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Kinetic characterization of PB1-F2-mediated immunopathology during highly pathogenic avian H5N1 influenza virus infection.

Leymarie O, Jouvion G, Hervé PL, Chevalier C, Lorin V, Lecardonnel J, Da Costa B, Delmas B, Escriou N, Le Goffic R - PLoS ONE (2013)

Bottom Line: Using a H5N1 HPAIV strain isolated from duck and its PB1-F2 knocked-out mutant, we characterized the dynamics of PB1-F2-associated host response in a murine model of lethal pneumonia.At day 8 pi, PB1-F2 expression was strongly associated with increased transcription of genes encoding chemokines and cytokines implicated in the recruitment of granulocytes, as well as expression of a number of genes encoding enzymes expressed by neutrophils.In summary, our study shows that PB1-F2 of H5N1 HPAIV markedly influences the expression of the host transcriptome in a different way than its H1N1 counterparts: H5N1 PB1-F2 first delays the initial immune response but increases the pulmonary inflammatory response during the late stages of infection.

View Article: PubMed Central - PubMed

Affiliation: Unité de Virologie et Immunologie Moléculaires, UR 892 INRA, Domaine de Vilvert, Jouy-en-Josas, France.

ABSTRACT
The PB1-F2 protein encoded by influenza A viruses can contribute to virulence, a feature that is dependent of its sequence polymorphism. Whereas PB1-F2 from some H1N1 viruses were shown to exacerbate the inflammatory response within the airways, the contribution of PB1-F2 to highly pathogenic avian influenza virus (HPAIV) virulence in mammals remains poorly described. Using a H5N1 HPAIV strain isolated from duck and its PB1-F2 knocked-out mutant, we characterized the dynamics of PB1-F2-associated host response in a murine model of lethal pneumonia. The mean time of death was 10 days for the two viruses, allowing us to perform global transcriptomic analyses and detailed histological investigations of the infected lungs at multiple time points. At day 2 post-infection (pi), while no histopathological lesion was observed, PB1-F2 expression resulted in a significant inhibition of cellular pathways involved in macrophage activation and in a transcriptomic signature suggesting that it promotes damage to the epithelial barrier. At day 4 pi, the gene profile associated with PB1-F2 expression revealed dysfunctions in NK cells activity. At day 8 pi, PB1-F2 expression was strongly associated with increased transcription of genes encoding chemokines and cytokines implicated in the recruitment of granulocytes, as well as expression of a number of genes encoding enzymes expressed by neutrophils. These transcriptomic data were fully supported by the histopathological analysis of the mice lungs which evidenced more severe inflammatory lesions and enhanced recruitment of neutrophils in the context of PB1-F2 expression, and thus provided a functional corroboration to the insight obtained in this work. In summary, our study shows that PB1-F2 of H5N1 HPAIV markedly influences the expression of the host transcriptome in a different way than its H1N1 counterparts: H5N1 PB1-F2 first delays the initial immune response but increases the pulmonary inflammatory response during the late stages of infection.

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Confrontation of pulmonary histological lesions after intranasal inoculation of wild type and Δ F2 viruses.Groups of 3 mice were infected intranasally using 200 TCID50 of WT or ΔF2 Nig06 virus. Mice were euthanized at 2, 4 and 8 days pi (dpi) and lungs were processed to allow histopathological examination. WT virus. (A, D) 2 dpi: no histological lesion. (B) 4 dpi: inflammatory infiltrate centered on alveoli/interstitial tissue, in the periphery of a bronchiole, (E) presence of infected cells in the inflammatory infiltrate. (C) 8 dpi: more severe inflammation in alveoli/insterstitial tissue associated with (F) a high number of infected cells. ΔF2 virus. (G, J) 2 dpi: no histological lesion. (H) 4 dpi: inflammatory infiltrates centered on bronchi/bronchioles, (K) Infected cells are identified in the inflammatory infiltrates. (I) 8 dpi: inflammatory lesions persist in the bronchi/bronchioles but are associated with more severe lesions in the alveoli/interstitium. (L) Infected cells could be detected in inflammatory lesions. (A-C, G-I: HE staining; D-F, J-L: immunohistochemistry with anti-H5N1 antinbodies; Δ: alveoli/interstitium, asterisk: bronchi/bronchioles). Time-course of neutrophil infiltration. Neutrophils were quantified within bronchi/bronchioles (M), alveoli (N) and interstitium (O) compartments by histological observation. Results presented are the mean graded scores of 3 replicate animals ± SEM.
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pone-0057894-g002: Confrontation of pulmonary histological lesions after intranasal inoculation of wild type and Δ F2 viruses.Groups of 3 mice were infected intranasally using 200 TCID50 of WT or ΔF2 Nig06 virus. Mice were euthanized at 2, 4 and 8 days pi (dpi) and lungs were processed to allow histopathological examination. WT virus. (A, D) 2 dpi: no histological lesion. (B) 4 dpi: inflammatory infiltrate centered on alveoli/interstitial tissue, in the periphery of a bronchiole, (E) presence of infected cells in the inflammatory infiltrate. (C) 8 dpi: more severe inflammation in alveoli/insterstitial tissue associated with (F) a high number of infected cells. ΔF2 virus. (G, J) 2 dpi: no histological lesion. (H) 4 dpi: inflammatory infiltrates centered on bronchi/bronchioles, (K) Infected cells are identified in the inflammatory infiltrates. (I) 8 dpi: inflammatory lesions persist in the bronchi/bronchioles but are associated with more severe lesions in the alveoli/interstitium. (L) Infected cells could be detected in inflammatory lesions. (A-C, G-I: HE staining; D-F, J-L: immunohistochemistry with anti-H5N1 antinbodies; Δ: alveoli/interstitium, asterisk: bronchi/bronchioles). Time-course of neutrophil infiltration. Neutrophils were quantified within bronchi/bronchioles (M), alveoli (N) and interstitium (O) compartments by histological observation. Results presented are the mean graded scores of 3 replicate animals ± SEM.

Mentions: To further characterize the effect of PB1-F2 in vivo, we performed thorough histological investigations of the lungs of C57BL/6 mice infected with 200 TCID50 of WT or ΔF2 Nig06 virus. The aim of the histopathological analysis was to decipher the consequences of PB1-F2 expression during C57BL/6 mouse infection at the tissue scale. Many lesion patterns were similar between WT and ΔF2 virus infection (Fig. 2). Both indeed displayed: (i) an absence of lesion and apparent infected cells 2 days pi (Fig. 2A, D, G, J), (ii) necrosis and infiltration of neutrophils, macrophages and lymphocytes 4 days pi (Fig. 2B, E, H, K), (iii) an increase in the severity of inflammatory lesions at day 8 pi (Fig. 2C, F, I, L) and (iv) the presence of viral antigen in epithelial cells, macrophages and cell debris 2 and 4 days pi. However, many differences were identified, suggesting that PB1-F2 expression has a significant impact in the pulmonary tissue during infection. These differences were: (i) the early targeting of bronchi/bronchioles by the ΔF2 virus, 4 days pi, characterized by the presence of the virus and a much higher infiltration of neutrophils (Fig. 2H, K and M), (ii) a higher number of infected cells in the bronchi/bronchioles of ΔF2-infected mice at day 8, (iii) a more severe inflammation in WT-infected mice at day 8 pi (Fig. 2C, F) associated with (iv) a lower number of infected cells and more severe lesions in the alveoli and interstitial tissues (Fig. 2C, F), where the recruitment of neutrophils was significantly more important (Fig. 2N, O).


Kinetic characterization of PB1-F2-mediated immunopathology during highly pathogenic avian H5N1 influenza virus infection.

Leymarie O, Jouvion G, Hervé PL, Chevalier C, Lorin V, Lecardonnel J, Da Costa B, Delmas B, Escriou N, Le Goffic R - PLoS ONE (2013)

Confrontation of pulmonary histological lesions after intranasal inoculation of wild type and Δ F2 viruses.Groups of 3 mice were infected intranasally using 200 TCID50 of WT or ΔF2 Nig06 virus. Mice were euthanized at 2, 4 and 8 days pi (dpi) and lungs were processed to allow histopathological examination. WT virus. (A, D) 2 dpi: no histological lesion. (B) 4 dpi: inflammatory infiltrate centered on alveoli/interstitial tissue, in the periphery of a bronchiole, (E) presence of infected cells in the inflammatory infiltrate. (C) 8 dpi: more severe inflammation in alveoli/insterstitial tissue associated with (F) a high number of infected cells. ΔF2 virus. (G, J) 2 dpi: no histological lesion. (H) 4 dpi: inflammatory infiltrates centered on bronchi/bronchioles, (K) Infected cells are identified in the inflammatory infiltrates. (I) 8 dpi: inflammatory lesions persist in the bronchi/bronchioles but are associated with more severe lesions in the alveoli/interstitium. (L) Infected cells could be detected in inflammatory lesions. (A-C, G-I: HE staining; D-F, J-L: immunohistochemistry with anti-H5N1 antinbodies; Δ: alveoli/interstitium, asterisk: bronchi/bronchioles). Time-course of neutrophil infiltration. Neutrophils were quantified within bronchi/bronchioles (M), alveoli (N) and interstitium (O) compartments by histological observation. Results presented are the mean graded scores of 3 replicate animals ± SEM.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3585811&req=5

pone-0057894-g002: Confrontation of pulmonary histological lesions after intranasal inoculation of wild type and Δ F2 viruses.Groups of 3 mice were infected intranasally using 200 TCID50 of WT or ΔF2 Nig06 virus. Mice were euthanized at 2, 4 and 8 days pi (dpi) and lungs were processed to allow histopathological examination. WT virus. (A, D) 2 dpi: no histological lesion. (B) 4 dpi: inflammatory infiltrate centered on alveoli/interstitial tissue, in the periphery of a bronchiole, (E) presence of infected cells in the inflammatory infiltrate. (C) 8 dpi: more severe inflammation in alveoli/insterstitial tissue associated with (F) a high number of infected cells. ΔF2 virus. (G, J) 2 dpi: no histological lesion. (H) 4 dpi: inflammatory infiltrates centered on bronchi/bronchioles, (K) Infected cells are identified in the inflammatory infiltrates. (I) 8 dpi: inflammatory lesions persist in the bronchi/bronchioles but are associated with more severe lesions in the alveoli/interstitium. (L) Infected cells could be detected in inflammatory lesions. (A-C, G-I: HE staining; D-F, J-L: immunohistochemistry with anti-H5N1 antinbodies; Δ: alveoli/interstitium, asterisk: bronchi/bronchioles). Time-course of neutrophil infiltration. Neutrophils were quantified within bronchi/bronchioles (M), alveoli (N) and interstitium (O) compartments by histological observation. Results presented are the mean graded scores of 3 replicate animals ± SEM.
Mentions: To further characterize the effect of PB1-F2 in vivo, we performed thorough histological investigations of the lungs of C57BL/6 mice infected with 200 TCID50 of WT or ΔF2 Nig06 virus. The aim of the histopathological analysis was to decipher the consequences of PB1-F2 expression during C57BL/6 mouse infection at the tissue scale. Many lesion patterns were similar between WT and ΔF2 virus infection (Fig. 2). Both indeed displayed: (i) an absence of lesion and apparent infected cells 2 days pi (Fig. 2A, D, G, J), (ii) necrosis and infiltration of neutrophils, macrophages and lymphocytes 4 days pi (Fig. 2B, E, H, K), (iii) an increase in the severity of inflammatory lesions at day 8 pi (Fig. 2C, F, I, L) and (iv) the presence of viral antigen in epithelial cells, macrophages and cell debris 2 and 4 days pi. However, many differences were identified, suggesting that PB1-F2 expression has a significant impact in the pulmonary tissue during infection. These differences were: (i) the early targeting of bronchi/bronchioles by the ΔF2 virus, 4 days pi, characterized by the presence of the virus and a much higher infiltration of neutrophils (Fig. 2H, K and M), (ii) a higher number of infected cells in the bronchi/bronchioles of ΔF2-infected mice at day 8, (iii) a more severe inflammation in WT-infected mice at day 8 pi (Fig. 2C, F) associated with (iv) a lower number of infected cells and more severe lesions in the alveoli and interstitial tissues (Fig. 2C, F), where the recruitment of neutrophils was significantly more important (Fig. 2N, O).

Bottom Line: Using a H5N1 HPAIV strain isolated from duck and its PB1-F2 knocked-out mutant, we characterized the dynamics of PB1-F2-associated host response in a murine model of lethal pneumonia.At day 8 pi, PB1-F2 expression was strongly associated with increased transcription of genes encoding chemokines and cytokines implicated in the recruitment of granulocytes, as well as expression of a number of genes encoding enzymes expressed by neutrophils.In summary, our study shows that PB1-F2 of H5N1 HPAIV markedly influences the expression of the host transcriptome in a different way than its H1N1 counterparts: H5N1 PB1-F2 first delays the initial immune response but increases the pulmonary inflammatory response during the late stages of infection.

View Article: PubMed Central - PubMed

Affiliation: Unité de Virologie et Immunologie Moléculaires, UR 892 INRA, Domaine de Vilvert, Jouy-en-Josas, France.

ABSTRACT
The PB1-F2 protein encoded by influenza A viruses can contribute to virulence, a feature that is dependent of its sequence polymorphism. Whereas PB1-F2 from some H1N1 viruses were shown to exacerbate the inflammatory response within the airways, the contribution of PB1-F2 to highly pathogenic avian influenza virus (HPAIV) virulence in mammals remains poorly described. Using a H5N1 HPAIV strain isolated from duck and its PB1-F2 knocked-out mutant, we characterized the dynamics of PB1-F2-associated host response in a murine model of lethal pneumonia. The mean time of death was 10 days for the two viruses, allowing us to perform global transcriptomic analyses and detailed histological investigations of the infected lungs at multiple time points. At day 2 post-infection (pi), while no histopathological lesion was observed, PB1-F2 expression resulted in a significant inhibition of cellular pathways involved in macrophage activation and in a transcriptomic signature suggesting that it promotes damage to the epithelial barrier. At day 4 pi, the gene profile associated with PB1-F2 expression revealed dysfunctions in NK cells activity. At day 8 pi, PB1-F2 expression was strongly associated with increased transcription of genes encoding chemokines and cytokines implicated in the recruitment of granulocytes, as well as expression of a number of genes encoding enzymes expressed by neutrophils. These transcriptomic data were fully supported by the histopathological analysis of the mice lungs which evidenced more severe inflammatory lesions and enhanced recruitment of neutrophils in the context of PB1-F2 expression, and thus provided a functional corroboration to the insight obtained in this work. In summary, our study shows that PB1-F2 of H5N1 HPAIV markedly influences the expression of the host transcriptome in a different way than its H1N1 counterparts: H5N1 PB1-F2 first delays the initial immune response but increases the pulmonary inflammatory response during the late stages of infection.

Show MeSH
Related in: MedlinePlus