Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2020 Sep 23:11:551413.
doi: 10.3389/fimmu.2020.551413. eCollection 2020.

Adenovirus 7 Induces Interlukin-6 Expression in Human Airway Epithelial Cells via p38/NF-κB Signaling Pathway

Affiliations

Adenovirus 7 Induces Interlukin-6 Expression in Human Airway Epithelial Cells via p38/NF-κB Signaling Pathway

Lifeng Qi et al. Front Immunol. .

Abstract

Human Adenovirus (AdV) infection is very common and usually has a significant impact on children. AdV-induced inflammation is believed to be one of the main causes of severe symptoms. However, an inflammatory response profile in the airway in AdV-infected children is still lacking, and the mechanism underlying AdV-induced inflammation in the airway is also poorly understood. In the current study, we determined the expression of a panel of inflammation cytokines in the airway samples from AdV 7 infected children and further investigated the molecular mechanism underlying AdV 7-induced cytokine expression. Our results showed that eight out of 13 tested inflammatory cytokines were significantly increased in nasal washes of AdV 7-infected children comparing to healthy control, with IL-6 showing the highest enhancement. AdV 7 infection of bronchial epithelial cell line and primary airway epithelial cells confirmed that AdV 7 increased IL-6 mRNA and protein expression in an infection dose-dependent manner. Promoter analysis revealed that AdV 7 infection transactivated IL-6 promoter and a NF-κB binding site in IL-6 promoter was involved in the transactivation. Further analysis showed that upon AdV 7 infection, NF-κB p65 was phosphorylated and translocated into nucleus and bound onto IL-6 promoter. Signaling pathway analysis revealed that p38/NF-κB pathway was involved in AdV 7 infection induced IL-6 elevation. Taken together, our study shows that AdV 7 infection triggers the expression of a range of inflammatory cytokines including IL-6 in the airway of infected children, and AdV 7 enhances IL-6 expression by transactivating IL-6 promoter via p38/NF-κB signaling pathway. Findings of our current study have provided more information toward a better understanding of AdV-induced airway inflammation, which might also benefit the development of intervention strategies.

Keywords: Adenovirus 7; Interlukin-6; airway epithelial cell; inflammation; p38/NF-κB signaling pathway.

PubMed Disclaimer

Figures

Figure 1
Figure 1
AdV 7 infection triggers the production of inflammatory cytokines in the airway in children. Nasal samples from AdV 7 infected and healthy children were harvested and inflammatory cytokines IL-1β, IFN-α2, IFN-γ, TNF-α, MCP-1, IL-6, IL-8, IL-10, IL-12p70, IL-17A, IL-18, IL-23, and IL-33 were measured by BioLegend's LEGENDplex bead-based immunoassay. ns, statistically not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.
Figure 2
Figure 2
AdV 7 infection induces IL-6 mRNA and protein expression in airway epithelial cells. Human normal bronchial epithelial cell line BEAS-2B and primary human airway epithelial cells (hAEC) were either mock-infected or infected with ascendant doses of AdV 7 for 24 h and then (A) IL-6 mRNA and (B) protein expression were measured by RT-PCR and ELISA, respectively. Data shown are mean ± SD of three independent experiments with each condition performed in duplicate. *p < 0.05; **p < 0.01.
Figure 3
Figure 3
A NF-κB binding site in the IL-6 promoter is involved in AdV 7-induced IL-6 transactivation. (A) Schematic illustration of all the IL-6 promoter-controlled luciferase reporter gene plasmids. (B) BEAS-2B cells were first transfected with (-1,000/+11)IL-6-Luc, and then either mock-infected or infected with ascendant doses of AdV 7 for 24 h. Afterwards, cells were lysed and luciferase activity was measured. Data shown are mean ± SD of three independent experiments with each condition performed in duplicate. *p < 0.05; **p < 0.01. (C,D) BEAS-2B cells were first infected with either (C) serially truncated IL-6 promoter plasmids or (D) site-directed mutated IL-6 promoter plasmids, and then were either mock-infected or infected with 1 MOI of AdV 7. Twenty-four hours later, cells were lysed, and luciferase activity was measure. Data shown are mean ± SD of three independent experiments with each condition performed in duplicate.
Figure 4
Figure 4
AdV 7 infection induces NF-κB binding onto IL-6 promoter. (A) BEAS-2B cells were either mock-infected or infected with 1 MOI AdV 7 for 24 h, and then ChIP assay was performed with either control IgG or anti-p65 antibody. The amplification of IL-6 promoter sequence from ChIP-pulldown was performed with PCR. One representative result is shown. (B) BEAS-2B cells were sequentially transfected with control siRNA or p65 siRNA and (-1,000/+11)IL-6-Luc, and then were mock-infected or infected with 1 MOI AdV 7. Twenty-four hours later, cells were lysed, and luciferase activity was measured. Data shown are mean ± SD of three independent experiments with each condition performed in duplicate. Ns, not statistically significant; **p < 0.01. (C) BEAS-2B cells were first transfected with either control siRNA or p65 siRNA, and then were mock-infected or infected with 1 MOI AdV 7. Twenty-four hours later, IL-6 concentration in the cell culture supernatant was quantified by ELISA. Data shown are mean ± SD of three independent experiments with each condition performed in duplicate. Ns, not statistically significant; *p < 0.05; **p < 0.01. (D,E) BEAS-2B cells were either mock-infected or infected with 1 MOI AdV 7, and then (D) cells were lysed, or (E) cell cytoplasmic and nuclear fractions were isolated, and then (D,E) the expression of p65 and p-p65 was determined by Western blot. One out of three results is sown.
Figure 5
Figure 5
AdV 7 infection induces IL-6 expression via p38/NF-κB signaling pathway. (A,B) BEAS-2B cells were first (A) transfected with (-1,000/+11)IL-6-Luc and then (A,B) mock-infected or infected with 1 MOI AdV 7 and treated with different signaling pathway inhibitors. Twenty-four hours later, (A) cells were lysed and luciferase activity was measured, or (B) IL-6 concentration in the supernatant was measured by ELISA. Data shown are mean ± SD of three independent experiments with each condition performed in duplicate. *p < 0.05; **p < 0.01; ***p < 0.001. (C,D) BEAS-2B cells were first transfected with control siRNA or p38 siRNA, and then were mock-infected or infected with 1 MOI AdV 7. Twenty-four hours later, (C) IL-6 concentration in the supernatant was measured by ELISA, or (D) cells were lysed and the expression of p38, p-p38, p65, and p-p65 was determined by Western blot. (C) Data shown are mean ± SD of three independent experiments with each condition performed in duplicate. Ns, not statistically significant; *p < 0.05. (D) One representative result out of three is shown.
Figure 6
Figure 6
Blocking p38/NF-κB signaling pathway suppresses AdV 7-induced IL-6 expression. (A,B) primary hAEC cells were first (A) treated with signaling pathway inhibitors or (B) transfected with control siRNA or p38 siRNA, and then (A,B) infected with 1 MOI AdV 7. Twenty-four hours later, IL-6 concentration was quantified by ELISA. Data shown are mean ± SD of three independent experiments with each condition performed in duplicate. All statistical comparisons were made against cells with AdV 7 infection only. Ns, not statistically significant; *p < 0.05; **p < 0.01.

Similar articles

Cited by

References

    1. Xie L, Zhang B, Xiao N, Zhang F, Zhao X, Liu Q, et al. . Epidemiology of human adenovirus infection in children hospitalized with lower respiratory tract infections in Hunan, China. J Med Virol. (2019) 91:392–400. 10.1002/jmv.25333 - DOI - PMC - PubMed
    1. Moro MR, Bonville CA, Suryadevara M, Cummings E, Faddoul D, Kobayaa H, et al. . Clinical features, adenovirus types, and local production of inflammatory mediators in adenovirus infections. Pediatric Infect Dis J. (2009) 28:376–80. 10.1097/INF.0b013e31819075a6 - DOI - PubMed
    1. Chen R-F, Lee C-Y. Adenoviruses types, cell receptors and local innate cytokines in adenovirus infection. Int Rev Immunol. (2014) 33:45–53. 10.3109/08830185.2013.823420 - DOI - PubMed
    1. Becroft D. Histopathology of fatal adenovirus infection of the respiratory tract in young children. J Clin Pathol. (1967) 20:561–9. 10.1136/jcp.20.4.561 - DOI - PMC - PubMed
    1. Chuang Y, Chiu C-H, Wong K-S, Huang J-G, Huang YC, Chang LY, et al. . Severe adenovirus infection in children. J Microbiol Immunol Infect. (2003) 36:37–40. - PubMed

Publication types

MeSH terms

LinkOut - more resources