Dataset Information

Lacticaseibacillus casei AMBR2 Restores Airway Epithelial Integrity in Chronic Rhinosinusitis With Nasal Polyps.

ABSTRACT:

Purpose

A defective epithelial barrier has been demonstrated in chronic rhinosinusitis with nasal polyps (CRSwNP). Lactobacilli are shown to restore epithelial barrier defects in gastrointestinal disorders, but their effect on the airway epithelial barrier is unknown. In this study, hence, we evaluated whether the nasopharyngeal isolates Lacticaseibacillus casei AMBR2 and Latilactobacillus sakei AMBR8 could restore nasal epithelial barrier integrity in CRSwNP.

Methods

Ex vivo trans-epithelial tissue resistance and fluorescein isothiocyanate-dextran 4 kDa (FD4) permeability of nasal mucosal explants were measured. The relative abundance of lactobacilli in the maxillary sinus of CRSwNP patients was analyzed by amplicon sequencing of the V4 region of the 16S rRNA gene. The effect of spray-dried L. casei AMBR2 and L. sakei AMBR8 on epithelial integrity was investigated in vitro in primary nasal epithelial cells (pNECs) from healthy controls and patients with CRSwNP as well as in vivo in a murine model of interleukin (IL)-4 induced barrier dysfunction. The activation of Toll-like receptor 2 (TLR2) was explored in vitro by using polyclonal antibodies.

Results

Patients with CRSwNP had a defective epithelial barrier which positively correlated with the relative abundance of lactobacilli-specific amplicons in the maxillary sinus. L. casei AMBR2, but not L. sakei AMBR8, increased the trans-epithelial electrical resistance (TEER) of pNECs from CRSwNP patients in a time-dependent manner. Treatment of epithelial cells with L. casei AMBR2 promoted the tight junction proteins occludin and zonula occludens-1 reorganization. Furthermore, L. casei AMBR2 prevented IL-4-induced nasal permeability in vivo and in vitro. Finally, the beneficial effect of L. casei AMBR2 on nasal epithelial cells in vitro was TLR2-dependent as blocking TLR2 receptors prevented the increase in TEER.

Conclusions

A defective epithelial barrier in CRSwNP may be associated with a decrease in relative abundance of lactobacilli-specific amplicons. L. casei AMBR2 would restore nasal epithelial integrity and can be a novel therapeutic strategy for CRSwNP.

SUBMITTER: Martens K

PROVIDER: S-EPMC8255346 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BACKGROUND:Chronic rhinosinusitis with nasal polyps (CRSwNP) is characterized by an alteration in airway epithelial cell functions including barrier function, wound repair mechanisms, mucociliary clearance. The mechanisms leading to epithelial cell dysfunction in nasal polyps (NPs) remain poorly understood. Our hypothesis was that among the inflammatory cytokines involved in NPs, IL-6 could alter epithelial repair mechanisms and mucociliary clearance. The aim of this study was to evaluate the in vitro effects of IL-6 on epithelial repair mechanisms in a wound repair model and on ciliary beating in primary cultures of Human Nasal Epithelial Cells (HNEC). METHODS:Primary cultures of HNEC taken from 38 patients during surgical procedures for CRSwNP were used in an in vitro model of wound healing. Effects of increasing concentrations of IL-6 (1 ng/mL, 10 ng/mL, and 100 ng/mL) and other ILs (IL-5, IL-9, IL-10) on wound closure kinetics were compared to cultures without IL-modulation. After wound closure, the differentiation process was characterized under basal conditions and after IL supplementation using cytokeratin-14, MUC5AC, and ?IV tubulin as immunomarkers of basal, mucus, and ciliated cells, respectively. The ciliated edges of primary cultures were analyzed on IL-6 modulation by digital high-speed video-microscopy to measure: ciliary beating frequency (CBF), ciliary length, relative ciliary density, metachronal wavelength and the ciliary beating efficiency index. RESULTS:Our results showed that: (i) IL-6 accelerated airway wound repair in vitro, with a dose-response effect whereas no effect was observed after other ILs-stimulation. After 24 h, 79% of wounded wells with IL6-100 were fully repaired, vs 46% in the IL6-10 group, 28% in the IL6-1 group and 15% in the control group; (ii) specific migration analyses of closed wound at late repair stage (Day 12) showed IL-6 had the highest migration compared with other ILs (iii) The study of the IL-6 effect on ciliary function showed that CBF and metachronal wave increased but without significant modifications of ciliary density, length of cilia and efficiency index. CONCLUSION:The up-regulated epithelial cell proliferation observed in polyps could be induced by IL-6 in the case of prior epithelial damage. IL-6 could be a major cytokine in NP physiopathology.

Project description:BackgroundThe aim of this study is to investigate the potential key genes related to Chronic rhinosinusitis with nasal polyps (CRSwNP).MethodsDatasets GSE36830 and GSE72713 were obtained from Gene Expression Omnibus. Dataset GSE36830 was used to identify differentially expressed genes in CRSwNP patients. GO, KEGG analysis, and PPI network analysis were applied to further investigate the function of DEGs in CRSwNP. GSEA was also performed to explore the mechanisms of DEGs. Dataset GSE72713 was applied to validate the key gene. Moreover, to detect the expression of target gene, nasal polyp tissues and middle turbinate specimens were collected from CRSwNP patients (n = 20) and controls (n = 20), respectively. RT-PCR, Western blot, and immunofluorescence staining were applied. HE and AB-PAS staining were used to assess the infiltration of inflammatory cells. The proliferation and migration ability of human nasal epithelial cells (HNEpCs) were tested via Cell Counting kit-8, wound healing assay and Transwell migration assay. Air-liquid interface was used to culture primary human nasal epithelial cells (pHNECs) from health controls and nasal polyp tissues of CRSwNP patients.ResultsA total of 1035 DEGs were identified, and 661 genes were up-regulated and 374 genes were down-regulated. According to PPI network analysis, the top 10 scored genes were identified. Among them, only EGF was down-regulated in CRSwNP. Meanwhile, GSEA result shows that EGF is significantly enriched in WNT activated receptor activity. CCK-8, wound healing assay, and transwell migration assay indicated that recombinant human EGF can promote the proliferation and migration of HNEpCs in vitro. Immunofluorescence staining shows that rhEGF can increase the expression of ZO-1 in pHNECs from nasal polyp tissues.ConclusionBioinformatics analysis and in vitro experiments were used to explore the pathogenesis of CRSwNP, and the results showed that EGF may play an important role in the protection of nasal epithelial barrier.