Euphorbium compositum SN improves the innate defenses of the airway mucosal barrier network during rhinovirus infection

Abstract

Background: Rhinoviruses (RV) are the major cause of common colds in healthy individuals and are associated with acute exacerbations in patients with chronic lung diseases. Yet, no vaccines or effective treatment against RV are available. This study investigated the effect of Euphorbium compositum SN (ECSN6), a multicomponent, multitarget medication made from natural ingredients, on the mucosal barrier network during RV infection.

Methods: Mucociliary-differentiated airway epithelial cell cultures were infected with RV or sham, and treated with 20% ECSN6 or placebo twice daily. Barrier integrity was assessed by measuring transepithelial resistance (TER), permeability to inulin, and expression and localization of intercellular junctions proteins (IJ). Ciliary beat frequency (CBF), expression of pro-inflammatory cytokines, antiviral interferons and mucins, and viral load were also measured. C57BL/6 mice were infected intranasally with RV or sham and treated with 40% ECSN6 or placebo twice daily. Inflammation of sinunasal mucosa, localization of E-cadherin, viral load and mucin gene expression were determined.

Results: ECSN6-treated, uninfected cell cultures showed small, but significant increase in TER over placebo, which was associated with enhanced localization of E-cadherin and ZO-1 to IJ. In RV-infected cultures, treatment with ECSN6, but not placebo prevented RV-induced (1) reduction in TER, (2) dissociation of E-cadherin and ZO-1 from the IJ, (3) mucin expression, and (4) CBF attenuation. ECSN6 also decreased RV-stimulated expression of pro-inflammatory cytokines and permeability to inulin. Although ECSN6 significantly increased the expression of some antiviral type I and type III interferons, it did not alter viral load. In vivo, ECSN6 reduced RV-A1-induced moderate inflammation of nasal mucosa, beneficially affected RV-A1-induced cytokine responses and Muc5ac mRNA expression and prevented RV-caused dissociation of E-cadherin from the IJ of nasal mucosa without an effect on viral clearance.

Conclusions: ECSN6 prevents RV-induced airway mucosal barrier dysfunction and improves the immunological and mucociliary barrier function. ECSN6 may maintain integrity of barrier function by promoting localization of tight and adherence junction proteins to the IJ. This in turn may lead to the observed decrease in RV-induced pro-inflammatory responses in vitro. By improving the innate defenses of the airway mucosal barrier network, ECSN6 may alleviate respiratory symptoms caused by RV infections.

Keywords: Apical junctional complex; Ciliary beat frequency; E-cadherin; Inflammation; Multicomponent; Multitarget; Transepithelial resistance.

Fig. 1

ECSN6 prevents RV-A1-induced reduction in TER and reduces permeability to inulin. Mucociliary-differentiated airway epithelial cultures were infected apically with RV-A1 or sham. The cultures were treated with placebo or 20% ECSN6 every 12 h starting from 2 h post-RV infection. A–C) TER was measured prior to RV infection (0 h) and at 24 h interval for up to 72 h during placebo or ECSN6 treatment. The data represent % change from TER at 0 h ± SEM from 3 experiments using cells from 3 donors (n = 3–12, cells from 3 donors). D) Apical surface of the cultures was washed 24 h post-infection, FITC-labeled inulin was added to the apical surface and fluorescence intensity was measured in the basolateral medium after 4 h. Data represents mean ± SEM calculated from cells obtained from 2 donors conducted in triplicates (n = 6). Statistical significance was conducted by ANOVA with Student-Newman-Keuls post-hoc analysis. * p < 0.05; ** p < 0.01; *** p < 0.001; ns = non-significant

Fig. 2

ECSN6 inhibits RV-A1-induced dissociation of E-cadherin and ZO-1. Mucociliary-differentiated airway epithelial cultures were infected apically with RV-A1 or sham. The cultures were treated with placebo or 20% ECSN6 every 12 h starting from 2 h post-RV infection. At 24 h post-infection, cultures were fixed in cold methanol, blocked with 1% BSA and incubated with antibodies to ZO-1 and E-cadherin. The bound antibodies were detected by Alexa Fluor 488-labeled anti-mouse IgG (ZO-1) and Alexa Fluor 594-labeled anti-rabbit IgG (E-cadherin). Nuclei were counterstained with DAPI and imaged using confocal microscopy. Arrows in RV-A1-infected placebo-treated cultures represent dissociation of ZO-1 and E-cadherin. Images are representative of 3 experiments

Fig. 3

ECSN6 reduces RV-A1-induced pro-inflammatory IL-6 and IL-8 cytokine secretion. Mucociliary-differentiated airway epithelial cultures were infected apically with RV-A1 or sham. The cultures were treated with placebo or 20% ECSN6 every 12 h starting from 2 h post-RV infection. Basolateral medium was collected at 24 h post-infection. IL-6, IL-8 and CCL-20 protein levels were determined by ELISA. Since there was a wide variability from donor to donor in IL-8 levels in uninfected cells (sham/placebo) the data were normalized to sham/placebo. For IL-6 and CCL-20 the basal levels did not vary widely, therefore absolute levels are presented. Data represent mean ± SEM calculated from cells obtained from 3 donors in triplicates or more (n = 8–9). ANOVA with Student-Newman-Keuls post-hoc analysis was conducted to determine the statistical significance. * p < 0.05; ** p < 0.01; *** p < 0.001; ns = non-significant

Fig. 4

ECSN6 inhibits RV-A1-stimulated MUC5AC expression. Mucociliary-differentiated airway epithelial cultures were infected apically with RV-A1 or sham. The cultures were treated with placebo or 20% ECSN6 at 2 and 12 h post-RV infection. At 24 h post-infection, total RNA extracted from the cells was converted to cDNA and mRNA expression of MUC5AC, MUC5B and GAPDH was determined by qPCR. The expression of genes was normalized to GAPDH. Data represent mean ± SEM calculated from cells obtained from 3 donors in one to two replicates (n = 5). ANOVA with Student-Newman-Keuls post-hoc analysis was conducted to determine the statistical significance. * p < 0.05; ** p < 0.01; *** p < 0.001; ns = non-significant. ANOVA with Student-Newman-Keuls post-hoc analysis showed no difference between groups of MUC5B expression

Fig. 5

ECSN6 prevents RV-A1-induced reduction in CBF. Mucociliary-differentiated airway epithelial cultures were infected apically with RV-A1 or sham and treated with placebo or 20% ECSN6 every 12 h starting from 2 h post-RV infection. (A) At 24 h post-infection, the cultures were subjected to high-speed microscopy and CBF was analyzed by Ciliarmove. Data represent mean ± SEM calculated from cells obtained from 3 donors with duplicates or triplicates (n = 6–8). ANOVA with Student-Newman-Keuls post-hoc analysis was conducted to determine the statistical significance. * p < 0.05; ** p < 0.01; *** p < 0.001; ns = non-significant. (B) Cell cultures were fixed in paraformaldehyde, blocked with 1% BSA and incubated with antibody to acetylated α-tubulin. The bound antibody was detected by Alexa Fluor 488-labeled anti-mouse IgG. Nuclei were counterstained with DAPI and imaged using confocal microscopy. Representative images of cell cultures at 24 h post-RV-A1 infection. Yellow arrows in RV-A1-infected placebo-treated culture represent clumping of cilia

Fig. 6

RV-A1-infected mice treated with ECSN6 show reduced inflammation and mucus secretion. Mice were infected with sham or RV-A1 and treated with placebo or 40% ECSN6 twice a day at 10 h interval for 24 h starting from 1 h after sham infection. The paraffin sections of snout in (A and C) were stained with H & E or in (B) with PAS. * represents sinunasal cavity and inflammatory milieu respectively in panel A; red and yellow arrows represent secreted mucus and goblet cells, respectively in panel B; and black arrow represent disorganization of cilia in panel C. Images are representative of 3–4 mice in each group

Fig. 7

ECSN6 attenuates RV-A1-induced expression of pro-inflammatory cytokines. Mice were infected with sham or RV-A1 and treated with placebo or 40% ECSN6 twice day at 10 h interval for up to 60 h starting from 1 h after RV-A1 infection. Mice were euthanized after 24–72 h post-infection and total RNA from sinunasal TRIZOL lysates was isolated and subjected RT-qPCR using gene-specific Taqman assays. The mRNA expression was normalized to β-actin. Data represent median with range from 3 experiments with a total of 6 mice per group (n = 6). ANOVA on ranks with Tukey post-hoc analysis. * p < 0.05; ** p < 0.01; *** p < 0.001; ns = non-significant. ANOVA on ranks with Tukey post-hoc analysis showed no difference between groups of Muc5B expression at 24 h and 72 h post-infection and Muc5ac expression at 72 h post-infection

Fig. 8

ECSN6 treatment prevents RV-A1-induced dissociation of E-cadherin. Mice were infected with sham or RV-A1 and treated with placebo or 40% ECSN6 twice day at 10 h interval for up to 60 h starting from 1 h after RV-A1 infection. (A) At 24 h post-infection, the paraffin sections of snout were prepared and immunostained with E-cadherin and imaged under a fluorescence microscope. Arrow in RV-A1-infected placebo-treated panel denotes dissociation of E-cadherin from the intercellular junctions of nasal epithelium. Images are representative of 3 to 4 mice per group. (B) Density of E-cadherin was measured by Image J and expressed as pixels/50 µm². Data represent median with range and statistical significance was determined by ANOVA on ranks with Tukey post-hoc analysis (n = 3 to 4; *p = < 0.05). (C) Total RNA from sinunasal TRIZOL lysates was isolated and subjected RT-qPCR using gene-specific Taqman assays after 24–72 h post-infection. The mRNA expression was normalized to β-actin. Data represent median with range from 3 experiments with a total of 6 mice per group. ANOVA on ranks with Tukey post-hoc analysis showed no difference between groups