Development of a Novel ex vivo Nasal Epithelial Cell Model Supporting Colonization With Human Nasal Microbiota

Parkinson’s disease (PD) is the most common neurodegenerative movement disorder, characterized by olfactory dysfunction in the early stages. α-Synuclein pathologies in the olfactory organs are shown to spread to the brain through the nose-brain axis. We first developed a nasal epithelial PD cellular model by treating RPMI-2650 cells with α-synuclein preformed fibrils (PFF). Upon uptake of PFF, RPMI-2650 cells showed mitochondrial proteome alteration and downregulation of parkin, which has previously been identified as a nasal biomarker of PD. Functional cluster analysis of differentially expressed genes in RPMI-2650 cells revealed various pathways affected by α-synuclein pathology, including the detection of chemical stimulus involved in sensory perception, olfactory receptor activity, and sensory perception of smell. Among genes that were most affected, we validated, by real-time quantitative PCR, the downregulation of MAP3K8, OR10A4, GRM2, OR51B6, and OR9A2, as well as upregulation of IFIT1B, EPN1, OR1D5, LCN, and OTOL1 in PFF-treated RPMI-2650 cells. Subsequent analyses of clinical samples showed a downregulation of OR10A4 and OR9A2 transcripts and an upregulation of IFIT1B in cells isolated from the nasal fluid of PD patients, as compared to those from the controls (cutoff value = 0.5689 for OR9A2, with 72.4% sensitivity and 75% specificity, and 1.4658 for IFIT1B, with 81.8% sensitivity and 77.8% specificity). Expression levels of these nasal PD markers were not altered in nasal fluid cells from SWEDD (scans without evidence of dopaminergic deficits) patients with PD-like motor symptoms. These nasal markers were significantly altered in patients of PD with hyposmia compared to the control hyposmic subjects. Our results validated the α-synuclein-treated nasal epithelial cell model to identify novel biomarkers for PD and suggest the utility of olfactory transcripts, along with olfactory dysfunction, in the diagnosis of PD.

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Delayed induction of type I and III interferons mediates nasal epithelial cell permissiveness to SARS-CoV-2

Nature Communications ◽

10.1038/s41467-021-27318-0 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Catherine F. Hatton ◽

Rachel A. Botting ◽

Maria Emilia Dueñas ◽

Iram J. Haq ◽

Bernard Verdon ◽

...

Keyword(s):

Epithelial Cell ◽

Host Response ◽

Cell Model ◽

Viral Gene Expression ◽

Cell Types ◽

Nasal Delivery ◽

Nasal Epithelium ◽

Viral Gene ◽

Type I ◽

Nasal Epithelial Cell

AbstractThe nasal epithelium is a plausible entry point for SARS-CoV-2, a site of pathogenesis and transmission, and may initiate the host response to SARS-CoV-2. Antiviral interferon (IFN) responses are critical to outcome of SARS-CoV-2. Yet little is known about the interaction between SARS-CoV-2 and innate immunity in this tissue. Here we apply single-cell RNA sequencing and proteomics to a primary cell model of human nasal epithelium differentiated at air-liquid interface. SARS-CoV-2 demonstrates widespread tropism for nasal epithelial cell types. The host response is dominated by type I and III IFNs and interferon-stimulated gene products. This response is notably delayed in onset relative to viral gene expression and compared to other respiratory viruses. Nevertheless, once established, the paracrine IFN response begins to impact on SARS-CoV-2 replication. When provided prior to infection, recombinant IFNβ or IFNλ1 induces an efficient antiviral state that potently restricts SARS-CoV-2 viral replication, preserving epithelial barrier integrity. These data imply that the IFN-I/III response to SARS-CoV-2 initiates in the nasal airway and suggest nasal delivery of recombinant IFNs to be a potential chemoprophylactic strategy.

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Delayed induction of type I and III interferons and nasal epithelial cell permissiveness to SARS-CoV-2

10.1101/2021.02.17.431591 ◽

2021 ◽

Author(s):

Catherine F Hatton ◽

Rachel A Botting ◽

Maria Emilia Dueñas ◽

Iram J Haq ◽

Bernard Verdon ◽

...

Keyword(s):

Epithelial Cell ◽

Host Response ◽

Cell Model ◽

Viral Gene Expression ◽

Cell Types ◽

Nasal Delivery ◽

Nasal Epithelium ◽

Viral Gene ◽

Type I ◽

Nasal Epithelial Cell

AbstractThe nasal epithelium is a plausible entry point for SARS-CoV-2, a site of pathogenesis and transmission, and may initiate the host response to SARS-CoV-2. Antiviral interferon responses are critical to outcome of SARS-CoV-2. Yet little is known about the interaction between SARS-CoV-2 and innate immunity in this tissue. Here we applied single-cell RNA sequencing and proteomics to a primary cell model of human primary nasal epithelium differentiated at air-liquid interface. SARS-CoV-2 demonstrated widespread tropism for nasal epithelial cell types. The host response was dominated by type I and III IFNs and interferon-stimulated gene products. Nevertheless, this response was notably delayed in onset compared to viral gene expression, and thus failed to impact substantially on SARS-CoV-2 replication. However, when provided prior to infection, recombinant IFNβ or IFNλ1 induced an efficient antiviral state that potently restricted SARS-CoV-2 viral replication, preserving epithelial barrier integrity. These data suggest nasal delivery of recombinant IFNs to be a potential chemoprophylactic strategy.

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The mystery behind the nostrils – technical clues for successful nasal epithelial cell cultivation

Annals of Anatomy - Anatomischer Anzeiger ◽

10.1016/j.aanat.2021.151748 ◽

2021 ◽

pp. 151748

Author(s):

Malik Aydin ◽

Ella A. Naumova ◽

Aliyah Bellm ◽

Ann-Kathrin Behrendt ◽

Federica Giachero ◽

...

Keyword(s):

Epithelial Cell ◽

Cell Cultivation ◽

Nasal Epithelial Cell

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Characterisation of morphological differences in well-differentiated nasal epithelial cell cultures from preterm and term infants at birth and one-year

PLoS ONE ◽

10.1371/journal.pone.0201328 ◽

2018 ◽

Vol 13 (12) ◽

pp. e0201328 ◽

Cited By ~ 5

Author(s):

Helen E. Groves ◽

Hong Guo-Parke ◽

Lindsay Broadbent ◽

Michael D. Shields ◽

Ultan F. Power

Keyword(s):

Epithelial Cell ◽

Cell Cultures ◽

Term Infants ◽

Nasal Epithelial Cell ◽

Epithelial Cell Cultures ◽

Well Differentiated ◽

Morphological Differences ◽

One Year

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si-SNHG5-FOXF2 inhibits TGF-β1-induced fibrosis in human primary endometrial stromal cells by the Wnt/β-catenin signalling pathway

Stem Cell Research & Therapy ◽

10.1186/s13287-020-01990-3 ◽

2020 ◽

Vol 11 (1) ◽

Author(s):

Limin Liu ◽

Guobin Chen ◽

Taoliang Chen ◽

Wenjuan Shi ◽

Haiyan Hu ◽

...

Keyword(s):

Cell Proliferation ◽

Stromal Cells ◽

Target Genes ◽

Cell Model ◽

Ex Vivo ◽

Signalling Pathway ◽

Endometrial Stromal Cells ◽

Downstream Target ◽

Related Proteins ◽

Tgf Β1

Abstract Background Intrauterine adhesions (IUAs) are manifestations of endometrial fibrosis characterized by inflammation and fibrinogen aggregation in the extracellular matrix (ECM). The available therapeutic interventions for IUA are insufficiently effective in the clinical setting for postoperative adhesion recurrence and infertility problems. In this study, we investigated whether si-SNHG5-FOXF2 can serve as a molecular mechanism for the inhibition of IUA fibrosis ex vivo. Methods FOXF2, TGF-β1 and collagen expression levels were measured by microarray sequencing analysis in three normal endometrium groups and six IUA patients. We induced primary human endometrial stromal cells (HESCs) into myofibroblasts (MFs) to develop an IUA cell model with various concentrations of TGF-β1 at various times. Downstream target genes of FOXF2 were screened by chromatin immunoprecipitation combined with whole-genome high-throughput sequencing (ChIP-seq). We investigated ECM formation, cell proliferation and Wnt/β-catenin signalling pathway-related proteins in primary HESCs with FOXF2 downregulation by quantitative reverse transcription-polymerase chain reaction (qRT-PCR), western blotting (WB), immunohistochemistry (IHC), flow cytometry, ethylenediurea (EdU) and CCK8 assays. We identified long noncoding RNAs (lncRNA) SNHG5 as the upstream regulatory gene of FOXF2 through RNA immunoprecipitation (RIP), RNA pulldown and fluorescence in situ hybridization (FISH). Finally, we examined FOXF2 expression, ECM formation, cell proliferation and Wnt/β-catenin signalling pathway-related proteins in primary HESCs upon FOXF2 downregulation. Results FOXF2 was highly expressed in the endometrium of patients with IUA. Treatment of primary HESCs with 10 ng/ml TGF-β1 for 72 h was found to be most effective for developing an IUA cell model. FOXF2 regulated multiple downstream target genes, including collagen, vimentin (VIM) and cyclin D2/DK4, by ChIP-seq and ChIP-PCR. FOXF2 downregulation inhibited TGF-β1-mediated primary HESC fibrosis, including ECM formation, cell proliferation and Wnt/β-catenin signalling pathway-related protein expression. We identified lncRNA SNHG5 as an upstream gene that directly regulates FOXF2 by RIP-seq, qRT-PCR, WB and FISH. SNHG5 downregulation suppressed FOXF2 expression in the IUA cell model, resulting in synergistic repression of the Wnt/β-catenin pathway, thereby altering TGF-β1-mediated ECM aggregation in endometrial stromal cells ex vivo. Conclusions Regulation of the Wnt/β-catenin signalling pathway and ECM formation by si-SNHG5-FOXF2 effectively inhibited the profibrotic effect of TGF-β1 on primary HESCs. This finding can provide a molecular basis for antagonizing TGF-β1-mediated fibrosis in primary HESCs.

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