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. 2014 Jul;88(13):7178-88.
doi: 10.1128/JVI.00254-14. Epub 2014 Apr 9.

Neutrophil depletion suppresses pulmonary vascular hyperpermeability and occurrence of pulmonary edema caused by hantavirus infection in C.B-17 SCID mice

Takaaki Koma  1 , Kumiko Yoshimatsu  1 , Noriyo Nagata  2 , Yuko Sato  2 , Kenta Shimizu  1 , Shumpei P Yasuda  1 , Takako Amada  1 , Sanae Nishio  1 , Hideki Hasegawa  2 , Jiro Arikawa  3
Affiliations

Neutrophil depletion suppresses pulmonary vascular hyperpermeability and occurrence of pulmonary edema caused by hantavirus infection in C.B-17 SCID mice

Takaaki Koma et al. J Virol. 2014 Jul.

Abstract

Hantavirus infections are characterized by vascular hyperpermeability and neutrophilia. However, the pathogenesis of this disease is poorly understood. Here, we demonstrate for the first time that pulmonary vascular permeability is increased by Hantaan virus infection and results in the development of pulmonary edema in C.B-17 severe combined immunodeficiency (SCID) mice lacking functional T cells and B cells. Increases in neutrophils in the lung and blood were observed when pulmonary edema began to be observed in the infected SCID mice. The occurrence of pulmonary edema was inhibited by neutrophil depletion. Moreover, the pulmonary vascular permeability was also significantly suppressed by neutrophil depletion in the infected mice. Taken together, the results suggest that neutrophils play an important role in pulmonary vascular hyperpermeability and the occurrence of pulmonary edema after hantavirus infection in SCID mice.

Importance: Although hantavirus infections are characterized by the occurrence of pulmonary edema, the pathogenic mechanism remains largely unknown. In this study, we demonstrated for the first time in vivo that hantavirus infection increases pulmonary vascular permeability and results in the development of pulmonary edema in SCID mice. This novel mouse model for human hantavirus infection will be a valuable tool and will contribute to elucidation of the pathogenetic mechanisms. Although the involvement of neutrophils in the pathogenesis of hantavirus infection has largely been ignored, the results of this study using the mouse model suggest that neutrophils are involved in the vascular hyperpermeability and development of pulmonary edema in hantavirus infection. Further study of the mechanisms could lead to the development of specific treatment for hantavirus infection.

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Figures

FIG 1
FIG 1
Percent body weight change in SCID mice following HTNV infection. Mice were inoculated intraperitoneally with Hantaan virus strain 76-118 cl-1 (HTNV+) or not treated (control). Values are means ± the standard errors (SE); there were five to eleven mice per group. Asterisks denote a significant difference from the control mice (**, P < 0.01 [Mann-Whitney U test]).
FIG 2
FIG 2
Mean viral genome load in SCID mice following HTNV infection. The viral load in each organ was measured by quantitative real-time PCR. Values are means + the SE. The number of mice tested is shown below the names of organs or serum.
FIG 3
FIG 3
Histopathological changes in the lung from HTNV-infected SCID mice at various times. In the lung, virus antigen-positive cells were detected and sequentially increased in the alveoli at 14, 21, 28, and 35 dpi. Low magnification, ×25; high magnification ×100. Br, bronchi; Al, alveoli; V, vein.
FIG 4
FIG 4
Pulmonary lesions in the lungs of HTNV-infected SCID mice on day 35 dpi. (A and B) HTNV antigens were detected using an E5/G6 MAb against the N of hantavirus (B, inset). Original magnifications: ×25 (A and B), ×100 (C and D), and ×250 (insets). Cellular proliferation was observed in the lung (A). Adhesion of neutrophils and degenerated and multilayered endothelial cells were seen in a middle-sized vein (A, inset). Br, bronchi; Al, alveoli; V, vein. Many virus antigen-positive cells were present in the alveolar area (B). Alveolar cells were positive for virus antigens (B, inset). (C and D) Pulmonary effusion, macrophages (arrows), and neutrophils (arrowheads) were observed in the alveoli. Inflammatory cells were increased in the alveolar wall. (E) Ratio of alveoli with exudate in each individual. There were four mice per group except for the 35 dpi group (n = 7). (F) Method for calculating the ratio of alveoli with exudate. To investigate the extent of pulmonary edema, the left lungs were sectioned at constant positions, and the number of alveoli with exudate was counted in more than 300 alveoli and in more than 25 high-power fields. Exudate is seen in asterisk-labeled alveoli in the lower panels. Since there is little exudate in a normal lung, an alveolus was considered to be an alveolus with exudate if the alveolus was filled with exudate of more than one-tenth of the area.
FIG 5
FIG 5
Histopathology in HTNV-infected SCID mice. Tissues were obtained from HTNV-infected SCID mice on day 35 dpi. Hematoxylin and eosin staining was performed (A to C, G to I, and M to N). HTNV antigens were detected using an E5/G6 MAb against the N of hantavirus (D to F, J to L, and P to Q). Original magnification, ×100. Virus antigen-positive cells were seen in the kidney (A and D, renal cortex; B and E, renal medulla), heart (C and F), spleen (G and J), liver (H and K), and brain (I and L, cerebral cortex; M and P, cerebellar cortex; N and Q, choroid plexus) without inflammatory reactions. Hemosiderin was stained blue by Berlin blue stain (J).
FIG 6
FIG 6
Total white blood cell counts (A) and neutrophil counts (B) in SCID mice following HTNV infection. Values are means ± the SE; there were four to six mice per group. Asterisks denote a significant difference from the control (i.e., uninfected) (*, P < 0.05; **, P < 0.01 [Mann-Whitney U test]). The dashed line represents average ± 2 standard deviations of control mice (n = 5).
FIG 7
FIG 7
Representative FACS plots (A) and percent positive expression (B) for lung homogenate stained with MAbs to Gr-1, CD11b, CD3, NK1.1, CD11c, and MHC class II at 28 dpi with HTNV. Values are means + the SE; there were four or five mice per group. Asterisks denote a significant difference from control (uninfected) mice (*, P < 0.05 [Mann-Whitney U test]).
FIG 8
FIG 8
Effect of neutrophil depletion on pulmonary edema. (A) Histopathological findings in lungs collected from HTNV-infected and uninfected SCID mice in the presence of anti-Gr-1 MAb or rat IgG at 33 dpi. Br, bronchi; Al, alveoli; V, vein. Upper and middle panels show hematoxylin and eosin staining; lower panels show immunohistochemistry using an E5/G6 MAb against the N of hantavirus. Original magnification, ×25 (upper panels) and ×100 (middle and lower panels). Gross observation of left lungs from mice in neutrophil depletion experiments (A, upper panels, inset). HTNV-infected mice with anti-Gr-1 MAb showed no pulmonary edema. (B) Ratio of alveoli with exudate in each individual in neutrophil depletion experiments using anti-Gr-1 MAb. There were three to five mice per group. (C) Ratio of alveoli with exudate and representative gross observation of left lungs from mice in macrophage depletion experiments using clodronate liposome. There were three mice per group. (D) Ratio of alveoli with exudate and representative gross observation of left lungs from mice in NKT cell depletion experiments using anti-NK1.1 MAb. There were three or four mice per group. Asterisks denote a significant difference between HTNV-infected mice with anti-Gr-1 MAb and with rat IgG (*, P < 0.05 [Mann-Whitney U test]).
FIG 9
FIG 9
Pulmonary vascular permeability in HTNV-infected mice. To assess changes in pulmonary vascular permeability, the Evans blue dye accumulation in lung tissue at 28 and 33 dpi was measured. Moreover, to investigate the involvement of neutrophils in pulmonary vascular permeability, the permeability was examined in mice with neutrophil depletion (anti-Gr-1 +) at 28 and 33 dpi. There were four mice per group except for the control group with Rat IgG (n = 3). Asterisks denote a significant difference between HTNV-infected mice with anti-Gr-1 MAb and with Rat IgG (*, P < 0.05 [Mann-Whitney U test]).
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