Early and sustained innate immune response defines pathology and death in nonhuman primates infected by highly pathogenic influenza virus

Abstract

The mechanisms responsible for the virulence of the highly pathogenic avian influenza (HPAI) and of the 1918 pandemic influenza virus in humans remain poorly understood. To identify crucial components of the early host response during these infections by using both conventional and functional genomics tools, we studied 34 cynomolgus macaques (Macaca fascicularis) to compare a 2004 human H5N1 Vietnam isolate with 2 reassortant viruses possessing the 1918 hemagglutinin (HA) and neuraminidase (NA) surface proteins, known conveyors of virulence. One of the reassortants also contained the 1918 nonstructural (NS1) protein, an inhibitor of the host interferon response. Among these viruses, HPAI H5N1 was the most virulent. Within 24 h, the H5N1 virus produced severe bronchiolar and alveolar lesions. Notably, the H5N1 virus targeted type II pneumocytes throughout the 7-day infection, and induced the most dramatic and sustained expression of type I interferons and inflammatory and innate immune genes, as measured by genomic and protein assays. The H5N1 infection also resulted in prolonged margination of circulating T lymphocytes and notable apoptosis of activated dendritic cells in the lungs and draining lymph nodes early during infection. While both 1918 reassortant viruses also were highly pathogenic, the H5N1 virus was exceptional for the extent of tissue damage, cytokinemia, and interference with immune regulatory mechanisms, which may help explain the extreme virulence of HPAI viruses in humans.

Fig. 1.

Log (TCID₅₀/mL) averaged from all lung lobes (4 out of 7, including left cranial, middle, caudal and accessory lobes) harvested. Detection limit of the plaque assay was 100 TCID₅₀/mL. Note: Tissues from day 6 death were not suitable for viral isolation.

Fig. 2.

Influenza antigen staining in lung tissues shows prolonged targeting of type II pneumocytes during the H5N1 infection (50×). In contrast, the middle insert illustrates targeting of type I pneumocytes in 1 of the 1918 recombinant groups (150×).

Fig. 3.

Stronger IFN, inflammatory, and innate immune transcriptional induction in the H5N1 group. Heat maps were generated with genes of interest after performing a 1-way ANOVA (P ≤ 0.01, Benjamini-Hochberg FDR) with statistical cut-off criteria of a 5-fold change in at least 2 experiments (P ≤ 0.01). Up-regulation relative to the reference pool is in red, down-regulation in green, and lack or statistically nonsignificant regulation in the darker shades of these colors. *, this transcriptional profile is from a non-lesion area of the animal euthanized on day 7, since tissues from H5N1 day 6 death were not suitable for RNA isolation.

Fig. 4.

Sustained inflammatory response in H5N1 infected macaques. (A) Mac 387 staining (10×) of lung sections with representative pathology and infection from each experimental group on days 1, 2, 4, and 7. This image illustrates the prolonged and more severe degree of granulocyte and macrophage infiltration in the H5N1 group and the intermediate phenotype of the 1918HANANS group for Mac387. (B) H&E staining (20×) of terminal bronchioles in the H5N1 and 1918HANA group on day 1, showing the severe damage to bronchiolar epithelium and severe infiltration of immune cells in the lumen of H5N1 animals.

Fig. 5.

Loss of dendritic cells due to apoptosis in H5N1 infected macaques. (A) CD83 staining in lungs and tracheobronchial lymph nodes was scored according to the following scale: 0 = none apparent; 0.5 = few/rare scattered cells; 1 = small numbers of positive cells, often in clusters; 2 = moderate increase in numbers of positive cells; 3 = moderate increase in numbers of positive cells; 4 = marked increase in interstitiae and/or BALT of the lung or in the paracortical zone of lymph nodes. Note: The entire section (min 7 sections per animal) was examined for CD83⁺ cells, translating into at least 30–60 high power fields. Day 6 death in H5N1 group is illustrated as day 7 for simplicity. A 2-way ANOVA was performed and revealed that groups were significantly different from one another (P < 0.001) over the entire course of the study, although not on any specific day. (B) CD83, activated caspase-3, and resulting double-labeling (100×) of apoptotic mature dendritic cells in lung tissue of an H5N1 animal on day 4. In most cases, apoptotic cells (caspase-3+) were identified as dendritic cells, mainly through morphology, due to the poor expression of CD83 in apoptotic cells.