Severe human lower respiratory tract illness caused by respiratory syncytial virus and influenza virus is characterized by the absence of pulmonary cytotoxic lymphocyte responses

Timothy P Welliver, Roberto P Garofalo, Yashoda Hosakote, Karen H Hintz, Luis Avendano, Katherine Sanchez, Luis Velozo, Hasan Jafri, Susana Chavez-Bueno, Pearay L Ogra, LuAnn McKinney, Jennifer L Reed, Robert C Welliver Sr, Timothy P Welliver, Roberto P Garofalo, Yashoda Hosakote, Karen H Hintz, Luis Avendano, Katherine Sanchez, Luis Velozo, Hasan Jafri, Susana Chavez-Bueno, Pearay L Ogra, LuAnn McKinney, Jennifer L Reed, Robert C Welliver Sr

Abstract

Background: Respiratory syncytial virus (RSV) and influenza virus are common causes of infantile lower respiratory tract infection (LRTI). It is widely believed that both viral replication and inappropriately enhanced immune responses contribute to disease severity. In infants, RSV LRTI is known to be more severe than influenza virus LRTI.

Methods: We compared cytokines and chemokines in secretions of infants surviving various forms of respiratory illness caused by RSV or influenza viruses, to determine which mediators were associated with more-severe illness. We analyzed lung tissue from infants with fatal cases of RSV and influenza virus LRTI to determine the types of inflammatory cells present. Autopsy tissues were studied for the lymphotoxin granzyme and the apoptosis marker caspase 3.

Results: Quantities of lymphocyte-derived cytokines were minimal in secretions from infants with RSV infection. Concentrations of most cytokines were greater in influenza virus, rather than RSV, infection. Lung tissues from infants with fatal RSV and influenza virus LRTI demonstrated an extensive presence of viral antigen and a near absence of CD8-positive lymphocytes and natural killer cells, with marked expression of markers of apoptosis.

Conclusions: Severe infantile RSV and influenza virus LRTI is characterized by inadequate (rather than excessive) adaptive immune responses, robust viral replication, and apoptotic crisis.

Conflict of interest statement

Potential conflicts of interest: none reported.

Figures

Table 1.
Table 1.
Demographic and clinical features of the study subjects.
Figure 1.
Figure 1.
Quantities of cytokines and chemokines in nasopharyngeal secretions of infants surviving respiratory syncytial virus (RSV) or influenza virus infection. Concentrations (log10) of each cytokine are illustrated for subjects with upper respiratory tract illness alone or bronchiolitis caused by each virus. The horizontal bars within boxes indicate the 50th percentile, the limits of boxes indicate the 25th and 75th percentiles, and the vertical lines indicate the third and 97th percentiles. The dotted horizontal line indicates the lower limit of detection of the assay. Statistical comparison was by t test (P values are shown above the bars). A, Quantities of interleukin (IL)—2, interferon (IFN)—γ, and IL-17. B, Quantities of IL-4, IL-5, and IL- 13. C, Quantities of IL-12, macrophage chemotactic protein (MCP)—1, and IL-6. D, Quantities of IL-1β, IL-7, and IL-10. E, Quantities of granulocyte colonystimulating factor (G-CSF), granulocyte-macrophage (GM)—CSF, and tumor necrosis factor (TNF)—α. F, Quantities of IL-8 and macrophage inflammatory protein (MIP)—1β.
Table 2.
Table 2.
Concentrations of T lymphocyte cytokines in nasopharyngeal secretions from surviving infants with lower respiratory tract infection grouped by day after onset of wheezing.
Figure 2.
Figure 2.
Immunohistochemical (IHC) staining for influenza virus and respiratory syncytial virus (RSV) antigen of bronchiolar and alveolar tissue from infants with lower respiratory tract infection (LRTI) or bronchiolitis and from healthy infants. Autopsy tissues were obtained from human infants with fatal cases of LRTI caused by either influenza virus (A, upper panels) or RSV (B, upper panels). Normal infant lung tissue (from an infant dying of asphyxia) is stained as a control (A and B, lower panels). Brown stain indicates the presence of viral antigen. Influenza virus antigen is found primarily in airway epithelium, whereas RSV antigen is present in both epithelium and in exfoliated epithelial cells plugging the airway. Scale bars show 15 μm; original magnification is ×40.
Figure 3.
Figure 3.
Immunohistochemical staining of lung tissue for CD4- and CD8-positive lymphocytes. Autopsy tissues were stained for cells bearing CD4 (upper panels) or CD8 (lower panels) surface antigens. Normal infant lung tissue is illustrated in the left column. Lung tissue from infants with fatal influenza and respiratory syncytial virus (RSV) infection is displayed in the middle and right columns, respectively. Human tonsilar tissue (not shown) demonstrated numerous cells with positive staining for CD4 and CD8 (positive control).
Figure 4.
Figure 4.
Immunohistochemical staining of lung tissue for CD56-positive lymphocytes. Autopsy tissues were studied for CD56 (NK lymphocyte) antigen. Tissues were obtained from an infant dying of asphyxia (normal lung, left panel) and from infants with fatal influenza virus (center panel) or respiratory syncytial virus (RSV) (right panel) infection. Tissue from an adult with lung cancer served as a positive control, demonstrating numerous antigen-positive cells.
Figure 5.
Figure 5.
Immunohistochemical staining of lung tissue for granzyme. Lung tissues were stained for the presence of granzyme, a lymphocyte product that mediates cytotoxic reactions against infected and stressed cells. Tissue was obtained from an infant dying of asphyxia (normal lung, upper left panel). An infant with fatal influenza virus infection is represented in the upper right panel. An infant with fatal respiratory syncytial virus (RSV) infection is represented in the lower left panel, and an adult with lung cancer is represented in the lower right panel.
Figure 6.
Figure 6.
Immunohistochemical staining of lung tissue for CD16. Lung tissues were studied for CD16 (expressed primarily on granulocytes and macrophages) antigen. Tissues were obtained from an infant dying of asphyxia (normal lung, left panel) and from infants with fatal influenza virus (center panel) or respiratory syncytial virus (RSV; right panel) infection.
Figure 7.
Figure 7.
Immunohistochemical staining of lung tissue for caspase 3. Lung tissues were stained for the presence of the apoptosis marker caspase 3. Tissue from an infant dying of asphyxia (normal lung, left panel) served as a negative control. Staining in cases of influenza virus infection was primarily observed in inflammatory cells (center panel). Subjects with respiratory syncytial virus (RSV) infection demonstrated positive staining in bronchiolar epithelial cells (right panel).

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