Cytokine and chemokine profiles in lung tissues from fatal cases of 2009 pandemic influenza A (H1N1): role of the host immune response in pathogenesis

Abstract

Pathological studies on fatal cases caused by 2009 pandemic influenza H1N1 virus (2009 pH1N1) reported extensive diffuse alveolar damage and virus infection predominantly in the lung parenchyma. However, the host immune response after severe 2009 pH1N1 infection is poorly understood. Herein, we investigated viral load, the immune response, and apoptosis in lung tissues from 50 fatal cases with 2009 pH1N1 virus infection. The results suggested that 7 of the 27 cytokines/chemokines showed remarkably high expression, including IL-1 receptor antagonist protein, IL-6, tumor necrosis factor-α, IL-8, monocyte chemoattractant protein-1, macrophage inflammatory protein 1-β, and interferon-inducible protein-10 in lung tissues of 2009 pH1N1 fatal cases. Viral load, which showed the highest level on day 7 of illness onset and persisted until day 17 of illness, was positively correlated with mRNA levels of IL-1 receptor antagonist protein, monocyte chemoattractant protein-1, macrophage inflammatory protein 1-β, interferon-inducible protein-10, and regulated on activation normal T-cell expressed and secreted. Apoptosis was evident in lung tissues stained by the TUNEL assay. Decreased Fas and elevated FasL mRNA levels were present in lung tissues, and cleaved caspase-3 was frequently seen in pneumocytes, submucosal glands, and lymphoid tissues. The pathogenesis of the 2009 pH1N1 virus infection is associated with viral replication and production of proinflammatory mediators. FasL and caspase-3 are involved in the pathway of 2009 pH1N1 virus-induced apoptosis in lung tissues, and the disequilibrium between the Fas and FasL level in lung tissues could contribute to delayed clearance of the virus and subsequent pathological damages.

Figure 1

Viral load in fresh-frozen lung tissues, as measured by RT-qPCR. The diagram displays quantification of the matrix gene (log₂ of the matrix gene/B2M housekeeping gene) in lung tissues of fatal cases over different durations. The Kruskal-Wallis test was performed to assess variance at different durations.

Figure 2

Profiles of cytokines and chemokines in lung tissues. The graphs depict the levels of IL-1RA, IL-6, TNF-α, IL-8, MIP-1β, MCP-1, IP-10, or RANTES protein expression in pH1N1, normal, pH1N1 with bacterial co-infection, or pH1N1 with nonbacterial co-infection groups. After fresh-frozen lung tissues were homogenized in lysis buffer, the supernatants were tested using a 27-cytokine magnetic bead-based array. Error bars represent means ± SEM. A U-test was performed to assess statistical significance.

Figure 3

Correlation between viral load and immune mediator mRNA levels. Relative quantification of gene expression (log₂ scale) was normalized to that of the B2M housekeeping gene for the viral matrix gene and immune mediator mRNAs (MCP-1, IP-10, RANTES, FasL, MIP-1β, and IL-1RA).

Figure 4

Apoptosis-associated gene mRNA levels, as determined by RT-qPCR. The column diagrams depict the relative quantification of FasL (A), FAS (B), and TRAIL (C) mRNA in lung tissues. Error bars represent means ± SEM. The U-test was performed to assess the statistical significance between 2009 pH1N1 cases and the normal group.

Figure 5

Histopathological characteristics and immunostaining of viral antigens and cytokines in lung and airway. Diffuse alveolar damage (A), infiltration of inflammatory cells in glands (B), and bronchopneumonia (C) using H&E staining. Viral nucleoprotein immunohistochemical staining (red) in nuclei and cytoplasm of pneumocytes lining alveoli (D) and submucosal gland cells (E). Negative IHC staining for IL-6 in normal lung tissue (F), IL-6 immunostaining (red) in nuclei and cytoplasm of pneumocytes (G) and submucosal gland cells (H), and after recombinant IL-6 absorption (I). IP-10 immunostaining (red) was present in the nuclei and cytoplasm of pneumocytes (J) and submucosal gland cells (K), and after recombinant IP-10 absorption (L). IL-8 immunostaining (red) present in the cytoplasm of pneumocytes (M) and submucosal gland cells (N), and after recombinant IL-8 absorption (O). Original magnifications: ×10 (A–C, F, I, L, and O); ×20 (D, E, G, H, J, K, M, and N); ×100 (insets D, E, G, H, J, K, M, and N).

Figure 6

Cleaved caspase-3 immunostaining and TUNEL assay on consecutive sections of lung and airway. Caspase-3 immunostaining (red) demonstrates cleaved capase-3 staining in nuclei and cytoplasm of submucosal glands (A), epithelium and pneumocytes (C and E), alveoli (G), or lymphoid tissue (I). By TUNEL assay, apoptotic bodies (brown) seen in submucosal glands (B), epithelium and pneumocytes (D and F), alveoli (H), or lymphoid tissue (J). Original magnifications: ×10 (C and D); ×20 (A, B, G, H, I, and J); ×40 (E and F); ×100 (insets C, D, I, and J).