IL-12 deficiency transiently improves viral clearance during the late phase of respiratory tract infection with influenza A virus in mice

Abstract

T helper 1-driven immune responses have been implicated in protective immunity against viral infections. Interleukin (IL)-12 is a heterodimeric proinflammatory cytokine formed by a p35 and a p40 subunit that can induce differentiation of naïve T cells towards a T helper 1-response. To determine the role of IL-12 in respiratory tract infection with influenza, p35 gene deficient (p35-/-) and normal wild type mice were intranasally infected with influenza A virus. IL-12 p35-/- mice displayed a transiently enhanced rather than an impaired viral clearance, as indicated by a 10-fold reduction in viral loads on day 8 after infection. Although interferon-gamma levels were significantly lower in the lungs of IL-12 p35-/- mice, their cellular immune responses were not altered, as reflected by similar T cell CD69 expression and influenza-specific T cell recruitment. Our data indicate that endogenous IL-12 impairs viral clearance during the late phase of influenza A virus infection in mice.

Fig. 1

IL-12 p40 expression after influenza virus infection. Pulmonary IL-12 p40 levels in total-lung homogenates were measured in influenza-virus-infected mice (open bars) and control mice (filled bars) on days 4, 8, 12 and 21 after intranasal inoculation (6–8 mice per group). Data are expressed in pg/g lung tissue (mean ± S.E.). ^*p < 0.05 vs. control mice.

Fig. 2

Viral load in the lungs of IL-12 p35^−/− mice. Viral load was determined on days 2, 4, 8 and 12 after influenza virus infection (seven mice per time-point) in IL-12 p35^−/− mice (open bars) and wildtype mice (filled bars). Viral load is expressed as viral RNA copies per lung. In control mice influenza could not be detected either (four mice per time-point, data not shown).

Fig. 3

Survival after influenza virus infection in wildtype mice and IL-12 p35^−/− mice. Survival after influenza infection in wildtype mice (squares) and IL-12 p35^−/− mice (triangles). All mice received 200 CCID₅₀ influenza virus intranasally on day 0. Mice were monitored at least twice a day after infection.

Fig. 4

Cytokine expression in the lungs of IL-12 p35^−/− mice. IL-6 (A), IFN-γ (B) and IL-12 p40 (C) levels in total-lung homogenates were measured in IL-12 p35^−/− mice (open bars) and wildtype mice (filled bars) on days 2, 4, 8 and 12 after influenza virus infection (7–8 mice per group). Data are expressed in pg/g lung tissue (mean ± S.E.). ^*p < 0.05 vs. wildtype mice. BD = below detection level.

Fig. 5

CD69 expression and influenza specific T-cell recruitment in IL-12 p35^−/− mice. Representative dotplots for wildtype and IL-12 p35^−/− mice are shown. CD69 expression on lung-derived CD4+ (A) and CD8+ (B) T cells in IL-12 p35^−/− mice (open bars) and wildtype mice (filled bars) on day 8 after influenza virus infection (six mice per group). Influenza-specific T cell recruitment was determined by tetramer-staining (C) in IL-12 p35^−/− mice (open bars) and wildtype mice (filled bars) on day 8 after viral infection (six mice per group). All data are expressed as percentage of total number of CD4+ or CD8+ T cells in the lungs. Total lymphocyte numbers in the lungs on day 8 after viral infection were similar in IL-12 p35^−/− mice and wildtype mice (data not shown).

Fig. 6

Memory T cell formation in the spleen of IL-12 p35^−/− mice. Representative dotplots for wildtype and IL-12 p35^−/− mice are shown. Influenza-specific T cell recruitment was determined by tetramer-staining (A) in IL-12 p35^−/− mice (open bars) and wildtype mice (filled bars) on day 21 after viral infection (six mice per group). CD44 expression on spleen-derived CD8+ (B) T cells in IL-12 p35^−/− mice (open bars) and wildtype mice (filled bars) on day 21 after influenza virus infection (six mice). Data are expressed as percentage of total number of CD8+ (lower graph) T cells in the lungs. Total lymphocyte numbers in the spleen on day 21 after viral infection were similar in IL-12 p35^−/− mice and wildtype mice (data not shown).

Fig. 7

Ig responses on day 12 after influenza infection in IL-12 p35^−/− mice. IgM, IgG1, IgG2a and IgG2b levels were measured in pooled sera of wildtype mice (squares) and IL-12 p35^−/− mice (triangles). Each data point represents the OD₄₅₀ at the specified dilution of the sera.

Fig. 8

Histopathology of the lungs of IL-12 p35^−/− mice. Increased lung inflammation during influenza pneumonia. Histopathological analysis of the lungs of IL-12 p35^−/− mice (B and D) and wildtype mice (A and C). Lungs were isolated on day 4 (A and B) and day 8 (C and D) after viral infection and prepared for histopathological analysis. Original magnification: 100×. Slides are representative for six mice per group.