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. 2015 Jan;89(1):730-42.
doi: 10.1128/JVI.02897-14. Epub 2014 Oct 29.

Impact and regulation of lambda interferon response in human metapneumovirus infection

Ma Del Rocío Baños-Lara  1 , Lindsey Harvey  1 , Alexander Mendoza  1 , Dawn Simms  1 , Vladimir N Chouljenko  2 , Nobuko Wakamatsu  1 , K Gus Kousoulas  2 , Antonieta Guerrero-Plata  3
Affiliations

Impact and regulation of lambda interferon response in human metapneumovirus infection

Ma Del Rocío Baños-Lara et al. J Virol. 2015 Jan.

Abstract

Human metapneumovirus (hMPV) is a respiratory paramyxovirus that is distributed worldwide and induces significant airway morbidity. Despite the relevance of hMPV as a pathogen, many aspects of the immune response to this virus are still largely unknown. In this report, we focus on the antiviral immune response, which is critical for viral clearance and disease resolution. Using in vitro and in vivo systems, we show that hMPV is able to induce expression of lambda interferon 1 (IFN-λ1), IFN-λ2, IFN-λ3, and IFN-λ4. The induction of IFN-λ expression by hMPV was dependent on interferon regulatory factor 7 (IRF-7) expression but not on IRF-3 expression. Treatment of hMPV-infected mice with IFN-λ reduced the disease severity, lung viral titer, and inflammatory response in the lung. Moreover, the IFN-λ response induced by the virus was regulated by the expression of the hMPV G protein. These results show that type III interferons (IFN-λs) play a critical protective role in hMPV infection.

Importance: Human metapneumovirus (hMPV) is a pathogen of worldwide importance. Despite the relevance of hMPV as a pathogen, critical aspects of the immune response induced by this virus remain unidentified. Interferons (IFNs), including IFN-λ, the newest addition to the interferon family, constitute an indispensable part of the innate immune response. Here, we demonstrated that IFN-λ exhibited a protective role in hMPV infection in vitro and in an experimental mouse model of infection.

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Figures

FIG 1
FIG 1
IFN-λ is differentially expressed in epithelial cells in hMPV and RSV infection. The expression of IFN-β, IFN-λ1, -λ2, -λ3, and -λ4 was determined in RSV- or hMPV-infected A549 cells by qRT-PCR. (A) Cells were infected with RSV or hMPV at MOIs of 0.1, 0.5, 1, and 3. IFN expression was assessed after 24 h of infection. Data shown are representative of the results of two experiments run in duplicate. (B) Cells were infected with virus at an MOI of 3. IFN-λ and hMPV or RSV N gene expression was determined at 12, 24, 48, and 72 h after infection. Viruses were also inactivated by UV light, as previously described (30). Data shown are representative of the results of 3 independent experiments run in duplicate. The bar graphs represent means ± standard errors of the means. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
FIG 2
FIG 2
hMPV is sensitive to the effect of IFN-λ. A549 cells were treated with 10 or 50 ng/ml of human recombinant IFN-λ1, -λ2, or -λ3 for 24 h, followed by hMPV infection (MOI of 3) and replenishment of the corresponding IFN-λ for additional 24 h. (A) Expression of the hMPV N gene was determined by qRT-PCR. Uninf, uninfected. (B) Released virus titers were determined on LLC-MK2 cell monolayers by methylcellulose plaque assay. The bar graph represents PFU means ± standard errors of the means per million cells. Data shown in the graphs are the means of the results of 3 independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
FIG 3
FIG 3
hMPV infection induces IFN-λ in vivo. (A and B) BALB/c mice were infected i.n. with 1 ×107 PFU of hMPV or RSV or were mock infected (Mock). Mice were sacrificed at the indicated time after infection, and BAL fluid and lung samples were collected. Production of IFN-λ2/3 (A) or IFN-β (B) in BAL fluid was determined by ELISA. (C) RSV or hMPV N gene expression was quantitated in lung tissue by qRT-PCR. Data are representative of the results of two independent experiments (n = 6 mice/group). The bar graphs represent means ± standard errors of the means. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, not significant.
FIG 4
FIG 4
IFN-λ2/3 production is dependent on IRF-7 expression. IRF-7−/− (A) or IRF-3−/− (B) mice were infected i.n. with 1 × 107 PFU of hMPV or RSV or were mock infected. C57BL/6 mice were used as controls (WT). After 24 h, mice were sacrificed and BAL fluid samples were collected. Production of IFN-λ2/3 was determined by ELISA. Data are representative of the results of 3 independent experiments (n = 6 to 7 mice/group). Bar graphs represent means ± standard errors of the means. *, P < 0.05; ns, not significant.
FIG 5
FIG 5
IFN-λ2 and IFN-λ3 treatment decreases hMPV lung viral titer in infected mice. BALB/c mice were treated i.n. with the indicated concentrations of murine recombinant IFN-λ2 (A and B) or IFN-λ3 (C and D) or with a combination of IFN-λ2 plus IFN-λ3 (E and F) for 24 h prior infection with 1 × 107 PFU of hMPV. Lung tissue was collected at day 4 after infection. (A, C, and E) Expression of the hMPV N gene was determined by qRT-PCR. (B, D, and F) Infectious viral particles were titrated on LLC-MK2 cell monolayers by a methylcellulose plaque assay. Data are representative of at least two experiments with similar results (n = 3 to 6 mice/group). The bar graphs represent means ± standard errors of the means. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
FIG 6
FIG 6
IFN-λ2 reduces disease severity in hMPV-infected mice. BALB/c mice were treated i.n. with 1, 3, or 5 μg of rmIFN-λ2 for 24 h and infected with 1 × 107 PFU of hMPV for 6 days. Mice were monitored daily for body weight loss (A) and illness score (B). Data are representative of two experiments with similar results (n = 4 mice/group). The bar graphs represent means ± standard errors of the means. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
FIG 7
FIG 7
IFN-λ modulates inflammatory response in the lung after hMPV infection. BALB/c mice were treated i.n. with rmIFN-λ2 for 24 h prior to infection with 1 × 107 PFU of hMPV. At the 7-day time point, lung tissue was collected, fixed for slide preparation, and stained with H&E. (A) Representative stained lung tissue sections are presented. Scale bar, 200 μm. The graph shows pathology scores of prepared slides. (B) Cytokine and chemokine profile determined by multiplex analysis in BAL fluid samples from same mice. Data are representative of two independent experiments with similar results (n = 4 to 8 mice/group). The bar graphs represent means ± standard errors of the means. *, P < 0.05; **, P < 0.01; ***, P < 0.001. G-CSF, granulocyte colony-stimulating factor.
FIG 8
FIG 8
hMPV G protein inhibits the expression of IFN-λ in vitro and in vivo. A549 cells were infected with full-length recombinant hMPV (rhMPV) and the recombinant mutant hMPV lacking G protein (rhMPVΔG) at different MOIs for 24 h. (A) The virus genotype was confirmed by analyzing the expression of hMPV N and G genes by qRT-PCR. (B) The expression of IFN-λ1, IFN-λ2/3, and IFN-λ4 was also determined by qRT-PCR. Data are representative of the results of three independent experiments. The effect of rhMPVΔG on the expression of IFN-λ2/3 in vivo was also determined by infecting BALB/c mice with 105 PFU of rhMPV or rhMPV ΔG. Lung tissue was collected after 24 h and analyzed for the expression of IFN-λ2/3 by qRT-PCR. Data are representative of two independent experiments with similar results (n = 4 to 6 mice/group). (C) RIG-I, MDA5, MyD88, and IRF-7 expression was determined in rhMPV- and rhMPVΔG-infected cells by qRT-PCR. Data are representative of the results of 4 independent experiments. The bar graphs represent means ± standard errors of the means. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
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