysfunction and TJ disruption. Chronic restraint stress in mice resulted in the elevation of intracellular Ca2+, activation of JNK and c-Src, and disruption of TJ in the colonic epithelium. Furthermore, corticosterone administration induced JNK and c-Src activation, TJ disruption and protein thiol oxidation in colonic mucosa. The present study demonstrates that oxidative stress is a common signal in the mechanism of TJ disruption in the intestinal epithelium by different types of cellular stress in vitro and bio behavioral stress in vivo.

Calcium Channels and Oxidative Stress Mediate a Synergistic Disruption of Tight Junctions by Ethanol and Acetaldehyde in Caco-2 Cell Monolayers.

Ethanol is metabolized into acetaldehyde in most tissues. In this study, we investigated the synergistic effect of ethanol and acetaldehyde on the tight junction integrity in Caco-2 cell monolayers. Expression of alcohol dehydrogenase sensitized Caco-2 cells to ethanol-induced tight junction disruption and barrier dysfunction, whereas aldehyde dehydrogenase attenuated acetaldehyde-induced tight junction disruption. Ethanol up to 150 mM did not affect tight junction integrity or barrier function, but it dose-dependently increased acetaldehyde-mediated tight junction disruption and barrier dysfunction. Src kinase and MLCK inhibitors blocked this synergistic effect of ethanol and acetaldehyde on tight junction. Ethanol and acetaldehyde caused a rapid and synergistic elevation of intracellular calcium. Calcium depletion by BAPTA or Ca2+-free medium blocked ethanol and acetaldehyde-induced barrier dysfunction and tight junction disruption. Diltiazem and selective knockdown of TRPV6 or CaV1.3 channels, by shRNA blocked ethanol and acetaldehyde-induced tight junction disruption and barrier dysfunction. Ethanol and acetaldehyde induced a rapid and synergistic increase in reactive oxygen species by a calcium-dependent mechanism. N-acetyl-L-cysteine and cyclosporine A, blocked ethanol and acetaldehyde-induced barrier dysfunction and tight junction disruption. These results demonstrate that ethanol and acetaldehyde synergistically disrupt tight junctions by a mechanism involving calcium, oxidative stress, Src kinase and MLCK.

Chronic ethanol feeding promotes azoxymethane and dextran sulfate sodium-induced colonic tumorigenesis potentially by enhancing mucosal inflammation.

BACKGROUND: Alcohol consumption is one of the major risk factors for colorectal cancer. However, the mechanism involved in this effect of alcohol is unknown. METHODS: We evaluated the effect of chronic ethanol feeding on azoxymethane and dextran sulfate sodium (AOM/DSS)-induced carcinogenesis in mouse colon. Inflammation in colonic mucosa was assessed at a precancerous stage by evaluating mucosal infiltration of neutrophils and macrophages, and analysis of cytokine and chemokine gene expression. RESULTS: Chronic ethanol feeding significantly increased the number and size of polyps in colon of AOM/DSS treated mice. Confocal microscopic and immunoblot analyses showed a significant elevation of phospho-Smad, VEGF and HIF1α in the colonic mucosa. RT-PCR analysis at a precancerous stage indicated that ethanol significantly increases the expression of cytokines IL-1α, IL-6 and TNFα, and the chemokines CCL5/RANTES, CXCL9/MIG and CXCL10/IP-10 in the colonic mucosa of AOM/DSS treated mice. Confocal microscopy showed that ethanol feeding induces a dramatic elevation of myeloperoxidase, Gr1 and CD68-positive cells in the colonic mucosa of AOM/DSS-treated mice. Ethanol feeding enhanced AOM/DSS-induced suppression of tight junction protein expression and elevated cell proliferation marker, Ki-67 in the colonic epithelium. CONCLUSION: This study demonstrates that chronic ethanol feeding promotes colonic tumorigenesis potentially by enhancing inflammation and elevation of proinflammatory cytokines and chemokines.

Rapid disruption of intestinal epithelial tight junction and barrier dysfunction by ionizing radiation in mouse colon in vivo: protection by N-acetyl-l-cysteine.

The goals of this study were to evaluate the effects of ionizing radiation on apical junctions in colonic epithelium and mucosal barrier function in mice in vivo. Adult mice were subjected to total body irradiation (4 Gy) with or without N-acetyl-l-cysteine (NAC) feeding for 5 days before irradiation. At 2-24 h postirradiation, the integrity of colonic epithelial tight junctions (TJ), adherens junctions (AJ), and the actin cytoskeleton was assessed by immunofluorescence microscopy and immunoblot analysis of detergent-insoluble fractions for TJ and AJ proteins. The barrier function was evaluated by measuring vascular-to-luminal flux of fluorescein isothiocyanate (FITC)-inulin in vivo and luminal-to-mucosal flux in vitro. Oxidative stress was evaluated by measuring protein thiol oxidation. Confocal microscopy showed that radiation caused redistribution of occludin, zona occludens-1, claudin-3, E-cadherin, and ß-catenin, as well as the actin cytoskeleton as early as 2 h postirradiation, and this effect was sustained for at least 24 h. Feeding NAC before irradiation blocked radiation-induced disruption of TJ, AJ, and the actin cytoskeleton. Radiation increased mucosal permeability to inulin in colon, which was blocked by NAC feeding. The level of reduced-protein thiols in colon was depleted by radiation with a concomitant increase in the level of oxidized-protein thiol. NAC feeding blocked the radiation-induced protein thiol oxidation. These data demonstrate that radiation rapidly disrupts TJ, AJ, and the actin cytoskeleton by an oxidative stress-dependent mechanism that can be prevented by NAC feeding.

Occludin deficiency promotes ethanol-induced disruption of colonic epithelial junctions, gut barrier dysfunction and liver damage in mice.

BACKGROUND: Disruption of epithelial tight junctions (TJ), gut barrier dysfunction and endotoxemia play crucial role in the pathogenesis of alcoholic tissue injury. Occludin, a transmembrane protein of TJ, is depleted in colon by alcohol. However, it is unknown whether occludin depletion influences alcoholic gut and liver injury. METHODS: Wild type (WT) and occludin deficient (Ocln(-/-)) mice were fed 1-6% ethanol in Lieber-DeCarli diet. Gut permeability was measured by vascular-to-luminal flux of FITC-inulin. Junctional integrity was analyzed by confocal microscopy. Liver injury was assessed by plasma transaminase, histopathology and triglyceride analyses. The effect of occludin depletion on acetaldehyde-induced TJ disruption was confirmed in Caco-2 cell monolayers. RESULTS: Ethanol feeding significantly reduced body weight gain in Ocln(-/-) mice. Ethanol increased inulin permeability in colon of both WT and Ocln(-/-) mice, but the effect was 4-fold higher in Ocln(-/-) mice. The gross morphology of colonic mucosa was unaltered, but ethanol disrupted the actin cytoskeleton, induced redistribution of occludin, ZO-1, E-cadherin and ß-catenin from the junctions and elevated TLR4, which was more severe in Ocln(-/-) mice. Occludin knockdown significantly enhanced acetaldehyde-induced TJ disruption and barrier dysfunction in Caco-2 cell monolayers. Ethanol significantly increased liver weight and plasma transaminase activity in Ocln(-/-) mice, but not in WT mice. Histological analysis indicated more severe lesions and fat deposition in the liver of ethanol-fed Ocln(-/-) mice. Ethanol-induced elevation of liver triglyceride was also higher in Ocln(-/-) mice. CONCLUSION: This study indicates that occludin deficiency increases susceptibility to ethanol-induced colonic mucosal barrier dysfunction and liver damage in mice.