Enhanced susceptibility of Ago1/3 double-null mice to influenza A virus infection

Abstract

RNA interference (RNAi) is a critical component of many cellular antiviral responses in plants, invertebrates, and mammals. However, its in vivo role in host protection from the negative-sense RNA virus influenza virus type A (flu) is unclear. Here we have examined the role of RNAi in host defense to flu by analyzing Argonaute 1 and 3 double-knockout mice deficient in components of the RNA-induced silencing complex. Compared to littermate controls, flu-infected double-knockout mice exhibited increased mortality, consistent with more severe alveolitis and pneumonitis. These data indicate that optimal resistance to flu requires Argonaute 1 and/or 3. Enhanced mortality of double-knockout mice was not associated either with increased viral replication or with differential pulmonary recruitment or function of innate and adaptive immune cells. Given the absence of detectable immune defects, our results support the notion that the enhanced flu susceptibility of double-knockout mice arises from an intrinsic impairment in the ability of lung cells to tolerate flu-elicited inflammation.

Fig 1

Susceptibility of WT and DKO mice to influenza virus A infection. WT (n = 25), Het (n = 34), and DKO (n = 29) mice were infected intranasally with 2,000 EID₅₀ of PR8. Mice were monitored daily for weight loss (A) and survival (B). *, P < 0.04, log-rank Mantel-Cox test. Data are from three independent experiments (mean and SEM).

Fig 2

Viral replication in flu-infected WT and DKO mouse lungs and TEC cultures. (A) Mice were infected intranasally with 2,000 EID₅₀ of PR8. Lungs were isolated at d3 (WT mice, n = 15; DKO mice, n = 16) and at d7 (WT mice, n = 18; DKO mice, n = 15). Viral titers in the lungs were determined by plaque-forming assay with MDCK cells. Data are from three independent experiments (mean and SEM). (B) WT and DKO mouse TECs were infected with PR8 at a multiplicity of infection of 0.01 in triplicate. Apical supernatants were collected at 16, 48, 72, and 96 h postinfection and assessed for viral titer by plaque-forming assay with MDCK cells. Data are from three independent experiments (mean and SEM).

Fig 3

Characterization of immune responses in WT and DKO mice. (A and B) Mice were infected intranasally with 2,000 EID₅₀ of PR8 (WT mice, n = 15; DKO mice, n = 16). BAL fluid was collected at d3 (A) and d7 (B) postinfection; cells were analyzed by FACS; and cell numbers for neutrophils (CD11b⁺, Gr1⁺, major histocompatibility complex class II negative), macrophages (Macs; CD11b⁺, Gr1⁺, major histocompatibility complex class II positive), TNF-iNOS-producing DCs (TIP DCs; Ly6C^high, CD11b⁺, CD11c⁺), and monocytic DCs (CD11b⁺, CD11c⁺) were enumerated. (C) Mice were infected intranasally with 2,000 EID₅₀ of PR8, and BAL fluid was collected at d7 postinfection (WT mice, n = 18; DKO mice, n = 15). Cells from BAL fluid and MLN were analyzed by FACS, and total numbers of CD4⁺ T cells, CD8⁺ CTLs, and tetramer-positive CTLs were enumerated. *, P < 0.05, Student's t test. (D) PR8-specific serum antibody titers at d7 postinfection (WT mice, n = 11; DKO mice, n = 9) were determined by ELISA. *, P < 0.02, two-way analysis of variance. Data in panels A to C are from three independent experiments (mean and SEM).

Fig 4

Lung histology of flu-infected WT and DKO mice. WT and DKO mice were infected intranasally with 2,000 EID₅₀ of PR8. Lungs were harvested for histopathological analysis at d5 (WT mice, n = 5; DKO mice, n = 8) and d7 (WT mice, n = 19; DKO mice, n = 14) postinfection. (A) Lung sections from d5 and d7 postinfection were immunohistochemically stained for flu antigen (top four panels, brown). Representative micrographs are shown. Magnification, ×5. Lung sections from d5 and d7 were stained with hematoxylin-eosin (bottom four panels). Representative micrographs are shown. Magnification, 200×. (B) Sections were scored blindly for flu antigen distribution (% involvement; top four panels) and the combined score for severity of alveolitis and interstitial pneumonitis (bottom four panels). Severity scoring criteria for lung lesions is the following: 0, no lesions; 1, minimal, focal to multifocal, inconspicuous; 2, mild, multifocal, conspicuous; 3, moderate, multifocal, prominent; 4, marked, diffuse or coalescing, lobar; 5, severe, diffuse consolidation, multilobar.