Background & Aims Epithelial tight junctions are compromised in gastrointestinal disease. Processes that contribute to the resulting barrier loss include endocytic occludin removal from the tight junction and reduced occludin expression. Nevertheless, the relatively-normal basal phenotype of occludin knockout (KO) mice has been taken as evidence that occludin does not contribute to gastrointestinal barrier function. We asked whether stress could unmask occludin functions within intestinal epithelia. Methods Wildtype (WT), universal and intestinal epithelial-specific occludin KO, and villin-EGFP-occludin transgenic mice as well as WT and occludin knockdown (KD) Caco-2_BBe cell monolayers were challenged with DSS, TNBS, staurosporine, 5-FU, or TNF. Occludin and caspase-3 expression were assessed in patient biopsies. Results Intestinal epithelial occludin loss limited severity of DSS- and TNBS-induced colitis due to epithelial resistance to apoptosis; activation of both intrinsic and extrinsic apoptotic pathways was blocked in occludin KO epithelia. Promoter analysis revealed that occludin enhances CASP3 transcription and, conversely, that occludin downregulation reduces caspase-3 expression. Analysis of biopsies from Crohn’s disease and ulcerative colitis patients and normal controls demonstrated that disease-associated occludin downregulation was accompanied by and correlated with reduced caspase-3 expression. In vitro, cytokine-induced occludin downregulation resulted in reduced caspase-3 expression and resistance to intrinsic and extrinsic pathway apoptosis, demonstrating an overall protective effect of inflammation-induced occludin loss. Conclusions The tight junction protein occludin regulates apoptosis by enhancing caspase-3 transcription. These data suggest that reduced epithelial caspase-3 expression downstream of occludin downregulation is a previously-unappreciated anti-apoptotic process that contributes to mucosal homeostasis in inflammatory conditions.

Objective Intestinal barrier loss is a Crohn’s disease (CD) risk factor. This may be related to increased expression and enzymatic activation of myosin light chain kinase 1 (MLCK1), which increases intestinal paracellular permeability and correlates with CD severity. Moreover, preclinical studies have shown that MLCK1 recruitment to cell junctions is required for tumour necrosis factor (TNF)-induced barrier loss as well as experimental inflammatory bowel disease progression. We sought to define mechanisms of MLCK1 recruitment and to target this process pharmacologically. Design Protein interactions between FK506 binding protein 8 (FKBP8) and MLCK1 were assessed in vitro. Transgenic and knockout intestinal epithelial cell lines, human intestinal organoids, and mice were used as preclinical models. Discoveries were validated in biopsies from patients with CD and control subjects. Results MLCK1 interacted specifically with the tacrolimus-binding FKBP8 PPI domain. Knockout or dominant negative FKBP8 expression prevented TNF-induced MLCK1 recruitment and barrier loss in vitro. MLCK1-FKBP8 binding was blocked by tacrolimus, which reversed TNF-induced MLCK1-FKBP8 interactions, MLCK1 recruitment and barrier loss in vitro and in vivo. Biopsies of patient with CD demonstrated increased numbers of MLCK1-FKBP8 interactions at intercellular junctions relative to control subjects. Conclusion Binding to FKBP8, which can be blocked by tacrolimus, is required for MLCK1 recruitment to intercellular junctions and downstream events leading to immune-mediated barrier loss. The observed increases in MLCK1 activity, MLCK1 localisation at cell junctions and perijunctional MLCK1-FKBP8 interactions in CD suggest that targeting this process may be therapeutic in human disease. These new insights into mechanisms of disease-associated barrier loss provide a critical foundation for therapeutic exploitation of FKBP8-MLCK1 interactions.

Objectives: Titanium implants are regarded as a promising treatment modality for replacing missing teeth. Osteointegration and antibacterial properties are both desirable characteristics for titanium dental implants. The aim of this study was to create zinc (Zn)-, strontium (Sr)-, and magnesium (Mg)-multidoped hydroxyapatite (HAp) porous coatings, including HAp, Zn-doped HAp, and Zn-Sr-Mg-doped HAp, on titanium discs and implants using the vapor-induced pore-forming atmospheric plasma spraying (VIPF-APS) technique. Methods: The mRNA and protein levels of osteogenesis-associated genes such as collagen type I alpha 1 chain (COL1A1), decorin (DCN), osteoprotegerin (TNFRSF11B), and osteopontin (SPP1) were examined in human embryonic palatal mesenchymal cells. The antibacterial effects against periodontal bacteria, including Porphyromonas gingivalis and Prevotella nigrescens, were investigated. In addition, a rat animal model was used to evaluate new bone formation via histologic examination and micro-computed tomography (CT). Results: The ZnSrMg-HAp group was the most effective at inducing mRNA and protein expression of TNFRSF11B and SPP1 after 7 days of incubation, and TNFRSF11B and DCN after 11 days of incubation. In addition, both the ZnSrMg-HAp and Zn-HAp groups were effective against P. gingivalis and P. nigrescens. Furthermore, according to both in vitro studies and histologic findings, the ZnSrMg-HAp group exhibited the most prominent osteogenesis and concentrated bone growth along implant threads. Significance: A porous ZnSrMg-HAp coating using VIPF-APS could serve as a novel technique for coating titanium implant surfaces and preventing further bacterial infection.

Objective: Immune checkpoint inhibitors (ICIs) have revolutionised cancer treatment and patients’ survival. However, ICIs also cause severe immune-related adverse events, notably colitis, resulting in ICIs therapy discontinuation and tumour immunotherapy failure. This study investigates long myosin light chain kinase 1 (MLCK1), a known regulator of tight junction and gut permeability, to elucidate the mechanisms underlying ICI-mediated colitis and identify approaches to reduce this toxicity. Design: This study employed an integrated approach, using clinical samples, in vivo models and in vitro organoid systems. Biopsies from patients with ICIs colitis were profiled using single-cell RNA sequencing (scRNA-seq) and spatial transcriptomics. To recapitulate human ICIs colitis, we used a wild mouse microbiota (WildR) model, alongside various genetically modified and tumour-bearing models (including melanoma and MC38). Furthermore, mechanisms were investigated through organoid-immune cell co-cultures. Finally, surface plasmon resonance, microscale thermophoresis, full-spectrum flow cytometry, bulk RNA sequencing, immunostaining, ELISA and gut permeability assays were performed to comprehensively delineate the underlying molecular mechanism. Results: Tight junction integrity was compromised in both human ICIs colitis and our WildR mouse model. We determined that this barrier dysfunction is driven by activation of the MLCK1-mediated leak pathway following ICI treatment. Using murine models, we identified tumour necrosis factor secreted by CD8 ⁺ and CD4 ⁺ T cells as an upstream regulator that induces colitis through this MLCK-dependent mechanism, as genetic deletion of MLCK preserved the tight junction structure and ameliorated the inflammation and ICIs colitis. Furthermore, a pharmacological screen identified the small molecule Epicatechin, which blocks MLCK1-FKBP8 interaction and inhibits the recruitment of MLCK1 to the perijunctional actomyosin ring and prevents the intestinal barrier loss. Finally, treatment with Epicatechin mitigated ICI-induced colitis without compromising the antitumour efficacy of the immunotherapy. Conclusions: These findings suggest that MLCK1-dependent tight junction regulation is essential for ICIs colitis, positioning barrier restoration as a potential therapeutic strategy.

Dysregulated serotonin/5‐hydroxytryptamine (5‐HT) metabolism and dense mucosal neurite distribution are associated with visceral hypersensitivity (VH), which plays a key role in irritable bowel syndrome (IBS) pain symptoms. The 5‐HT receptor subtype 7 (5‐HT₇) is involved in neuroplasticity. We aim to investigate the analgesic effects of 5‐HT₇ antagonists in mouse models and explore downstream changes of nerve growth factor (NGF) and brain‐derived neurotrophic factor (BDNF) in the enteric neurons. Selective 5‐HT₇ antagonists [CYY1005 (CYY), JNJ‐18038683 (JNJ) or SB269970 (SB7)] were orogavaged to an IBS‐like mouse model of postinflammatory VH after resolution from trinitrobenzene sulfonic acid‐induced colitis. Visceromotor response, mucosal neurite outgrowth, and neurotrophin levels were assessed. Orogavage of CYY had stronger analgesic effects than JNJ or SB7 in the IBS‐like mice. Higher density of 5‐HT₇‐expressing mucosal nerve fibres was associated with increased NGF and BDNF immunostaining in the submucosal plexus of IBS‐like mice compared to those of sham mice. No difference in the serotonergic neurons of spinal ganglia and brain regions was observed between IBS‐like and sham mice. Moreover, CYY treatment for 10 days decreased the colonic neurotrophin levels and reduced pain sensation in IBS‐like mice. Serotonin‐induced neurite elongation was inhibited by 5‐HT₇ antagonists in mouse primary submucosal neuron cultures and human SH‐SY5Y cell lines. Neutralizing antibodies to neurotrophins also diminished the serotonin‐induced neurite outgrowth. Lastly, 5‐HT₇ activation upregulated neurotrophin expression in neurons via Cdk5/mTOR/Cdc42 signalling. In conclusion, 5‐HT₇ antagonists attenuated neurotrophin overproduction in the submucosal nerve plexus, leading to lesser mucosal innervation and reduced intestinal nociception in IBS‐like mouse models. image Key points: Aberrant serotonin/5‐hydroxytryptamine (5‐HT) metabolism and dense mucosal neurites are linked with irritable bowel syndrome (IBS) pain symptoms. Current treatments are ineffective for IBS pain management. Previous studies showed 5‐HT receptor subtype 7 (5‐HT₇)‐expressing nerve fibres in the colonoscopic biopsy of IBS patients and colonic mucosa of IBS‐like mouse models. Moreover, 5‐HT₇ activation is involved in neuroplasticity in neural cell lines in vitro. Although visceral pain has been studied extensively in spinal afferents, the involvement of enteric neurons in intestinal nociception remains to be determined. Orogavage of 5‐HT₇ antagonist reduced mucosal neurite outgrowth and decreased neurotrophin overproduction in submucosal nerves, resulting in lower intestinal pain. Activation of 5‐HT₇ promotes neurite elongation in primary submucosal nerve cultures. The findings implicate a potential role of submucosal plexus in 5‐HT₇‐dependent visceral hypersensitivity.