Preterm infants are prone to gastrointestinal complications such as infectious diseases and necrotizing enterocolitis, which are associated with intestinal inflammation and increased intestinal permeability. Intestinal epithelial barrier (IEB) function is known to be immature in preterm neonates; however, our understanding of how the IEB develops - particularly the formation of junctional complexes - remains limited. Here, we analyzed intestinal tissue specimens from healthy resection margins of six very immature preterm infants who underwent bowel resection due to focal intestinal perforation (at the chronological age of 25 to 28 weeks' gestation), and compared the composition of tight junctions, adherens junctions and desmosomes to that of adults. Using immunostaining, our observations show, that tight junction proteins Claudin-2, Claudin-3 and Occludin, adherens junction proteins E-cadherin and β-Catenin as well as desmosomal proteins Plakoglobin and Plakophilin-2 appeared as mature as in adults as early as 25 weeks' gestation. However, Claudin-1, -4 and -5, as well as ZO-1 staining patterns increased and became more defined with increasing gestational age, suggesting junctional maturation during gestational week 26 and 27. Desmosomal protein Desmocollin-2 was increased until 26 weeks' gestation whereas Desmoglein-2 and Desmoplakin expression was immature at 28 weeks' gestation compared to the expression in adults. Our study provides the first sequential characterization of junctional protein maturation across all major IEB components early in life in human samples. These findings may help identify key mechanisms underlying intestinal barrier-associated pathologies in preterm infants.

Head and neck squamous cell carcinomas (HNSCC) are characterized by poor prognosis, primarily due to early metastatic spread. The junctional plaque protein plakoglobin (JUP), a key desmosomal component, contributes not only to cell–cell adhesion but also to intracellular signaling processes that regulate proliferation, migration, and metastasis. Although JUP has been implicated in tumor progression in other cancer types, its role in HNSCC remains largely undefined. In this study, clinical data from The Cancer Genome Atlas (TCGA) were analyzed to determine the prognostic relevance of JUP expression in HNSCC. Functional studies were performed in HPV-negative (FaDu) and HPV-positive (UPCI-SCC-154) cells following siRNA-mediated JUP knockdown, assessing proliferation, wound healing, and signaling activity. Pharmacological inhibition experiments were conducted to evaluate pathway specificity. Low JUP expression was significantly associated with advanced metastatic stage and reduced overall survival in HNSCC patients. In vitro, JUP-deficient cells exhibited accelerated wound closure and increased proliferation. Mechanistically, loss of JUP led to activation of the phosphoinositide 3-kinase (PI3K)/AKT signaling pathway, and the enhanced motility phenotype could be reversed by pharmacological PI3K inhibition. Taken together, these findings identify plakoglobin as a negative regulator of PI3K signaling in HNSCC. Loss of JUP promotes tumor cell motility and proliferation, underscoring its potential value as a prognostic biomarker and therapeutic target in head and neck cancer.

Necrotizing enterocolitis (NEC) is a major cause of mortality in preterm infants. Its pathophysiology remains poorly understood but intestinal epithelial barrier dysfunction contributes to the disease. We characterized junctional proteins in intestinal specimens from preterm infants. Samples from 27 patients with NEC and 20 patients with focal intestinal perforation (FIP) from the center of the specimens (affected) or the macroscopically healthy resection margins whenever available (non-affected) were collected. NEC patients displayed higher mortality and more commonly occurrence of impaired glucose homeostasis, patent ductus arteriosus, anemia and antibiotic treatment compared to FIP patients.Discrimination between NEC and FIP was not possible in affected areas based on H.E. staining using a newly developed scoring system. Immunofluorescence revealed reduced Claudin-3 in affected NEC samples and decreased Claudin-4 in affected FIP and all NEC samples. E-cadherin and Desmoglein-2 were reduced in a subgroup of the affected NEC samples. Plakophilin-2 was decreased in intestine affected by FIP and unaffected intestine in patients with NEC. In affected areas of NEC, Plakophilin-2 was completely lost. Plakoglobin reduction in affected NEC samples correlated with poor survival. This study provides novel insights into changes of junctional proteins in NEC, suggesting Claudin-3 and Plakophilin-2 as diagnostic markers to differentiate FIP from NEC and reduced Plakoglobin as a prognostic marker.

Enteropathogenic E. coli (EPEC) attaches to host intestinal epithelial cells, resulting in severe illness and diarrhoea. Different cell models have been used to study EPEC infection, but a direct comparison of infection of cell lines and primary cells is lacking. In this study, we compare EPEC infection in primary epithelial cells with HeLa cells. Jejunal organoid-derived cells contain differentiated intestinal epithelial cells and form a tight monolayer. Upon infection, they retain integrity over longer time periods than HeLa cells. Attachment of EPEC to host cells and innate immune response is strongly delayed in organoid-derived monolayers. These results indicate that host cell factors determine the outcome of infection. Future identification of these host factors will aid the development of new therapeutics.

Desmosomes play an underexplored role in intestinal homeostasis and are linked to the pathogenesis of inflammatory bowel diseases. We found a novel function of the desmosomal plaque protein Plakoglobin (JUP) in initiating the innate immune response to facilitate intestinal inflammation. Tissue samples from Crohn's disease (CD) patients revealed a loss of JUP, which was mirrored in a mouse model of dextran sodium sulfate-induced (DSS) colitis. Inducible intestinal epithelial-specific knock-out of Jup (iVilCreERT2Jupfl/fl) in mice resulted in increased submucosal infiltration of macrophages and neutrophils, along with activation of the inflammasome. This was paralleled by p38MAPK phosphorylation while loss intestinal epithelial barrier function was absent. In DSS-colitis, epithelial Jup-deficiency impaired recovery and enhanced IL23/IL17-signaling. Intestinal organoids lacking Jup demonstrated NLRP1 inflammasome activation, indicated by increased IL1β and IL18 levels, which was attenuated by p38MAPK inhibition. In silico analysis and co-immunoprecipitation confirmed a direct interaction between JUP and p38MAPK, revealing a regulatory mechanism where JUP limits inflammasome signaling in intestinal epithelial cells. These effects were blunted by NLRP1/3 inhibitor ADS032.. These findings identify JUP as a critical modulator of epithelial innate immunity in the gut. The loss of JUP in tissues from CD patients underscores its potential relevance in disease pathology.

Mucosal healing is critical to maintain and restore intestinal homeostasis in inflammation. Previous data provide evidence that glial cell line-derived neurotrophic factor (GDNF) restores epithelial integrity by largely undefined mechanisms. Here, we assessed the role of GDNF for mucosal healing. In dextran sodium sulphate (DSS)-induced colitis in mice application of GDNF enhanced recovery as revealed by reduced disease activity index and histological inflammation scores. In biopsy-based wounding experiments GDNF application in mice improved healing of the intestinal mucosa. GDNF-induced epithelial recovery was also evident in wound assays from intestinal organoids and Caco2 cells. These observations were accompanied by an increased number of Ki67-positive cells in vivo after GDNF treatment, which were present along elongated proliferative areas within the crypts. In addition, the intestinal stem cell marker and R-spondin receptor LGR5 was significantly upregulated following GDNF treatment in all experimental models. The effects of GDNF on cell proliferation, LGR5 and Ki67 upregulation were blocked using the RET-specific inhibitor BLU-667. Downstream of RET-phosphorylation, activation of Src kinase was involved to mediate GDNF effects. GDNF promotes intestinal wound healing by promoting cell proliferation. This is mediated by RET-dependent activation of Src kinase with consecutive LGR5 upregulation, indicating activation of the stem cell niche.

Inflammatory bowel diseases (IBD) such as Crohn ′s disease (CD) have a complex aetiology with alterations of both the intestinal epithelial barrier and the IL23/IL17 immune response. Here, we investigated the role of a novel mutation in the desmosomal cadherin desmoglein 2 gene (DSG2) in the pathogenesis of IBD. DSG2 is known to regulate intestinal epithelial barrier integrity. Genetic analysis of a CD patient revealed a novel likely pathogenic DSG2 mutation leading to a truncated protein lacking part of the intracellular domain. We generated an enterocyte-specific mouse model, recapitulating the human mutation to study how the cytoplasmic truncation of Dsg2 affects intestinal barrier properties systemically. Moreover, we analysed the intestinal genetic profile in these mice and compared it to IBD patients. We describe a first CD patient with a rare mutation in the DSG2 gene causing cytoplasmic truncation with affects Dsg2 mobility. Mice with enterocyte-specific Dsg2 truncation suffered from a lethal intestinal barrier defect and presented a skewed IL17 response similar to CD patients. We identified the desmosomal cadherin Dsg2 as a regulator of the skewed IL17 response. These data indicate that desmosomes regulate inflammation similar to psoriasis which explains why the same novel immune therapies are effective for both diseases. ### Competing Interest Statement The authors have declared no competing interest.

Loss of intestinal epithelial barrier function is a hallmark in digestive tract inflammation. The detailed mechanisms remain unclear due to the lack of suitable cell-based models in barrier research. Here we performed a detailed functional characterization of human intestinal organoid cultures under different conditions with the aim to suggest an optimized ex-vivo model to further analyse inflammation-induced intestinal epithelial barrier dysfunction. Differentiated Caco2 cells as a traditional model for intestinal epithelial barrier research displayed mature barrier functions which were reduced after challenge with cytomix (TNFα, IFN-γ, IL-1ß) to mimic inflammatory conditions. Human intestinal organoids grown in culture medium were highly proliferative, displayed high levels of LGR5 with overall low rates of intercellular adhesion and immature barrier function resembling conditions usually found in intestinal crypts. WNT-depletion resulted in the differentiation of intestinal organoids with reduced LGR5 levels and upregulation of markers representing the presence of all cell types present along the crypt-villus axis. This was paralleled by barrier maturation with junctional proteins regularly distributed at the cell borders. Application of cytomix in immature human intestinal organoid cultures resulted in reduced barrier function that was accompanied with cell fragmentation, cell death and overall loss of junctional proteins, demonstrating a high susceptibility of the organoid culture to inflammatory stimuli. In differentiated organoid cultures, cytomix induced a hierarchical sequence of changes beginning with loss of cell adhesion, redistribution of junctional proteins from the cell border, protein degradation which was accompanied by loss of epithelial barrier function. Cell viability was observed to decrease with time but was preserved when initial barrier changes were evident. In summary, differentiated intestinal organoid cultures represent an optimized human ex-vivo model which allows a comprehensive reflection to the situation observed in patients with intestinal inflammation. Our data suggest a hierarchical sequence of inflammation-induced intestinal barrier dysfunction starting with loss of intercellular adhesion, followed by redistribution and loss of junctional proteins resulting in reduced barrier function with consecutive epithelial death.

Previous data provided evidence for a critical role of desmosomes to stabilize intestinal epithelial barrier (IEB) function. These studies suggest that desmosomes not only contribute to intercellular adhesion but also play a role as signaling hubs. The contribution of desmosomal plaque proteins plakophilins (PKP) in the intestinal epithelium remains unexplored. The intestinal expression of PKP2 and PKP3 was verified in human gut specimens, human intestinal organoids as well as in Caco2 cells whereas PKP1 was not detected. Knock-down of PKP2 using siRNA in Caco2 cells resulted in loss of intercellular adhesion and attenuated epithelial barrier. This was paralleled by changes of the whole desmosomal complex, including loss of desmoglein2, desmocollin2, plakoglobin and desmoplakin. In addition, tight junction proteins claudin1 and claudin4 were reduced following the loss of PKP2. Interestingly, siRNA-induced loss of PKP3 did not change intercellular adhesion and barrier function in Caco2 cells, while siRNA-induced loss of both PKP2 and PKP3 augmented the changes observed for reduced PKP2 alone. Moreover, loss of PKP2 and PKP2/3, but not PKP3, resulted in reduced activity levels of protein kinase C (PKC). Restoration of PKC activity using Phorbol 12-myristate 13-acetate (PMA) rescued loss of intestinal barrier function and attenuated the reduced expression patterns of claudin1 and claudin4. Immunostaining, proximity ligation assays and co-immunoprecipitation revealed a direct interaction between PKP2 and PKC. In summary, our in vitro data suggest that PKP2 plays a critical role for intestinal barrier function by providing a signaling hub for PKC-mediated expression of tight junction proteins claudin1 and claudin4.

Breakdown of endothelial barrier integrity determines organ dysfunction and outcome of patients with sepsis. Increased levels of soluble vascular endothelial (VE)-cadherin fragments (sVE-cadherin) have previously been linked with inflammation-induced loss of endothelial barrier function. We provide evidence for a causative role of sVE-cadherin to induce loss of endothelial barrier function. In patients with sepsis, sVE-cadherin levels were associated with organ dysfunction and the need for volume resuscitation. Similarly, LPS-induced systemic inflammation in rats with microvascular dysfunction was paralleled by augmented sVE-cadherin levels. Newly generated recombinant human sVE-cadherin (extracellular domains EC1-5) induced loss of endothelial barrier function in both human microvascular endothelial cells in vitro and in rat mesenteric microvessels in vivo and reduced microcirculatory flow. sVE-cadherin(EC1-5) disturbed VE-cadherin-mediated adhesion and perturbed VE-protein tyrosine phosphatase (VE-PTP)/VE-cadherin interaction resulting in RhoGEF1-mediated RhoA activation. VE-PTP inhibitor AKB9778 and Rho-kinase inhibitor Y27632 blunted all sVE-cadherin(EC1-5)-induced effects, which uncovers a pathophysiological role of sVE-cadherin via dysbalanced VE-PTP/RhoA signaling.

Aim Increased levels of soluble Vascular endothelial (VE)-cadherin fragments (sVE-cadherin) have previously been linked with inflammation-induced loss of endothelial barrier function. We tested whether sVE-cadherin is critically involved in the onset of endothelial barrier dysfunction. Methods and Results Application of recombinant human sVE-cadherin (extracellular domains EC1-5) on human microvascular endothelial cells in vitro and in a rat model in vivo induced loss of endothelial barrier function and reduced microcirculatory flow. sVE-cadherin EC1-5 led to decreased localization of VE-cadherin at cell borders. Additionally, sVE-cadherin EC1-5 perturbed VE-protein tyrosine phosphatase (VE-PTP)/VE-cadherin interaction. VE-PTP inhibitor AKB9778 blunted all sVE-cadherin EC1-5 -induced effects in vitro and in vivo . Downstream effects involve VE-PTP-dependent RhoA activation which was attenuated by AKB9778. Rho-kinase inhibitor Y27632 blocked sVE-cadherin EC1-5 -induced loss of endothelial barrier function. Conclusion sVE-cadherin disrupts endothelial barrier function by dismantling the VE-cadherin complex at cell borders via VE-PTP-dependent RhoA activation. This uncovers a novel pathophysiological role of sVE-cadherin in the context of endothelial barrier dysfunction in inflammation.

Abstract Background Due to the rising incidence and socioeconomic relevance of Inflammatory Bowel Disease (IBD) worldwide, new therapeutic strategies are necessary to improve patient care. Since immunosuppressants are partially insufficient with relevant rates of side effects, novel concepts of treatment need to be developed. Mucosal and/or histological healing represent potential criteria to measure disease activity and are mentioned in current guidelines as favorable prognostic factors regarding disease outcomes. In previous studies, the soluble factor Glial cell line-Derived Neurotrophic Factor (GDNF) has been shown to be significantly reduced in tissue specimens from IBD patients and to be critically involved in intestinal epithelial barrier maturation. Therefore, the goal of this study was to further analyze the potential role of GDNF in IBD. Methods Intestinal wound healing was assessed in endoscopic biopsy-based wound assays as well as dextran sodium sulphate (DSS)-induced colitis in vivo. During the experiments, C57Bl/6 mice were either injected with GDNF or sodium chloride intraperitoneal while tissue samples were used for detailed molecular analyses. Further, scratch wound assays were performed in Caco2 monolayers to identify molecular pathways and cell mechanisms in vitro. Results Intraperitoneal injection of GDNF resulted in significantly enhanced wound closure in vivo. During the DSS-induced colitis model, mice treated with GDNF demonstrated significantly improved recovery from colitis in comparison to mice injected with sodium chloride. Similarly, application of GDNF in a scratch wound assay significantly enhanced wound closure in vitro. Mechanistically, molecular analysis revealed that GDNF application resulted in significantly increased cell proliferation. The effect of GDNF on cell proliferation was based on upregulation of LGR5-positive cells with increased phosphorylation of pSrc. Further studies demonstrated that the effect of GDNF on wound healing and cell proliferation could be inhibited by application of a Src-inhibitor. Conclusion GDNF significantly improves intestinal mucosal healing in vivo and in vitro which results in significantly enhanced recovery from colitis. While this effect is driven by increased cell proliferation due to upregulation of LGR5-positive cells and increased phosphorylation of pSrc, previous studies already demonstrated the critical impact of GDNF on epithelial barrier function. Following that, GDNF might have great potential in IBD treatment and could be a new target for future therapeutic approaches.

An impaired intestinal epithelial barrier (IEB) is a hallmark in the pathogenesis of inflammatory bowel diseases (IBD), including ulcerative colitis and Crohn's disease. Most of the IBD patients are treated with anti-inflammatory reagents including glucocorticoids, anti TNFα (tumor necrosis factor alpha) antibodies and mesasalazine. While the Immune-modulating effects of those therapies are well documented, we hypothesized whether there are direct effects of those reagents on enterocytes and the IEB. To address this hypothesis we used Caco2 cells as well as 2D and 3D murine organoids and performed analysis of intestinal epithelial barrier function. Inflammation-induced alterations were mimicked using cytomix for 24h (TNFα, Interleukin 1 beta and Interferon gamma). The enterocytes were treated with 100ng/ml anti-TNFα antibody Infliximab, 1mM prednisolone or 5mM mesasalazine, respectively following the challenge with cytomix. Epithelial permeability as revealed by measurements of 4kDa FITC Dextran flux and of transepithelial electric resistance showed that Infliximab and a pre-incubation with prednisolone for 24h attenuated inflammation-induced breakdown of the IEB. This was not the case after application of mesasalazine or a simultaneous incubation of prednisolone. The functional changes in intestinal epithelial barrier function following incubation with Cytomix were paralleled by alterations in the expression of "leaky" tight junction protein Claudin2, as revealed by Western blots. Furthermore, immunostainings demonstrated an altered distribution of barrier sealing proteins such as Claudin1, Desmoglein2 and E-Cadherin. All of these effects were blocked by infliximab and prednisolone. In summary, our data indicate that infliximab and prednisolone have direct effects on the IEB that may contribute to the efficacy in the treatment of IBD.

Enteric glial cells (EGCs) of the enteric nervous system are critically involved in the maintenance of intestinal epithelial barrier function (IEB). The underlying mechanisms remain undefined. Glial cell line-derived neurotrophic factor (GDNF) contributes to IEB maturation and may therefore be the predominant mediator of this process by EGCs. Using GFAPcre x Ai14floxed mice to isolate EGCs by Fluorescence-activated cell sorting (FACS), we confirmed that they synthesize GDNF in vivo as well as in primary cultures demonstrating that EGCs are a rich source of GDNF in vivo and in vitro. Co-culture of EGCs with Caco2 cells resulted in IEB maturation which was abrogated when GDNF was either depleted from EGC supernatants, or knocked down in EGCs or when the GDNF receptor RET was blocked. Further, TNFα-induced loss of IEB function in Caco2 cells and in organoids was attenuated by EGC supernatants or by recombinant GDNF. These barrier-protective effects were blunted when using supernatants from GDNF-deficient EGCs or by RET receptor blockade. Together, our data show that EGCs produce GDNF to maintain IEB function in vitro through the RET receptor.

Inflammation-induced reduction of intestinal desmosomal cadherin Desmoglein 2 (Dsg2) is linked to changes of tight junctions (TJ) leading to impaired intestinal epithelial barrier (IEB) function by undefined mechanisms. We characterized the interplay between loss of Dsg2 and upregulation of pore-forming TJ protein Claudin2. Intraperitoneal application of Dsg2-stablising Tandem peptide (TP) attenuated impaired IEB function, reduction of Dsg2 and increased Claudin2 in DSS-induced colitis in C57Bl/6 mice. TP blocked loss of Dsg2-mediated adhesion and upregulation of Claudin2 in Caco2 cells challenged with TNFα. In Dsg2-deficient Caco2 cells basal expression of Claudin2 was increased which was paralleled by reduced transepithelial electrical resistance and by augmented phosphorylation of AKTSer473 under basal conditions. Inhibition of phosphoinositid-3-kinase proved that PI-3-kinase/AKT-signaling is critical to upregulate Claudin2. In immunostaining PI-3-kinase dissociated from Dsg2 under inflammatory conditions. Immunoprecipitations and proximity ligation assays confirmed a direct interaction of Dsg2 and PI-3-kinase which was abrogated following TNFα application. In summary, Dsg2 regulates Claudin2 expression by sequestering PI-3-kinase to the cell borders in intestinal epithelium.