Acute Respiratory Distress Syndrome Induced by Avian Influenza A (H5N1) Virus in Mice Tong Xu, Jian Qiao, Lihong Zhao, Guirong Wang, Guimei He, Kai Li, Yong Tian, Mingyu Gao, Jianlin Wang, Huiyu Wang, and Changgui Dong Department of Pathophysiology, College of Veterinary Medicine, China Agricultural University, Beijing, People’s Republic of China
Rationale and Objective: The acute respiratory distress syndrome (ARDS) caused by avian influenza H5N1 viral infection has been reported in many humans since this virus was found to infect humans in Hong Kong in 1997, but no studies regarding an animal model of ARDS with H5N1 viral infection have been found in the literature. Here we present a mouse model of ARDS induced by H5N1 virus. Methods: Six- to 8-wk-old BALB/c mice were inoculated intranasally (50 l) with 1 ⫻ 102 50% mouse infectious doses of A/Chicken/ Hebei/108/2002 (H5N1) virus. Lung injury was assessed by observation of lung water content and histopathology. Arterial blood gas, white blood cell count in bronchial alveolar lavage fluid, and tumor necrosis factor-␣ and interleukin-6 in bronchoalveolar lavage fluid and serum were measured at the indicated time points. Results: Our data showed that H5N1 viral infection in mice resulted in typical ARDS, which was characterized by the following features: (1 ) about 80% of mice (13 of 16) dead on Days 6 to 8 postinoculation; (2 ) highly edematous lungs and dramatically increased lung wet:dry weight ratios and lung wet weight:body weight ratios; (3 ) inflammatory cellular infiltration, alveolar and interstitial edema, and hemorrhage in lungs; (4 ) progressive and severe hypoxemia; and (5 ) significant increase in neutrophils, tumor necrosis factor-␣, and interleukin-6 in BALF. Conclusion: These results suggested that we successfully established a mouse model of ARDS with H5N1 viral infection, which may benefit further investigation into the pathogenesis of human ARDS induced by H5N1 virus. Keywords: acute respiratory distress syndrome; avian influenza A H5N1 virus; cytokine
Acute respiratory distress syndrome (ARDS) is a clinical syndrome characterized by damage to the alveolus–capillary interface, usually secondary to an intense inflammatory response of the host lung to infectious or noninfectious insult (1, 2). ARDS may be directly caused by gastric acid, smog, or toxic gas aspiration and thoracic trauma, but in some cases it may occur indirectly due to hemorrhagic shock, burns, and septicemia (3, 4). Severe acute respiratory syndrome (SARS) virus and avian influenza A H5N1 viral infection of the lung has resulted in high mortality among humans because of the complication of ARDS. Therefore, infectious factors, most of which are viruses, have become one of the most important causes of ARDS in humans (5–7). Although the receptor specificity of avian influenza A viruses was believed to prevent their direct transmission to humans, an
(Received in original form November 15, 2005; accepted in final form August 16, 2006 ) Supported by the National Natural Science Foundation of China (grant 30571398). Correspondence and requests for reprints should be addressed to Jian Qiao, M.D., Department of Pathophysiology, College of Veterinary Medicine, China Agricultural University, Beijing 100094, PR China. E-mail:
[email protected] Am J Respir Crit Care Med Vol 174. pp 1011–1017, 2006 Originally Published in Press as DOI: 10.1164/rccm.200511-1751OC on August 17, 2006 Internet address: www.atsjournals.org
outbreak of avian influenza caused by H5N1 virus in Hong Kong in 1997 demonstrated that an avian influenza virus could cross the species barrier to infect humans and result in high mortality among patients (8, 9). Among humans, the infection appears to have been acquired directly from infected birds, but there also exists the possibility of potential genetic reassortment of H5N1 virus, resulting in the ability to effectively infect humans, with possible consecutive human-to-human transmission (10). Therefore, experts of the World Health Organization (Geneva, Switzerland) believed that once this kind of genetic reassortment happened, avian influenza H5N1 virus would cause a human influenza pandemic, leading to catastrophic consequences (11, 12). Most patients infected with H5N1 virus present with clinical signs of influenza-like illness, such as pyrexia (temperature, 38.5⬚C or more), sore throat, and myalgia or headache; develop progressive pneumonia; and die of ARDS (13, 14). Several previous studies have demonstrated that H5N1 virus replicates in the respiratory tract of humans and leads to severe lung lesions with histopathologic features of ARDS (10, 13, 14). Thus, ARDS induced by H5N1 viral infection might be one of the most important reasons for patient death. Because clinical experience with avian H5N1 disease in humans is limited, and knowledge regarding animal models of ARDS induced by H5N1 viral infection is not available, it is urgent to develop an animal model to study the pathogenesis of ARDS induced by H5N1 virus. Here we present a mouse model of ARDS induced by H5N1 viral infection, with the objective of facilitating studies of the pathogenesis of avian H5N1 disease in humans.
METHODS Virus The virus was isolated from chickens in Hebei Province of China in January 2002, and identified as avian influenza A H5N1 virus by means of hemagglutination inhibition and neuraminidase inhibition tests. The isolate was designated as A/Chicken/Hebei/108/2002 (H5N1) (Chicken/ HB/108). The complete genome sequences (DQ343152, DQ349116, DQ351860, DQ351861, DQ351866, DQ351867, DQ351872, and DQ351873) of the virus can be obtained from GenBank. The virus caused 100% (8 of 8) mortality of 4-wk-old specific pathogen–free chickens within 2 d of intravenous infection with 0.2 ml of infectious allantoic fluid (AF) at 1:10 dilution. On the basis of the criteria of viral virulence (15), this virus is a highly pathogenic avian influenza virus. Our previous studies showed that this virus was also highly lethal to mice: when 6- to 8-wk-old BALB/c mice were inoculated intranasally with 50 l of 10-fold dilutions of Chicken/HB/108 H5N1 infectious AF, 100% of the mice (16 of 16) died 4 to 6 d postinoculation. The 50% mouse infectious dose (MID50) was determined according to methods described by Lu and coworkers (16). In brief, seven mice in each group were inoculated with serial 10-fold dilutions of virus. Four days later, three mice from each group were killed, and lungs were collected and homogenized in 1 ml of cold phosphate-buffered saline. Solid debris was pelleted by centrifugation, and tissues were titrated for viral infectivity in eggs. After that, AF samples from eggs were harvested and 50% egg infectious dose (EID50) titers were determined by testing hemagglutination activity. Titration endpoints were calculated by the
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method of Reed and Muench (17) and were expressed as the EID50 value corresponding to 1 MID50 (our unpublished data). Virus was propagated in 10-d-old embryonated hen eggs at 37⬚C for 32 h. Third-passage virus was gradient purified and stored at –70⬚C until use.
Animals and Inoculation with Virus Six- to 8-wk-old female specific pathogen–free BALB/c mice (Beijing Laboratory Animal Research Center, Beijing, People’s Republic of China) were housed in microisolator cages ventilated under negative pressure with HEPA-filtered air. During the experiment, animals had access to food and water ad libitum. Initial MID50 studies indicated that the dose of 1 ⫻ 102 MID50 of Chicken/HB/108 H5N1 virus was optimal because the course of the disease was prolonged and the infected mice presented the obvious signs of respiratory illness. If infected with a higher dose of H5N1 virus (1 ⫻ 103 MID50), nearly all the mice died by 4 to 6 d postinoculation, and when inoculated with a lower dose of the virus (1 ⫻ 101 MID50), only a few mice presented with clinical signs and died. Therefore, in this investigation, the mice were lightly anesthetized with diethyl ether and then inoculated intranasally (50 l) with 1 ⫻ 102 MID50 of Chicken/HB/108 H5N1 virus diluted in sterile saline. Mock-infected control animals were inoculated intranasally (50 l) with an equivalent dilution of noninfectious AF. All manipulations were performed under biosafety level 3⫹ (BSL-3⫹) laboratory conditions. The study was approved by the Animal Care Committee of China Agricultural University (Beijing, People’s Republic of China).
Lung Histopathology Sixteen mice in the infected group were monitored daily for morbidity, as measured by weight loss, and mortality for 14 d postinoculation. In another experiment, three mice of each group were weighed and killed on Days 1, 3, 5, 6, 7, 8, and 14 postinoculation. Left lobes of lungs were fixed in buffered 10% formalin and then embedded in paraffin. Fivemicrometer-thick sections were stained with hematoxylin–eosin for light microscopy.
Virus Titration Virus titration was performed as previously described (16). Whole lungs, kidneys, brains, livers, spleens, and hearts were collected and homogenized in cold phosphate-buffered saline on Days 1, 3, 5, 6, 7, 8, and 14 postinoculation. Clarified homogenates were titrated for viral infectivity in embryonated chicken eggs from initial dilutions of 1:10 (lung) or 1:2 (other organs). Viral titers were expressed as mean log10 EID50 per milliliter ⫾ standard deviation (1 MID50 is about 1 ⫻ 104 EID50).
Assessment of Lung Water Content The right lung was weighed before and after oven desiccation at 80⬚C to determine the lung wet weight:body weight ratio and lung wet:dry weight ratio, which were taken as indicators of lung edema (18).
leukocyte staining and erythrocyte hemolysis. The number of leukocytes in BALF was counted with a hemacytometer under a microscope. The remaining BALF was centrifuged (300 ⫻ g, 10 min). Cell differential counts were determined by Wright staining of a spun sample, on the basis of morphologic criteria, under a light microscope with evaluation of at least 200 cells per slide. All slides were counted twice by different observers blinded to the status of the animal. The supernatant was stored at –70⬚C until measurement of cytokines.
Peripheral Blood Leukocyte Counts Heparinized blood samples (50 l) were collected from mice in each group at various time points. Cell numbers for three individual mice were determined in triplicate by counting with a hemocytometer. For differential counts, two blood smears from each mouse were stained with Wright stain, and the numbers of monocytes, polymorphonuclear leukocytes (PMNs), and lymphocytes were determined. At least 100 cells were counted for each slide at a magnification of ⫻1,000 (22).
Measurement of Tumor Necrosis Factor-␣ and Interleukin-6 The concentrations of tumor necrosis factor (TNF)-␣ and interleukin (IL)-6 were measured in BALF and serum, using ELISA kits (Sigma, St. Louis, MO).
Statistics All data are expressed as means ⫾ SD. Statistical analysis was performed with the SPSS statistical software package for Windows, version 13.0 (SPSS, Inc., Chicago, IL). Differences between groups were examined for statistical significance by two-tailed Student t test. A p value less than 0.05 was considered statistically significant.
RESULTS Clinical and Gross Pathologic Observations
Some mice showed slightly altered gait, inactivity, ruffled fur, inappetence, and weight loss (18.4 ⫾ 1.2 vs. 20.2 ⫾ 0.2 g, H5N1infected mice vs. control mice) on Day 3 postinoculation. By Day 6 postinoculation, most of the mice abruptly presented the clinical signs of respiratory disease, including visual signs of labored respirations and respiratory distress, and exhibited more severe inappetence, emaciation, and weight loss (15.1 ⫾ 1.5 vs. 21.3 ⫾ 0.4 g). Distention of the abdomen was also seen in about 10% of mice on Days 4 to 6 postinoculation. About 80% of the mice (13 of 16) died between Days 6 and 8 postinoculation. Gross observation of infected mice demonstrated the lungs to be highly edematous, with profuse areas of hemorrhage (see Figure 1). Retention of gas in the stomach and intestine was
Arterial Blood Gas Analysis Blood gas analysis was performed as described by Fagan and coworkers (19). Four mice from each group were anesthetized with pentobarbital sodium on Days 3, 5, 6, 8, and 14 postinoculation. Arterial blood samples (0.3 ml) were withdrawn into a heparinized syringe by percutaneous left ventricular sampling of lightly anesthetized mice spontaneously breathing room air. Blood gas analysis was immediately performed with an IL 1640 pH/blood gas/electrolytes analyzer (Instrumentation Laboratory, Lexington, MA).
Bronchial Alveolar Lavage and Cell Counts in Bronchial Alveolar Lavage Fluid After collection of blood sample, bronchoalveolar lavage (BAL) was performed immediately after sacrifice of the animal by cervical dislocation on the days indicated. The procedure was performed as described by Majeski and coworkers (20) and Nick and coworkers (21). In brief, the lungs were lavaged twice with a total volume of 1.0 ml of saline (4⬚C) inserted through an endotracheal tube. The rate of recovery of BAL fluid (BALF) was greater than 90% for all the animals tested. After the amount of fluid recovered was recorded, an aliquot of BALF was diluted 1:1 with 0.01% crystal violet dye and 2.7% acetic acid for
Figure 1. Gross pathology of H5N1 virus–infected lung. (A ) H5N1 virus– infected lung 6 d postinoculation, displaying highly edematous, congestive, and hemorrhagic changes. (B ) Noninfectious allantoic fluid–inoculated control lung 6 d postinoculation.
Xu, Qiao, Zhao, et al.: ARDS Induced by Avian Influenza A H5N1 Virus in Mice
found in about 10% of infected mice. No obvious gross lesions were observed in the hearts, livers, or kidneys in infected mice. Histopathologic Lesions in Lungs of H5N1-infected Mice
Infected mice displayed a similar histopathologic pattern, including an initial peribronchiolar patchy pneumonia on Days 3 to 5 postinoculation and predominantly peribronchiolar lesions, fully developed bronchiolitis, and bronchopneumonia by Day 8 postinoculation. Kinetic observations of lung lesions of H5N1 virus– infected mice are shown in Figure 2. On Day 3 postinoculation, lung lesions of infected mice were characterized by interstitial edema around the small blood vessels (Figure 2A, solid arrows), dropout of mucous epithelium adhering to the surface of bronchioles (Figures 2A and 2B, open arrows), and edema and inflammatory cellular infiltration into alveolar walls near the bronchioles (solid arrow in Figure 2B). On Day 5 postinoculation
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there was interstitial pneumonia (Figures 2C and 2D), which showed edema and thickening and inflammatory cellular infiltration of the alveolar walls (solid arrows). Dropout of mucous epithelium from the bronchioles is also seen at higher magnification (open arrow) in Figure 2D. On Day 6 postinoculation, there was bronchiolitis (Figures 2E and 2F, solid arrows) with inflammatory cellular infiltrate and erythrocytes in the bronchioles, and peribronchiolitis with edema and inflammatory cellular infiltrate around the bronchioles (Figure 2F). Bronchopneumonia (Figures 2E and 2F) was also observed, with alveolar lumens flooded with edema fluid mixed with fibrin, erythrocytes, and inflammatory cells (Figures 2E and 2F, open arrows), especially at the base of the lungs. Lesions in the lungs of infected mice became more severe on Day 8 postinoculation. More severe bronchiolitis, peribronchiolitis, and bronchopneumonia were found (see Figures 2G and 2H). There was prominent dropout of bronchial
Figure 2. Temporal observation of pulmonary inflammatory response as shown by hematoxylin–eosin staining after H5N1 viral infection. On Day 3 postinoculation, there was interstitial edema about small blood vessels (A; solid arrows), a dropout of mucous epithelium in bronchioles (A and B; open arrows), and edema and thickening of alveolar walls (B; solid arrow). On Day 5 postinoculation, interstitial pneumonitis (C and D; solid arrows) and a dropout of mucous epithelium in bronchioles (C and D; open arrows) were observed. (E and F ) On Day 6 postinoculation, bronchiolitis (solid arrows), peribronchiolitis (solid arrows), and bronchopneumonia (open arrows) with alveolar lumens flooded with edema fluid mixed with erythrocytes and inflammatory cells were noted. (G and H ) On Day 8 postinoculation, the lung lesions became more severe and there were predominantly peribronchiolar lesions, fully developed bronchiolitis (solid arrows), and severe bronchopneumonia, and severe hemorrhage was also found in alveolar space adjacent to bronchioles (open arrow). Original magnification: (A, C, E, and G ) ⫻10; (B, D, F, and H ) ⫻40.
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Figure 3. Replication of influenza A (H5N1) virus in the lungs of mice. Mice were infected with 1 ⫻ 102 50% mouse infectious doses of Chicken/HB/108 virus, tissues were collected on various days postinoculation, and the virus was titrated in embryonated eggs. Mean viral titers based on three mice per group are expressed as log10 50% egg infectious dose (EID50) per milliliter ⫾ SD. The limit of virus detection was 101.2 EID50/ml for lungs.
epithelium and a great number of neutrophils, fibrin, and suppurative exudates infiltrating the bronchioles (Figures 2G and 2H, solid arrows), but no bacteria were seen in sections of pulmonary tissue, stained with hematoxylin–eosin or Gram stain, in any of the mice, excluding the possible involvement of secondary bacterial infections. Severe hemorrhage (Figure 2H, open arrow) into the alveolar space adjacent to bronchioles was also observed. In comparison, lungs from mock-infected control mice had no apparent histologic changes.
infected mice had dropped by up to 75% on Day 6 postinoculation and remained low until the death of most mice. Only in 20% of the infected mice did the sum of leukocytes and lymphocytes recover from Day 8 postinoculation, and these mice survived (see Figure 5B). White Blood Cell Summary and Differential Counts in BALF
Table 2 shows the time course of white blood cell (WBC) summary and differential counts in BALF on Days 1, 3, 5, 6, 7, 8,
Replication of H5N1 Virus in Mouse Tissues
Viral infection resulted in high titers of virus in the lungs on Days 1 to 7 postinoculation (see Figure 3). Peak viral titer appeared on Day 6 postinoculation, reaching 7.8 log10 EID50/ml. However, virus was below the detectable level on Day 8 postinoculation. Virus was also isolated from the liver, kidneys, and heart on Days 3 to 6 postinoculation, and from the brain on Days 5 to 7 postinoculation (data not shown). Lung Water Content: Edema
Figure 4A shows the effect of H5N1 viral infection on lung wet:dry weight ratios, which did not change obviously within 3 d postinoculation, but dramatically increased on Days 5 to 8 postinoculation (p ⬍ 0.05). The change in lung wet weight:body weight ratios was similar to the change in lung wet:dry weight ratios, shown in Figure 4B, with the peak value nearly fourfold that in the control group on Day 7 postinoculation. Arterial Blood Gas Analysis
Table 1 shows the change in arterial blood gas parameters in mice over time. Infected mice showed a slightly decreased partial pressure of arterial oxygen (PaO2), saturation of arterial oxygen (SaO2), and pH value and slightly increased partial pressure of arterial carbon dioxide (PaCO2) 3 d postinoculation. Most of the infected mice presented apparent clinical signs of respiratory distress from 6 d postinoculation, and blood gas analysis also showed that PaO2 and SaO2 dramatically decreased in the infected mice as compared with the controls (p ⬍ 0.05). The lowest PaO2 was 6.15 (4.83–7.47) kPa (median and range; p ⬍ 0. 05) on Day 8 postinoculation. Peripheral Blood Leukocyte Counts
Figure 5A shows a progressive reduction in the number of leukocytes on Days 1 to 7 postinoculation in infected mice. The leukopenia of infected mice was detected from Day 3 postinoculation and was statistically significant on Days 3 to 7 postinoculation relative to the leukocyte counts of mice in the mock-infected group. The lowest value appeared on Day 7 postinoculation. Differential blood count revealed that the lymphocytes of virus-
Figure 4. Lung wet:dry weight ratios (A ) and lung wet weight:body weight ratios (B ) of virus-infected mice. Open bars, control group; solid bars, inoculation group. *p ⬍ 0.05 and **p ⬍ 0.01 compared with those in control mice.
Xu, Qiao, Zhao, et al.: ARDS Induced by Avian Influenza A H5N1 Virus in Mice
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TABLE 1. ARTERIAL BLOOD GAS ANALYSIS PaO2 (kPa) Time (d ) 3 5 6 8 14
Infection* 11.2 9.12 6.83 7.15 11.7
⫾ ⫾ ⫾ ⫾ ⫾
1.06 1.27† 1.46‡ 1.83‡ 1.83
PaCO2 (kPa)
Control§ 12.37 12.29 12.36 12.38 12.39
⫾ ⫾ ⫾ ⫾ ⫾
1.46 1.34 0.96 1.23 1.05
Infection* 5.41 5.93 7.16 7.31 5.47
⫾ ⫾ ⫾ ⫾ ⫾
0.21 0.18 0.61 0.46 0.52
pH
Control§ 5.32 5.27 5.23 5.22 5.26
⫾ ⫾ ⫾ ⫾ ⫾
Infection*
0.59 0.43 0.77† 0.46† 0.71
7.38 7.24 7.19 7.18 7.32
⫾ ⫾ ⫾ ⫾ ⫾
0.051 0.067 0.072 0.058 0.053
SaO2 (% ) Control§
7.36 7.36 7.35 7.37 7.36
⫾ ⫾ ⫾ ⫾ ⫾
0.062 0.049 0.045 0.043 0.078
Infection* 88.7 83.7 79.5 73.4 89.1
⫾ ⫾ ⫾ ⫾ ⫾
1.81 3.42† 4.32‡ 2.12‡ 2.15
Control§ 91.2 94.2 92.5 91.3 91.4
⫾ ⫾ ⫾ ⫾ ⫾
1.14 0.98 1.73 1.27 2.76
Values are expressed as means ⫾ SD. * Mice infected with 1 ⫻ 102 MID50 of Chicken/HB/108 virus. † p ⬍ 0.05 compared with control group. ‡ p ⬍ 0.01 compared with control group. § Mock-infected mice inoculated with noninfectious allantoic fluid.
and 14 postinoculation. The number of WBCs in infected mice increased gradually from Day 3 postinoculation, and reached its peak by Day 7 postinoculation, which was eightfold that of the control group. PMNs in BALF increased dramatically from Days 1 to 8 postinoculation and its peak was 26-fold greater than that of the control group on Day 8 postinoculation.
Infection with H5N1 Results in Increased TNF-␣ and IL-6 in BALF and Serum
Concentrations of TNF-␣ and IL-6 in BALF and serum were measured on Days 1, 3, 5, 6, 7, 8, and 14 after infection with H5N1 virus. As shown in Figure 6A, levels of TNF-␣ in infected mice significantly increased on Days 5 to 8 postinoculation in BALF (p ⬍ 0.05) compared with those of the controls. TNF-␣ levels also rose significantly in serum on Days 5 to 8 postinoculation (Figure 6B), but the alteration was not as significant as that in BALF. IL-6 levels were dramatically increased in both BALF and serum on Days 5 to 8 postinoculation (see Figures 6C and 6D).
DISCUSSION
Figure 5. Kinetic changes in circulating leukocytes (A ) and blood differential counts (B ) after H5N1 viral infection. (A ) Solid circles, control; solid triangles, inoculation. (B ) Open bars, monocytes; dotted bars, polymorphonuclear leukocytes; solid bars, lymphocytes.
As of February 13, 2006, the World Health Organization reported that more than 90 humans had died of H5N1 viral infection in southeast Asia, China, Turkey, and Nigeria since 2003 (23). Most patients with confirmed infection with influenza A (H5N1) virus died of respiratory failure complicated with ARDS, which is characterized mainly by diffuse alveolar damage, vascular congestion, infiltration of leukocytes into the interstitial areas, and severe refractory hypoxemia (10, 13, 24). In this report, we describe a mouse model of ARDS induced by H5N1 infection, which demonstrated that BALB/c mice become acutely ill when inoculated intranasally with 1 ⫻ 102 MID50 of Chicken/HB/108 H5N1 virus. First, we found that most of the infected mice exhibited clinical signs of respiratory disease, including visually prominent signs of respiratory distress, with about 80% of the mice dying between Days 6 and 8 postinoculation. Second, gross observations showed that the infected mice had highly edematous lungs, also demonstrated by the dramatically increased lung wet weight:body weight ratios and lung wet:dry weight ratios. Third, the infected mice displayed a similar histopathologic pattern, including an initial peribronchiolar patchy pneumonia on Days 3 to 5 postinoculation and predominantly peribronchiolar lesions, fully developed bronchiolitis, and bronchopneumonia by Day 8 postinoculation. Pathologic lesions in the lungs were characterized by inflammatory cellular infiltration, interstitial and alveolar edema, and hemorrhage. Last, on Day 6 postinoculation, when most of the infected mice suddenly presented prominent clinical signs of respiratory distress, the PaO2 decreased dramatically and the PaCO2 increased significantly. Especially on Day 8 postinoculation, the lowest PaO2 in infected mice was only 50% of that in control mice. Thus, the change in arterial blood gas demonstrated that most of the infected mice developed progressive and severe hypoxemia consistent with the time course of clinical signs and pulmonary lesions of ARDS. Our data suggested that we successfully induced prominent ARDS in mice with H5N1 viral infection.
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AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE VOL 174 2006 TABLE 2. WHITE BLOOD CELL SUMMARY AND DIFFERENTIAL COUNTS IN BRONCHOALVEOLAR LAVAGE FLUID WBC Count (% )
WBC Summary Time (d ) 1 3 5 6 7 8 14
Group
(1 ⫻ 10 /ml)
Macrophages
Infection* Control† Infection* Control† Infection* Control† Infection* Control† Infection* Control† Infection* Control† Infection* Control§
⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾
⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾
8
1.86 1.38 2.94 1.45 4.47 1.92 6.17 1.78 12.16 1.62 9.12 1.59 2.09 1.42
0.32 0.57 0.46‡ 0.23 0.68§ 0.39 1.76§ 0.52 2.78§ 0.37 4.64§ 0.26 0.62 0.41
59.75 90.2 49.6 88.3 32.5 89.7 31.25 89.3 23.37 90.8 26.2 91.4 66.4 93.8
‡
3.30 8.30 7.41‡ 4.8 4.2§ 7.3 5.12§ 4.34 2.05§ 1.93 1.41§ 2.71 6.2 1.04
Lymphocytes 37.0 8.61 43.0 11.8 55.25 7.24 48.75 8.64 43.7 7.47 46.2 6.87 32.6 5.93
⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾
4.24 1.23 4.69‡ 3.2 3.95§ 3.95 5.53§ 1.19 2.22§ 2.19 1.8§ 2.25 4.6 1.43
PMNs 1.625 0.78 3.29 0.95 7.25 1.12 12.25 1.68 24.75 1.07 28.25 0.85 1.13 0.84
⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾
0.48‡ 0.31 1.26§ 0.74 3.59§ 0.42 1.47§ 1.29 5.73§ 0.24 4.5§ 0.24 0.07 0.27
Definition of abbreviations: PMNs ⫽ polymorphonuclear leukocytes; WBC ⫽ white blood cell. Values are expressed as means ⫾ SD. * Mice infected with 1 ⫻ 102 MID50 of Chicken/HB/108 virus. † Mock-infected mice inoculated with noninfectious allantoic fluid. ‡ p ⬍ 0.05 compared with controls. § p ⬍ 0.01 as compared with controls.
Several studies have demonstrated that respiratory infections lead to impairment of the alveolar epithelium and endothelium, which increases the thickness of alveolar–capillary membranes and decreases the number of ventilated alveoli, resulting in hypoxia (25, 26). In this model, our data demonstrated that Chicken/ HB/108 H5N1 virus replicated extensively in the lungs, which had the highest viral replication titers of all the organs, similar to the pattern presented by the HK/483 H5N1 virus isolated from a human patient in Hong Kong in 1997 (7). However, it was interesting that viral titers in the lungs were declining at the time of infected mouse death. Sakai and coworkers also found that titers decreased in mice infected with influenza A/PR/8/34 virus from Day 4 to 6 postinoculation, and that most mice began to die from Day 6 (27). These data suggested that inflammatory injury rather than uncontrolled infection might be a determinant of the fatal outcome. It is believed that ARDS is a clinical syndrome usually secondary to an intense, neutrophil-predominant host inflammatory response (2). After stimulation by proinflammatory cytokines,
the activated neutrophils release free radicals, inflammatory mediators, and proteases, which lead to lung lesions (25). In H5N1 virus–infected mice, we observed that circulating leukocytes dramatically decreased in blood and that a great number of inflammatory cells infiltrated the lungs. In addition, the neutrophils in BALF increased nearly 30-fold compared with the control mice on Day 8 postinoculation. Moreover, we also observed histopathologic changes in other organs (e.g., brain, spleen, and kidneys). Although these organs presented congestion and mild hemorrhage, no obvious inflammatory cellular infiltration was found compared with the lungs (data not shown). Therefore, these results suggested that a great number of leukocytes, especially neutrophils, were recruited from the blood stream and sequestrated mainly in the lungs, and might be involved in the host inflammation response and severe pulmonary lesions induced by H5N1 viral infection. TNF-␣ is believed to be one of the important inflammatory cytokines involved in the development of ARDS. Lee and coworkers demonstrated that H5N1 virus isolated from humans
Figure 6. Tumor necrosis factor (TNF)-␣ and interleukin (IL)-6 concentrations in bronchoalveolar lavage fluid (BALF) and serum of mice infected with Chicken/HB/108 virus. TNF-␣ in BALF (A ) and in serum (B ); IL-6 in BALF (C ) and in serum (D ). Open bars, control group; solid bars, inoculation group. *p ⬍ 0.05, **p ⬍ 0.01 compared with those in control mice.
Xu, Qiao, Zhao, et al.: ARDS Induced by Avian Influenza A H5N1 Virus in Mice
induced high levels of TNF-␣ in primary human macrophage culture (28). To and coworkers found that the levels of TNF-␣ increased significantly in patients who died of H5N1 viral infection (29). Our data showed that Chicken/HB/108 H5N1 viral infection also induced high levels of TNF-␣ in the lungs of mice, but that TNF-␣ in serum did not change significantly, which indicates that TNF-␣ may be produced mainly in the lungs. In addition, IL-6 levels also dramatically increased in both BALF and serum. The role of elevations of these cytokines in the inflammation of the lungs during H5N1 viral infection remains to be investigated. Although primates and ferrets are sensitive to intranasal H5N1 viral infection and reproduce several clinical features of the human disease (30, 31), the additional symptoms that are associated with human H5N1 infection, such as lymphopenia, have not been reported (31). Furthermore, availability, cost, and ethical constraints may limit the utility of nonhuman primates for such research (31). In this report, our research demonstrated that Chicken/HB/108 H5N1 virus replicated efficiently in the respiratory tracts of BALB/c mice without prior adaptation, and that the mouse model could recapitulate most of the clinical and pathologic changes, including the lymphopenia, observed in human ARDS induced by H5N1 viral infection. Therefore, this model system not only makes it possible to investigate the pathogenesis of ARDS based on H5N1 viral infection, but also can provide a basis for studies on potential therapeutic intervention for human ARDS induced by H5N1 virus. In summary, our data showed that we successfully developed a mouse model of ARDS induced by H5N1 viral infection, which will benefit further investigation into the pathogenesis of human ARDS induced by H5N1 virus.
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Conflict of Interest Statement : None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript.
22. Acknowledgment : The authors thank Professor Qiyu Gao and Dr. Deyuan Ou for technical assistance in histopathologic observation.
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