Integration of clinical data, pathology, and cDNA microarrays in influenza virus-infected pigtailed macaques (Macaca nemestrina)

Abstract

For most severe viral pandemics such as influenza and AIDS, the exact contribution of individual viral genes to pathogenicity is still largely unknown. A necessary step toward that understanding is a systematic comparison of different influenza virus strains at the level of transcriptional regulation in the host as a whole and interpretation of these complex genetic changes in the context of multifactorial clinical outcomes and pathology. We conducted a study by infecting pigtailed macaques (Macaca nemestrina) with a genetically reconstructed strain of human influenza H1N1 A/Texas/36/91 virus and hypothesized not only that these animals would respond to the virus similarly to humans, but that gene expression patterns in the lungs and tracheobronchial lymph nodes would fit into a coherent and complete picture of the host-virus interactions during infection. The disease observed in infected macaques simulated uncomplicated influenza in humans. Clinical signs and an antibody response appeared with induction of interferon and B-cell activation pathways, respectively. Transcriptional activation of inflammatory cells and apoptotic pathways coincided with gross and histopathological signs of inflammation, with tissue damage and concurrent signs of repair. Additionally, cDNA microarrays offered new evidence of the importance of cytotoxic T cells and natural killer cells throughout infection. With this experiment, we confirmed the suitability of the nonhuman primate model in the quest for understanding the individual and joint contributions of viral genes to influenza virus pathogenesis by using cDNA microarray technology and a reverse genetics approach.

FIG. 1.

Timeline of study. Two animals (one experimental and one control) had a 4-day endpoint, and two others had a 7-day endpoint.

FIG. 2.

cDNA microarray of selected genes pertaining to interferon pathways and upregulated concurrently with appearance of clinical signs in experimental animals. Lane L4, experimental versus control lungs at day 4; lane L7, experimental versus control lungs at day 7. Genes in this bioset were extracted from the cluster in appendix A, i.e., they were differentially regulated by 1.5-fold or more in at least two experiments (P ≤ 0.05) and were clustered according to a hierarchical algorithm. Red bars represent genes that were induced by the experiment; green bars represent genes that were repressed by the experiment; and darker colors represent lesser differential expression.

FIG. 3.

Histopathology of lung tissue at day 4 postinoculation and cDNA microarrays of selected genes relevant to neutrophil and monocyte-macrophage function. The left slide shows uninfected lungs from the control animal; the right slide shows the lungs of the infected animal at day 4 postinoculation, showing pneumonia with mild alveolar septal and luminal infiltration of lymphocytes, neutrophils (black arrows), eosinophils, and macrophages (blue arrows), hypertrophy (red arrow), and hyperplasia (mitotic figures) in many pneumocytes with dark basophilic nuclei and cytoplasm but little or no apparent transudation to the alveoli. Magnification, ×70. The genes in these biosets were extracted from the cluster in appendix A, i.e., they were differentially regulated by 1.5-fold or more in at least two experiments (P ≤ 0.05) and were clustered according to a hierarchical algorithm. Red bars represent genes that were induced by the experiment; green bars represent genes that were repressed by the experiment; and darker colors represent lesser differential expression. The genes in the heat maps whose border color corresponds to a label on the histology slides are highly relevant to but not necessarily exclusive to the function of the labeled cells.

FIG. 4.

Histopathology of lung tissue at day 7 postinoculation and cDNA microarrays of selected genes relevant to neutrophil and monocyte-macrophage function. The left slide shows uninfected lungs from the control animal; the right slide (both sections) shows the lungs of the infected animal at day 7 postinoculation, showing pneumonia with alveolar flooding, hypertrophy, and hyperplasia of pneumocytes (red arrow), interstitial and alveolar infiltration by macrophages (blue arrows), lymphocytes, and a few neutrophils (black arrows), alveolar-septal necrosis (N), and areas of alveolar occlusion due to severe fibrin transudation and intra-alveolar cell migration. Hematoxylin and eosin stain. Magnification, ×70. The genes in these biosets were extracted from the cluster in appendix A, i.e., they were differentially regulated by 1.5-fold or more in at least two experiments (P ≤ 0.05) and were clustered according to a hierarchical algorithm. Red bars represent genes that were induced by the experiment; green bars represent genes that were repressed by the experiment; and darker colors represent lesser differential expression. The genes in the heat maps whose border color corresponds to a label on the histology slides are highly relevant to but not necessarily exclusive to the function of the labeled cells.

FIG. 5.

Histopathology of tracheobronchial lymph nodes. The left slide shows a quiescent node from the control animal; the middle panel shows the node from the infected animal at day 4 postinoculation, showing high cell density, dominance of small dense lymphocytes, macrophages with abundant cytoplasm (blue arrows), and golden to brown-black pigment granules and prominent high endothelial venules (green arrows); the right panel shows the node from the infected animal at day 7 postinoculation, showing diffuse edema, many activated macrophages (blue arrows) characterized by basophilic cytoplasm and nuclei with finely dispersed chromatin and a large central, magenta nucleolus (not visible at this magnification), dendritic cells (purple arrows), and hypertrophy of endothelial cells with large vesiculated nuclei (not visible at this magnification) in blood vessels (green arrows). Hematoxylin and eosin stain. Magnification, ×70.

FIG. 6.

Immunohistochemistry of lung and tracheal tissue. The left top panel shows lungs from the control animal at day 4 postinoculation, showing no influenza virus-positive cells; the left middle panel shows lungs from the infected animal at day 4 postinoculation, showing scattered influenza virus-positive pneumocytes (red arrows) and macrophages (blue arrows); the left lower panel shows lungs from the infected animal at day 7 postinfection showing scattered weak signals in macrophages (blue arrows) representing phagocytosed material from influenza virus-infected cells rather than productive infection and pneumocytes (red arrows); the right upper panel shows the trachea of the control animal at day 7 post-sham inoculation, showing no influenza virus-positive cells; the right middle panel shows the trachea from the infected animal at day 4 postinoculation, showing influenza virus-positive cells (red arrows) in addition to nonspecific binding of antibody to the basement membrane, a common occurrence enhanced by plasma transudation; note the collapse or disintegration of mucus goblet cells and atrophy of cilia due to inflammation; the right lower panel shows the trachea from the infected animal at day 7 postinoculation, showing influenza virus-positive cells (red arrows) present in a patchy pattern; note the collapse or disintegration of mucus goblet cells and the atrophy of cilia due to inflammation. Magnifications, ×100.

FIG. 7.

Percentages of upregulated genes in functional categories. The percentages of clustered genes [differentially regulated by 1.5-fold or more in at least two experiments (P ≤ 0.05)] involved in general innate immune response, interferon pathways, immune cell migration, antigen presentation on MHC complexes, and presence and/or activation of neutrophils, monocytes-macrophages, dendritic cells, NK cells, T cells, and B cells and upregulated at days 4 and 7 in either lungs (upper panel) or lymph nodes (lower panel).

FIG. 8.

Regulation of genes relevant to apoptosis and oxidative stress. Lane L4, experimental versus control lungs at day 4; lane L7, experimental versus control lungs at day 7; lane TB4, experimental versus control tracheobronchial lymph nodes at day 4; lane TB7, experimental versus control tracheobronchial lymph nodes at day 7. The genes in this bioset [differentially regulated by 1.5-fold or more in at least two experiments (P ≤ 0.05)] were clustered according to a hierarchical algorithm; green bars represent genes that were repressed by the experiment, and darker colors represent lesser differential expression.