scholarly journals Epithelial-Specific TLR4 Knockout Challenges Current Evidence of TLR4 Homeostatic Control of Gut Permeability

2021 ◽  
pp. 1-11
Author(s):  
Elise E. Crame ◽  
Joanne M. Bowen ◽  
Kate R. Secombe ◽  
Janet K. Coller ◽  
Maxime François ◽  
...  
Keyword(s):  
Tight Junction ◽  
Immune Cells ◽  
Conditional Knockout ◽  
Current Evidence ◽  
Intestinal Epithelial ◽  
Tlr4 Expression ◽  
Tissue Resistance ◽  
Junction Protein ◽  
Health And Disease ◽  
Histological Staining

<b><i>Introduction:</i></b> Toll-like receptor 4 (TLR4) is a highly conserved immunosurveillance protein of innate immunity, displaying well-established roles in homeostasis and intestinal inflammation. Current evidence shows complex relationships between TLR4 activation, maintenance of health, and disease progression; however, it commonly overlooks the importance of site-specific TLR4 expression. This omission has the potential to influence translation of results as previous evidence shows the differing and distinct roles that TLR4 exhibits are dependent on its spatiotemporal expression. <b><i>Methods:</i></b> An intestinal epithelial TLR4 conditional knockout (KO) mouse line (<i>Tlr4</i><sup><i>ΔIEC</i></sup>, <i>n</i> = 6–8) was utilized to dissect the contribution of epithelial TLR4 expression to intestinal homeostasis with comparisons to wild-type (WT) (<i>n</i> = 5–7) counterparts. Functions of the intestinal barrier in the ileum and colon were assessed with tissue resistance in Ussing chambers. Molecular and structural comparisons in the ileum and colon were assessed via histological staining, expression of tight junction proteins (occludin and zonular occludin 1 [ZO-1]), and presence of CD11b-positive immune cells. <b><i>Results:</i></b> There was no impact of the intestinal epithelial TLR4 KO, with no differences in (1) tissue resistance–ileum (mean ± standard error of mean [SEM]): WT 22 ± 7.2 versus <i>Tlr4</i><sup><i>ΔIEC</i></sup> 20 ± 5.6 (Ω × cm<sup>2</sup>) <i>p</i> = 0.831, colon WT 30.8 ± 3.6 versus <i>Tlr4</i><sup><i>ΔIEC</i></sup> 45.1 ± 9.5 <i>p</i> = 0.191; (2) histological staining (overall tissue structure); and (3) tight junction protein expression (% area stain, mean ± SEM)–ZO-1: ileum–WT 1.49 ± 0.155 versus <i>Tlr4</i><sup><i>ΔIEC</i></sup> 1.17 ± 0.07, <i>p</i> = 0.09; colon–WT 1.36 ± 0.26 versus <i>Tlr4</i><sup><i>ΔIEC</i></sup> 1.12 ± 0.18 <i>p</i> = 0.47; occludin: ileum–WT 1.07 ± 0.12 versus <i>Tlr4</i><sup><i>ΔIEC</i></sup> 0.95 ± 0.13, <i>p</i> = 0.53; colon–WT 1.26 ± 0.26 versus <i>Tlr4</i><sup><i>ΔIEC</i></sup> 1.02 ± 0.16 <i>p</i> = 0.45. CD11b-positive immune cells (% area stain, mean ± SEM) in the ileum were mildly decreased in WT mice: WT 0.14 ± 0.02 versus <i>Tlr4</i><sup><i>ΔIEC</i></sup> 0.09 ± 0.01 <i>p</i> = 0.04. However, in the colon, there was no difference in CD11b-positive immune cells between strains: WT 0.53 ± 0.08 versus <i>Tlr4</i><sup><i>ΔIEC</i></sup> 0.49 ± 0.08 <i>p</i> = 0.73. <b><i>Conclusions:</i></b> These data have 2 important implications. First, these data refute the assumption that epithelial TLR4 exerts physiological control of intestinal physiology and immunity in health. Second, and most importantly, these data support the use of the <i>Tlr4</i><sup><i>ΔIEC</i></sup> line in future models interrogating health and disease, confirming no confounding effects of genetic manipulation.

scholarly journals Layilin is critical for mediating hyaluronan 35 kDa-induced intestinal epithelial tight junction protein ZO-1 in vitro and in vivo

2018 ◽  
Vol 66 ◽  
pp. 93-109 ◽  
Author(s):  
Yeojung Kim ◽  
Gail A. West ◽  
Greeshma Ray ◽  
Sean P. Kessler ◽  
Aaron C. Petrey ◽  
...  
Keyword(s):  
Tight Junction ◽  
Tight Junction Protein ◽  
Intestinal Epithelial ◽  
Junction Protein ◽  
Epithelial Tight Junction

Infection of T84 cells with enteropathogenic Escherichia coli alters barrier and transport functions

1996 ◽  
Vol 270 (4) ◽  
pp. G634-G645 ◽  
Author(s):  
D. J. Philpott ◽  
D. M. McKay ◽  
P. M. Sherman ◽  
M. H. Perdue
Keyword(s):  
Escherichia Coli ◽  
Tight Junction ◽  
Diarrheal Disease ◽  
Chloride Secretion ◽  
Coli Strain ◽  
Intestinal Epithelial ◽  
Enteropathogenic Escherichia Coli ◽  
T84 Cells ◽  
Junction Protein ◽  
Barrier Defect

The effect of enteropathogenic Escherichia coli (EPEC) infection on electrophysiology of T84 cell monolayers was examined. After 18 h of infection with EPEC (E2348), transepithelial electrical resistance was decreased (30 +/- 5% of uninfected values) compared with monolayers infected with a nonpathogenic E. coli strain (104 +/- 13%). Resistance of monolayers infected with EPEC mutant strain CVD206, deficient in attaching and effacing lesion formation, was partially reduced (66 +/- 10%). In addition, permeability of EPEC-infected T84 monolayers increased compared with uninfected cells. Associated with these changes was an altered distribution of the tight junction protein, ZO-1. Taken together, these findings suggest that the barrier defect induced by EPEC was at the level of the tight junction. Adenosine 3'5'-cyclic monophosphate-stimulated chloride secretion was also diminished in EPEC-infected cells, whereas Ca2+ -dependent chloride secretion was not different from uninfected cells. These findings indicate that EPEC infection alters intestinal epithelial barrier and transport functions. Furthermore, these results provide a possible mechanism for EPEC-induced diarrheal disease.

scholarly journals Lactobacillus plantarum inhibits intestinal epithelial barrier dysfunction induced by unconjugated bilirubin

2010 ◽  
Vol 104 (3) ◽  
pp. 390-401 ◽  
Author(s):  
Yukun Zhou ◽  
Huanlong Qin ◽  
Ming Zhang ◽  
Tongyi Shen ◽  
Hongqi Chen ◽  
...  
Keyword(s):  
Tight Junction ◽  
Lactobacillus Plantarum ◽  
Laser Scanning Microscopy ◽  
Unconjugated Bilirubin ◽  
Epithelial Barrier ◽  
Intestinal Epithelial ◽  
Tight Junction Proteins ◽  
Intestinal Epithelial Barrier ◽  
Junction Protein ◽  
Junction Proteins

Although a large number of in vitro and in vivo tests have confirmed that taking probiotics can improve the intestinal barrier, few studies have focused on the relationship between probiotics and the intestinal epithelial barrier in hyperbilirubinaemia. To investigate the effects of and mechanisms associated with probiotic bacteria (Lactobacillus plantarum; LP) and unconjugated bilirubin (UCB) on the intestinal epithelial barrier, we measured the viability, apoptotic ratio and protein kinase C (PKC) activity of Caco-2 cells. We also determined the distribution and expression of tight junction proteins such as occludin, zonula occludens (ZO)-1, claudin-1, claudin-4, junctional adhesion molecule (JAM)-1 and F-actin using confocal laser scanning microscopy, immunohistochemistry, Western blotting and real-time quantitative PCR. The present study demonstrated that high concentrations of UCB caused obvious cytotoxicity and decreased the transepithelial electrical resistance (TER) of the Caco-2 cell monolayer. Low concentrations of UCB inhibited the expression of tight junction proteins and PKC but could induce UDP-glucuronosyltransferases 1 family-polypeptide A1 (UGT1A1) expression. UCB alone caused decreased PKC activity, serine phosphorylated occludin and ZO-1 levels. After treatment with LP, the effects of UCB on TER and apoptosis were mitigated; LP also prevented aberrant expression and rearrangement of tight junction proteins. Moreover, PKC activity and serine phosphorylated tight junction protein levels were partially restored after treatment with LP, LP exerted a protective effect against UCB damage to Caco-2 monolayer cells, and it restored the structure and distribution of tight junction proteins by activating the PKC pathway. In addition, UGT1A1 expression induced by UCB in Caco-2 cells could ameliorate the cytotoxicity of UCB.

scholarly journals Tight Junction Protein Claudin-7 Is Essential for Intestinal Epithelial Stem Cell Self-Renewal and Differentiation

2020 ◽  
Vol 9 (4) ◽  
pp. 641-659 ◽  
Author(s):  
Tiaosi Xing ◽  
Lesley Jasmine Benderman ◽  
Stephiya Sabu ◽  
Joel Parker ◽  
Jeffrey Yang ◽  
...  
Keyword(s):  
Stem Cell ◽  
Tight Junction ◽  
Tight Junction Protein ◽  
Intestinal Epithelial ◽  
Self Renewal ◽  
Epithelial Stem Cell ◽  
Junction Protein

scholarly journals The serine protease-mediated increase in intestinal epithelial barrier function is dependent on occludin and requires an intact tight junction

2016 ◽  
Vol 311 (3) ◽  
pp. G466-G479 ◽  
Author(s):  
Natalie J. Ronaghan ◽  
Judie Shang ◽  
Vadim Iablokov ◽  
Raza Zaheer ◽  
Pina Colarusso ◽  
...  
Keyword(s):  
Epithelial Cell ◽  
Tight Junction ◽  
Serine Protease ◽  
Barrier Function ◽  
Serine Proteases ◽  
Cell Fractionation ◽  
Intestinal Epithelial ◽  
Tight Junction Proteins ◽  
Junction Protein ◽  
Junction Proteins

Barrier dysfunction is a characteristic of the inflammatory bowel diseases (IBD), Crohn's disease and ulcerative colitis. Understanding how the tight junction is modified to maintain barrier function may provide avenues for treatment of IBD. We have previously shown that the apical addition of serine proteases to intestinal epithelial cell lines causes a rapid and sustained increase in transepithelial electrical resistance (TER), but the mechanisms are unknown. We hypothesized that serine proteases increase barrier function through trafficking and insertion of tight junction proteins into the membrane, and this could enhance recovery of a disrupted monolayer after calcium switch or cytokine treatment. In the canine epithelial cell line, SCBN, we showed that matriptase, an endogenous serine protease, could potently increase TER. Using detergent solubility-based cell fractionation, we found that neither trypsin nor matriptase treatment changed levels of tight junction proteins at the membrane. In a fast calcium switch assay, serine proteases did not enhance the rate of recovery of the junction. In addition, serine proteases could not reverse barrier disruption induced by IFNγ and TNFα. We knocked down occludin in our cells using siRNA and found this prevented the serine protease-induced increase in TER. Using fluorescence recovery after photobleaching (FRAP), we found serine proteases induce a greater mobile fraction of occludin in the membrane. These data suggest that a functional tight junction is needed for serine proteases to have an effect on TER, and that occludin is a crucial tight junction protein in this mechanism.

Piezo1 regulates intestinal epithelial function by affecting the tight junction protein claudin-1 via the ROCK pathway

Life Sciences ◽  
2021 ◽  
Vol 275 ◽  
pp. 119254
Author(s):  
Yudong Jiang ◽  
Jun Song ◽  
Yan Xu ◽  
Caiyuan Liu ◽  
Wei Qian ◽  
...  
Keyword(s):  
Tight Junction ◽  
Tight Junction Protein ◽  
Intestinal Epithelial ◽  
Junction Protein

scholarly journals PSIII-36 The endocytic pathway plays a major role in the regulation of tight junction remodeling during nutrient starvation in jejunal IPEC-J2 cells

2020 ◽  
Vol 98 (Supplement_4) ◽  
pp. 366-366
Author(s):  
Enkai Li ◽  
Kola Ajuwon
Keyword(s):  
Epithelial Cells ◽  
Tight Junction ◽  
Intestinal Epithelial Cells ◽  
Endocytic Pathway ◽  
Tight Junction Protein ◽  
Protein Abundance ◽  
Intestinal Epithelial ◽  
Bafilomycin A1 ◽  
Prolonged Starvation ◽  
Junction Protein

Abstract Postweaning pigs are subjected to nutrient deprivation during which intestinal epithelial cells undergo increased turnover. To preserve intestinal function, intestinal epithelial cells must activate adaptive mechanisms that allow them to cope with starvation-induced stress; most importantly, the preservation of intestinal barrier function. The objective of this study was to investigate the underlying mechanisms involved in starvation-induced alteration of tight junction protein abundance and function in IPEC-J2 cells. Cells were subjected to total nutrient starvation in Krebs-Ringer bicarbonate (KRB) buffer for 0, 3, 6, 12 and 24 h. Abundance of tight junction proteins was determined by RT-PCR, western blotting and immunofluorescence. Compared with control group (0 h), the protein expression of claudin 1, claudin 3 and claudin 4 protein was downregulated up to 6 h of starvation and then increased thereafter (P &lt; 0.01). However, there was no change in the protein level of occludin and ZO-1. To determine the contribution of the lysosome and the ubiquitin proteasome pathways to regulation of tight junction protein abundance, the lysosome (Bafilomycin A1) and the proteasome (MG132) inhibitors were used in nutrient starved cells. Results showed the degradation of claudin 1, 3 and 4 up to 6 h of starvation was through the lysosomal pathway. Surprisingly, re-synthesis of claudins 4 and claudin 3 after prolonged starvation (12 and 24 h) was prevented when cells were treated with bafilomycin A1 and MG132, respectively. The autophagy-lysosome pathway inhibitors (Wortmannin and MHY1485) and endosome-lysosome pathway inhibitors (Dynasore and Pitstop 2) were further used to determine the specific roles of these pathways. In summary, the degradation of claudin 3 and claudin 4 during short-term starvation (up to 6 h) was through the dynamin-dependent endocytic pathway. However, re-synthesis of these proteins after prolonged starvation relies on both the lysosome and proteasome pathways.

scholarly journals The role of tight junction proteins in ovarian follicular development and ovarian cancer

Reproduction ◽  
2018 ◽  
Vol 155 (4) ◽  
pp. R183-R198 ◽  
Author(s):  
Lingna Zhang ◽  
Tao Feng ◽  
Leon J Spicer
Keyword(s):  
Ovarian Cancer ◽  
Growth Factor ◽  
Tight Junction ◽  
Protein Structures ◽  
Follicular Development ◽  
Current Evidence ◽  
Serum Factors ◽  
Cell Functions ◽  
Junction Protein ◽  
Ovarian Follicular Development

Tight junctions (TJ) are protein structures that control the transport of water, ions and macromolecules across cell layers. Functions of the transmembrane TJ protein, occluding (OCLN) and the cytoplasmic TJ proteins, tight junction protein 1 (TJP1; also known as zona occludens protein-1), cingulin (CGN) and claudins (CLDN) are reviewed, and current evidence of their role in the ovarian function is reviewed. Abundance ofOCLN,CLDNsandTJP1mRNA changed during follicular growth.In vitrotreatment with various growth factors known to affect ovarian folliculogenesis indicated thatCGN,OCLNandTJP1are hormonally regulated. The summarized studies indicate that expression of TJ proteins (i.e.,OCLN,CLDN,TJP1andCGN) changes with follicle size in a variety of vertebrate species but whether these changes in TJ proteins are increased or decreased depends on species and cell type. Evidence indicates that autocrine, paracrine and endocrine regulators, such as fibroblast growth factor-9, epidermal growth factor, androgens, tumor necrosis factor-α and glucocorticoids may modulate these TJ proteins. Additional evidence presented indicates that TJ proteins may be involved in ovarian cancer development in addition to normal follicular and luteal development. A model is proposed suggesting that hormonal downregulation of TJ proteins during ovarian follicular development could reduce barrier function (i.e., selective permeability of molecules between theca and granulosa cells) and allow for an increase in the volume of follicular fluid as well as allow additional serum factors into the follicle that may directly impact granulosa cell functions.

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