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. 2011 May;7(5):e1002070.
doi: 10.1371/journal.ppat.1002070. Epub 2011 May 26.

MDA5 and TLR3 initiate pro-inflammatory signaling pathways leading to rhinovirus-induced airways inflammation and hyperresponsiveness

Qiong Wang  1 , David J Miller, Emily R Bowman, Deepti R Nagarkar, Dina Schneider, Ying Zhao, Marisa J Linn, Adam M Goldsmith, J Kelley Bentley, Umadevi S Sajjan, Marc B Hershenson
Affiliations

MDA5 and TLR3 initiate pro-inflammatory signaling pathways leading to rhinovirus-induced airways inflammation and hyperresponsiveness

Qiong Wang et al. PLoS Pathog. 2011 May.

Abstract

Rhinovirus (RV), a single-stranded RNA picornavirus, is the most frequent cause of asthma exacerbations. We previously demonstrated in human bronchial epithelial cells that melanoma differentiation-associated gene (MDA)-5 and the adaptor protein for Toll-like receptor (TLR)-3 are each required for maximal RV1B-induced interferon (IFN) responses. However, in vivo, the overall airway response to viral infection likely represents a coordinated response integrating both antiviral and pro-inflammatory pathways. We examined the airway responses of MDA5- and TLR3-deficient mice to infection with RV1B, a minor group virus which replicates in mouse lungs. MDA5 null mice showed a delayed type I IFN and attenuated type III IFN response to RV1B infection, leading to a transient increase in viral titer. TLR3 null mice showed normal IFN responses and unchanged viral titers. Further, RV-infected MDA5 and TLR3 null mice showed reduced lung inflammatory responses and reduced airways responsiveness. Finally, RV-infected MDA5 null mice with allergic airways disease showed lower viral titers despite deficient IFN responses, and allergic MDA5 and TLR3 null mice each showed decreased RV-induced airway inflammatory and contractile responses. These results suggest that, in the context of RV infection, binding of viral dsRNA to MDA5 and TLR3 initiates pro-inflammatory signaling pathways leading to airways inflammation and hyperresponsiveness.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. RV1B-induced expression of type I and III IFNs in MDA5−/− mice.
MDA5−/− and their control mice were inoculated with sham, UV-irradiated RV1B (UV RV1B) or intact RV1B. Lungs were harvested at 4, 24, 48, and 96 h after infection. A–D. The expression of IFN-α, IFN-β, IFN-λ2 and IFN-λ3 at each time point was determined by qPCR. E. IFN-β protein production was measured by ELISA at 24 h post-infection. The expression of each target gene was normalized to GAPDH. Data represent mean±SEM for 3–7 mice.
Figure 2
Figure 2. RV1B titer and copy number in MDA5−/− mice.
MDA5−/− and their control mice were infected with RV1B. Lungs were harvested at 4, 24, 48, and 96 h after infection. A. Lung titer at 24 h post-infection was determined by plaque assay. B. RV1B copy number at each time point was determined by qPCR. RV copy number was normalized to 18S rRNA. Data represent mean±SEM for 3–7 mice (*p<0.05, one-wayANOVA).
Figure 3
Figure 3. RV1B-induced chemokine expression in MDA5−/− and TLR3−/− mice.
MDA5−/−, TLR3−/− and their strain control mice were inoculated with sham, UV-irradiated RV1B (UV RV1B) or intact RV1B. Lungs were harvested 24 h after infection. A–D. The expression of CXCL1/KC, CXCL2/MIP-2, CCL2/MCP-1, CXCL10/IP-10 and CCL11/eotaxin-1 was determined by qPCR. E–F. Protein production of CXCL1/KC and CXCL2/MIP-2 was measured by ELISA and bioplex assay. The expression of each target gene was normalized to GAPDH. Data represent mean±SEM for 3–7 mice (*p<0.05, one-way ANOVA).
Figure 4
Figure 4. RV1B-induced pro-inflammatory cytokine expression in MDA5−/− and TLR3−/− mice.
MDA5−/−, TLR3−/−, and their strain control mice were inoculated with sham, UV-irradiated RV1B (UV RV1B) or intact RV1B. Lungs were harvested 24 h after infection. A–F. The expression of CXCL1/KC, CXCL2/MIP-2, CCL2/MCP-1, CXCL10/IP-10 and CCL11/eotaxin-1 was determined by qPCR. The expression of each target gene was normalized to GAPDH. Data represent mean±SEM for 3–7 mice (*p<0.05, one-way ANOVA).
Figure 5
Figure 5. Airway inflammation in RV1B-infected MDA−/− and TLR3−/− mice.
MDA5−/− (A), TLR3−/− (B) and their control mice were infected with RV1B. Twenty-four h after infection, lungs were fixed and stained with hematoxylin and eosin (original magnification, 100×).
Figure 6
Figure 6. Airway inflammation and responsiveness in RV1B-infected MDA5−/− and TLR3−/− mice.
MDA5−/−, TLR3−/−, and their control mice were infected with RV1B. Twenty-four h after infection, RV1B-induced neutrophil infiltration was determined in MDA5−/− mice (A) and TLR3−/− mice (B) along with their respective controls. Data represent mean±SEM for 3–7 mice, *p<0.05, one-way ANOVA). Total respiratory system resistance of MDA5−/− (C) and TLR3−/− (D) mice were determined by plethysmography. Data represent mean±SEM for 3–7 mice (*p<0.05, two-way ANOVA).
Figure 7
Figure 7. RV1B-induced IFN responses and viral titer in OVA-treated MDA5−/− mice.
OVA-treated MDA5−/− mice and control mice were infected with sham or RV1B. Total lungs were harvested at 24 post infection. A–C The expression of IFN-β, IFN-λ2, IFN-λ3 was determined by qPCR. D. Total lung titer at 24 h post infection was determined by plaque assay. The expression of each target gene was normalized to GAPDH. Data represent mean ± SEM for five-seven mice.
Figure 8
Figure 8. RV1B-induced pro-inflammatory cytokine expression in OVA-treated MDA5−/− mice.
OVA-sensitized and -challenged MDA5−/− and control mice were inoculated with sham or RV1B. Lungs were harvested at 24 and 96 h after infection. A–F. The expression of CXCL1/KC, CXCL2/MIP-2, IL-6, CCL2/MCP-1, IFN-γ and CCL11/eotaxin-1 was determined by qPCR. The expression of each target gene was normalized to GAPDH. Data represent mean±SEM for 4–7 mice (*p<0.05, one-way ANOVA).
Figure 9
Figure 9. Lung inflammation in OVA-treated RV1B-infected MDA5−/− mice: histology and airways responsiveness.
MDA5−/− mice and their control mice were sensitized and challenged with OVA and then infected with RV1B. A. Twenty-four h after infection, lungs were fixed and stained with hematoxylin and eosin (original magnification, 100×). B. Twenty-four h after infection, lungs were digested by collagenase. The number of infiltrated neutrophils were counted. C. Total respiratory system resistance one day after infection was determined by plethysmography. D. Four days after infection, MDA5 null mice showed significantly reduced lung eosinophils. E. Total respiratory system resistance four days after infection. Data represent mean±SEM for 6 mice (*p<0.05, two-way ANOVA).
Figure 10
Figure 10. Airway responses in RV1B-infected, OVA-sensitized and -challenged TLR3 null mice.
A, B. mRNA expression of IFN-β and IFN-λ2 24 and 96 h after infection was determined by qPCR. The expression of each target gene was normalized to GAPDH. C. Viral titer measured 24 h after RV1B infection of allergic wild-type and TLR3−/− mice. D, E. Lungs were digested by collagenase and the number of infiltrated neutrophils and macrophages were counted. F. Total respiratory system resistance one day after infection was determined by plethysmography. Data represent mean±SEM for 4 mice (*different from wild-type RV1B-infected mice, p<0.05, one- or two-way ANOVA; †different from sham-inoculated mice, p<0.05, one-way ANOVA).
Figure 11
Figure 11. RV1B-induced pro-inflammatory cytokine expression in OVA-treated TLR3−/− mice.
OVA-sensitized and -challenged TLR3−/− and control mice were inoculated with sham or RV1B. Lungs were harvested at 24 and 96 h after infection. A–F. The expression of CXCL1/KC, CXCL2/MIP-2, IL-6, CCL2/MCP-1, IFN-γ and CCL11/eotaxin-1 was determined by qPCR. The expression of each target gene was normalized to GAPDH. Data represent mean±SEM for 4 mice (*different from wild-type RV1B-infected mice, p<0.05, one-way ANOVA; †different from sham-inoculated mice, p<0.05, one-way ANOVA).
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