Altered Tight Junction Protein Research Articles

MYO5B levels are linked to microRNAs and UNC45A in colorectal cancer

Background: Colorectal cancer (CRC) is currently the third most prevalent cancer worldwide and ranks as the second deadliest. Colon cancer is characterized by disruption in cellular structure and polarity, processes that are commonly regulated by cell traffcking. The molecular motor Myosin 5b (Myo5b) is a key component of intracellular traffcking in the intestinal epithelium. Myo5b moves along F-actin tracks to transport cargo to and from the apical membrane. When Myo5b function is impaired, it leads to significant changes in gastrointestinal epithelial cells, including hyper-proliferation, alterations in tight junction proteins, and reduced differentiation. For Myo5b to function properly it must be properly folded; a process regulated by the myosin chaperone Unc45a. Recent work demonstrates that Myo5b levels are decreased in gastric cancer and during the progression of colorectal tumors. However, the mechanism driving this altered Myo5b expression in colorectal cancer remains unknown. We hypothesize that microRNAs regulate the expression of Unc45a, which subsequently impacts Myo5b function, promoting colorectal cancer. Methods and Results: Analysis of high-throughput RNA-sequencing data from the Cancer Genome Atlas (TCGA) revealed hyper-methylation of the Myo5b gene in CRC tumors (n=286) compared to controls (n=41). Findings in gastric cancer have shown that hyper-methylation leads to the silencing of Myo5b gene expression. Consistent with these findings, we observe a significant reduction in Myo5b gene expression in CRC tumors. Immunostaining of CRC tumor arrays and analysis from the Clinical Proteomic Tumor Analysis Consortium (CPTAC) demonstrated a corresponding decrease in Myo5b protein levels. For comparison, we examined Myo5b levels in normal human colonic organoids and the colon cancer cell lines T84,HT-29, HT29-MTX, HCT-8 and HCT-116. Using qPCR, we confirmed a significant reduction in Myo5b gene expression in cancer cells compared to human colonic organoids. We postulated that even if Myo5b was expressed in CRC, it might not function properly if Unc45a function was perturbed. Using the same databases, we found that while Unc45a gene expression levels remain unchanged, its protein levels were significantly reduced. Analysis of the miRMap data reveled multiple microRNAs that could target Unc45a. The top 10 Unc45a-targeting microRNAs were all found to be elevated in CRC compared to normal control tissue, suggesting that microRNAs may lead to reduced Unc45a protein levels. The functional importance of decreased Myo5b in CRC was underscored by the observation that CRC patients with lower levels of Myo5b had reduced survival compared to those CRC patients with higher levels of Myo5b. Additionally, the loss of Myo5b correlated with increased ribosome biogenesis, a key driver of tumor proliferation. Conclusions: These findings indicate that reduced Myo5b in CRC may be attributed to gene methylation and impaired protein folding by Unc45a. The loss of Myo5b holds significance, as it is associated with poorer prognosis and linked with cancer progression. National Diabetes and Digestive and Kidney Diseases Grant R43 DK109820, RO1 DK048370, KO1 DK121869, MUSC start up funds, Histochemical 2022 Cornerstone Grant, and NIDDK T32. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Octreotide attenuates intestinal barrier damage by maintaining basal autophagy in Caco2 cells.

The intestinal mucosal barrier is of great importance for maintaining the stability of the internal environment, which is closely related to the occurrence and development of intestinal inflammation. Octreotide (OCT) has potential applicable clinical value for treating intestinal injury according to previous studies, but the underlying molecular mechanisms have remained elusive. This article is based on a cell model of inflammation induced by lipopolysaccharide (LPS), aiming to explore the effects of OCT in protecting intestinal mucosal barrier function. A Cell Counting Kit‑8 assay was used to determine cell viability and evaluate the effectiveness of OCT. Gene silencing technology was used to reveal the mediated effect of somatostatin receptor 2 (SSTR2). The changes in intestinal permeability were detected through trans‑epithelial electrical resistance and fluorescein isothiocyanate‑dextran 4 experiments, and the alterations in tight junction proteins were detected using immunoblotting and reverse transcription fluorescence‑quantitative PCR technology. Autophagosomes were observed by electron microscopy and the dynamic changes of the autophagy process were characterized by light chain (LC)3‑II/LC3‑I conversion and autophagic flow. The results indicated that SSTR2‑dependent OCT can prevent the decrease in cell activity. After LPS treatment, the permeability of monolayer cells decreased and intercellular tight junctions were disrupted, resulting in a decrease in tight junction protein zona occludens 1 in cells. The level of autophagy‑related protein LC3 was altered to varying degrees at different times. These abnormal changes gradually returned to normal levels after the combined application of LPS and SSTR2‑dependent OCT, confirming the role of OCT in protecting intestinal barrier function. These experimental results suggest that OCT maintains basal autophagy and cell activity mediated by SSTR2 in intestinal epithelial cells, thereby preventing the intestinal barrier dysfunction in inflammation injury.

Bergenin ameliorates chemotherapy-induced neuropathic pain in rats by modulating TRPA1/TRPV1/NR2B signalling

Chemotherapy-induced neuropathic pain (CINP) is one of the most prominent and incapacitating complication associated with chemotherapeutic regimens. The exact mechanisms underlying CINP are not fully understood yet, which hampers the development of effective therapeutics. The current study has been designed to investigate the effect of bergenin on CINP and dissect the underlying cellular and molecular mechanisms. Behavioural responsiveness assays were conducted in rats before and after CINP induction and at different time points post-bergenin treatment. We also measured alterations in tight junction proteins, pro-inflammatory cytokines, microglia activity, transient receptor potential (TRP) channels (TRPV1, TRPA1 and TRPM8) and N-methyl-D-aspartate receptor subtype 2 (NR2B) in dorsal root ganglion (DRG) and spinal tissues of neuropathic rats. Bergenin treatment leads to a significant and dose-dependent reduction in evoked and spontaneous ongoing pain without causing central side effects in neuropathic rats. Furthermore, treatment with bergenin and gabapentin did not affect the baseline pain threshold in healthy, non-chemotherapy-treated rats, as evaluated through tail-flick and tail-clip assays. Chemotherapy administration leads to a significant activation of TRP channels, concurrent with microglial activation, disruption of spinal cord tight junction proteins, and subsequent infiltration of pro-inflammatory cytokines, as well as NR2B activation. Notably, bergenin treatment effectively reversed all of these alterations, with the exception of TRPM8, in both the DRG and spinal cord of neuropathic rats. Findings from the present study suggests that bergenin mitigates neuropathic pain by modulating the TRPA1/TRPV1/NR2B signalling and presents a promising therapeutic avenue for the treatment of chemotherapy-induced neuropathic pain.

Microvascular blood-brain barrier alterations in isolated brain capillaries of mice over-expressing alpha-synuclein (Thy1-aSyn line 61)

Dysfunction of the blood-brain barrier (BBB) is suggested to play a critical role in the pathological mechanisms of Parkinson's disease (PD). PD-related pathology such as alpha-synuclein accumulation and inflammatory processes potentially affect the integrity of the BBB early in disease progression, which in turn may alter the crosstalk of the central and peripheral immune response. Importantly, BBB dysfunction could also affect drug response in PD. Here we analyzed microvascular changes in isolated brain capillaries and brain sections on a cellular and molecular level during disease progression in an established PD mouse model that overexpresses human wild-type alpha-synuclein (Thy1-aSyn, line 61). BBB alterations observed in Thy1-aSyn mice included reduced vessel density, reduced aquaporin-4 coverage, reduced P-glycoprotein expression, increased low-density lipoprotein receptor-related protein 1 expression, increased pS129-alpha-synuclein deposition, and increased adhesion protein and matrix metalloprotease expression together with alterations in tight junction proteins. Striatal capillaries presented with more dysregulated BBB integrity markers compared to cortical capillaries. These alterations of BBB integrity lead, however, not to an overt IgG leakage in brain parenchyma. Our data reveals intricate alterations in key proteins of BBB function together with histological evidence for altered structure of the brain vasculature. Thy1-aSyn mice represent a useful model to investigate therapeutic targeting of BBB alterations in synucleinopathies.

Regulation of tight junction proteins and cell death by peroxisome proliferator-activated receptor γ agonist in brainstem of hypertensive rats.

The decrease in tight junction proteins and their adapter proteins in the hypertensive brain is remarkable. Here, we aimed to investigate tight junction proteins and peroxisome proliferator-activated receptor (PPARγ) activation as well as inflammation factors and cell death proteins in the brainstem of hypertension models, namely spontaneously hypertensive rats (SHR) and borderline hypertensive rats (BHR). At first, SHR and BHR groups were treated with PPARγ agonist, pioglitazone. Then, occludin, claudin-1, claudin-2, claudin-12, ZO-1, and NF-κB p65 gene expression levels; pIKKβ, NF-κB p65, TNF, IL-1β, caspase-3, caspase-9 levels, and PARP-1 cleavage were evaluated. Significantly lower pIKKβ, NF-κB p65, TNF, and IL-1β levels were measured in pioglitazone-treated SHR. Results from this study confirm higher occludin (1.35-fold), claudin-2 (7.45-fold), claudin-12 (1.12-fold), and NF-κB p65 subunit (4.76-fold) expressions in the BHR group when compared to the SHR group. Pioglitazone was found effective in terms of regulating gene expression in SHR. Pioglitazone significantly increased occludin (8.17-fold), claudin-2 (2.41-fold), and claudin-12 (1.85-fold) mRNA levels, which were accompanied by decreased cleaved caspase-3, caspase-9 levels, PARP-1 activation, and proinflammatory factor levels in SHR (p ˂ 0.05). Our work has led us to conclude that alterations in tight junction proteins, particularly occludin, and cell death parameters in the brainstem following PPARγ activation may contribute to neuroprotection in essential hypertension.

Host Gut-Resistome and Gut-Immune Axes are the key to Non-cholera Vibrioinfection severity in aging and heat stress

Abstract Global incidences of periodic heat waves and exposures are on the rise and that also have impacted non-cholera Vibrioabundance in coastal waters. We hypothesized that aging and a parallel heat stress (HS) may potentiate the risk of non-cholera Vibrioinfections in the host and exacerbate the pathology. To study the underlying pathological changes in possible co-exposure of HS and subsequent non-cholera Vibriochallenge, an aging mouse model was used. Mice were exposed to HS at 40°C for three hours/day for 15 days. This was followed by a Vibrio vulnificuschallenge 24 hours post HS exposure. Results showed that HS exposure decreased the diversity of the host gut microbiome and altered the relative abundance of many important taxa as assessed by Whole Meta-Genome Sequencing (WMS). Gut leaching was evident via the alteration of tight junction proteins and concomitant endotoxemia was evident. Decrease in abundance of Akkermansia muciniphila, a species well known for protection of gut barrier integrity was observed. Serum CRP levels, a clinical indicator of septicemia was elevated in the HS mice following Vibrio vulnificusinfection, over and above the Vibrio-only or HS-only group. Significant amplification of gut leaching, gastrointestinal and systemic inflammation (IL-1β, IL-6, TNF-α), T-cell senescence and altering IgA levels were also noted in the HS+ Vibriogroup. WMS also showed an altered host resistome signature and abundance of genes harboring the antimicrobial resistance to tetracycline group of antibiotics, a common antibiotic used for treatment of non-cholera Vibrioinfections. In summary, HS augmented susceptibility to non-cholera vibriosis via host variability in resistome patterns and altered gut-immune mediators. This study was supported by NIH/NIEHS Grant 1P01ES028942-01#Project 4 Toxicology awarded to Dr. S. Chatterjee.

MYO5B levels are linked to microRNAs and UNC45A in colorectal cancer

Background: Colorectal cancer (CRC) is currently the third most common cancer worldwide and the second most deadly cancer. CRC is characterized by the loss of cellular architecture and altered polarity; features which can be controlled by cell-trafficking. In the intestine key cell trafficking pathways are controlled by Myosin 5b (MYO5B). Loss of functional MYO5B leads to profound changes in gastrointestinal epithelial cells including increased proliferation, alterations in tight junction proteins, and decreased differentiation. For MYO5B to be functional, it must be folded into the correct structure. This folding is accomplished by the chaperone UNC45A. Recent work indicates that MYO5B is decreased in gastric cancer and during colorectal tumor progression. However, the mechanism resulting in altered MYO5B expression in colorectal cancer is currently unknown. Hypothesis: We hypothesize that microRNAs regulate the expression of UNC45A which in turn can impact MYO5B function to promote colorectal cancer. Methods & Results: Analysis of high-throughput RNA-sequencing data in the Cancer Genome Atlas revealed that the MYO5B gene was hyper-methylated in CRC tumors (n=286) compared to controls (n=41). In gastric cancer, hyper-methylation leads to silencing of MYO5B gene expression. Consistent with findings in gastric cancer, we found that MYO5B gene expression was significantly reduced in CRC tumors. Immunostaining of CRC tumor arrays and analysis of the Clinical Proteomic Tumor Analysis Consortium demonstrated that MYO5B was also reduced at the protein level. For comparison, we examined MYO5B levels in normal human colonic organoid monolayers, immortalized human colon NCM-460 cells, and colon cancer cell lines T84 and HT29-MTX cells. By qPCR, we found that MYO5B gene expression was significantly reduced in the cancer cells compared to the human colonic organoids, confirming our database analysis. We reasoned that even if MYO5B was generated in CRC, it might not be functional if UNC45A was not present. Using the same databases, we found that UNC45A levels were unchanged at the level of gene expression, but significantly reduced at the level of protein. Analysis of miRMap data revealed that multiple microRNAs can target UNC45A. The top 10 UN45A-targeting microRNAs were all found to be increased in CRC compared to normal control tissue; suggesting that UNC45A protein may be diminished due to microRNA mediated cleavage. The functional significance of decreased MYO5B in CRC was highlighted by the fact that patients with lower levels of MYO5B had decreased survival compared to those with higher levels of MYO5B. Additionally, loss of MYO5B correlated with increased ribosome biogenesis; a key driver of tumor proliferation. Conclusions: These data indicate the MYO5B is likely decreased in CRC due to gene methylation and improper protein folding by UNC45A. Loss of MYO5B is significant since it is associated with a worse prognosis and c This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Candida tropicalis Infection Modulates the Gut Microbiome and Confers Enhanced Susceptibility to Colitis in Mice.

Background & AimsWe previously showed that abundance of Candida tropicalis is significantly greater in Crohn’s disease patients compared with first-degree relatives without Crohn’s disease. The aim of this study was to determine the effects and mechanisms of action of C tropicalis infection on intestinal inflammation and injury in mice.MethodsC57BL/6 mice were inoculated with C tropicalis, and colitis was induced by administration of dextran sodium sulfate in drinking water. Disease severity and intestinal permeability subsequently were evaluated by endoscopy, histology, quantitative reverse-transcription polymerase chain reaction, as well as 16S ribosomal RNA and NanoString analyses (NanoString Technologies, Seattle, WA).ResultsInfected mice showed more severe colitis, with alterations in gut mucosal helper T cells (Th)1 and Th17 cytokine expression, and an increased frequency of mesenteric lymph node–derived group 2 innate lymphoid cells compared with uninfected controls. Gut microbiome composition, including changes in the mucin-degrading bacteria, Akkermansia muciniphila and Ruminococcus gnavus, was altered significantly, as was expression of several genes affecting intestinal epithelial homeostasis in isolated colonoids, after C tropicalis infection compared with uninfected controls. In line with these findings, fecal microbiome transplantation of germ-free recipient mice using infected vs uninfected donors showed altered expression of several tight-junction proteins and increased susceptibility to dextran sodium sulfate–induced colitis.ConclusionsC tropicalis induces dysbiosis that involves changes in the presence of mucin-degrading bacteria, leading to altered tight junction protein expression with increased intestinal permeability and followed by induction of robust Th1/Th17 responses, which ultimately lead to an accelerated proinflammatory phenotype in experimental colitic mice.

Effect of Xiaoyaosan on Colon Morphology and Intestinal Permeability in Rats With Chronic Unpredictable Mild Stress.

PurposeIn our present study, a rat depression model induced by 6 weeks of chronic unpredictable mild stress (CUMS) was established, and we investigated how Xiaoyaosan affects the intestinal permeability of depressed rats and alterations in tight-junction proteins (TJs) involved in this process.MethodsThe rat depression model was established using CUMS for 6 consecutive weeks. A total of 40 healthy male Sprague-Dawley rats were randomly sorted into four groups: the control group, CUMS group, Xiaoyaosan group, and fluoxetine group. All groups, excluding the control group, were subjected to the 6-week CUMS program to generate the depression model. Body weight, food intake, and behaviors were observed during the modeling period. Histopathological alterations of colon tissue were evaluated by hematoxylin-eosin staining (H&E), and mucus-containing goblet cells were detected by periodic acid-Schiff (PAS) staining. The ultrastructural morphology of colonic mucosa was observed by transmission electron microscopy. Furthermore, immunohistochemistry (IHC) and quantitative reverse transcription polymerase chain reaction (qRT-PCR) were used to determine the expression of TJs. The concentrations of 5-hydroxytryptamine (5-HT) in the hypothalamus and colon were also assessed using enzyme-linked immunosorbent assay (ELISA).ResultsTreatment of depressed rats with Xiaoyaosan alleviated depression-like behaviors as demonstrated by increases in the total distance traveled, the number of entries into the central area in the open field test, the duration spent in the central area, and sucrose preference. Xiaoyaosan treatment also increased body weight gain and food intake in depressed rats. Moreover, Xiaoyaosan treatment effectively improved the colonic pathological and ultrastructural changes, upregulated the expression of ZO-1, occludin, and claudin-1 in the colon, and increased 5-HT levels in the hypothalamus and colonic mucosa.ConclusionsXiaoyaosan treatment attenuates depression-like behaviors caused by CUMS and ameliorates CUMS-induced abnormal intestinal permeability, which may be associated with the expression of TJs. These results suggest that Xiaoyaosan exerts an antidepressant effect that may be related to an improvement of intestinal barrier function via the brain-gut axis.

Idiopathic pulmonary fibrosis is associated with tight junction protein alterations

Idiopathic pulmonary fibrosis (IPF) is a chronic disease characterized by an abnormal healing response to injury of the alveolar epithelium. Tight junctions provide a physical barrier at the apical intercellular space between epithelial cells and regulate paracellular flux. While tight junction alterations are known to contribute to barrier dysfunction in a number of disease states, the role of tight junction proteins in IPF is poorly defined. To determine a potential role for tight junction protein alterations in IPF, we performed immunohistochemical staining for tight junction proteins ZO-1, occludin, claudin-2, claudin-3, claudin-4, claudin-5, and claudin-18. Staining intensity and localization were compared between IPF and control lung tissues. IPF was associated with type II pneumocyte hyperplasia and altered tight junction protein expression. While there was no difference in the expression of ZO-1, claudin-3, or claudin-5, between IPF and normal control, there was an overall increase in claudin-2 expression in bronchiolar and alveolar epithelium and a decrease in claudin-4 expression in type II pneumocytes. There was also increased occludin and decreased claudin-18 expression in pneumocytes overlying fibroblastic foci. These findings suggest that epithelial barrier alterations may be important to the pathogenesis of IPF.

Dysbiosis-Associated Enteric Glial Cell Immune-Activation and Redox Imbalance Modulate Tight Junction Protein Expression in Gulf War Illness Pathology.

About 14% of veterans who suffer from Gulf war illness (GWI) complain of some form of gastrointestinal disorder but with no significant markers of clinical pathology. Our previous studies have shown that exposure to GW chemicals resulted in altered microbiome which was associated with damage associated molecular pattern (DAMP) release followed by neuro and gastrointestinal inflammation with loss of gut barrier integrity. Enteric glial cells (EGC) are emerging as important regulators of the gastrointestinal tract and have been observed to change to a reactive phenotype in several functional gastrointestinal disorders such as IBS and IBD. This study is aimed at investigating the role of dysbiosis associated EGC immune-activation and redox instability in contributing to observed gastrointestinal barrier integrity loss in GWI via altered tight junction protein expression. Using a mouse model of GWI and in vitro studies with cultured EGC and use of antibiotics to ensure gut decontamination we show that exposure to GW chemicals caused dysbiosis associated change in EGCs. EGCs changed to a reactive phenotype characterized by activation of TLR4-S100β/RAGE-iNOS pathway causing release of nitric oxide and activation of NOX2 since gut sterility with antibiotics prevented this change. The resulting peroxynitrite generation led to increased oxidative stress that triggered inflammation as shown by increased NLRP-3 inflammasome activation and increased cell death. Activated EGCs in vivo and in vitro were associated with decrease in tight junction protein occludin and selective water channel aquaporin-3 with a concomitant increase in Claudin-2. The tight junction protein levels were restored following a parallel treatment of GWI mice with a TLR4 inhibitor SsnB and butyric acid that are known to decrease the immunoactivation of EGCs. Our study demonstrates that immune-redox mechanisms in EGC are important players in the pathology in GWI and may be possible therapeutic targets for improving outcomes in GWI symptom persistence.

Environmental microcystin targets the microbiome and increases the risk of intestinal inflammatory pathology via NOX2 in underlying murine model of Nonalcoholic Fatty Liver Disease

With increased climate change pressures likely to influence harmful algal blooms, exposure to microcystin, a known hepatotoxin and a byproduct of cyanobacterial blooms can be a risk factor for NAFLD associated comorbidities. Using both in vivo and in vitro experiments we show that microcystin exposure in NAFLD mice cause rapid alteration of gut microbiome, rise in bacterial genus known for mediating gut inflammation and lactate production. Changes in the microbiome were strongly associated with inflammatory pathology in the intestine, gut leaching, tight junction protein alterations and increased oxidative tyrosyl radicals. Increased lactate producing bacteria from the altered microbiome was associated with increased NOX-2, an NADPH oxidase isoform. Activationof NOX2 caused inflammasome activation as shown by NLRP3/ASCII and NLRP3/Casp-1 colocalizations in these cells while use of mice lacking a crucial NOX2 component attenuated inflammatory pathology and redox changes. Mechanistically, NOX2 mediated peroxynitrite species were primary to inflammasome activation and release of inflammatory mediators. Thus, in conclusion, microcystin exposure in NAFLD could significantly alter intestinal pathology especially by the effects on microbiome and resultant redox status thus advancing our understanding of the co-existence of NAFLD-linked inflammatory bowel disease phenotypes in the clinic.

Brain Infection by Hepatitis E Virus Probably via Damage of the Blood-Brain Barrier Due to Alterations of Tight Junction Proteins.

Extrahepatic injury, particularly neurologic dysfunctions such as Guillain-Barré syndrome, neurologic amyotrophy, and encephalitis/meningoencephalitis/myositis were associated with HEV infection, which was supported by both clinical and laboratory studies. Thus, it is crucial to figure out how the virus invades into the central nervous system (CNS). In this study, CNS lesions were determined in rabbits and Mongolian gerbils inoculated with genotype 4 HEV. Junctional proteins were detected in HEV infected primary human brain microvascular cells (HBMVCs). Viral encephalitis associated perivascular cuffs of lymphocytes and microglial nodules were observed in HEV infected rabbits. Both positive- and negative-strand of HEV RNA was detected in brain and spinal cord in rabbits intraperitoneally infected with HEV at 28 dpi (days postinoculation), but not in rabbits gavaged with HEV. HEV ORF2 protein was further examined in both brain and spinal cord sections of infected rabbits, with positive signals located mainly in neural cells and perivascular areas. Ultrastructural study showed thickened and reduplicated basement membranes of capillary endothelium in HEV RNA positive brain tissues. In vitro study showed loss of tight junction proteins including Claudin5, Occludin, and ZO-1 (zonula occludens-1) in HBMVCs inoculated with HEV for 48 h. These findings indicated that disruption of the blood-brain barrier (BBB) might be potential mechanisms of HEV invasion into the CNS. It provides new insights to further study HEV associated neurologic disorders and will be helpful for seeking potential therapeutics for HEV infection in the future.

Abstract # 2071 A rodent model of anxiety: The effect of perinatal immune challenges on gastrointestinal inflammation and integrity

Background Anxiety disorders affect close to 10% of the world’s population, accounting for 30% of the global non-fatal disease burden, and costing the global economy US$1 trillion each year. Although their aetiology remains unknown, it has been suggested that anxiety disorders occur in response to altered gastrointestinal (GI) functioning, as the presence of a GI disorder will most often precede the diagnosis of an anxiety disorder. The current study aimed to determine whether the well-characterized animal model of neuropsychopathology, the maternal immune activation (MIA) model, produced GI inflammation and integrity disruptions, in association with anxiety-like behaviour. Method Pregnant Wistar rats were exposed to the viral mimetic PolyI:C on gestational days (GD) 10 and 19. Evidence of GI inflammation and barrier integrity was assessed in the small and large intestine in both neonatal and adult offspring. Results Neonatal offspring exposed to MIA exhibited an increased intestinal inflammatory profile with evidence of disruptions to GI tight junction protein mRNA. In addition, adult offspring exhibited altered tight junction protein mRNA expression and an increase in anxiety-like behaviours. Conclusion These results indicate that the rat MIA model, which is well documented to produce behavioural, neurochemical and neuroanatomical abnormalities, also produces GI inflammation and integrity disruptions. We suggest that this model may be a useful tool to elucidate biological pathways associated with anxiety disorders.

Severe Burn-Induced Intestinal Epithelial Barrier Dysfunction Is Associated With Endoplasmic Reticulum Stress and Autophagy in Mice.

The disruption of intestinal barrier plays a vital role in the pathophysiological changes after severe burn injury, however, the underlying mechanisms are poorly understood. Severe burn causes the disruption of intestinal tight junction (TJ) barrier. Previous studies have shown that endoplasmic reticulum (ER) stress and autophagy are closely associated with the impairment of intestinal mucosa. Thus, we hypothesize that ER stress and autophagy are likely involved in burn injury-induced intestinal epithelial barrier dysfunction. Mice received a 30% total body surface area (TBSA) full-thickness burn, and were sacrificed at 0, 1, 2, 6, 12 and 24 h postburn. The results showed that intestinal permeability was increased significantly after burn injury, accompanied by the damage of mucosa and the alteration of TJ proteins. Severe burn induced ER stress, as indicated by increased intraluminal chaperone binding protein (BIP), CCAAT/enhancer-binding protein homologous protein (CHOP) and inositol-requiring enzyme 1(IRE1)/X-box binding protein 1 splicing (XBP1). Autophagy was activated after burn injury, as evidenced by the increase of autophagy related protein 5 (ATG5), Beclin 1 and LC3II/LC3I ratio and the decrease of p62. Besides, the number of autophagosomes was also increased after burn injury. The levels of p-PI3K(Ser191), p-PI3K(Ser262), p-AKT(Ser473), and p-mTOR were decreased postburn, suggesting that autophagy-related PI3K/AKT/mTOR pathway is involved in the intestinal epithelial barrier dysfunction following severe burn. In summary, severe burn injury induces the ER stress and autophagy in intestinal epithelia, leading to the disruption of intestinal barrier.

Modelling an autism risk factor in rats. The impact of perinatal immune challenges on gastrointestinal inflammation and integrity

A growing subset of Autism Spectrum Disorder (ASD) sufferers present with comorbid gastrointestinal (GI) abnormalities. Research suggests these abnormalities may be involved in the aetiology, pathogenesis and pathophysiology of ASD, however, the biological pathways involved are unknown. As human prenatal infection has been associated with an increased risk of ASD in offspring, animal models of maternal immune activation (MIA) have provided researchers a translational tool to investigate the biological abnormalities underlying neurodevelopmental disorders such as ASD. The current study aimed to determine if MIA would produce ASD-like GI inflammation and integrity disruptions. Pregnant rats were exposed to Poly-I:C (MIA) at gestational day (GD) 10 and 19. A segment of distal large intestine was extracted from offspring on postnatal days (P) 7, 21 and 84. Tissue was examined for expression of tight junction protein and proinflammatory cytokine mRNA. Adult offspring were examined for anxiety-related behaviour. P7 MIA offspring exhibited alterations in tight junction proteins as well as proinflammatory immune markers. P21 and P84 offspring exhibited subtle alterations in tight junction proteins however no alterations in inflammatory markers were observed. Adult MIA offspring exhibited increased anxiety-related behaviour. MIA alters expression of pro-inflammatory cytokines and GI tight junction proteins during early development, with alterations diminishing throughout adulthood. Early changes in GI integrity may impact the trajectory of brain development leading to ASD.

Adiponectin controls the apoptosis and the expression of tight junction proteins in brain endothelial cells through AdipoR1 under beta amyloid toxicity

Alzheimer’s disease (AD) is the most common neurodegenerative disease, characterized by excessive beta amyloid (Aβ) deposition in brain, leading to blood–brain barrier (BBB) disruption. The mechanisms of BBB disruption in AD are still unclear, despite considerable research. The adipokine adiponectin is known to regulate various metabolic functions and reduce inflammation. Though adiponectin receptors have been reported in the brain, its role in the central nervous system has not been fully characterized. In the present study, we investigate whether adiponectin contributes to the tight junction integrity and cell death of brain endothelial cells under Aβ-induced toxicity conditions. We measured the expression of adiponectin receptors (AdipoR1 and AdipoR2) and the alteration of tight junction proteins in in vivo 5xFAD mouse brain. Moreover, we examined the production of reactive oxygen species (ROS) and the loss of tight junction proteins such as Claudin 5, ZO-1, and inflammatory signaling in in vitro brain endothelial cells (bEnd.3 cells) under Aβ toxicity. Our results showed that Acrp30 (a globular form of adiponectin) reduces the expression of proinflammatory cytokines and the expression of RAGE as Aβ transporters into brain. Moreover, we found that Acrp 30 attenuated the apoptosis and the tight junction disruption through AdipoR1-mediated NF-κB pathway in Aβ-exposed bEnd.3 cells. Thus, we suggest that adiponectin is an attractive therapeutic target for treating BBB breakdown in AD brain.

CD8+ T Cells Induce Fatal Brainstem Pathology during Cerebral Malaria via Luminal Antigen-Specific Engagement of Brain Vasculature.

Cerebral malaria (CM) is a severe complication of Plasmodium falciparum infection that results in thousands of deaths each year, mostly in African children. The in vivo mechanisms underlying this fatal condition are not entirely understood. Using the animal model of experimental cerebral malaria (ECM), we sought mechanistic insights into the pathogenesis of CM. Fatal disease was associated with alterations in tight junction proteins, vascular breakdown in the meninges / parenchyma, edema, and ultimately neuronal cell death in the brainstem, which is consistent with cerebral herniation as a cause of death. At the peak of ECM, we revealed using intravital two-photon microscopy that myelomonocytic cells and parasite-specific CD8+ T cells associated primarily with the luminal surface of CNS blood vessels. Myelomonocytic cells participated in the removal of parasitized red blood cells (pRBCs) from cerebral blood vessels, but were not required for the disease. Interestingly, the majority of disease-inducing parasite-specific CD8+ T cells interacted with the lumen of brain vascular endothelial cells (ECs), where they were observed surveying, dividing, and arresting in a cognate peptide-MHC I dependent manner. These activities were critically dependent on IFN-γ, which was responsible for activating cerebrovascular ECs to upregulate adhesion and antigen-presenting molecules. Importantly, parasite-specific CD8+ T cell interactions with cerebral vessels were impaired in chimeric mice rendered unable to present EC antigens on MHC I, and these mice were in turn resistant to fatal brainstem pathology. Moreover, anti-adhesion molecule (LFA-1 / VLA-4) therapy prevented fatal disease by rapidly displacing luminal CD8+ T cells from cerebrovascular ECs without affecting extravascular T cells. These in vivo data demonstrate that parasite-specific CD8+ T cell-induced fatal vascular breakdown and subsequent neuronal death during ECM is associated with luminal, antigen-dependent interactions with cerebrovasculature.

Modified Pulsatilla decoction attenuates oxazolone-induced colitis in mice through suppression of inflammation and epithelial barrier disruption.

Inflammatory bowel diseases (IBDs) are chronic inflammatory gastrointestinal disorders caused by a dysregulated mucosal immune response and epithelial barrier disruption. Conventional treatment of IBD is currently limited to overcoming patient symptoms and is often associated with severe adverse effects from the drugs used. Modified Pulsatilla decoction has been used previously to treat ulcerative colitis (UC) in clinical practice in China, however, the underlying mechanism in the treatment of UC remains to be elucidated. In the present study, the efficiency and mechanisms of modified Pulsatilla decoction in the treatment of oxazolone-induced colitis were investigated. Assessment of clinical colitis and histological examination found that the administration of modified Pulsatilla decoction attenuated the severity of oxazolone-induced colitis in mice. Measurement of cytokine concentration, western blotting and reverse transcription-quantitative polymerase chain reaction demonstrated modified Pulsatilla decoction treatment significantly reduced the secretion of pro-inflammatory cytokines and restored alterations in tight junction proteins in the colon tissues. In addition, modified Pulsatilla decoction suppressed the activation of the nuclear factor-κB signaling pathway. Thus, the findings of the present study demonstrated that modified Pulsatilla decoction offers an effective therapeutic approach for the treatment of IBD and revealed the underlying mechanisms of action offered by modified Pulsatilla decoction.

Modulatory effect of perforin gene dosage during pathogen associatated blood brain barrier (BBB) disruption

Abstract Neuroinflammation is a putative mechanism of pathogen associated blood brain barrier (BBB) disruption. However, mechanisms of immune mediated vascular permeability remain an active topic of research. Our lab has defined a role for CD8 T cells and perforin in BBB tight junction protein alterations during neuroinflammation. Within the human population, perforin is highly polymorphic, the significance of which is unknown. Therefore, we investigated the capacity of T cells with a single perforin allele, which reduces perforin mRNA and protein expression by half, in modulating BBB disruption in a murine model. We also conducted a genetic analysis to determine the significance of perforin polymorphisms in humans. In vivo, we assessed perforin+/+, perforin+/− and perforin−/− mice using the peptide induced fatal syndrome (PIFS) model for CD8 T cell mediated BBB disruption. Using 3D volumetric analysis of gadolinium enhanced, T1 weighted MRI we determined that perforin alleles are additive in increasing BBB disruption. This finding was not the result of differing immune cell infiltration or virus load. In parallel, we conducted a population genetic analysis of the human perforin allele. Using the Exome Aggregation Consortium and primary literature, we categorized over 450 perforin polymorphisms. Perforin polymorphism rates directly correlated with equatorial distance and environmental pathogen richness. Therefore, our studies demonstrate a single perforin allele alters BBB disruption severity. We hypothesize that diversity at the human perforin allele is a form of inclusive fitness by contributing to differential susceptibility to pathogen associated CNS vascular permeability.