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Mouse-Passaged Severe Acute Respiratory Syndrome-Associated Coronavirus Leads to Lethal Pulmonary Edema and Diffuse Alveolar Damage in Adult but Not Young Mice

2008; Elsevier BV; Volume: 172; Issue: 6 Linguagem: Inglês

10.2353/ajpath.2008.071060

ISSN

1525-2191

Autores

Noriyo Nagata, Naoko Iwata, Hideki Hasegawa, Shuetsu Fukushi, Ayako Harashima, Yuko Sato, Masayuki Saijo, Fumihiro Taguchi, Shigeru Morikawa, Tetsutaro Sata,

Tópico(s)

Long-Term Effects of COVID-19

Resumo

Advanced age is a risk factor of severe acute respiratory syndrome (SARS) in humans. To understand its pathogenesis, we developed an animal model using BALB/c mice and the mouse-passaged Frankfurt 1 isolate of SARS coronavirus (SARS-CoV). We examined the immune responses to SARS-CoV in both young and adult mice. SARS-CoV induced severe respiratory illness in all adult, but not young, mice on day 2 after inoculation with a mortality rate of 30 to 50%. Moribund adult mice showed severe pulmonary edema and diffuse alveolar damage accompanied by virus replication. Adult murine lungs, which had significantly higher interleukin (IL)-4 and lower IL-10 and IL-13 levels before infection than young murine lungs, rapidly produced high levels of proinflammatory chemokines and cytokines known to induce macrophage and neutrophil infiltration and activation (eg, tumor necrosis factor-α). On day 2 after inoculation, young murine lungs produced not only proinflammatory cytokines but also IL-2, interferon-γ, IL-10, and IL-13. Adult mice showed early and acute excessive proinflammatory responses (ie, cytokine storm) in the lungs after SARS-CoV infection, which led to severe pulmonary edema and diffuse alveolar damage. Intravenous injection with anti-tumor necrosis factor-α antibody 3 hours after infection had no effect on SARS-CoV infection. However, intraperitoneal interferon-γ injection protected adult mice from the lethal respiratory illness. The experimental model described here may be useful for elucidating the pathophysiology of SARS and for evaluating therapies to treat SARS-CoV infection. Advanced age is a risk factor of severe acute respiratory syndrome (SARS) in humans. To understand its pathogenesis, we developed an animal model using BALB/c mice and the mouse-passaged Frankfurt 1 isolate of SARS coronavirus (SARS-CoV). We examined the immune responses to SARS-CoV in both young and adult mice. SARS-CoV induced severe respiratory illness in all adult, but not young, mice on day 2 after inoculation with a mortality rate of 30 to 50%. Moribund adult mice showed severe pulmonary edema and diffuse alveolar damage accompanied by virus replication. Adult murine lungs, which had significantly higher interleukin (IL)-4 and lower IL-10 and IL-13 levels before infection than young murine lungs, rapidly produced high levels of proinflammatory chemokines and cytokines known to induce macrophage and neutrophil infiltration and activation (eg, tumor necrosis factor-α). On day 2 after inoculation, young murine lungs produced not only proinflammatory cytokines but also IL-2, interferon-γ, IL-10, and IL-13. Adult mice showed early and acute excessive proinflammatory responses (ie, cytokine storm) in the lungs after SARS-CoV infection, which led to severe pulmonary edema and diffuse alveolar damage. Intravenous injection with anti-tumor necrosis factor-α antibody 3 hours after infection had no effect on SARS-CoV infection. However, intraperitoneal interferon-γ injection protected adult mice from the lethal respiratory illness. The experimental model described here may be useful for elucidating the pathophysiology of SARS and for evaluating therapies to treat SARS-CoV infection. 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However, the course of infection in these animals is shorter than that in humans. As in humans, an advanced age correlates positively and independently with adverse outcomes and is a predictor of mortality in animal models.6Booth CM Matukas LM Tomlinson GA Rachlis AR Rose DB Dwosh HA Walmsley SL Mazzulli T Avendano M Derkach P Ephtimios IE Kitai I Mederski BD Shadowitz SB Gold WL Hawryluck LA Rea E Chenkin JS Cescon DW Poutanen SM Detsky AS Clinical features and short-term outcomes of 144 patients with SARS in the greater Toronto area.JAMA. 2003; 289: 2801-2809Crossref PubMed Scopus (1159) Google Scholar, 7Donnelly CA Ghani AC Leung GM Hedley AJ Fraser C Riley S Abu-Raddad LJ Ho LM Thach TQ Chau P Chan KP Lam TH Tse LY Tsang T Liu SH Kong JH Lau EM Ferguson NM Anderson RM Epidemiological determinants of spread of causal agent of severe acute respiratory syndrome in Hong Kong.Lancet. 2003; 361: 1761-1766Abstract Full Text Full Text PDF PubMed Scopus (783) Google Scholar, 8Lee N Hui D Wu A Chan P Cameron P Joynt GM Ahuja A Yung MY Leung CB To KF Lui SF Szeto CC Chung S Sung JJ A major outbreak of severe acute respiratory syndrome in Hong Kong.N Engl J Med. 2003; 348: 1986-1994Crossref PubMed Scopus (1975) Google Scholar, 9Peiris JS Chu CM Cheng VC Chan KS Hung IF Poon LL Law KI Tang BS Hon TY Chan CS Chan KH Ng JS Zheng BJ Ng WL Lai RW Guan Y Yuen KY HKU/UCH SARS Study Group Clinical progression and viral load in a community outbreak of coronavirus-associated SARS pneumonia: a prospective study.Lancet. 2003; 361: 1767-1772Abstract Full Text Full Text PDF PubMed Scopus (1980) Google Scholar, 10Tsui PT Kwok ML Yuen H Lai ST Severe acute respiratory syndrome: clinical outcome and prognostic correlates.Emerg Infect Dis. 2003; 9: 1064-1069Crossref PubMed Scopus (186) Google Scholar Moreover, SARS-CoV isolates replicate better in aged BALB/c mice than in younger mice.29Roberts A Paddock C Vogel L Butler E Zaki S Subbarao K Aged BALB/c mice as a model for increased severity of severe acute respiratory syndrome in elderly humans.J Virol. 2005; 79: 5833-5838Crossref PubMed Scopus (175) Google Scholar It is likely that the correlation between poor outcome and advanced age reflects the weakened immune responses of the elderly, in particular their impaired cytokine responses. This is significant because cytokines regulate the immune response to infection. Indeed, analysis of the cytokine responses of elderly individuals to respiratory infections that lead to severe pulmonary diseases (eg, Listeria monocytogenes, respiratory syncytial virus, influenza virus)30Sandmand M Bruunsgaard H Kemp K Andersen-Ranberg K Pedersen AN Skinhoj P Pedersen BK Is ageing associated with a shift in the balance between type 1 and type 2 cytokines in humans?.Clin Exp Immunol. 2002; 127: 107-114Crossref PubMed Scopus (135) Google Scholar, 31Looney RJ Falsey AR Walsh E Campbell D Effect of aging on cytokine production in response to respiratory syncytial virus infection.J Infect Dis. 2002; 185: 682-685Crossref PubMed Scopus (69) Google Scholar, 32Antonini JM Roberts JR Clarke RW Yang HM Barger MW Ma JY Weissman DN Effect of age on respiratory defense mechanisms: pulmonary bacterial clearance in Fischer 344 rats after intratracheal instillation of Listeria monocytogenes.Chest. 2001; 120: 240-249Crossref PubMed Scopus (45) Google Scholar, 33Boukhvalova MS Yim KC Kuhn KH Hemming JP Prince GA Porter DD Blanco JC Age-related differences in pulmonary cytokine response to respiratory syncytial virus infection: modulation by anti-inflammatory and antiviral treatment.J Infect Dis. 2007; 195: 511-518Crossref PubMed Scopus (55) Google Scholar have revealed unbalanced Th1-type and Th2-type responses. We recently succeeded in establishing a rat model of SARS using rat-passaged SARS-CoV.34Nagata N Iwata N Hasegawa H Fukushi S Yokoyama M Harashima A Sato Y Saijo M Morikawa S Sata T Participation of both host and virus factors in induction of severe acute respiratory syndrome (SARS) in F344 rats infected with SARS coronavirus.J Virol. 2007; 81: 1848-1857Crossref PubMed Scopus (52) Google Scholar Although the rat-passaged SARS-CoV was not lethal, it induced more severe pathological lesions in adult F344 rats than in young rats. We found that the severe inflammation in the adult rats was associated with high levels of inflammatory cytokines in the serum and lung homogenates, especially interleukin (IL)-6, along with low levels of the immunosuppressive cytokine IL-10. IL-6 is an inflammatory cytokine that is produced by monocytes, leukocytes, endothelial cells, fibroblasts, and alveolar epithelial cells. SARS patients have significantly elevated serum IL-6 levels.19Zhang Y Li J Zhan Y Wu L Yu X Zhang W Ye L Xu S Sun R Wang Y Lou J Analysis of serum cytokines in patients with severe acute respiratory syndrome.Infect Immun. 2004; 72: 4410-4415Crossref PubMed Scopus (279) Google Scholar IL-10 is produced by macrophages, Th2 lymphocytes, and B cells and inhibits tumor necrosis factor (TNF)-α production and neutrophil activation in lipopolysaccharide-induced acute lung injury, thereby suppressing lung tissue injury.35Inoue G Effect of interleukin-10 (IL-10) on experimental LPS-induced acute lung injury.J Infect Chemother. 2000; 6: 51-60Abstract Full Text PDF PubMed Scopus (58) Google Scholar It has been reported that serum IL-10 levels increase in SARS patients during the convalescence phase.19Zhang Y Li J Zhan Y Wu L Yu X Zhang W Ye L Xu S Sun R Wang Y Lou J Analysis of serum cytokines in patients with severe acute respiratory syndrome.Infect Immun. 2004; 72: 4410-4415Crossref PubMed Scopus (279) Google Scholar In this study, we established a new and more useful experimental small animal model for SARS by using BALB/c mice and mouse-passaged SARS-CoV. This model allows us to better characterize the virus-host relationship and determine which immune responses are antiviral and which are pathogenic. Here, we sought to determine why SARS-CoV infection is more frequently lethal in elderly patients by comparing SARS-CoV-infected adult and young mice in terms of their pulmonary pathology and immune responses. The Frankfurt 1 isolate of SARS-CoV used in this study was kindly supplied by Dr. John Ziebuhr, Institute of Virology and Immunology, University of Würzburg, Würzburg, Germany. The virus was propagated twice in Vero E6 cells purchased from American Type Cell Collection (Manassas, VA) that were cultured in Eagle's minimal essential medium (MEM) containing 5% fetal bovine serum, 50 IU of penicillin G, and 50 μg of streptomycin per ml. Titers of this stock virus were expressed as 50% of the tissue culture infectious dose (TCID50)/ml on Vero E6 cells, which was calculated according to the Behrens-Kärber method. Work with infectious SARS-CoV was performed under biosafety level 3 conditions. Compared to the original virus, the Frankfurt 1 isolate used in our laboratory has one amino acid change at position 641 (His to Tyr) in the S protein and another in open reading frame (ORF) 1a 429 (Ala to Ser). These changes presumably arose during the passage through Vero E6 cells. Female 4-week-old or 6-month-old BALB/c mice were purchased from Japan SLC (Shizuoka, Japan) and maintained in specific pathogen-free facilities. On experimental infection, these animals were housed in biosafety level 3 animal facilities. These animal experiments were approved by the Animal Care and Use Committee of the National Institute of Infectious Diseases, Tokyo, Japan. The Frankfurt 1 isolate of SARS-CoV was serially passaged 10 times in 4-week-old female BALB/c mice, as follows. After intranasal inoculation, three mice were sacrificed on day 3 after inoculation and their bronchoalveolar wash fluids were collected. These bronchoalveolar fluids were then used to inoculate three additional BALB/c mice, whose bronchoalveolar fluids on day 3 after inoculation were used to inoculate fresh mice. After 10 such passages in mice, the lungs were removed under sterile conditions, washed three times, and homogenized in 1 ml of phosphate buffer containing 0.1% bovine serum albumin, 20 IU of penicillin G, 20 μl of streptomycin, and 1 μg of amphotericin B per ml. The lung homogenates were centrifuged at 1000 × g for 20 minutes, and 1 ml of the supernatants in 10 ml of MEM containing 2% fetal bovine serum were used to infect Vero E6 cells. After 1 hour of adsorption, the inoculum was removed and MEM containing 2% fetal bovine serum was added. The cell cultures were incubated at 37°C with 5% CO2 for 2 days and then treated once with freeze-thawing. After centrifugation at 1000 × g for 20 minutes, the supernatants (referred to here as F-musX-VeroE6) were used as the virus inoculum. Compared to the original virus, F-musX-VeroE6 has amino acid mutations in the S protein at positions 480 (Asp to Glu) and 641 (His to Tyr); The latter change is identical to one of the mutations found in the Frankfurt 1 isolate. In the completely sequenced genome, F-musX-VeroE6 also has two additional mutations in ORF1a 3534 (Phe to Leu) and ORF1ab 5172 (Thr to Ile). The mutation in ORF1a 429 found in the Frankfurt 1 isolate was not present. Mice were anesthetized by intraperitoneal injection with a 0.1 ml/10 g body weight mixture of 1.0 mg ketamine and 0.02 mg xylazine. The animals were then inoculated intranasally in the left nostril with the Frankfurt 1 isolate or F-musX-VeroE6 (2 × 106 TCID50 in 20 μl) and observed for clinical signs. Body weight was measured daily for 10 or 21 days. Infected animals were also sacrificed at various time points after inoculation to analyze virus replication, hematology, cytokine expression, and pathology (n = 3 in each group). Twenty percent (w/v) tissue homogenates of the lung, maxilla (including the nasal cavity), cervical lymph node, spleen, liver, and kidney were prepared in MEM containing 2% fetal bovine serum, 50 IU penicillin G, 50 μg streptomycin, and 2.5 μg amphotericin B per ml (MEM-2FBS). Bronchoalveolar and nasal wash fluids were also collected for analysis of virus replication. Viral infectivity titers of respiratory tract and wash fluids were determined as described above. Virus isolation from other tissues was performed by blind passage after freezing and thawing the first-round passage using Vero E6 cells. Total blood cell counts in peripheral blood collected in sodium-heparinized tubes were measured by an autoanalyzer (Cell Tuck; Nihon Koden, Tokyo, Japan). Neutrophil, lymphocyte, monocyte, eosinophil, and basophil counts were determined by microscopic analysis. Antibodies used for flow cytometry were anti-CD4-phycoerythrin-Cy5 (eBioscience, San Diego, CA), anti-CD8β-phycoerythrin (Santa Cruz Biotechnology, Santa Cruz, CA), and anti-pan-NK cells-fluorescein isothiocyanate (eBioscience). Cells incubated with these surface-binding antibodies were fixed in 2% paraformaldehyde in phosphate-buffered saline (PBS) and subjected to flow cytometry (EPICS Elite; Beckman Coulter, Fullerton, CA) using EXPO cytometer software (Beckman Coulter). Homogenized lung tissue samples were diluted 1:1 with cell extraction buffer [10 mmol/L Tris, pH 7.4, 100 mmol/L NaCl, 1 mmol/L EDTA, 1 mmol/L EGTA, 1 mmol/L NaF, 20 mmol/L Na4P2O7, 2 mmol/L Na3VO4, 1% Triton X-100, 10% glycerol, 0.1% sodium dodecyl sulfate, and 0.5% deoxycholate (BioSource International, Inc., Camarillo, CA)], incubated for 30 minutes on ice with vortexing at 10 minute intervals, and then centrifuged at 15,000 × g for 10 minutes at 4°C. Supernatants were diluted 1:5 in assay diluent of the Mouse Cytokine 20-Plex antibody bead kit (BioSource International). Sera and the 20% lung homogenate supernatants were subjected to ultraviolet irradiation for 10 minutes to inactivated virus infectivity and stored at −80°C until they were used to determine the presence of mouse cytokines, namely, basic fibroblast growth factor, GM-CSF, interferon (IFN)-γ, IL-1α, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12p40/p70, IL-13, IL-17, IP-10, keratinocyte chemoattractant (KC), monocyte chemoattractant protein 1 (MCP-1), MIG, MIP-1α, TNF-α, and vascular endothelial growth factor (VEGF), by using the Mouse Cytokine 20-Plex antibody bead kit and Luminex 100TM (Luminex Co., Austin, TX). Animals (n = 3 in each group) were anesthetized and perfused with 2 ml of 10% phosphate-buffered formalin. Fixed lung, heart, kidney, liver, spleen, small and large intestine, brain, spinal cord, and maxilla (including nasal cavity) tissues were routinely embedded in paraffin, sectioned, and stained with hematoxylin and eosin. Maxilla samples were decalcified in phosphate-buffered saline (pH 7.4) plus 10% EDTA before being embedded. Immunohistochemical detection of the SARS-CoV antigens was performed on paraffin-embedded sections, as follows. After deparaffinizing with xylene, sections were rehydrated in ethanol and immersed in PBS. Antigens were retrieved by hydrolytic autoclaving for 20 minutes at 121°C in 10 mmol/L sodium citrate-sodium chloride buffer (pH 6.0). After cooling, the sections were immersed in PBS. Endogenous peroxidase was blocked by incubation in 1% hydrogen peroxide in methanol for 30 minutes. After washing in PBS, the sections were incubated with normal rabbit serum for 5 minutes, and then with rabbit antibody against SARS-CoV32Antonini JM Roberts JR Clarke RW Yang HM Barger MW Ma JY Weissman DN Effect of age on respiratory defense mechanisms: pulmonary bacterial clearance in Fischer 344 rats after intratracheal instillation of Listeria monocytogenes.Chest. 2001; 120: 240-249Crossref PubMed Scopus (45) Google Scholar, 36Fukushi S Mizutani T Saijo M Matsuyama S Miyajima N Taguchi F Itamura S Kurane I Morikawa S Vesicular stomatitis virus pseudotyped with severe acute respiratory syndrome coronavirus spike protein.J Gen Virol. 2005; 86: 2269-2274Crossref PubMed Scopus (95) Google Scholar overnight at 4°C. After three washes in PBS, the sections were incubated with biotin-conjugated anti-rabbit IgG for 30 minutes at 37°C, followed by reaction with streptavidin-peroxidase for 30 minutes at room temperature. Peroxidase activity was detected by development with diaminobenzidine containing hydrogen peroxide. Nuclei were counterstained by hematoxylin. SARS-CoV- and mock-infected adult and young mice were euthanized 1, 3, and 5 days after inoculation by exsanguination under excess ether anesthesia, after which the lungs were harvested for pathological examination (three mice per group). Mock infection was performed by using MEM containing 2% fetal bovine serum. For staining with anti-Mac-3 and anti-surfactant D (SP-D) antibodies and to detect SARS-CoV antigens, the lungs were fixed with 4% paraformaldehyde in PBS at 4°C for 15 to 18 hours and embedded in paraffin according to the manufacturer's instructions (BD Biosciences Pharmingen, San Diego, CA). The paraffin-embedded sections were then subjected to a double-immunofluorescence staining method37Liem NT Nakajima N Phat LLP Sato Y Thach HN Hung PV San LT Katano H Kumasaka T Oka T Kawachii S Matsushita T Sata T Kudo K Suzuki K H5N1-infected cells in lung with diffuse alveolar damage in exudative phase from a fatal case in Vietnam.Jpn J Infect Dis. 2008; 61: 157-160PubMed Google Scholar using a polyclonal rabbit antibody against SARS-CoV36Fukushi S Mizutani T Saijo M Matsuyama S Miyajima N Taguchi F Itamura S Kurane I Morikawa S Vesicular stomatitis virus pseudotyped with severe acute respiratory syndrome coronavirus spike protein.J Gen Virol. 2005; 86: 2269-2274Crossref PubMed Scopus (95) Google Scholar and the SKOT9 monoclonal mouse antibody against nucleocapsid protein38Ohnishi K Sakaguchi M Kaji T Akagawa K Taniyama T Kasai M Tsunetsugu-Yokota Y Oshima M Yamamoto K Takasuka N Hashimoto S Ato M Fujii H Takahashi Y Morikawa S Ishii K Sata T Takagi H Itamura S Odagiri T Miyamura T Kurane I Tashiro M Kurata T Yoshikura H Takemori T Immunological detection of severe acute respiratory syndrome coronavirus by monoclonal antibodies.Jpn J Infect Dis. 2005; 58: 88-94PubMed Google Scholar or a monoclonal rat anti-Mac-3 antibody against mouse mononuclear phagocytes (BD Biosciences Pharmingen)

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