Chimeric Claudins: A New Tool to Study Tight Junction Structure and Function

The tight junction (TJ) is a structure composed of multiple proteins, both cytosolic and membranal, responsible for cell–cell adhesion in polarized endothelium and epithelium. The TJ is intimately connected to the cytoskeleton and plays a role in development and homeostasis. Among the TJ’s membrane proteins, claudins (CLDNs) are key to establishing blood–tissue barriers that protect organismal physiology. Recently, several crystal structures have been reported for detergent extracted recombinant CLDNs. These structural advances lack direct evidence to support quaternary structure of CLDNs. In this article, we have employed protein-engineering principles to create detergent-independent chimeric CLDNs, a combination of a 4-helix bundle soluble monomeric protein (PDB ID: 2jua) and the apical—50% of human CLDN1, the extracellular domain that is responsible for cell–cell adhesion. Maltose-binding protein-fused chimeric CLDNs (MBP-CCs) used in this study are soluble proteins that retain structural and functional aspects of native CLDNs. Here, we report the biophysical characterization of the structure and function of MBP-CCs. MBP-fused epithelial cadherin (MBP-eCAD) is used as a control and point of comparison of a well-characterized cell-adhesion molecule. Our synthetic strategy may benefit other families of 4-α-helix membrane proteins, including tetraspanins, connexins, pannexins, innexins, and more.     

Evidence for the Involvement of RhoA Signaling in the Ethanol-Induced Increase in Intestinal Epithelial Barrier Permeability

In this work, we investigated the potential role of the small G protein RhoA in ethanol-induced tight junction (TJ) protein disassembly and increased intestinal epithelial barrier (IEB) permeability. Our study used Caco-2 cells as an in vitro IEB model and RhoA short hairpin RNA (shRNA) interference to establish whether RhoA plays a role in ethanol-induced TJ opening. RhoA shRNA interference partially inhibited epithelial leakage and restored normal transepithelial electrical resistance (TEER) values in the IEB. Moreover, RhoA shRNA interference prevented a shift in occludin distribution from insoluble to soluble fractions. Additionally, RhoA shRNA interference inhibited the ethanol-induced expression of zonula occludens-1 (ZO-1). Finally, RhoA shRNA interference inhibited an ethanol-induced increase in RhoA activity. The contributions of RhoA to an ethanol-induced increase in IEB permeability are associated with TJ disassembly.     

An Experimental Workflow for Studying Barrier Integrity,Permeability, and Tight Junction Composition and Localization in a Single Endothelial Cell Monolayer: Proof of Concept

Endothelial and epithelial barrier function is crucial for the maintenance of physiological processes. The barrier paracellular permeability depends on the composition and spatial distribution of the cell-to-cell tight junctions (TJ). Here, we provide an experimental workflow that yields several layers of physiological data in the setting of a single endothelial cell monolayer. Human umbilical vein endothelial cells were grown on Transwell filters. Transendothelial electrical resistance (TER) and 10 kDa FITC dextran flux were measured using Alanyl-Glutamine (AlaGln) as a paracellular barrier modulator. Single monolayers were immunolabelled for Zonula Occludens-1 (ZO-1) and Claudin-5 (CLDN5) and used for automated immunofluorescence imaging. Finally, the same monolayers were used for single molecule localization microscopy (SMLM) of ZO-1 and CLDN5 at the nanoscale for spatial clustering analysis. The TER increased and the paracellular dextran flux decreased after the application of AlaGln and these functional changes of the monolayer were mediated by an increase in the ZO-1 and CLDN5 abundance in the cell–cell interface. At the nanoscale level, the functional and protein abundance data were accompanied by non-random increased clustering of CLDN5. Our experimental workflow provides multiple data from a single monolayer and has wide applicability in the setting of paracellular studies in endothelia and epithelia.     

β-Conglycinin Reduces the Tight Junction Occludin and ZO-1 Expression in IPEC-J2

Soybean allergy presents a health threat to humans and animals. The mechanism by which food/feed allergen β-conglycinin injures the intestinal barrier has not been well understood. In this study, the changes of epithelial permeability, integrity, metabolic activity, the tight junction (TJ) distribution and expression induced by β-conglycinin were evaluated using IPEC-J2 model. The results showed a significant decrease of trans-epithelial electrical resistance (TEER) (p < 0.001) and metabolic activity (p < 0.001) and a remarkable increase of alkaline phosphatase (AP) activity (p < 0.001) in a dose-dependent manner. The expression levels of tight junction occludin and ZO-1 were decreased (p < 0.05). The reduced fluorescence of targets and change of cellular morphology were recorded. The tight junction occludin and ZO-1 mRNA expression linearly declined with increasing β-conglycinin (p < 0.001).     

A Novel Perspective and Approach to Intestinal Octreotide Absorption: Sinomenine-Mediated Reversible Tight Junction Opening and Its Molecular Mechanism

In this work, we assessed the effects of sinomenine (SN) on intestinal octreotide (OCT) absorption both in Caco-2 cell monolayers and in rats. We also investigated the molecular mechanisms of tight junction (TJ) disruption and recovery by SN-mediated changes in the claudin-1 and protein kinase C (PKC) signaling pathway. The data showed that exposure to SN resulted in a significant decrease in the expression of claudin-1, which represented TJ weakening and paracellular permeability enhancement. Then, the recovery of TJ after SN removal required an increase in claudin-1, which demonstrated the transient and reversible opening for TJ. Meanwhile, the SN-mediated translocation of PKC-α from the cytosol to the membrane was found to prove PKC activation. Finally, SN significantly improved the absolute OCT bioavailability in rats and the transport rate in Caco-2 cell monolayers. We conclude that SN has the ability to enhance intestinal OCT absorption and that these mechanisms are related at least in part to the important role of claudin-1 in SN-mediated, reversible TJ opening via PKC activation.     

Molecular Characterization of the Extracellular Domain of Human Junctional Adhesion Proteins

The junction adhesion molecule (JAM) family of proteins play central roles in the tight junction (TJ) structure and function. In contrast to claudins (CLDN) and occludin (OCLN), the other membrane proteins of the TJ, whose structure is that of a 4α-helix bundle, JAMs are members of the immunoglobulin superfamily. The JAM family is composed of four members: A, B, C and 4. The crystal structure of the extracellular domain of JAM-A continues to be used as a template to model the secondary and tertiary structure of the other members of the family. In this article, we have expressed the extracellular domains of JAMs fused with maltose-binding protein (MBP). This strategy enabled the work presented here, since JAM-B, JAM-C and JAM4 are more difficult targets due to their more hydrophobic nature. Our results indicate that each member of the JAM family has a unique tertiary structure in spite of having similar secondary structures. Surface plasmon resonance (SPR) revealed that heterotypic interactions among JAM family members can be greatly favored compared to homotypic interactions. We employ the well characterized epithelial cadherin (E-CAD) as a means to evaluate the adhesive properties of JAMs. We present strong evidence that suggests that homotypic or heterotypic interactions among JAMs are stronger than that of E-CADs.     

Potential Protection Effect of ER Homeostasis of N6-(2-Hydroxyethyl)adenosine Isolated from Cordyceps cicadae in Nonsteroidal Anti-Inflammatory Drug-Stimulated Human Proximal Tubular Cells

Nonsteroidal anti-inflammatory drugs (NSAIDs) belong to a class of universally and commonly used anti-inflammatory analgesics worldwide. A diversity of drawbacks of NSAIDs have been reported including cellular oxidative stress, which in turn triggers the accumulation of unfolded proteins, enhancing endoplasmic reticulum stress, and finally resulting in renal cell damage. Cordyceps cicadae (CC) has been used as a traditional medicine for improving renal function via its anti-inflammatory effects. N⁶-(2-hydroxyethyl)adenosine (HEA), a physiologically active compound, has been reported from CC mycelia (CCM) with anti-inflammatory effects. We hypothesize that HEA could protect human proximal tubular cells (HK–2) from NSAID-mediated effects on differential gene expression at the mRNA and protein levels. To verify this, we first isolated HEA from CCM using Sephadex^® LH–20 column chromatography. The MTT assay revealed HEA to be nontoxic up to 100 µM toward HK–2 cells. The HK–2 cells were pretreated with HEA (10–20 µM) and then insulted with the NSAIDs diclofenac (DCF, 200 µM) and meloxicam (MXC, 400 µM) for 24 h. HEA (20 µM) effectively prevented ER stress by attenuating ROS production (p < 0.001) and gene expression of ATF–6, PERK, IRE1α, CDCFHOP, IL1β, and NFκB within 24 h. Moreover, HEA reversed the increase of GRP78 and CHOP protein expression levels induced by DCF and MXC, and restored the ER homeostasis. These results demonstrated that HEA treatments effectively protect against DCF- and MXC-induced ER stress damage in human proximal tubular cells through regulation of the GRP78/ATF6/PERK/IRE1α/CHOP pathway.     

Receptor-Loaded Virion Endangers GPCR Signaling: Mechanistic Exploration of SARS-CoV-2 Infections and Pharmacological Implications

SARS-CoV-2 exploits the respiratory tract epithelium including lungs as the primary entry point and reaches other organs through hematogenous expansion, consequently causing multiorgan injury. Viral E protein interacts with cell junction-associated proteins PALS1 or ZO-1 to gain massive penetration by disrupting the inter-epithelial barrier. Conversely, receptor-mediated viral invasion ensures limited but targeted infections in multiple organs. The ACE2 receptor represents the major virion loading site by virtue of its wide tissue distribution as demonstrated in highly susceptible lung, intestine, and kidney. In brain, NRP1 mediates viral endocytosis in a similar manner to ACE2. Prominently, PDZ interaction involves the entire viral loading process either outside or inside the host cells, whereas E, ACE2, and NRP1 provide the PDZ binding motif required for interacting with PDZ domain-containing proteins PALS1, ZO-1, and NHERF1, respectively. Hijacking NHERF1 and β-arrestin by virion loading may impair specific sensory GPCR signalosome assembling and cause disordered cellular responses such as loss of smell and taste. PDZ interaction enhances SARS-CoV-2 invasion by supporting viral receptor membrane residence, implying that the disruption of these interactions could diminish SARS-CoV-2 infections and be another therapeutic strategy against COVID-19 along with antibody therapy. GPCR-targeted drugs are likely to alleviate pathogenic symptoms-associated with SARS-CoV-2 infection.     

Characterization of a putative phosphorylation switch: adaptation of SPOT synthesis to analyze PDZ domain regulation mechanisms

Transient macromolecular complexes are often formed by protein-protein interaction domains (e.g., PDZ, SH2, SH3, WW), which are often regulated (positively or negatively) by phosphorylation. To address the in vitro analysis of PDZ domain regulation by such phosphorylation, we improved the inverted peptide method. This method is based on standard SPOT synthesis, followed by inversion of the peptide under acidic conditions to generate the free C termini necessary for PDZ domain ligand recognition. The benefit of the newly introduced acidic conditions is the preservation of the incorporated phosphate group during peptide synthesis. Furthermore, the improved method is more robust and shows an increased signal-to-noise ratio. As representative examples, we used the AF6, ERBIN, and SNA1 (alpha-1-syntrophin) PDZ domains to analyze the influence of ligand-position-dependent phosphorylation. We could clearly demonstrate severe down-regulation by phosphorylation of the PDZ ligand position -2 (<50 %) and slightly less at position -1 ( approximately 50 %). These results are specific and reproducible for all three PDZ domains. Finally, we confirmed the influence of negative regulation by using the protein kinase BCR as the AF6 PDZ domain ligand. For the first time, this approach allows the SPOT synthesis technique to be used to screen large libraries of phosphorylated peptides in vitro. This should ultimately help in the identification of phosphorylation-dependent regulation mechanisms in vivo.     

Identification of Novel Fragments Binding to the PDZ1-2 Domain of PSD-95

Inhibition of PSD-95 has emerged as a promising strategy for the treatment of ischemic stroke, as shown with peptide-based compounds that target the PDZ domains of PSD-95. In contrast, developing potent and drug-like small molecules against the PSD-95 PDZ domains has so far been unsuccessful. Here, we explore the druggability of the PSD-95 PDZ1-2 domain and use fragment screening to investigate if this protein is prone to binding small molecules. We screened 2500 fragments by fluorescence polarization (FP) and validated the hits by surface plasmon resonance (SPR), including an inhibition counter-test, and found four promising fragments. Three ligand efficient fragments were shown by ¹H,¹⁵N HSQC NMR to bind in the small hydrophobic P⁰ pockets of PDZ1-2, and one of them underwent structure-activity relationship (SAR) studies. Overall, we demonstrate that fragment screening can successfully be applied to PDZ1-2 of PSD-95 and disclose novel fragments that can serve as starting points for optimization towards small-molecule PDZ domain inhibitors.     

Tumor Necrosis Factor Alpha Effects on the Porcine Intestinal Epithelial Barrier Include Enhanced Expression of TNF Receptor 1

Tumor necrosis factor alpha (TNFα) has been shown to impair the intestinal barrier, inducing and maintaining inflammatory states of the intestine. The aim of the current study was to analyze functional, molecular and regulatory effects of TNFα in a newly established non-transformed jejunal enterocyte model, namely IPEC-J2 monolayers. Incubation with 1000 U/mL TNFα induced a marked decrease in transepithelial electrical resistance (TEER), and an increase in permeability for the paracellular flux marker [³H]-D-mannitol compared to controls. Immunoblots revealed a significant decrease in tight junction (TJ) proteins occludin, claudin-1 and claudin-3. Moreover, a dose-dependent increase in the TNF receptor (TNFR)-1 was detected, explaining the exponential nature of pro-inflammatory effects, while TNFR-2 remained unchanged. Recovery experiments revealed reversible effects after the removal of the cytokine, excluding apoptosis as a reason for the observed changes. Furthermore, TNFα signaling could be inhibited by the specific myosin light chain kinase (MLCK) blocker ML-7. Results of confocal laser scanning immunofluorescence microscopy were in accordance with all quantitative changes. This study explains the self-enhancing effects of TNFα mediated by MLCK, leading to a differential regulation of TJ proteins resulting in barrier impairment in the intestinal epithelium.     

Pharmacological Evaluation and Preliminary Pharmacokinetics Studies of a New Diclofenac Prodrug without Gastric Ulceration Effect

Long-term nonsteroidal anti-inflammatory drugs (NSAIDs) therapy has been associated with several adverse effects such as gastric ulceration and cardiovascular events. Among the molecular modifications strategies, the prodrug approach is a useful tool to discover new safe NSAIDs. The 1-(2,6-dichlorophenyl)indolin-2-one is a diclofenac prodrug which demonstrated relevant anti-inflammatory properties without gastro ulceration effect. In addition, the prodrug decreases PGE₂ levels, COX-2 expression and cellular influx into peritoneal cavity induced by carrageenan treatment. Preliminary pharmacokinetic studies have shown in vivo bioconversion of prodrug to diclofenac. This prodrug is a new nonulcerogenic NSAID useful to treat inflammatory events by long-term therapy.     

Effects of soybean agglutinin on intestinal barrier permeability and tight junction protein expression in weaned piglets

This study was developed to provide further information on the intestinal barrier permeability and the tight junction protein expression in weaned piglets fed with different levels of soybean agglutinin (SBA). Twenty-five weaned crossbred barrows (Duroc × Landrace × Yorkshire) were selected and randomly allotted to five groups, each group with five replicates. The piglets in the control group were not fed with leguminous products. 0.05, 0.1, 0.15 and 0.2% SBA was added to the control diet to form four experimental diets, respectively. After the experimental period of 7 days (for each group), all the piglets were anesthetized with excess procaine and slaughtered. The d-lactic acid in plasma and the Ileal mucosa diamine oxidase (DAO) was analyzed to observe the change in the intestinal permeability. The tight junction proteins occludin and ZO-1 in the jejunum tissue distribution and relative expression were detected by immunohistochemistry and Western Blot. The results illustrated that a high dose of SBA (0.1-0.2%) could increase the intestinal permeability and reduce piglet intestinal epithelial tight junction protein occludin or ZO-1 expression, while low dose of SBA (0.05% of total diet) had no significant affects. The contents of DAO, d-lactic acid, occludin or ZO-1, had a linear relationship with the SBA levels (0-0.2%) in diets. The high dose SBA (0.1-0.2%) could increase the intestinal permeability and reduce piglet intestinal epithelial tight junction protein occludin or ZO-1 expression, while low dose of SBA (0.05% of total diet) had no affects.     

Investigation on the Interactions between Self-Assembled β-Sheet Peptide Nanofibers and Model Cell Membranes

Self-assembled peptide nanofibers (NFs) obtained from β-sheet peptides conjugated with drugs, including antigenic peptides, have recently attracted significant attention. However, extensive studies on the interactions of β-sheet peptide NFs with model cell membranes have not been reported. In this study, we investigated the interactions between three types of NFs, composed of PEG-peptide conjugates with different ethylene glycol (EG) lengths (6-, 12- and 24-mer), and dipalmitoylphosphatidylcholine (DPPC) Langmuir membranes. When increasing the EG chain length, those interactions significantly decreased considering measurements in the presence of the NFs of: (i) changes in surface pressure of the DPPC Langmuir monolayers and (ii) surface pressure–area (π–A) compression isotherms of DPPC. Because the observed trend was similar to the EG length dependency with regard to cellular association and cytotoxicity of the NFs that was reported previously, the interaction of NFs with phospholipid membranes represented a crucial factor to determine the cellular association and toxicity of the NFs. In contrast to NFs, no changes were observed with varying EG chain length on the interaction of the building block peptide with the DPPC membrane. The results obtained herein can provide a design guideline on the formulation of β-sheet peptide NFs, which may broaden its potential.     

Regional Differences in the Permeability Barrier of the Skin—Implications in Acantholytic Skin Diseases

The chemical milieu, microbiota composition, and immune activity show prominent differences in distinct healthy skin areas. The objective of the current study was to compare the major permeability barrier components (stratum corneum and tight junction (TJ)), investigate the distribution of (corneo)desmosomes and TJs, and measure barrier function in healthy sebaceous gland-rich (SGR), apocrine gland-rich (AGR), and gland-poor (GP) skin regions. Molecules involved in cornified envelope (CE) formation, desquamation, and (corneo)desmosome and TJ organization were investigated at the mRNA and protein levels using qRT-PCR and immunohistochemistry. The distribution of junction structures was visualized using confocal microscopy. Transepidermal water loss (TEWL) functional measurements were also performed. CE intracellular structural components were similarly expressed in gland-rich (SGR and AGR) and GP areas. In contrast, significantly lower extracellular protein levels of (corneo)desmosomes (DSG1 and CDSN) and TJs (OCLN and CLDN1) were detected in SGR/AGR areas compared to GP areas. In parallel, kallikrein proteases were significantly higher in gland-rich regions. Moreover, gland-rich areas were characterized by prominently disorganized junction structures ((corneo)desmosomes and TJs) and significantly higher TEWL levels compared to GP skin, which exhibited a regular distribution of junction structures. According to our findings, the permeability barrier of our skin is not uniform. Gland-rich areas are characterized by weaker permeability barrier features compared with GP regions. These findings have important clinical relevance and may explain the preferred localization of acantholytic skin diseases on gland-rich skin regions (e.g., Pemphigus foliaceus, Darier’s disease, and Hailey–Hailey disease).     

A Comparative Study of the Effects of Platinum (II) Complexes on β-Amyloid Aggregation: Potential Neurodrug Applications

Herein the effects of three platinum complexes, namely (SP-4-2)-(2,2′-bipyridine)dichloridoplatinum(II), Pt-bpy, (SP-4-2)-dichlorido(1,10-phenanthroline) platinum(II), Pt-phen, and (SP-4-2)-chlorido(2,2′:6′,2′′-terpyridine)platinum(II) chloride, Pt-terpy, on the aggregation of an amyloid model system derived from the C-terminal domain of Aβ peptide (Aβ_21–40) were investigated. Thioflavin T (ThT) binding assays revealed the ability of Pt(II) compounds to repress amyloid aggregation in a dose-dependent way, whereas the ability of Aβ_21–40 peptide to interfere with ligand field of metal complexes was analyzed through UV-Vis absorption spectroscopy and electrospray ionization mass spectrometry. Spectroscopic data provided micromolar EC₅₀ values and allowed to assess that the observed inhibition of amyloid aggregation is due to the formation of adducts between Aβ_21–40 peptide and complexes upon the release of labile ligands as chloride and that they can explore different modes of coordination toward Aβ_21–40 with respect to the entire Aβ_1–40 polypeptide. In addition, conformational studies through circular dichroism (CD) spectroscopy suggested that Pt-terpy induces soluble β-structures of monomeric Aβ_21–40, thus limiting self-recognition. Noticeably, Pt-terpy demonstrated the ability to reduce the cytotoxicity of amyloid peptide in human SH-SY5Y neuroblastoma cells. Presented data corroborate the hypothesis to enlarge the application field of already known metal-based agents to neurodegenerative diseases, as potential neurodrugs.     

Importance of a Conserved Lys/Arg Residue for Ligand/PDZ Domain Interactions as Examined by Protein Semisynthesis

PDZ domains are ubiquitous small protein domains that are mediators of numerous protein–protein interactions, and play a pivotal role in protein trafficking, synaptic transmission, and the assembly of signaling‐transduction complexes. In recent years, PDZ domains have emerged as novel and exciting drug targets for diseases (in the brain in particular), so understanding the molecular details of PDZ domain interactions is of fundamental importance. PDZ domains bind to a protein partner at either a C‐terminal peptide or internal peptide motifs. Here, we examined the importance of a conserved Lys/Arg residue in the ligand‐binding site of the second PDZ domain of PSD‐95, by employing a semisynthetic approach. We generated six semisynthetic PDZ domains comprising different proteogenic and nonproteogenic amino acids representing subtle changes of the conserved Lys/Arg residue. These were tested with four peptide interaction partners, representing the two different binding modes. The results highlight the role of a positively charged amino acid in the β1–β2 loop of PDZ domains, and show subtle differences for canonical and noncanonical interaction partners, thus providing additional insight into the mechanism of PDZ/ligand interaction.     

Netrin-1 as a Multitarget Barrier Stabilizer in the Peripheral Nerve after Injury

The blood–nerve barrier and myelin barrier normally shield peripheral nerves from potentially harmful insults. They are broken down during nerve injury, which contributes to neuronal damage. Netrin-1 is a neuronal guidance protein with various established functions in the peripheral and central nervous systems; however, its role in regulating barrier integrity and pain processing after nerve injury is poorly understood. Here, we show that chronic constriction injury (CCI) in Wistar rats reduced netrin-1 protein and the netrin-1 receptor neogenin-1 (Neo1) in the sciatic nerve. Replacement of netrin-1 via systemic or local administration of the recombinant protein rescued injury-induced nociceptive hypersensitivity. This was prevented by siRNA-mediated knockdown of Neo1 in the sciatic nerve. Mechanistically, netrin-1 restored endothelial and myelin, but not perineural, barrier function as measured by fluorescent dye or fibrinogen penetration. Netrin-1 also reversed the decline in the tight junction proteins claudin-5 and claudin-19 in the sciatic nerve caused by CCI. Our findings emphasize the role of the endothelial and myelin barriers in pain processing after nerve damage and reveal that exogenous netrin-1 restores their function to mitigate CCI-induced hypersensitivity via Neo1. The netrin-1-neogenin-1 signaling pathway may thus represent a multi-target barrier protector for the treatment of neuropathic pain.     

A Novel Netrin-1-Derived Peptide Enhances Protection against Neuronal Death and Mitigates of Intracerebral Hemorrhage in Mice

It has been reported that Netrin-1 is involved in neuroprotection following injury to the central nervous system. However, the minimal functional domain of Netrin-1 which can preserve the neuroprotection but avoid the major side effects of Netrin remains elusive. Here, we investigated the neuroprotective effect of a peptide E1 derived from Netrin-1′s EGF3 domain (residues 407–422). We found that it interacts with deleted colorectal carcinoma (DCC) to activate focal adhesion kinase phosphorylation exhibiting neuroprotection. The administration of the peptide E1 was able to improve functional recovery through reduced apoptosis in an experimental murine model of intracerebral hemorrhage (ICH). In summary, we reveal a functional sequence of Netrin-1 that is involved in the recovery process after ICH and identify a candidate peptide for the treatment of ICH.     

Vitamin D Reverses Disruption of Gut Epithelial Barrier Function Caused by Campylobacter jejuni

Infections by the zoonotic foodborne bacterium Campylobacter jejuni (C. jejuni) are among the most frequent causes of bacterial gastroenteritis worldwide. The aim was to evaluate the relationship between epithelial barrier disruption, mucosal immune activation, and vitamin D (VD) treatment during C. jejuni infection, using intestinal epithelial cells and mouse models focused on the interaction of C. jejuni with the VD signaling pathway and VD treatment to improve C. jejuni-induced barrier dysfunction. Our RNA-Seq data from campylobacteriosis patients demonstrate inhibition of VD receptor (VDR) downstream targets, consistent with suppression of immune function. Barrier-preserving effects of VD addition were identified in C. jejuni-infected epithelial cells and IL-10^−/− mice. Furthermore, interference of C. jejuni with the VDR pathway was shown via VDR/retinoid X receptor (RXR) interaction. Paracellular leakiness of infected epithelia correlated with tight junction (TJ) protein redistribution off the TJ domain and apoptosis induction. Supplementation with VD reversed barrier impairment and prevented inhibition of the VDR pathway, as shown by restoration of transepithelial electrical resistance and fluorescein (332 Da) permeability. We conclude that VD treatment restores gut epithelial barrier functionality and decreases bacterial transmigration and might, therefore, be a promising compound for C. jejuni treatment in humans and animals.