Gene expression analysis of host innate immune responses during Lethal H5N1 infection in ferrets

Abstract

How viral and host factors contribute to the severe pathogenicity of the H5N1 subtype of avian influenza virus infection in humans is poorly understood. We identified three clusters of differentially expressed innate immune response genes in lungs from H5N1 (A/Vietnam/1203/04) influenza virus-infected ferrets by oligonucleotide microarray analysis. Interferon response genes were more strongly expressed in H5N1-infected ferret lungs than in lungs from ferrets infected with the less pathogenic H3N2 subtype. In particular, robust CXCL10 gene expression in H5N1-infected ferrets led us to test the pathogenic role of signaling via CXCL10's cognate receptor, CXCR3, during H5N1 influenza virus infection. Treatment of H5N1-infected ferrets with the drug AMG487, a CXCR3 antagonist, resulted in a reduction of symptom severity and delayed mortality compared to vehicle treatment. We contend that unregulated host interferon responses are at least partially responsible for the severity of H5N1 infection and provide evidence that attenuating the CXCR3 signaling pathway improves the clinical course of H5N1 infection in ferrets.

FIG. 1.

Four prominent gene expression clusters identified in H3N2- and H5N1-infected ferrets. Sequential Student's t tests (time point versus mock) identified genes significantly differentially expressed (P ≤ 0.05 and ≥2-fold change) for at least one time point and group during H3N2 and H5N1 infection in ferrets. These genes were combined with significantly differentially expressed genes (≥2-fold change for at least one time point and group and P ≤ 0.05) identified by EDGE analysis of differential gene expression between H3N2- and H5N1-infected ferrets. As shown, 2,295 significantly different genes were analyzed by one-way (by gene) hierarchical clustering (red, upregulated; blue, downregulated). The most significant signaling pathway(s) (IFN and IFN/acute-phase response signaling) or network (cell growth and proliferation, gene expression, immune response, and infectious disease) according to IPA for the resulting clusters are noted. M, mock. E, end point as described in Materials and Methods.

FIG. 2.

Complement cascade activation in the lungs of influenza virus-infected ferrets. (A) One-way hierarchical clustering of complement-related genes selected by IPA from the three innate-immunity-related gene clusters in Fig. 1 (red, upregulated; blue, downregulated). (B) qRT-PCR analysis of SERPING1 gene expression performed in triplicate. Error bars indicate standard deviations. (C) qRT-PCR analysis of FCN1 performed in triplicate. Error bars indicate standard deviations, and the Mann-Whitney P value is shown. (D) IPA canonical pathway modeling of the complement system using the microarray analysis of gene expression data from the lungs of influenza virus-infected ferrets at the end point (red, upregulated; blue, downregulated). M, mock. E, end point as described in Materials and Methods.

FIG. 3.

IFN signaling pathway activation in the lungs of influenza virus-infected ferrets. (A) One-way hierarchical clustering of IRGs selected by IPA from the three innate immunity-related gene clusters in Fig. 1 (red, upregulated; blue, downregulated). (B) qRT-PCR analysis of STAT1 performed in triplicate. Error bars indicate standard deviations. (C) Left, qRT-PCR analysis of CXCL10. The Mann-Whitney P value is shown. Right, qRT-PCR analysis of CXCL9. Analyses were performed in triplicate, and error bars indicate standard deviations. (D) IPA modeling of the IFN signaling pathway using the microarray analysis of gene expression data from the lungs of influenza virus-infected ferrets at the end point (red, upregulated; blue, downregulated). M, mock. E, end point as described in Materials and Methods.

FIG. 4.

AMG487 treatment improves respiratory function and delays mortality in H5N1-infected ferrets. Ferrets were infected with H5N1 as described in Materials and Methods and received either 1.65 mg/kg AMG487 in a 3-ml volume of PBS (n = 8) or PBS vehicle (n = 9) intraperitoneally every 12 h starting at 24 h postinfection and continuing until the end point. Weight loss, activity, temperature, and SpO2 (pulse oximetry) were monitored daily from 2 dpi. (a) Change in body temperature relative to baseline means (P ≤ 0.001 by two-way ANOVA). (b) SpO2 (P ≤ 0.005 by two-way ANOVA). (c) Weight loss (percentage of original body mass; P ≤ 0.001 by two-way ANOVA). (d) Mean daily activity scores: 0, normal; 1, alert, playful when stimulated; 2, alert, not playful when stimulated; 3, neither alert nor playful when stimulated (P ≤ 0.05 by two-way ANOVA). (e) Kaplan-Meier survival curves for AMG487 (Exp.)- versus vehicle (Control)-treated ferrets. Curves are significantly different (P ≤ 0.05) by log rank analysis. (f) Lung viral titers in three ferrets from each treatment group euthanized at 6 dpi (*, P ≤ 0.05 by Student's t test). (g and h) Representative hematoxylin- and eosin-stained lung sections at 6 dpi (magnification, ×4). Error bars represent standard errors of the means in all panels.