Differential interferon responses to influenza A and B viruses in primary ferret respiratory epithelial cells

Abstract

Influenza B viruses (IBV) cocirculate with influenza A viruses (IAV) and cause periodic epidemics of disease, yet antibody and cellular responses following IBV infection are less well understood. Using the ferret model for antisera generation for influenza surveillance purposes, IAV resulted in robust antibody responses following infection, whereas IBV required an additional booster dose, over 85% of the time, to generate equivalent antibody titers. In this study, we utilized primary differentiated ferret nasal epithelial cells (FNECs) which were inoculated with IAV and IBV to study differences in innate immune responses which may result in differences in adaptive immune responses in the host. FNECs were inoculated with IAV (H1N1pdm09 and H3N2 subtypes) or IBV (B/Victoria and B/Yamagata lineages) and assessed for 72 h. Cells were analyzed for gene expression by quantitative real-time PCR, and apical and basolateral supernatants were assessed for virus kinetics and interferon (IFN), respectively. Similar virus kinetics were observed with IAV and IBV in FNECs. A comparison of gene expression and protein secretion profiles demonstrated that IBV-inoculated FNEC expressed delayed type-I/II IFN responses and reduced type-III IFN secretion compared to IAV-inoculated cells. Concurrently, gene expression of Thymic Stromal Lymphopoietin (TSLP), a type-III IFN-induced gene that enhances adaptive immune responses, was significantly downregulated in IBV-inoculated FNECs. Significant differences in other proinflammatory and adaptive genes were suppressed and delayed following IBV inoculation. Following IBV infection, ex vivo cell cultures derived from the ferret upper respiratory tract exhibited reduced and delayed innate responses which may contribute to reduced antibody responses in vivo.IMPORTANCEInfluenza B viruses (IBV) represent nearly one-quarter of all human influenza cases and are responsible for significant clinical and socioeconomic impacts but do not pose the same pandemic risks as influenza A viruses (IAV) and have thus received much less attention. IBV accounts for greater severity and deaths in children, and vaccine efficacy remains low. The ferret can be readily infected with human clinical isolates and demonstrates a similar course of disease and immune responses. IBV, however, generates lower antibodies in ferrets than IAV following the challenge. To determine whether differences in initial innate responses following infection may affect the development of robust adaptive immune responses, ferret respiratory tract cells were isolated, infected with IAV/IBV, and compared. Understanding the differences in the initial innate immune responses to IAV and IBV may be important in the development of more effective vaccines and interventions to generate more robust protective immune responses.

Keywords: TSLP; ferret; focus-forming assay; influenza B; innate; interferon; respiratory tract.

Fig 1

Replication kinetics of IAV/IBV in FNEC. (A) Replication kinetics of IAV [H1N1PDM09 “CA” (A/California/07/2009) and H3N2 “KS” (A/Kansas/14/2017)] and IBV [B-Victoria lineage “BR” (B/Brisbane/60/2008) and B-Yamagata lineage “PH” (B/Phuket/3073/2013)] infection in FNEC at 33°C (mutiplicity of infection = 0.3) for 72 h. (A) H1PDM09 = black squares, H3N2 = open squares, B-Victoria = black triangles, and B-Yamagata = open triangles. Each point represents the average FFU from each experiment at each timepoint. The average (horizontal line) of all experiments and SDs between experiments are shown. The significance between viruses at each timepoint was determined using one-way ANOVA (Sidak’s multiple comparison test). No significant differences were found. (B) Comparison of IAV (CA/KS) and IBV (BR/PH) in black and white circles, respectively. All points are from four independent assays in FNEC conducted in triplicate. The limit of detection, indicated by a dotted line, was 10^3.3 FFU/mL. Significance between groups was determined using one-way ANOVA (Sidak’s multiple comparison test). No significant differences were found.

Fig 2

Heatmap of early/late gene expression in IAV/IBV-infected FNEC. Influenza A and B infected (33°C, MOI = 0.3) FNEC from four independent experiments performed in triplicate. Average fold gene expression, compared to uninfected FNEC, blue = downregulated and red = upregulated. (A) Comparison of IAV: CA and KS, H1N1PDM09 and H3N2, respectively; IBV: BR and PH, B-Victoria and B-Yamagata lineages, respectively. Temporal RNA expression from 12 to 72 h postinfection. All gene groups (Table 1) are separated by dotted lines or solid lines for IFN. (B) Average gene expression comparison of all IAV “A” to all IBV “B” infected FNEC by type followed by an hour postinfection. IFN gene regulation demarcated between dotted lines. Up to two outliers were removed using statistical analysis (Grubb’s test) from 12 replicates/gene from four independent experiments if necessary.

Fig 3

Regulation of genetic functional categories by IAV and IBV in FNEC. Comparison of temporal changes in gene expression following IAV and IBV infection of FNEC. Individual points for each gene/category are shown for each timepoint 12 (white circles), 24 (light gray circles). 48 (dark gray circles), and 72 h (black circles) from individual samples used in Fig. 2 are presented. Panels comparing specific functional categories: (A) Inflammatory response “INFLAM” genes (CCL2, MCP1, CCL5, IL-1A, IL-1B, IL-6, IL-8, and TNFA). Significant differences between IAV and IBV at 24 (P = 0.00007) and 48 h (P = 0.0016). (B) Interferon response “IFN” genes (IFNA, IFNB, IFNG, and IFNL3). Significant differences between IAV and IBV at 24 (P = 0.0019) and 48 h (P = 0.0046). (C) T-effector response “T-EFF” genes (IL-4, IL-12, and IL-17). Significant differences between IAV and IBV at 24 (P = 0.0011) and 48 h (P = 0.019). (D) T-regulatory response “T-REG” genes (FOXP3, IL-10, and TGFB1). Significant differences between IAV and IBV at 24 (P = 0.002) and 72 h (P = 0.005). (E) T_H1 response “TH1” genes (CXCL10, CXCL11, and CXCL9). Significant differences between IAV and IBV at 12 (P = 0.0018), 24 (P = 0.0009), and 48 h (P = 0.005). (F) T_H2 response “TH2” gene (IL-2). Significant differences between IAV and IBV at 24 (P = 0.02) and 48 h (P = 0.021). Significance at each timepoint was determined using multiple unpaired t-test.

Fig 4

IFN gene expression following IAV/IBVinfection of FNEC. Influenza A and B infected (33°C, MOI = 0.3) FNEC from four independent experiments performed in triplicate. Individual fold (Log2) IFN gene expression with a geometric mean (horizontal bar), compared to uninfected FNEC, over 72 h. Black circles (IAV; CA and KS) and white circles (IBV; BR and PH). (A) Type-I, IFNA. Significant differences between IAV and IBV at 72 h (P = 0.002). (B) Type-I, IFNB. Significant differences between IAV and IBV at 72 h (P = 0.002). (C) Type-II, IFNG. Significant differences between IAV and IBV at 48 h (P = 0.014). (D) Type-III, IFNL3. Significant differences between IAV and IBV at 72 h (P = 0.0154). Data from four independent experiments using one-way ANOVA for determination of significance between IAV and IBV.

Fig 5

Type-III IFN protein levels following IAV/B-infection of FNEC. Type-III IFN secretion by IAV and IBV-infected FNEC. Average IFNL levels (pg/mL) secreted from the basolateral side of infected FNEC from 12 to 72 h postinfection. IAV vs IBV P = 0.0238 (one-way ANOVA). Significant differences for timepoints: IAV vs IBV (24 h P = 0.0165 and 48 h P = 0.049). The limit of detection is indicated by a dotted line.

Fig 6

TSLP gene expression following IAV/IBV infection of FNEC. Influenza A and B infected (33°C, MOI = 0.3) FNEC from four independent experiments performed in triplicate. Individual fold (Log2) TSLP gene expression over 72 h postinoculation is shown. Black circles (IAV; CA and KS) and white circles (IBV; BR and PH). Significant differences between IAV and IBV at 48 h (P = 0.037). Data from four independent experiments were analyzed to determine the significance between IAV and IBV.