key: cord-0005617-57q69icp
authors: Zou, Zhen; Yan, Yiwu; Shu, Yuelong; Gao, Rongbao; Sun, Yang; Li, Xiao; Ju, Xiangwu; Liang, Zhu; Liu, Qiang; Zhao, Yan; Guo, Feng; Bai, Tian; Han, Zongsheng; Zhu, Jindong; Zhou, Huandi; Huang, Fengming; Li, Chang; Lu, Huijun; Li, Ning; Li, Dangsheng; Jin, Ningyi; Penninger, Josef M.; Jiang, Chengyu
title: Angiotensin-converting enzyme 2 protects from lethal avian influenza A H5N1 infections
date: 2014-05-06
journal: Nat Commun
DOI: 10.1038/ncomms4594
sha: 4bc55ca2806bde632c4d929ff7da7f97009aa85f
doc_id: 5617
cord_uid: 57q69icp

The potential for avian influenza H5N1 outbreaks has increased in recent years. Thus, it is paramount to develop novel strategies to alleviate death rates. Here we show that avian influenza A H5N1-infected patients exhibit markedly increased serum levels of angiotensin II. High serum levels of angiotensin II appear to be linked to the severity and lethality of infection, at least in some patients. In experimental mouse models, infection with highly pathogenic avian influenza A H5N1 virus results in downregulation of angiotensin-converting enzyme 2 (ACE2) expression in the lung and increased serum angiotensin II levels. Genetic inactivation of ACE2 causes severe lung injury in H5N1-challenged mice, confirming a role of ACE2 in H5N1-induced lung pathologies. Administration of recombinant human ACE2 ameliorates avian influenza H5N1 virus-induced lung injury in mice. Our data link H5N1 virus-induced acute lung failure to ACE2 and provide a potential treatment strategy to address future flu pandemics. SUPPLEMENTARY INFORMATION: The online version of this article (doi:10.1038/ncomms4594) contains supplementary material, which is available to authorized users.

T he H5N1 avian influenza virus has spread throughout the world, primarily infecting poultry and migratory birds 1,2 . Since 2003, more than 600 people worldwide have been identified to be infected with avian H5N1 influenza virus with higher mortality rate than that of seasonal flu and even the Spanish flu (http://www.who.int/influenza/human_animal_ interface/EN_GIP_20131008CumulativeNumberH5N1cases.pdf). Human-to-human transmission of H5N1 viruses is rare and human infections have been primarily limited to people in very close contact with infected birds 2,3 . Many viruses, including H5N1, mutate readily and produce strains that are no longer manageable using vaccines or currently known antiviral drugs 4 . The apparent high lethality of H5N1 infections and their potential social impact make it paramount to identify common molecular disease mechanisms as well as new treatment options for H5N1 infections 5 .

The renin-angiotensin system (RAS) has an important role in maintaining blood pressure, cardiac homeostasis and fluid and salt balance [6] [7] [8] . Angiotensin-converting enzyme (ACE) and its later discovered sibling ACE2 share homology in their catalytic domain and have opposite functions in the RAS 9 . ACE cleaves angiotensin I to generate angiotensin II, which is a key effector peptide and exerts multiple biological functions related to cardiovascular and renal biology 10 . In contrast, ACE2 cleaves a single amino acid residue from Angiotensin II to generate Ang1-7 and thereby negatively regulates the RAS by reducing Angiotensin II level 11, 12 . Our previous studies using ACE2knockout mice have demonstrated that ACE2 protects against acute lung injury (ALI) triggered by acid aspiration and sepsis 13 . Furthermore, ACE2 has been identified as the essential in vivo receptor for the SARS-Corona virus. SARS-CoV infections and the in vivo administration of the SARS-CoV spike protein reduce ACE2 expression 14 , suggesting that the RAS is involved in the pathogenesis of ALI and SARS-CoV infections 14 . Importantly, the administration of recombinant ACE2 has been proven beneficial in improving lung pathologies associated with SARS-CoV spike-, acid aspiration-and sepsis-induced ALI in different species 13, 14 . Based on these data, ACE2 has now entered phase II clinical testing for the treatment of ALI in humans.

Human infections of influenza A virus subtype H5N1, also known as 'bird flu', causes severe pneumonia and ALI, ultimately resulting in death owing to acute respiratory distress syndrome (ARDS), the most severe form of ALI [15] [16] [17] [18] . In multiple species, the same clinical syndrome of acute lung failure/ARDS has been observed for various pathogenic conditions including sepsis, gastric acid aspiration or pulmonary infections with SARS-CoV and H5N1 bird flu 13, 14, [19] [20] [21] . In our accompanying paper, we observed elevation of serum angiotensin II in H7N9-infected patients, and more importantly, we showed that serum angiotensin II level was linked to disease severity and outcome 22 . We therefore wanted to determine whether ACE2 is also involved in H5N1-induced lung injury. Here we show that ACE2 plays an important role in H5N1 virus-induced ALI. In mice, experimental treatment with recombinant hACE2 ameliorates the disease and increases survival rate following H5N1 infection.

Serum level of angiotensin II is increased in H5N1 patients. To determine whether the RAS is indeed deregulated in humans with severe influenza, serum samples from 26 H5N1-infected patients were collected and divided into three groups; 0-7 days (n ¼ 8), 8-14 days (n ¼ 13) and more than 15 days (n ¼ 5) after illness onset. The level of angiotensin II, as a marker for RAS activation 23, 24 , was significantly increased in the avian influenza A H5N1-infected patients when compared with healthy controls (n ¼ 10) (Fig. 1a, Table 1 ). Our data also indicated that serum angiotensin II levels might be linked to the severity and lethality of bird flu infections in patients (Fig. 1b) .

To directly demonstrate that flu infections alter the RAS, we infected mice with live avian influenza A H5N1 virus. The infection of mice with the highly pathogenic H5N1 virus strain resulted in the rapid downregulation of ACE2 expression in the lungs 24 h after infection (n ¼ 3, Fig. 2a ) and a concurrent increase of systemic angiotensin II levels in the serum 72 h after infection (n ¼ 5-6 per group, Fig. 2b ). ACE expression was apparently not affected in the lungs of H5N1-infected mice (Fig. 2a) . ACE2 expression in the lungs and the serum angiotensin II levels remained unchanged in mice challenged with the less pathogenic H1N1 virus (Fig. 2a,b) . Furthermore, sera collected from infected mice on day 3 and day 5 showed that the changes in angiotensin II levels correlated with death (n ¼ 14) and survival (n ¼ 4) of H5N1-infected mice (Fig. 2c) . Thus, similar to our human data, in our experimental mouse models the downregulation of ACE2 and elevation of serum angiotensin II Ang II concentration (pg ml level may also be correlated with the disease severity and lethality of bird flu infections.

ACE2 deficiency exacerbates H5N1-induced lung injury. To determine whether ACE2 has a functional role in ALI induced by infections with live H5N1 avian flu viruses, we examined mice that are genetically deficient in ACE2 (ref. 25) . H5N1 virusinfected ACE2-knockout mice (n ¼ 10) died significantly faster than the wild-type controls (n ¼ 10) (Fig. 3a) . The H5N1 viral load detected in mice lung tissue of ACE2-knockout mice was significantly higher than that of wild-type mice lung 5 days after infection (n ¼ 3-5 per time point, Fig. 3b ,c, Supplementary  Fig. 1a) . Moreover, the wet-to-dry lung tissue ratio, which is a measure of lung edema, was markedly higher in the ACE2knockout mice 3 days after infection (n ¼ 4-6 per group, Fig. 3d ), supporting the hypothesis that ACE2 has a protective role against H5N1 virus-induced acute lung failure. H5N1 challenge also resulted in increased inflammatory cell infiltration, and a more severe lung injury score (including increased alveolar wall thickness, formation of hyaline membranes and proteinaceous debris filling the airspaces), in the ACE2-knockout mice (n ¼ 3 per group, Fig. 3e ). Thus, the genetic inactivation of ACE2 expression results in a lower survival rate and more severe ALI following respiratory H5N1 infections.

Recombinant hACE2 protects against H5N1 infection. Finally, we investigated whether recombinant human ACE2 can alleviate H5N1-induced pathologies. The administration of recombinant ACE2 protein (3 h prior to, 8 h and 3 days after infection, i.p. 100 mg kg À 1 ) prolonged the overall survival time of mice infected with H5N1 virus (Fig. 4a ) and decreased the viral replication in mice lungs after H5N1 viral infection (n ¼ 3-5 per time point, Fig. 4b , Supplementary Fig. 1b) . The weight loss of surviving hACE2 protein-treated mice was recovered ( Supplementary  Fig. 2 ). Treatment with recombinant hACE2 also markedly reduced the serum level of angiotensin II (n ¼ 3-4 per time point, Fig. 4c ) and the development of edema 4 days after infection Fig. 4d ). The lung histopathology induced by H5N1 flu infection was also ameliorated (n ¼ 3, Fig. 4e ). Moreover, hACE2 treatment improved the impaired lung functions of mice challenged with an inactivated H5N1 virus, as assessed via lung elastance ( Supplementary Fig. 3 ). Of note, lung elastance is the reciprocal of lung compliance, a measure of the change in pressure per unit change in volume representing the stiffness of the lungs 26 . To assess whether administration of recombinant hACE2 is also effective after virus infection, we treated mice with recombinant hACE2 protein 1 h, 1 day and 2 days after H5N1 infection (i.p. 100 mg kg À 1 ). Recombinant human ACE2 again ameliorated ALI induced in H5N1 infected mice ( Supplementary  Fig. 4 ). Further studies are required to assess the therapeutic efficacy of ACE2. Taken together, these data show that avian influenza A H5N1 virus infections cause severe acute lung failure, at least in part, by functionally altering the RAS via ACE2 expression and that H5N1-induced lung injury can be alleviated by the administration of recombinant human ACE2 protein.

Our data establish an important role for ACE2 and the RAS in H5N1 virus-induced lung injury and death. The discovery that the severe acute lung injuries in patients with acid aspiration, sepsis or SARS-CoV infection and in those infected with H5N1 viruses share a common molecular mechanism, namely the downregulation of ACE2 leading to RAS activation, suggests that this pathogenesis mechanism may also explain other causes of ALI/ARDS. Further studies should include measuring other Supplementary Fig. 7) . Bar graphs present quantitative analyses of ACE and ACE2 protein levels, which are presented as the mean ACE-and ACE2-to-b-actin ratios±s.e.m. (n ¼ 3 mice per treatment). **Po0.01 (two-tailed t-test). (b) Angiotensin (Ang) II levels in the serum of vehicle control and virus-challenged mice 72 h after virus or vehicle instillation. Angiotensin II levels were determined using radioimmunoassays (data are shown as mean±s.e.m.; n ¼ 5-6 mice per group). **Po0.01 (two-tailed t-test). (c) Angiotensin II levels in sera on day 3 (72 h) or day 5 (120 hrs) after infection with live H5N1 virus (10 6 TCID 50 ). Angiotensin II levels were determined using radioimmunoassays. Death group, n ¼ 14, Survival group, n ¼ 4, data are shown as mean±s.e.m., *Po0.05 (two-tailed t-test). Each experiment was repeated at least three times.

potential biomarkers in H5N1-infected patients. Previous studies have reported the NS1 protein of influenza can induce disease pathology 27, 28 ; however, we could not detect significant changes in ACE2 mRNA or protein expression following overexpression of the H5N1 proteins PB1, PB2, PA, NP, HA, NA, M1, M2, NS1 and NS2 ( Supplementary Fig. 5 , full blots are provided as Supplementary Fig. 6) . Thus, the mechanism by which H5N1 infections result in ACE2 downmodulation requires further elucidation.

Given that flu viruses have high mutation rates, the identification of ACE2 as a common regulator of lung injury may also be useful to alleviate ALI/ARDS owing to mutated, newly emerging flu viruses including avian origin influenza virus H7N9 and/or currently unknown lethal lung pathogens [29] [30] [31] [32] [33] [34] . Importantly, our data identify ACE2 as a potential novel mode of action to alleviate the symptoms of acute lung pathologies in individuals infected with highly lethal avian influenza A H5N1 viruses.

Influenza viruses and cells. The influenza viruses used in this study were A/New Caledonia/20/1999(H1N1) and A/chicken/Jilin/9/2004(H5N1). Live virus experiments were performed in biosafety level 3 facilities following governmental and institutional guidelines. All animal experiments were conducted with the Institutional Animal Care and Use Committee approval from the Institute of Military Veterinary, Academy of Military Medical Sciences. The viruses were propagated via inoculation into 10-to 11-day-old SPF embryonated fowl eggs via the allantoic route. Hemagglutinating allantoic fluid was collected from the eggs, and the viruses were then directly used as live viruses or inactivated using formaldehyde (only lung elastance assays were performed with inactivated virus). MDCK cells and 293T cells were purchased from Peking Union Medical College Cell Culture Center and cultured in DMEM (Gibco) supplemented with 10% FBS, 100 U ml À 1 penicillin, and 100 mg ml À 1 streptomycin at 37°C. 25 and were backcrossed onto a C57BL/6J background. C57BL/6J mice were purchased from Vital River, Beijing. All mice were housed in a specific pathogen-free facility. Lung injury was induced via intratracheal instillation of allantoic fluid (AF) vehicle or virus (10 6 TCID 50 ) 14 . In the rescue experiments, recombinant human ACE2 protein (100 mg kg À 1 , Apeiron Biologics) was injected intraperitoneally 3 h prior to, as well as 8 h and 3 day or 1 h, 1 day and 2 days after vehicle control or virus instillation.

Assessment of lung injury. Three or four days after the instillation of either the vehicle control or flu viruses, the mice were euthanized. The lungs were removed from the thoracic cavity and placed in a glass vial containing approximately 50 ml of fixative. Formalin-fixed mouse lungs were embedded in paraffin, coronally sectioned, and mounted on glass slides using standard techniques. Sections were stained with hematoxylin and eosin. For each mouse, a slide containing one lung section was examined independently by three pathologists blinded to the treatments and genotypes. The numbers of inflammatory cells were counted in 100 microscopic fields, and lung injury scores were quantified 36 inclusive of the alveolar wall thickness, hyaline membranes, and proteinaceous debris filling the airspaces, which gives an overall score of between 0 and 1. To determine serum angiotensin II levels, blood was collected into 1. ARTICLE collected at each time point via tail puncture. Radioimmunoassays were used to measure angiotensin II in the serum 37 . For the assessment of pulmonary edema, the wet weight of the lungs was determined and then the lungs were dried in a 65°C oven for 48 h to obtain the dry weight. Survival data were determined for each cohort and plotted as Kaplan-Meier survival curves.

Lung elastance measurements. Mice were intratracheally treated with either the vehicle control or inactivated virus (10 mg g À 1 ) after anesthesia. Lung elastance was measured via BUXCO Pulmonary Function Testing (PFT) every 30 min during spontaneous breathing periods for 4 h. For the rescue experiments, recombinant human ACE2 protein (100 mg kg À 1 ) was intraperitoneally injected 20 min prior to instillation of the vehicle control or H5N1 virus infections.

Western blotting. For Western blotting, lung tissues were obtained 24 h after intratracheal instillation of the vehicle control or H1N1 and H5N1 viruses (10 6 TCID 50 ). Tissues were homogenized in ice-cold lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1.0% Triton X-100, 20 mM EDTA, 1 mM Na3VO4, 1 mM NaF, and protease inhibitors), the tissue lysates separated by SDS-PAGE, and the proteins transferred onto a nitrocellulose membrane. The membranes were incubated with antibodies against ACE, ACE2, and b-actin, followed by incubation with horseradish peroxidase-conjugated secondary antibodies. The anti-ACE antibody (2E2, sc-23908, 1:1,000) was purchased from Santa Cruz Biotechnology, Inc. The anti-ACE2 antibody (clone #460502, 1:300) was from R&D systems. The anti-b-actin antibody (clone AC-15, A5441, 1:10,000) was purchased from Sigma-Aldrich. The anti-Flag-Tag mouse mAb (LK-ab002-100, 1:5,000) was from Multisciences for validation the plasmid transfection efficiency in 293T cell line. The anti-ACE2 antibody (K465, 1:500) was purchased from Bioworld. Horseradish peroxidase (HRP)-conjugated secondary antibodies and Western Blotting Luminol Reagent were from Santa Cruz Biotechnology. Antibody binding was visualized using Kodak detection system and analyzed using Quantity One software.

Real-time quantitative PCR analysis. For real-time quantitative PCR, lung tissues were obtained at the indicated time points after the intratracheal instillation of H5N1 viruses (10 6 TCID 50 ) and 293T cells were grown in 12-well plates and samples analyzed 48 h after transfection with control plasmid or H5N1 virus protein-coding plasmids using X-treme GENE HP DNA Transfection Reagent (Roche) according to manufacturer's instruction (see Supplementary Fig. 5 for a list of the viral protein-coding plasmids). The total RNA of lung tissues and cells were isolated using Trizol (Invitrogen). Complementary DNA (cDNA) was synthesized from 1.5 mg of total RNA with the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems). PCR amplification assays were performed with the Light-Cycler 480 SYBR Green I Master (Roche Applied Science, Cat.No.04707516001, USA) on a LightCycler 480 PCR System. PCR products were confirmed by sequencing. Lung tissue samples were normalized to b-actin levels, while 293T-cell samples were normalized to human glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as a reference. The specific primers used were as follows: M1 forward: 5 0 -CTCTCTATCATCCCGTCAG-3 0 ; M1 reverse: 5 0 -GTCTTGTCTTTAGCC ATTCC-3 0 ; M2 forward: 5 0 -ATTGTGGATTCTTGATCGTC-3 0 ; M2 reverse: 5 0 -TGACAAAATGACCATCGTC-3 0 ; mice beta-actin forward: 5 0 -CTCTCCCT CACGCCATCC-3 0 ; mice beta-actin reverse: 5 0 -CGCACGATTTCCCTCTCAG-3 0 ; ACE2 forward: 5 0 -CAGTTGATTGAAGATGTGGAA-3 0 ; ACE2 reverse: 5 0 -TGA TATAGGAAGGATAGGCATT-3 0 ; human GAPDH forward: 5 0 -CGGAGTAACG GATTTGGTC-3 0 ; human GAPDH reverse: 5 0 -TGGGTGGAATCATATTGGAA CAT-3 0 .

H5N1 patients and the assessment of patient sera. Sera were collected from 26 H5N1-infected flu patients and 10 healthy volunteers. H5N1 infections were confirmed by the Chinese Center for Disease Control and Prevention (China CDC) according to the Chinese guidelines for diagnosis and treatment of human avian influenza (http://www.gov.cn/zlzt/gzqlg/content_107633.htm) and the WHO case definitions for human infections with influenza A(H5N1) virus (http://www.who.int/ influenza/resources/documents/case_definition2006_08_29/en/). Information about patients and healthy volunteers used as controls are presented in Table 1 . Sera were collected by the China CDC after informed consent was obtained, and the study was approved by the institutional review board of China CDC. Angiotensin II levels in the sera were determined using ELISA according to the manufacturer's instructions (RapidBio Lab).

Angiotensin II values in H5N1-infected patients are shown as scatter diagram, the bar represents the median value. The data were analyzed using a Mann-Whitney U test. Mouse data are shown as the mean values ± s.e.m. Measurements at single time points were analyzed using ANOVA and, if they demonstrated significance, further analyzed using a two-tailed t-test. Time courses were analyzed using a repeated measures (mixed model) ANOVA with Bonferroni post-t-tests. All statistical tests were performed using GraphPad Prism 5.0 (GraphPad Software, San Diego, CA, USA). Po0.05 was considered indicative of statistical significance.

Wild bird migration across the Qinghai-Tibetan plateau: a transmission route for highly pathogenic H5N1

Highly pathogenic avian influenza (H5N1): pathways of exposure at the animal-human interface, a systematic review

Qualitative and quantitative analyses of influenza virus receptors in trachea and lung tissues of humans, mice, chickens and ducks

H5N1 Oseltamivir-resistance detection by real-time PCR using two high sensitivity labeled TaqMan probes

Avian influenza: public health and food safety concerns

The biochemistry of the renin-angiotensin system

Peptidyl dipeptidase A: angiotensin I-converting enzyme

Angiotensin-converting enzyme 2--a new cardiac regulator

Angiotensinconverting enzyme 2 (ACE2) in disease pathogenesis

Apelin is a positive regulator of ACE2 in failing hearts

Devil and angel in the renin-angiotensin system: ACE-angiotensin II-AT1 receptor axis vs. ACE2-angiotensin-(1-7)-Mas receptor axis

ACE2: more of Ang-(1-7) or less Ang II?

Angiotensin-converting enzyme 2 protects from severe acute lung failure

A crucial role of angiotensin converting enzyme 2 (ACE2) in SARS coronavirus-induced lung injury

Human infection with highly pathogenic H5N1 influenza virus

Acute respiratory distress syndrome and pneumonia: a comprehensive review of clinical data

Clinical characteristics of 26 human cases of highly pathogenic avian influenza A (H5N1) virus infection in China

Avian influenza H5N1: an update on molecular pathogenesis

Identification of oxidative stress and Toll-like receptor 4 signaling as a key pathway of acute lung injury

Inhibition of autophagy ameliorates acute lung injury caused by avian influenza A H5N1 infection

Haunted with and hunting for viruses

Angiotensin II plasma levels are linked to disease severity and predict fatal outcomes in H7N9-infected patients

The reninangiotensin system: a target of and contributor to dyslipidemias, altered glucose homeostasis, and hypertension of the metabolic syndrome

Physiology of local renin-angiotensin systems

Angiotensin-converting enzyme 2 is an essential regulator of heart function

Bench-to-bedside review: chest wall elastance in acute lung injury/acute respiratory distress syndrome patients

Species-specific contribution of the four C-terminal amino acids of influenza A virus NS1 protein to virulence

A new influenza virus virulence determinant: the NS1 protein four C-terminal residues modulate pathogenicity

Realities and enigmas of human viral influenza: pathogenesis, epidemiology and control

Heterologous interactions between NS1 proteins from different influenza A virus subtypes/strains

Methionine-101 from one strain of H5N1 NS1 protein determines its IFN-antagonizing ability and subcellular distribution pattern

Environmental connections of novel avian-origin H7N9 influenza virus infection and virus adaptation to the human

Clinical findings in 111 cases of influenza A (H7N9) virus infection. New Engl

Lessons learnt from the human infections of avian-origin influenza A H7N9 virus: live free markets and human health

The cotton rat provides a useful small-animal model for the study of influenza virus pathogenesis

An official American Thoracic Society workshop report: features and measurements of experimental acute lung injury in animals

Evidence for an intrinsic angiotensin system in the canine pancreas

We thank the Chinese influenza surveillance network for providing access to their collection of patient sera. This work was supported by National Natural Science