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Comparative Study
. 2014 Apr 9;45(1):42.
doi: 10.1186/1297-9716-45-42.

Innate immune response to a H3N2 subtype swine influenza virus in newborn porcine trachea cells, alveolar macrophages, and precision-cut lung slices

Mario Delgado-Ortega, Sandrine Melo, Darsaniya Punyadarsaniya, Christelle Ramé, Michel Olivier, Denis Soubieux, Daniel Marc, Gaëlle Simon, Georg Herrler, Mustapha Berri, Joëlle Dupont, François Meurens  1
Affiliations
Comparative Study

Innate immune response to a H3N2 subtype swine influenza virus in newborn porcine trachea cells, alveolar macrophages, and precision-cut lung slices

Mario Delgado-Ortega et al. Vet Res. .

Abstract

Viral respiratory diseases remain of major importance in swine breeding units. Swine influenza virus (SIV) is one of the main known contributors to infectious respiratory diseases. The innate immune response to swine influenza viruses has been assessed in many previous studies. However most of these studies were carried out in a single-cell population or directly in the live animal, in all its complexity. In the current study we report the use of a trachea epithelial cell line (newborn pig trachea cells - NPTr) in comparison with alveolar macrophages and lung slices for the characterization of innate immune response to an infection by a European SIV of the H3N2 subtype. The expression pattern of transcripts involved in the recognition of the virus, interferon type I and III responses, and the host-response regulation were assessed by quantitative PCR in response to infection. Some significant differences were observed between the three systems, notably in the expression of type III interferon mRNA. Then, results show a clear induction of JAK/STAT and MAPK signaling pathways in infected NPTr cells. Conversely, PI3K/Akt signaling pathways was not activated. The inhibition of the JAK/STAT pathway clearly reduced interferon type I and III responses and the induction of SOCS1 at the transcript level in infected NPTr cells. Similarly, the inhibition of MAPK pathway reduced viral replication and interferon response. All together, these results contribute to an increased understanding of the innate immune response to H3N2 SIV and may help identify strategies to effectively control SIV infection.

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Figures

Figure 1
Figure 1
Selected candidate reference genes and their expression stability. The stability of the reference genes has been assessed in a mix of non-infected and infected newborn pig trachea cells (NPTr), porcine alveolar macrophages (PAMs) and precision-cut lung slices (PCLS) at the different time points. Gene expression stability of candidate reference genes was analyzed by the geNorm application. Threshold for eliminating a gene was ≥ 1.0 for PCLS and ≥ 0.5 for NPTr cells and PAMs. The three most stable reference genes are depicted in green.
Figure 2
Figure 2
Relative expression of M-viral RNA and of host transcripts in infected newborn pig trachea cells. The NPTr cells were infected with the H3N2 SIV strain at different time points (1 h, 3 h, 8 h, and 24 h). Green dots stand for non-infected NPTr cells and red dots for infected cells (individual values (dots) and median value (bar), n = 6 wells per condition). Comparisons were made using one way ANOVA test and Tukey’s post-test. Differences were considered significant when P < 0.05 (*), P < 0.01 (**) or P < 0.001 (***).
Figure 3
Figure 3
Relative expression of various viral and host transcripts in infected porcine alveolar macrophages (PAMs). The cells were infected with the H3N2 SIV strain at different time points (1 h, 3 h, 8 h, and 24 h). Green dots stand for non-infected PAMs and red dots for infected PAMs (each individual value (n = 7) represents the data obtained with PAMs prepared from one pig, bar represents the median value). Comparisons were made using one way ANOVA test and Tukey’s post-test. Differences were considered significant when P < 0.05 (*), P < 0.01 (**) or P < 0.001 (***).
Figure 4
Figure 4
Immunostaining of infected Precision-cut lung slices (PCLS). The slices were infected by the H3N2 SIV strain. Cryosections were prepared after 3, 8, 24, and 48 h of infection and image data were collected using a laser-scanning confocal microscope. Infected cells were stained with an anti-nucleoprotein polyclonal antibody (green) for the detection of SIV. Ciliated cells were stained using an anti-beta-tubulin monoclonal antibody (red). Cell nuclei of prepared slides were stained by incubation with 4′,6′-diamidino-2-phenylindole (DAPI, blue). Scale bar = 20 μm.
Figure 5
Figure 5
Relative expression of M-vRNA and of host transcripts in infected precision-cut lung slices (PCLS). The slices were infected with the H3N2 SIV strain at different time points (3 h, 8 h, and 24 h). Green dots stand for non-infected PCLS and red dots for infected PCLS (minimum one slice/pig for each time point, total 5 pigs, n = 5-12 and median value). Comparisons were made using one way ANOVA test and Tukey’s post-test. Differences were considered significant when P < 0.05 (*), P < 0.01 (**) or P < 0.001 (***).
Figure 6
Figure 6
Western blots in infected NPTr cells. Western blots for phospho-ERK1/2 (pERK1/2) (A), phospho-p38 (pp38) (B), phospho-JAK2 (pJAK2) (C), and phospho-Akt (pAkt) (D) were performed in newborn porcine trachea (NPTr) cells infected with H3N2 SIV at different time points (0, 5, 10, 30, 60, and 240 min). Total ERK1/2, p38 and JAK2 are shown as loading controls and did not change with each condition over time. Data are presented as means ± SEM, (n = 3). Results are representative of three independent experiments. Different letters indicate significant differences (one way ANOVA, P < 0.05) as compared to control (time 0).
Figure 7
Figure 7
Western blots in infected NPTr cells in presence or absence of chemical inhibitor. Western blots for phospho-ERK1/2 (pERK1/2), phospho-p38 (pp38) and phospho-JAK2 (pJAK2) in newborn porcine trachea (NPTr) cells infected with H3N2 SIV at 30 min in presence or in absence of chemical inhibitors UO126 (ERK1/2), SB202190 (p38), and JAK inhibitor (JAK Inhibitor I). The control group was cultured in presence of DMSO, data are represented as means ± SEM, (n = 3). Results are representative of three independent experiments. Different letters indicate significant differences (one way ANOVA, P < 0.05) as compared to control (without inhibitor and virus).
Figure 8
Figure 8
Relative expression of SOCS1 transcripts in infected NPTr cells in presence of chemical inhibitors. The relative expression of SOCS1 transcripts was measured in newborn porcine trachea (NPTr) cells infected with H3N2 SIV in presence of chemical inhibitors UO126 (ERK1/2), SB202190 (p38), and JAK Inhibitor I (JAK/STAT) at different time points (1 h, 3 h, 8 h, and 24 h). The control groups were cultured either in the presence of 0.1% DMSO (C, green) or in the presence of inhibitor (I, blue). Infected cells were either untreated (V, red) or inhibitor-treated (I + V, orange). Comparisons were made using one way ANOVA test and Tukey’s post-test (n = 5, mean ± SEM). Differences were considered significant when P < 0.05 (*), P < 0.01 (**) or P < 0.001 (***).
Figure 9
Figure 9
Relative expression of various viral and host transcripts in infected NPTr cells in presence of JAK Inhibitor I. The relative expression of various viral and host transcripts was measured in newborn porcine trachea (NPTr) cells infected with H3N2 SIV in presence of JAK Inhibitor I at different time points (1 h, 3 h, 8 h, and 24 h). The control groups were cultured either in the presence of 0.1% DMSO (C, green) or in the presence of inhibitor (I, blue). Infected cells were either untreated (V, red) or inhibitor-treated (I + V, orange). Comparisons were made using one way ANOVA test and Tukey’s post-test (n = 5, mean ± SEM). Differences were considered significant when P < 0.05 (*), P < 0.01 (**) or P < 0.001 (***).
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