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. 2014 Sep;95(Pt 9):1870-1879.
doi: 10.1099/vir.0.066258-0. Epub 2014 May 30.

Highly pathogenic avian influenza A H5N1 and pandemic H1N1 virus infections have different phenotypes in Toll-like receptor 3 knockout mice

Y H Connie Leung  1 , John M Nicholls  2 , Chuk Kwan Ho  1 , Sin Fun Sia  1 , Chris K P Mok  3   1 , Sophie A Valkenburg  1 , Peter Cheung  1 , Kenrie P Y Hui  1 , Renee W Y Chan  1 , Y Guan  4   1 , S Akira  5 , J S Malik Peiris  4   3   1
Affiliations

Highly pathogenic avian influenza A H5N1 and pandemic H1N1 virus infections have different phenotypes in Toll-like receptor 3 knockout mice

Y H Connie Leung et al. J Gen Virol. 2014 Sep.

Abstract

Toll-like receptors (TLRs) play an important role in innate immunity to virus infections. We investigated the role of TLR3 in the pathogenesis of H5N1 and pandemic H1N1 (pH1N1) influenza virus infections in mice. Wild-type mice and those defective in TLR3 were infected with influenza A/HK/486/97 (H5N1) or A/HK/415742/09 (pH1N1) virus. For comparison, mice defective in the gene for myeloid differential factor 88 (MyD88) were also infected with the viruses, because MyD88 signals through a TLR pathway different from TLR3. Survival and body weight loss were monitored for 14 days, and lung pathology, the lung immune-cell profile, viral load and cytokine responses were studied. H5N1-infected TLR3(-/-) mice had better survival than H5N1-infected WT mice, evident by significantly faster regain of body weight, lower viral titre in the lung and fewer pathological changes in the lung. However, this improved survival was not seen upon pH1N1 infection of TLR3(-/-) mice. In contrast, MyD88(-/-) mice had an increased viral titre and decreased leukocyte infiltration in the lungs after infection with H5N1 virus and poorer survival after pH1N1 infection. In conclusion, TLR3 worsens the pathogenesis of H5N1 infection but not of pH1N1 infection, highlighting the differences in the pathogenesis of these two viruses and the different roles of TLR3 in their pathogenesis.

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Figures

Fig. 1.
Fig. 1.
Survival and weight loss in mice after challenge with 1 LD50 HPAI H5N1 or pH1N1 virus. Survival of WT, TLR3−/− and MyD88−/− mice after challenge with HPAI H5N1 (a) or pH1N1 (b) virus. TLR3−/− mice had a significantly improved survival compared with WT mice after challenge with H5N1, but not with pH1N1. MyD88−/− mice had a poorer survival after challenge with pH1N1, but there was no significant difference between H5N1-infected MyD88−/− and WT mice. Percentage weight loss of WT, TLR3−/− and MyD88−/− mice after challenge with H5N1 (c) or pH1N1 (d) virus; asterisks denote statistically significant differences between WT and TLR3−/− mice. The data represent means±sem (TLR3−/−, n = 20; WT, n = 22; MyD88−/−, n = 18 for H5N1 infection. TLR3−/−, n = 14; WT, n = 12; and MyD88−/−, n = 7 for pH1N1 infection). Statistical significance of differences is denoted as: *, P≤0.05; **, P≤0.01; ***, P≤0.001.
Fig. 2.
Fig. 2.
Viral titres and serum antibodies in mice infected with H5N1 or pH1N1. (a) Viral titre in lungs of TLR3−/−, MyD88−/− and WT mice after challenge with 1 LD50 of H5N1 at days 3, 7, 9 and 10 p.i. The viral titre in TLR3−/− mice was significantly lower than in WT mice at day 10 p.i. Viral titre in MyD88−/− was significantly higher than in WT mice at days 7 and 9 p.i. (b) Serum antibody against A/HK/486/1999 H5N1 virus in TLR3−/− and WT mice at days 5 and 7 p.i. The dotted line indicates the detection limit. (c) Viral titres in lungs of pH1N1-infected TLR3−/−, MyD88−/− and WT mice. At days 3 and 7 p.i., the viral titre in MyD88−/− mice was significantly higher than in WT mice. Viral titre in MyD88−/− mice at day 9 p.i. was not determined as most of these mice had succumbed to the infection by this time. The data represent means±sem (n =  3–7 per group). Viral titre was determined in MDCK cells. Statistical significance of differences is denoted as: *, P≤0.05; **, P≤0.01. nd, Not determined.
Fig. 3.
Fig. 3.
Lung histology and immunohistochemistry of H5N1- or pH1N1-infected mice at day 3 p.i. (a) Immunohistochemistry of lung sections stained with F4/80 (macrophage, ×80), MCA1171G (neutrophil, ×80), CD3 (mainly T cells, ×80), Hb65 (NP, ×200) and haematoxylin and eosin (H&E, ×80) from TLR3−/− and MyD88−/− mice compared with wild-type mice after H5N1 infection (first to third row of panel) and from TLR3−/− compared with WT mice after pH1N1 infection (fourth and fifth row of panel). Pictures are representatives of three mice per group. Arrows indicate cells with positive staining. (b) Quantification of macrophage (F4/80+), neutrophil (MCA1171G+) and T cell (CD3+) infiltration per 100 mm2 of cross-sectional area of lung sections counted by Aperigo Image Scope after H5N1 and pH1N1 infection at day 3 p.i. (c) The number of neutrophils, monocyte, dendritic, NK T and NK cells per 10 000 cells, after infection of H5N1 at day 3 p.i. in WT, TLR3−/− and MyD88−/− mice. The data were analysed by flow cytometry on a FACS LSRII. The data represent means±sem (n = 3). Statistical significance of differences is denoted as: *, P≤0.05; **, P≤0.01; ***, P≤0.001.
Fig. 4.
Fig. 4.
Lung histology, histological score and lung-to-body weight ratio of H5N1- and pH1N1-infected mice. Haematoxylin and eosin staining of lung sections from WT (a), TLR3−/− (b) and MyD88−/− (c) mice after infection of H5N1 on day 10 p.i. (×80). (d) The histopathology score of the peribronchial, perivascular and lung parenchyma, and the overall lung histopathology score of WT, TLR3−/− and MyD88−/− mice at day 10 p.i. after H5N1 influenza virus infection. The overall lung histopathology score of both TLR3−/− and MyD88−/− mice was significantly lower than that of WT mice. TLR3−/− mice had less parenchymal, perivascular and peribronchial inflammation compared with WT mice; while MyD88−/− mice had less perivascular and peribronchial inflammation, parenchymal inflammation was comparable to WT mice. (e) Lung-to-body weight ratio of H5N1-infected TLR3−/− and MyD88−/− mice compared with H5N1-infected WT mice at day 10 p.i. The lung-to-body weight ratios of MyD88−/− mice were significantly higher than those of WT mice. Haematoxylin and eosin staining of lung sections (×80) from WT (f), and TLR3−/− mice (g) and the histopathology scores of the peribronchial, perivascular and lung parenchyma, and overall lung histopathology scores of WT and TLR3−/− mice at day 10 p.i. after infection with pH1N1 (h). Pictures a, b, c, f and g are representatives of three mice per group. The data represent means±sem (n = 3 per group). Statistical significance of differences is denoted as: *, P≤0.05; **, P≤0.01; ***, P≤0.001.
Fig. 5.
Fig. 5.
Lung cytokine and chemokine levels in mice infected with H5N1 (a) or pH1N1 (b) at day 3 p.i. Cytokines and chemokines were measured from lung homogenates using R&D Systems DuoSet ELISAs. The data represent means±SEM (n = 3 per group). Asterisks indicate statistically significant differences between groups. *, P≤0.05; **, P≤0.01; ***, P ≤ 0.001.
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