Journal: Frontiers in Immunology

Article Title: Airway Epithelial Cells Generate Pro-inflammatory Tenascin-C and Small Extracellular Vesicles in Response to TLR3 Stimuli and Rhinovirus Infection

doi: 10.3389/fimmu.2019.01987

Figure Lengend Snippet: RV infection induces TN-C release in vitro and poly(I:C) induces TN-C in vivo , with increased release observed in bronchial epithelial cells from people with asthma. (A,B) Cell-free supernatants from NANA and AA PBECs, infected with RV-1B and RV-16 for 6 or 24 h, were obtained from the ALLIANCE study. (A) Cell-free supernatants were analyzed by western blot using antibodies specific to TN-C (one representative blot shown), with media samples (M), RV-1B (1B), and RV-16 samples (16). (B) Densitometry of the large >250 kDa variant was performed in ImageJ and normalized to protein concentration (determined by a bicinchoninic acid assay). Data shown are mean ± SEM with each replicate carried out using an independent PBEC donor ( n = 4). (C,D) Under recovery anesthesia, adult C57BL/6 mice were treated intranasally with 50 μl PBS or 100 μg poly(I:C) in 50 μl PBS for up to 48 h. The mice were then sacrificed, and BALF was collected by washing the lungs with 3 ml of PBS. (C) 150 μl of mouse BALF was TCA precipitated to a final volume of 20 μl and the presence of TN-C at 24 and 48 h was analyzed by western blot (48 h blot shown). (D) Densitometry of the small ~250 kDa variant was then performed using ImageJ software and normalized to neutrophil cell count. Data shown are mean ± SEM from a single experiment, with each point a separate mouse (3 mice for PBS treatment and 7 mice for poly(I:C) treatment). Significant differences in TN-C secretion are indicated by # p < 0.05; ** p < 0.01; *** p < 0.001; analyzed by two way ANOVA with Tukey's post-hoc test.

Article Snippet: The BEAS-2B epithelial cell line, and primary bronchial epithelial cells (PBECs) isolated from healthy humans, were purchased from ATCC and Promocell (Heidelberg, Germany) and cells were maintained as described ( , ).

Techniques: Infection, In Vitro, In Vivo, Western Blot, Variant Assay, Protein Concentration, Acid Assay, Software, Cell Counting

Journal: Frontiers in Immunology

Article Title: Airway Epithelial Cells Generate Pro-inflammatory Tenascin-C and Small Extracellular Vesicles in Response to TLR3 Stimuli and Rhinovirus Infection

doi: 10.3389/fimmu.2019.01987

Figure Lengend Snippet: RV induced TN-C release from bronchial epithelial cells is triggered by TLR3 activation and is not RV-serotype specific. PBECs (A–C) and BEAS-2B cells (D) were treated with poly(I:C) (25 μg/ml), RV-1B (MOI 0.6), or RV-16 (MOI 1.5) for the indicated times. Cell-free supernatants were analyzed by ELISA to measure TN-C release. Data shown are mean ± SEM ( N = 3–5) with each replicate a separate BEAS-2B cell passage or independent PBEC donor. Significant differences in TN-C release are indicated by * p < 0.05; ** p < 0.01; *** p < 0.001; analyzed by Kruskal-Wallis one-way ANOVA with Dunn's post-hoc test (A,C) or two way repeated measures ANOVA with Tukey's post-hoc test (B,D) .

Article Snippet: The BEAS-2B epithelial cell line, and primary bronchial epithelial cells (PBECs) isolated from healthy humans, were purchased from ATCC and Promocell (Heidelberg, Germany) and cells were maintained as described ( , ).

Techniques: Activation Assay, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Immunology

Article Title: Airway Epithelial Cells Generate Pro-inflammatory Tenascin-C and Small Extracellular Vesicles in Response to TLR3 Stimuli and Rhinovirus Infection

doi: 10.3389/fimmu.2019.01987

Figure Lengend Snippet: Induction of epithelial cell death is not sufficient to induce PBEC TN-C release. PBECs were grown to confluence and infected with RV-1B (MOI 0.6) or stimulated with poly(I:C) (25 μg/ml) or staurosporine (5 μg/ml) for the indicated times. Metabolic activity was measured by MTT assay in response to RV and staurosporine (A) and poly(I:C) and staurosporine (B) . The presence of TN-C was analyzed by western blot (M for Media, R for RV-1B, P for poly(I:C), and S for staurosporine; one representative blot shown; (C,D) . Densitometry of the large >250 kDa variant in response to RV (E) and poly(I:C) (F) was performed in ImageJ software. Values are expressed as mean ± SEM ( N = 3–4) with each replicate representing an independent PBEC donor. Significant differences in cell viability (# compared to media control which was assigned 100% and * compared to poly(I:C)/RV) and TN-C release are indicated by, * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001; ## p < 0.01; #### p < 0.0001, analyzed by two way ANOVA with Dunnett's post-hoc test. Analysis for MTT assay was performed on raw values.

Article Snippet: The BEAS-2B epithelial cell line, and primary bronchial epithelial cells (PBECs) isolated from healthy humans, were purchased from ATCC and Promocell (Heidelberg, Germany) and cells were maintained as described ( , ).

Techniques: Infection, Activity Assay, MTT Assay, Western Blot, Variant Assay, Software, Control

Journal: Frontiers in Immunology

Article Title: Airway Epithelial Cells Generate Pro-inflammatory Tenascin-C and Small Extracellular Vesicles in Response to TLR3 Stimuli and Rhinovirus Infection

doi: 10.3389/fimmu.2019.01987

Figure Lengend Snippet: The proposed mechanism of TN-C release following RV infection of bronchial epithelial cells. Upon viral infection, RV is internalized to the early endosome and uncoats. The single stranded RNA exits the endosome (early or late endosome depending on the RV serotype) and forms a temporary double stranded RNA intermediate (recognized by TLR3 in the cytoplasm). It is not yet clear how the endosomal TLR recognizes the cytoplasmic RNA. Poly(I:C) enters the endosome upon addition to cells and stimulates the TLR3 pathway. The amount of RV/Poly(I:C)-dependent cell associated TN-C upregulation may depend on existing basal levels of expression in the cell, with PBECs expressing higher TN-C levels at baseline compared to BEAS-2B cells. Furthermore, RV-induced TN-C release occurs in response to major and minor serotypes of RV, and is not an indirect consequence of cell cytotoxicity, as poly(I:C) induces TN-C release despite no significant cell death. Activation of the TLR3 dependent pathway (and other pathways upon viral infection) induce the release of TNFα and TGFβ, known transcriptional regulators of TN-C through the MAPK/ERK and NF-κB/p65 pathways. It is postulated that this triggers the signaling cascade required for the expression and release of TN-C in PBECs. Black lines denote events determined by experiments with both poly(I:C) and RV, the dashed line indicates events that occur in RV infection and the dotted line indicates events determined by experiments with poly(I:C) only.

Article Snippet: The BEAS-2B epithelial cell line, and primary bronchial epithelial cells (PBECs) isolated from healthy humans, were purchased from ATCC and Promocell (Heidelberg, Germany) and cells were maintained as described ( , ).

Techniques: Infection, Expressing, Activation Assay

Journal: bioRxiv

Article Title: The RNA binding protein Insulin Growth factor 2 binding Protein 3 (IGF2BP3) modulates IL-13/IL-4 signalling in human bronchial epithelial cells and is dysregulated in type 2 disease

doi: 10.1101/2025.07.19.665669

Figure Lengend Snippet: IGF2BP3 is increased in type 2 disease. A. Left: normalized expression levels of IGF2BP3 mRNA in severe asthma (SA) vs healthy control (HC) primary BECs at total and polyribosome-bound levels; centre: IGF2BP3 normalized microarray expression data comparing airway epithelial cells from healthy non-atopic (HNA) and atopic asthma (AA) children; right IGF2BP3 normalized microarray expression data comparing nasal lavage samples from children during Picornavirus-induced exacerbation (EX) vs 7-14 days post-exacerbation (post-EX). B. Normalized count levels comparing type 2 high vs type 2 low nasal brushings for genes that showed IGF2BP3-dependent IL-13-modulation in our Frac-seq data . C: Heatmap showing the normalized expression values of the common mRNA signature between differentially expressed genes in the GALA II cohort and our Frac-seq IGF2BP3 and IL-13-dependent DEGs. D. Model for IGF2BP3 effects on type 2 responses. Decreasing IGF2BP3 levels leads to increased stability of IL13RA1 and IL4R mRNA and increased surface expression of IL4R and IL13Rα1, leading to increased STAT6 phosphorylation and increased IL-13 (and possibly IL-4) transcriptional responses, contributing to an overall increased type 2 response. In disease, IGF2BP3 is upregulated potentially to supress excessive T2 responses.

Article Snippet: Primary bronchial epithelial cells (BECs) were commercially sourced (PromoCell), grown and expanded in growth factor-supplemented medium (PromoCell) on collagen-coated plates.

Techniques: Expressing, Control, Microarray, Phospho-proteomics

Journal: bioRxiv

Article Title: The RNA binding protein Insulin Growth factor 2 binding Protein 3 (IGF2BP3) modulates IL-13/IL-4 signalling in human bronchial epithelial cells and is dysregulated in type 2 disease

doi: 10.1101/2025.07.19.665669

Figure Lengend Snippet: Decreasing IGF2BP3 levels increases IL-13/IL-4-driven transcriptional responses. RT-qPCR results of primary BECs transfected with siRNAs against IGF2BP3 (siIGF2BP3) or scrambled control (siControl) and stimulated with IL-13 or IL-4. A: RT-qPCR results for IGF2BP3 knock down and the major signalling mediators of IL-13 and IL-4. B: RT-qPCR results for chemokines. C: RT-qPCR results for pro-inflammatory IL6 and IL8 mRNAs. Data (n=3) depicted as mean ±SEM. One-tailed ratio t-tests. * P ≤ 0.05; ** P ≤ 0.01.

Article Snippet: Primary bronchial epithelial cells (BECs) were commercially sourced (PromoCell), grown and expanded in growth factor-supplemented medium (PromoCell) on collagen-coated plates.

Techniques: Quantitative RT-PCR, Transfection, Control, Knockdown, One-tailed Test