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. 2015 Oct;53(4):525-35.
doi: 10.1165/rcmb.2014-0334OC.

The Induction of Pattern-Recognition Receptor Expression against Influenza A Virus through Duox2-Derived Reactive Oxygen Species in Nasal Mucosa

Hyun Jik Kim  1   2 , Chang-Hoon Kim  3   2 , Min-Ji Kim  4 , Ji-Hwan Ryu  4 , Sang Yeop Seong  3 , Sujin Kim  4 , Su Jin Lim  1 , Michael J Holtzman  5   6 , Joo-Heon Yoon  3   2   7   4
Affiliations

The Induction of Pattern-Recognition Receptor Expression against Influenza A Virus through Duox2-Derived Reactive Oxygen Species in Nasal Mucosa

Hyun Jik Kim et al. Am J Respir Cell Mol Biol. 2015 Oct.

Abstract

We studied the relative roles of Duox2-derived reactive oxygen species (ROS) in host defense against influenza A virus (IAV) infection in normal human nasal epithelial cells and mouse nasal mucosa. We found that Duox2 primarily generated ROS rapidly after IAV infection in normal human nasal epithelial cells and that knockdown of Duox2 aggravated IAV infection. In addition, Duox2-derived ROS enhancement significantly suppressed IAV infection in nasal epithelium. In particular, Duox2-derived ROS were required for the induction of retinoic acid-inducible gene (RIG)-I and melanoma differentiation-associated protein 5 (MDA5) transcription. After intranasal IAV inoculation into mice, viral infection was significantly aggravated from 3 days postinoculation (dpi) in the nasal mucosa, and the IAV viral titer was highest at 7 dpi. Both RIG-I and MDA5 messenger RNA levels increased dominantly in mouse nasal mucosa from 3 dpi; consistent with this, RIG-I and MDA5 proteins were also induced after IAV infection. RIG-I and MDA5 messenger RNA levels were induced to a lower extent in the nasal mucosa of the mice that were inoculated with Duox2 short hairpin RNA, and the IAV viral titer was significantly higher in nasal lavage. Taken together, Duox2-derived ROS are necessary for the innate immune response and trigger the induction of RIG-I and MDA5 to resist IAV infection in human nasal epithelium and mouse nasal mucosa.

Keywords: Duox2; influenza A virus; melanoma differentiation–associated protein 5; reactive oxygen species; retinoic acid–inducible gene-I.

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Figures

Figure 1.
Figure 1.
Normal human nasal epithelial (NHNE) cells were susceptible to influenza A virus (IAV) infection. NHNE cells from five healthy volunteers were inoculated with WS/33 (H1N1) for 10 and 30 minutes, 1, 2, and 8 hours, and 1, 2, and 3 days at an multiplicity of infection (MOI) of 1. (A) Real-time PCR showed that the IAV messenger RNA (mRNA) level was elevated from 1 day postinoculation (dpi) and was highest at 3 dpi. (B) Plaque assay also showed that viral titer was significantly higher from 1 dpi. Results are presented here as the mean ± SD from five independent experiments. *P < 0.05 compared with levels in mock-infected cells. GAPDH, glyceraldehyde 3-phosphate dehydrogenase.
Figure 2.
Figure 2.
The mRNA levels of three nicotinamide adenine dinucleotide phosphate oxidase (Nox) subtypes are preferentially induced to produce reactive oxygen species (ROS) after IAV infection. NHNE cells were inoculated with WS/33 (H1N1) for 10 and 30 minutes, 1, 2, and 8 hours, and 1, 2, and 3 days at an MOI of 1. Real-time PCR showed that Nox4 (A), dual oxidase (Duox) 1 (B), and Duox2 (C) mRNA levels are induced from 10 minutes after infection. Results are presented here as the mean ± SD from five independent experiments. *P < 0.05 compared with mRNA levels in mock-infected cells.
Figure 3.
Figure 3.
Duox2 is mainly involved in IAV-induced intracellular ROS generation in the nasal epithelium. NHNE cells were transfected with control (Cont) short hairpin RNA (shRNA), Nox4 shRNA, Duox1 shRNA, and Duox2 shRNA to suppress endogenous mRNA expression for 48 hours, and plaque assay was performed to measure changes in IAV viral titers after the suppression of Nox4-, Duox1-, and Duox2-induced intracellular ROS generation (A and B). After transfecting control shRNA and Duox2 shRNA into NHNE cells, Western blot analysis was performed to measure changes in IAV nucleoprotein (NP) after the suppression of Duox2-derived intracellular ROS generation (C). NHNE cells were transfected with pCMV-Duox2 and Duoxa2 overexpression vectors to enhance Duox2-derived intracellular ROS. RT-PCR showed that both Duox2 and Duoxa2 mRNA levels were significantly induced (D), and the amount of intracellular ROS also increased (E) after transfection with Duox2 and Duoxa2 overexpression vectors. Plaque assay and Western blot analysis were performed to measure changes in IAV viral titers (F) and IAV NP (G) after the enhancement of Duox2-derived intracellular ROS. The fluorescence intensity and Western blot analysis data are representative of five independent experiments, and results are presented here as the mean ± SD from five independent experiments. *P < 0.05 compared with levels in IAV-infected cells or cells transfected with control shRNA or pCMV vector. DCF, 2′,7′-dichlorofluorescein.
Figure 4.
Figure 4.
The mRNA levels of Toll-like receptor (TLR) 3, retinoic acid–inducible gene (RIG)-I, and melanoma differentiation–associated protein 5 (MDA5) are preferentially induced to recognize IAV in nasal epithelium. NHNE cells were inoculated with WS/33 (H1N1) for 10 and 30 minutes, 1, 2, and 8 hours, and 1, 2, and 3 days, at an MOI of 1. RT-PCR (A) and real-time PCR showed that TLR3 (B), RIG-I (C), and MDA5 (D) mRNA levels are induced from 8 hours after infection. Results are presented here as the mean ± SD from five independent experiments. *P < 0.05 compared with mRNA levels in mock-infected cells. PI day, postinfection day.
Figure 5.
Figure 5.
The transcription of RIG-I and MDA5 is preferentially augmented by Duox2-derived ROS after IAV infection. NHNE cells were transfected with control shRNA and Duox2 shRNA, and real-time PCR was performed to measure changes in IAV-induced TLR3 (A), RIG-I (B), and MDA5 (C) gene expression. IAV-induced RIG-I and MDA5 mRNA levels were significantly attenuated in cells transfected with Duox2 shRNA. IAV-induced TLR3 mRNA levels were not changed after the suppression of Duox2-derived intracellular ROS in NHNE cells. The gene expression levels of both RIG-I (D) and MDA5 (E) were considerably elevated in cells in which Duox2-derived ROS levels were increased through the use of Duox2 and Duoxa2 overexpression vectors. The results are presented here as the mean ± SD from five independent experiments. *P < 0.05 compared with levels in IAV-infected cells or cells transfected with control shRNA or pCMV vector.
Figure 6.
Figure 6.
IAV infection in vivo. Wild-type (WT) mice were infected with 213 pfu IAV WS/33 (H1N1) and assessed for loss of body weight (A) (circles, no infection mice, n = 3; triangles, infection mice, n = 3, *P < 0.05 compared with mean body weight of no infection mice) and viral titer from nasal lavage (NAL) fluid of infected mice (n = 5) (B) over the postinfection period (the results are presented here as the mean ± SD from the NAL fluid from five mice). Hematoxylin and eosin (H&E) micrographs of nose (coronal) sections obtained from WT mice infected with 213 pfu IAV on Days 0, 7, and 14. The H&E micrographs are representative of nose sections from five mice (C) and polymorphonuclear neutrophils (PMNs) were counted in subepithelium of nasal mucosa (D). *P < 0.05 compared the number of PMNs in nasal mucosa of mice at PI 7 and PI 14. R, right; L, left; S, septum. Boxed areas in C are shown enlarged in bottom images.
Figure 7.
Figure 7.
IAV infection is enhanced in Duox2 mutant mice, and Duox2-induced ROS are required for inducing IAV recognition receptors. WT mice were pretreated with control shRNA (shCont; n = 5) or Duox2 shRNA (shDuox2; n = 5) mice, and mice infected with 213 pfu IAV WS/33 (H1N1) were assessed for Duox2 gene expression on nasal mucosa (A) and survival rate over the postinfection period (B). Cell lysates from mice nasal mucosa and NAL fluid were obtained at 7 dpi, and real-time PCR and plaque assays were performed to compare viral mRNA levels (C) or viral titers (D) between shCont mice and shDuox2 mice (open circle, shCont mice; solid circle, shDuox2 mice). WT mice (n = 5) infected with 213 pfu IAV WS/33 (H1N1) were assessed for mRNA levels of TLR3, TLR7, TLR9, RIG-I, and MDA5, and cell lysates of mouse nasal mucosa and NAL fluid were obtained at 3, 7, 10, and 14 dpi (E) (white bar, TLR3; light gray bar, TLR7; gray bar, TLR9; dark gray bar, RIG-I; black bar, MDA5). Real-time PCR showed that both RIG-I and MDA5 mRNA levels were significantly induced after IAV infection in the mouse nasal mucosa. Western blot analysis was also performed using cell lysates to compare RIG-I and MDA5 protein expression levels at 7 dpi (F). shDuox2 mice (n = 5) were infected with 213 pfu IAV WSN/33 (H1N1) and were assessed for RIG-I and MDA5 mRNA levels using real-time PCR (G) and protein levels using western blot analysis (H) at 7 dpi. Western blot analysis results are representative of five mice, and PCR results are presented here as the mean ± SD from five independent experiments. *P < 0.05 compared with levels in shCont mice and shDuox2 mice. PRR, pattern recognition receptor.
Figure 8.
Figure 8.
Schematic picture of the innate immune response against IAV infection in nasal epithelial cells. IAV infection triggers Duox2-derived and mitochondrial ROS generation, resulting in mediating IFN-β and λ secretion in NHNE cells. In particular, Duox2-derived ROS are primarily involved in the rapid induction of IAV recognition receptors, especially RIG-I and MDA5, and Duox2-derived ROS would be necessary for IAV sensing in human nasal epithelium.
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References

    1. Kawai T, Akira S. Pathogen recognition with Toll-like receptors. Curr Opin Immunol. 2005;17:338–344. - PubMed
    1. Yoneyama M, Kikuchi M, Natsukawa T, Shinobu N, Imaizumi T, Miyagishi M, Taira K, Akira S, Fujita T. The RNA helicase RIG-I has an essential function in double-stranded RNA-induced innate antiviral responses. Nat Immunol. 2004;5:730–737. - PubMed
    1. Gitlin L, Benoit L, Song C, Cella M, Gilfillan S, Holtzman MJ, Colonna M. Melanoma differentiation–associated gene 5 (MDA5) is involved in the innate immune response to Paramyxoviridae infection in vivo. PLoS Pathog. 2010;6:e1000734. - PMC - PubMed
    1. García-Sastre A, Biron CA. Type 1 interferons and the virus–host relationship: a lesson in détente. Science. 2006;312:879–882. - PubMed
    1. Wu S, Metcalf JP, Wu W. Innate immune response to influenza virus. Curr Opin Infect Dis. 2011;24:235–240. - PubMed

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