Angiotensin-(1-7) decreases inflammation and lung damage caused by betacoronavirus infection in mice

Angiotensin-(1-7) decreases inflammation and lung damage caused by betacoronavirus infection in mice | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Angiotensin-(1-7) decreases inflammation and lung damage caused by betacoronavirus infection in mice Erick Bryan de Sousa Lima, Antônio Felipe Silva Carvalho, Isabella Zaidan, and 15 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4529565/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 18 Sep, 2024 Read the published version in Inflammation Research → Version 1 posted 10 You are reading this latest preprint version Abstract Objective: Pro-resolving molecules, including the peptide Angiotensin-(1-7) [Ang-(1-7)], have potential adjunctive therapy for infections. Here we evaluate the actions of Ang-(1-7) in betacoronavirus infection in mice. Methods: C57BL/6 mice were infected intranasally with the murine betacoronavirus MHV-3 and K18-hACE2 mice were infected with SARS-CoV-2. Mice were treated with Ang-(1-7) (30 μg/mouse, i.p.) at 24-, 36-, and 48-hours post-infection (hpi) or at 24, 36, 48, 72, and 96 h. For lethality evaluation, one additional dose of Ang-(1-7) was given at 120 hpi. At 3- and 5-days post- infection (dpi) blood cell, inflammatory mediators, viral loads, and lung histopathology were evaluated. Results: Ang-(1-7) rescued lymphopenia in MHV-infected mice, and decreased airways leukocyte infiltration and lung damage at 3- and 5-dpi. The levels of pro-inflammatory cytokines and virus titers in lung and plasma were decreased by Ang-(1-7) during MHV infection. Ang-(1-7) improved lung function and increased survival rates in MHV-infected mice. Notably, Ang-(1-7) treatment during SARS-CoV-2 infection restored blood lymphocytes to baseline, decreased weight loss, virus titters and levels of inflammatory cytokines, resulting in improvement of pulmonary damage and clinical scores. Conclusion: Ang-(1-7) protected mice from lung damage and death during betacoronavirus infections by modulating inflammation, hematological parameters and enhancing viral clearance. Covid-19 Coronavirus MHV-3 SARS-CoV-2 Angiotensin-(1-7) Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 INTRODUCTION The global pandemic of coronavirus disease 2019 (COVID-19) caused by the extremely contagious severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has led to nearly 700 million cases and 7 million deaths worldwide as of May of 2024 ( https://www.worldometers.info/coronavirus/ ). In most cases, respiratory infection by SARS-CoV-2 are asymptomatic or cause mild respiratory symptoms. Yet, COVID-19 can cause acute respiratory distress syndrome (ARDS) and severe systemic inflammation leading to death [ 1 ]. Comprehensive studies using clinical and experimental approaches, have been employed to investigate the pathogenesis of betacoronavirus infection, which is associated with a systemic dysregulated host immune response with bystander tissue damage, increased morbidity and mortality [ 2 ]. Indeed, infection with SARS-CoV-2 can lead to compromise of alveolar structure, and local and systemic pro-inflammatory cytokines production [ 3 , 4 ]. In addition, severe COVID-19 patients display a lower blood lymphocyte count compared to non-severe patients [ 5 , 6 , 7 ] indicating weakened immunity, and potentially worsening the prognosis [ 8 , 9 ]. The main receptor for SARS-CoV-2 and other coronaviruses is the angiotensin converting enzyme 2. This enzyme is an important component of the renin-angiotensin system (RAS). More specifically, ACE-2 is a key enzyme for angiotensin-(1–7) [(Ang-(1–7)] production, a mediator involved in the RAS non-classical pathway [ 10 ] and possesses both anti-inflammatory and pro-resolving actions in the lungs [ 11 ]. The biological effects of Ang-(1–7) are mainly mediated by Mas receptor (MasR) and have been shown to antagonize the pro-inflammatory actions of Ang II [ 11 , 12 ]. RAS dysregulation has been linked to inflammatory response exacerbation and development of severe acute respiratory syndrome, seen during severe pulmonary infections [ 13 ], such as influenza A virus (IAV) infection [ 14 , 15 , 16 ]. The impact of dysbalanced RAS responses in COVID-19 pathogenesis is yet to be explored [ 17 , 18 , 19 ]. Management of COVID-19 severe disease include antiviral, anticoagulant and anti-inflammatory steroids [ 20 ]. Indeed, regulation of the disbalanced inflammation evoked by SARS-CoV-2 has proved to be effective in protection of severe disease [ 21 ]. Over the years, the Ang-(1–7) hormone peptide has been associated with a number of pharmacological mechanisms in modulation of the inflammatory response by decreasing secretion of proinflammatory cytokine and leukocyte influx to the inflammatory sites and inducing key steps of resolution of inflammation, including apoptosis, efferocytosis and clearance of pathogen [ 11 ]. Despite the growing evidence supporting the anti-inflammatory and pro-resolving bioactions of Ang-(1–7), studies addressing the effect of the peptide during viral infection, especially in coronavirus infection in mice are lacking. Here, we have investigated the effect of Ang-(1–7) in inflammatory response triggered by murine and human betacoronaviruses and found that Ang-(1–7) modulates the inflammatory response during infection, while restores lymphopenia and decrease virus loads and lung damage, resulting in improvement of lung function. These finds have a translational potential and reinforce clinical studies in SARS-CoV-2-infected patients suggesting that Ang-(1–7) could serve as an anti-inflammatory/pro-resolving molecule to be used as an adjunctive therapy along with antivirals in the treatment of COVID-19. MATERIAL AND METHODS Cell, Virus, and Plaque Assay L929 (ATCC CCL-1) and Calu-3 (ATCC HTB-55) cells were cultured at 37°C with 5% CO2 in high-glucose Dulbecco's Modified Eagle Medium (DMEM) for L929 cells or Minimal Essential Medium (MEM) for Calu-3 cells. Both media were supplemented with 7% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 mg/ml streptomycin. The MHV-3 strain was provided and sequenced (GenBank accession number MW620427.1.) by Dr. Clarice Arns and Dr. Ricardo Durães-Carvalho from the State University of Campinas (UNICAMP, Brazil), and propagated in L929 cells. The SARS-CoV-2 gamma variant (P1 lineage; #EPI_ISL_1060902, hCoV-19/Brazil/AM-L70-71-CD1739/2020) was isolated from nasopharyngeal swabs of COVID-19 confirmed cases on Vero E6 cells. For viral titration, 100 µL of serially diluted virus suspensions, plasma samples, and lung and liver tissue homogenates were inoculated onto confluent monolayers of L929 cells (for MHV-3) or Calu-3 cells (for SARS-CoV-2) grown in 24-well plates. After gentle agitation for 1 h (4 × 15 min), the samples were harvested, and the culture medium was replaced by DMEM containing 1.6% carboxymethylcellulose, 2% FBS, and 1% penicillin-streptomycin-glutamine and maintained at 37ºC and 5% CO 2 for 2 days (for MHV-3) or 3 days (for SARS-CoV-2). The cells were fixed with 10% neutral-buffered formalin for 1 h and stained with 0.1% crystal violet. Viral titers were determined as plaque-forming units (PFU) expressed as Log/PFU per g of tissue. Ethical statement and Mouse models For the experiments using MHV-3, male and female C57BL/6 mice aged six to eight weeks were obtained from the Central Animal House at UFMG and housed in the animal facilities of the Biochemistry and Immunology Department at the same institution. In the SARS-CoV-2 experiments, we used male and female transgenic mice expressing the human ACE-2 receptor (K18-hACE2 mice from Jackson Laboratories) aged 10–12 weeks. These experiments were conducted in the Biosafety Level 3 (BSL-3) multiuser facility at the Institute of Biological Sciences, UFMG. All mice were kept under controlled conditions, with free access to food and water, at a temperature of 29–30°C, following a 12-hour light/dark cycle, and maintained at 50–58% humidity. All the procedures performed in this study adhered to the Brazilian Guideline for the Care and Use of Animals in Teaching or Scientific Research Activities, with approval from the Animal Ethics Committees of UFMG (protocol number 159/2021, 191/2021 and CTNBio No 8.842/2023). MHV-3 infection Mice were anesthetized intraperitoneally with ketamine (50 mg/kg, Syntec, São Paulo, Brazil) and xylazine (5 mg/kg, Syntec). To induce a SARS-like disease, the mice received an intranasal inoculation of 10³ PFU of MHV-3 in 30 µL of 0.9% sterile saline solution (Equiplex, São Paulo, Brazil) [ 22 ]. Control group mice were administered intranasally with the same volume of 0.9% sterile saline. If any mouse experienced a weight loss exceeding 25%, euthanasia was performed to prevent suffering. Three and five days post-infection, blood samples were collected from the abdominal vena cava of anesthetized mice using heparinized tubes. Euthanasia was then performed using an overdose of anesthetics (ketamine 240 mg/kg and xylazine 45 mg/kg, i.p.). Bronchoalveolar lavage fluid (BAL) was collected by instilling 1 mL of phosphate-buffered saline (PBS) through a tracheal catheter, withdrawing and re-instilling the fluid twice more. This process was repeated, and the lavages were pooled. The BAL was centrifuged (5 minutes, 300 × g, 4°C), and the supernatant was collected for protein level analysis using the Bradford assay. Part of the resuspended cell pellet was used for total and differential leukocyte counting. Following the BAL collection, the lungs were harvested for subsequent viral titration, cytokine assays, and histopathological examination. To determination of survival curves mice were infected with 10 2 PFU of MHV-3 and continuously monitoring of body weight loss and clinical signs of disease for up to 10 days. SARS-CoV-2 infection Mice were anesthetized intraperitoneally using a mixture of ketamine (50 mg/kg, Syntec) and xylazine (5 mg/kg, Syntec) before being intranasally inoculated with 2x10 4 PFU of the SARS-CoV-2 gamma strain in 30 µL of 0.9% sterile saline solution [ 23 , 24 ]. Control group mice were administered the same volume of 0.9% sterile saline intranasally. Over the next five days, mice were observed daily for changes in body weight and clinical score, encompassing ruffled fur, back arching, weight loss and activity level. Euthanasia was performed on any mice that experienced more than 25% weight loss to prevent undue suffering. Three and five days after inoculation, mice were anesthetized for blood cell collection from the vena cava. Afterwards, mice were euthanized using an overdose of anesthetics (ketamine 240 mg/kg and xylazine 45 mg/kg, i.p.). Lungs were then harvested and subjected to different analyses: routine histology, ELISA and virus titration. Angiotensin-(1–7) treatment Ang-(1–7) was sourced as a synthetic peptide from Bachem Inc., with its purity exceeding 99% as confirmed by high-performance liquid chromatography (HPLC). The peptide was diluted (saline + 0.02% DMSO) and administered intraperitoneally (i.p) at a dose of 30 µg/mouse, with the administration protocol varying based on the specific experiment, as detailed in the figures. The dose of Ang-(1–7) was based on previous studies in murine models of infection [ 25 , 26 ]. For the 3-day post-inoculation (3dpi) treatment regimen, Ang-(1–7) administration started 24 hours after MHV-3 or SARS-CoV-2 inoculation and continued at 36- and 48-hours later. In the 5-dpi treatment protocol, the peptide was administered at 24, 36, 48, 72, and 96 hours post-inoculation. In survival curve studies, mice received the peptide at 24, 36, 48, 72, 96, and 120 hours post-inoculation, with treatments continuing until death was observed. The vehicle group received saline + 0.02% DMSO. For Sars-CoV-2 a group of infected mice was treated with Remdesivir (25mg/kg, ip., 2x/day - GILEAD Sciences, São Paulo, Brazil), starting 6 hours after virus inoculation, as a positive antiviral control group of the experiment. Hematological Evaluation The numbers of total leucocytes, monocytes, granulocytes, lymphocytes, and circulating platelets were determined in blood samples using the Celltac MEK-6500K hemocytometer (Nihon Kohden, Indaiatuba, São Paulo, Brazil). BAL protein measurement To evaluate edema formation and lung tissue damage, protein concentration in the bronchoalveolar lavage fluid (BAL) was quantified using the Bradford assay (Bio-Rad, Hercules, California, USA). In this procedure, the working reagent was diluted fivefold and incubated with BSA standards or BAL samples. Following incubation at room temperature, absorbance was measured at 595 nm. Cytokine Assay Lung homogenates were prepared by homogenizing lung samples in cold cytokine extraction buffer, containing 100 mM Tris (pH 7.4), 150 mM NaCl, 1 mM EGTA, 1 mM EDTA, 1 mM Triton X-100, 1% sodium deoxycholate, 0.5% NP-40, and a protease inhibitor cocktail (1%). The homogenate was centrifuged at 3,000× g for 10 minutes at 4°C, and the supernatant was collected for further analysis. The concentrations of TNF, IFN-γ, IL-10, IL-6, CCL2, and CXCL1 in the lung homogenate supernatant and in plasma samples were measured using mouse ELISA kits (DuoSet ELISA System, R&D Systems Inc., Minneapolis, MN, USA) according to the manufacturer's instructions. Histopathology Lung samples were fixed in 4% neutral buffered formalin for 48 hours, followed by dehydration in ethanol and embedding in paraffin. Sections of 5 µm thickness were cut and stained with hematoxylin and eosin (H&E) for examination under light microscopy. A pathologist, blinded to the experimental groups, performed a histopathological assessment based on the criteria described by Andrade et al. (2021). The evaluation included scoring of airway (0–4), vascular (0–4) and parenchymal inflammation (0–5), and polymorphonuclear (PMN) infiltrate (0–5), culminating in a total possible score of 18 points. Immunohistochemistry Paraffin-embedded MHV-3 lung samples were sectioned (5µm thickness) and used for detecting viral double strand RNA (dsRNA) by immunohistochemistry. Sections were dewaxed, hydrated and submitted to antigen retrieval with boiling (95ºC) EDTA solution (EDTA 0,37M, pH 8,0) for 20 minutes. The endogenous peroxidase activity was blocked with a 0.3% hydrogen peroxide solution, and then a protein block solution (Abcam ab64226) was used to inhibit nonspecific reactions. Slides were then treated with Fc-blocking reagents from the Mouse-on-Mouse kit (Vector labs, California, USA), and incubated with the primary anti-dsRNA antibody (1:50, Merck MABE1134, Clone rJ2) overnight. Then, the secondary antibody (biotinylated goat anti-mouse IgG from the Mouse-on-Mouse kit) was applied for 30 minutes, followed by avidin-peroxidase treatment. The reaction was visualized using a DAB chromogenic solution and counterstained with hematoxylin. Assessment of Pulmonary Function in MHV-3 Infected Mice To evaluate lung function in mice infected with MHV-3, invasive forced spirometry was utilized. Mice were anesthetized with ketamine and xylazine and then tracheostomized. They were placed in a body plethysmograph and connected to a computer-controlled ventilator (Forced Pulmonary Maneuver System; Buxco Research Systems, Wilmington, NC, USA). This setup allowed for the measurement of various pulmonary parameters under mechanical ventilation, including flow respiratory, forced Expiratory volume at 20 milliseconds and lung resistance (Rl), as determined by resistance and compliance tests. To ensure the accuracy of the results, suboptimal maneuvers were discarded. Each mouse underwent at least three acceptable maneuvers for each test, providing reliable mean values for all the numerical parameters. Calu-3 cells infection Calu-3 cells, purchased from Banco de Células do Rio de Janeiro (BCRJ), code 0264, were infected with SARS-CoV-2 (Wuhan strain) at an MOI of 0.1. Cells were infected at densities of 2 × 10 5 cells/well in 24-well plates for 1 h at 37°C. After 1 h, cell monolayers were washed, supernatant removed and 1 mL RPMI supplemented with Angio 1–7 at different concentrations (50, 100 and 200 nM) was added. Calu-3 cells infected and untreated or treated with vehicle only (saline + 0.02% DMSO) were kept as controls. After 48 h, the supernatants were collected, and viable virus titers determined via plaque assay in permissive Vero cells. Results are expressed as Log10 P.F.U per mL of supernatant. Statistical analysis All results are presented as the mean ± SEM. Data were analyzed by one-way ANOVA, and differences between groups were assessed using the Tukey post-test. When only two groups were evaluated Student’s t-test was used. A P value < 0.05 was considered significant. Calculations were performed using the Prism 8.0 software for Windows (GraphPad Software, San Diego, CA). RESULTS Ang-(1–7) modulates inflammation and rescued lymphopenia induced by MHV-infection To evaluate the therapeutic effect of Ang-(1–7) during coronavirus infection we first employed a model of murine coronavirus (MHV-3) infection [ 22 ] that resembles relevant clinical aspects of severe COVID-19, including lymphopenia and features of macrophage activation syndrome and cytokine storm [ 22 ]. Thus, mice were treated intraperitoneally as shown in Fig. 1 a, and euthanized at 3 dpi, at the peak of lung disease in the model [ 22 ]. Intranasal infection of mice with MHV-3 led to leukopenia (Fig. 1 b) primarily due to a decreased number of lymphocytes (Fig. 1 c) in blood of infected mice. Of interest, Ang-(1–7) treatment restored leukocyte blood numbers, by increasing lymphocytes and granulocyte counts in the blood at 3 dpi (Fig. 1 d). Thrombocytopenia was a feature of the infection and Ang-(1–7) did not modify the blood platelets counts in infected mice at this timepoint evaluated (Fig. 1 e). Notably, treatment with Ang-(1–7) reduced leukocyte infiltration to the alveoli of infected mice (Fig. 1 f-h), which was mainly characterized by monocytes/macrophages (Fig. 1 g). Neutrophils were barely detected in the alveolar space of MHV-infected mice at 3 dpi (Fig. 1 i). Akin to the anti-inflammatory effect of Ang-(1–7) in other models of viral infection [ 14 ], treatment of MHV-infected mice decrease the lung levels IL-6, TNF, CXCL-1 and IFN-γ (Fig. 1 j). To test whether serial administration of Ang-(1–7) could affect systemic signs seen at 5-day post-MHV infection, we applied a longer therapeutical protocol (scheme in Fig. 2 a). In keeping with our previous findings, longer Ang-(1–7) treatment restored blood leukocytes/lymphocytes (Fig. 2 b-c) and increased neutrophils (Fig. 2 d) numbers [ 13 ]. Notably, longer Ang-(1–7) treatment partially restored the virus-induced thrombocytopenia (Fig. 2 e) and decreased the numbers of monocytes/macrophages numbers into the airways (Fig. 2 f-h). Ang-(1–7) decreased the lung and systemic levels of inflammatory cytokines/chemokines (Fig. 2 k-l). Collectively, these results demonstrate that treatment with Ang-(1–7) modulates the local and systemic inflammatory response and rescued lymphopenia and thrombocytopenia throughout the MHV infection in mice. Ang-(1–7) decreases viral load and damage/mechanical dysfunction in lungs of MHV-3 infected mice Next, we addressed whether Ang-(1–7) treatment could affect viral load in MHV-infected mice. Viral double-strand RNA (dsRNA) was detected by immunostaining of lung sections at 3 days post-infection (dpi) (Fig. 3 a). Importantly, in lung slices from Ang-(1–7)-treated mice the staining was lower (Fig. 3 a). In agreement with the immunohistochemistry analysis, plate unit forming (PFU) assay showed reduced viral titers in the lungs, liver, and plasma of Ang-(1–7)-treated mice at 3 dpi and 5 dpi (Fig. 3 b-e) when compared to vehicle-treated infected-animals. Histopathological scores of lungs of infected mice evaluated at 3 dpi showed pronounced tissue damage that was significantly reduced after Ang-(1–7) treatment (Fig. 4 a-b). In addition, total protein levels in bronchoalveolar lavage fluid, an indirect measurement of edema, were reduced after Ang-(1–7) treatment compared to the vehicle group (Fig. 4 c). Moreover, Ang-(1–7) improved lung function of infected mice as evaluated by flow parameters, forced expiratory volume, and pulmonary resistance (Fig. 4 d-f). In summary, these data suggest that Ang-(1–7) treatment reduced viral loads while protecting mice from inflammation-related lung damage and dysfunction caused by MHV-infection. Ang-(1–7) decreases lethality of MHV-infected mice Given the protective effect of Ang-(1–7) during MHV infection, we questioned if Ang-(1–7)-mediated immunomodulation and viral control could result in improvement of lethality in mice. To test that, infected mice were treated for 5 days with Ang-(1–7) (scheme in Fig. 5 a) and were monitored daily for weight loss and lethality. Infected animals began to lose weight progressively after 2–3 days post-infection (dpi) (Fig. 5 b) and succumbed to the infection between 6 and 8 dpi (Fig. 5 b). Treatment with Ang-(1–7) prevented the weight loss observed from the 5 dpi (Fig. 5 c). Of note, treated animals exhibited progressive weight gain until the end of the experimental analysis. Notably, Ang-(1–7) partially rescued mice from MHV-induced lethality (100% versus 40%, vehicle to Ang-(1–7) respectively - Fig. 5 d). Ang-(1–7) restores lymphopenia, improves clinical parameters, reduces viral loads, and protects mice from lung injury induced by SARS-CoV-2 in hK18ACE2 mice To validate our findings in animals actually infected with SARS-CoV-2, we infected transgenic mice expressing the human angiotensin I-converting enzyme 2 (ACE2) receptor (K18-hACE2 mice) with SARS-CoV-2 and treated infected-mice with Ang-(1–7) (scheme in Fig. 6 a). In agreement with previous studies [ 22 ], SARS-CoV-2 infection in K18-hACE2 mice led to intense weight loss of mice (Fig. 6 c). In addition, as for the MHV infection, blood lymphocyte counts were significantly reduced with SARS-CoV-2 infection (Fig. 6 b). Ang-(1–7) treatment promoted a modest recovery in lymphocyte circulation starting at 3 dpi (Fig. 6 b) and continued treatment led to a significant recovery of lymphocytes in the blood at 5 dpi (Fig. 6 b). Notably, treatment with Ang-(1–7) promoted significant recovery of the weight of infected mice on days 4–5 after infection compared to the vehicle-treated group (Fig. 6 c-d). In addition, SARS-CoV-2 infected mice treated with Ang-(1–7) showed improved clinical scores at 5 dpi (Fig. 6 e). Importantly, pulmonary viral loads in the lungs were significantly reduced after Ang-(1–7) treatment as evidenced by plaque-forming assays (Fig. 6 .f). Lung levels of IL-6, TNF, and CXCL1 were significantly reduced in Ang-(1–7) treated mice in comparison to the vehicle treated mice (Fig. 6 g). Lungs histopathological analyses of SARS-CoV-2 infected mice showed tissue damage that was decreased by Ang-(1–7) treatment (Fig. 7 a-b). Lastly, we questioned whether Ang-(1–7) could impact viral replication directly by employing an in vitro infection with SARS-CoV-2. Of importance, no direct antiviral effect was observed after treating lung epithelial Calu-3 cells infected by SARSCOV-2 with Ang-(1–7) (supplementary Fig. 1). Taken together, our results indicate that treatment with Ang-(1–7) protects mice against SARS-CoV-2 by taming inflammation and damage at the same time it reduces viral titers. DISCUSSION RAS activation results in the production of angiotensin II, which upon binding to AT1 receptor evoke effects as vasoconstriction, inflammation, and oxidative stress [ 10 ]. Conversely, angiotensin-converting enzyme (ACE) 2 counteracts these effects by degrading angiotensin II into Ang-(1–7), which, upon activation of the MAS receptor, elicits anti-inflammatory, antioxidative, vasodilatory, and pro-resolving responses [ 11 , 12 , 14 , 26 ]. Early in COVID-19 pandemic several reviews claimed a putative benefic effect of Ang-(1–7) [ 27 , 28 , 29 ], given the known actions of this peptide. However, while higher systemic levels of Ang II were consistently documented in COVID-19 patients [ 30 , 31 ] compared to healthy donors, divergent findings of Ang-(1–7) measurement were reported in severely ill hospitalized COVID-19 patients, with some exhibiting elevated Ang-(1–7) concentrations while others show decreased levels [ 32 , 33 ]. In addition, two randomized clinical trials using synthetic Ang-(1–7) (TXA-127) and an angiotensin II type 1 receptor-biased ligand (TRV-027) showed no clinical benefit for patients with severe COVID-19 [ 34 ], as initially hypothesized. Nevertheless, some concerns regarding the administration protocol have been raised by experts in the field [ 35 ]. In this regard, recent results from a clinical trial using a Mas-receptor activation by 20-hydroxyecdysone (BIO101) has shown positive results in severe COVID-19 by significantly reducing the risk of death or respiratory failure and supporting the use of MasR activators/agonists during disease [ 36 ]. The protective bioactions of the ACE2/Ang (1–7)/MasR axis in pre-clinical models of lung injury, including influenza virus infection [ 13 , 14 , 15 , 16 ] and the lack of specific pre-clinical date regarding the role Ang-(1–7) in coronavirus diseases, emphasize the urgent need to understand the effects of Ang-(1–7) during betacoronavirus infection for better planning of novel clinical studies in humans. Here, we have investigated the effect of the delayed administration of Ang-(1–7) on the immunopathology evoked by two betacoronavirus, MHV-3 and SARS-CoV-2, in mice. Importantly, this pro-resolving peptide afforded significant protection in infected mice by taming the inflammatory response, restoring lymphopenia, and decreasing viral loads and lung damage. Notably, Ang-(1–7) improved lung function and partially rescued mice from morbidity and lethality. MHV-3 is a betacoronavirus that infects mice and can be used in BSL-2 safety conditions. Infection with MHV-3 caused severe acute respiratory syndrome in C57BL/6 mice with efficient viral replication in the lungs, tissue damage associated with inflammation, and compromised respiratory function [ 22 ]. SARS-CoV-2 triggered similar host inflammatory responses, characterized by an overabundance of pro-inflammatory cytokines, leading to the infiltration of immune cells into both the bronchioalveolar space and lung interstitial compartment [ 1 , 2 , 3 ]. These events contribute to lung tissue damage, ultimately leading to reduced lung compliance, and impaired lung function [ 37 ]. In this context, mice infected with MHV-3 exhibited elevated edema and heightened infiltration of inflammatory cells to the airways, along with hyperplasia and disruption of tissue architecture, characteristics associated with acute respiratory distress syndrome [ 22 ]. In the present study, the treatment with Ang-(1–7) improved clinical parameters and protected mice from death, resulting in higher percentage of survival in Ang-(1–7)-treated mice. Mechanistically, Ang-(1–7) treatment decreased monocyte/macrophage infiltration to the airways coupled to decreased levels of cytokines, viral RNA and PFUs in the lungs of MHV-infected mice compared to vehicle-treated group. The effects of Ang-(1–7) taming inflammation and promoting viral clearance were associated with improvement of lung function and respiratory mechanics, in agreement with the data obtained from Ang-(1–7)-treated IAV-infected mice [ 14 ]. In addition to its beneficial role on lung inflammation and function, Ang-(1–7) reduced virus titers in plasma, when evaluated at the time point of viremia (5dpi) in this model [ 22 ]. Therefore, our findings suggest that administration of Ang-(1–7) is protective during coronavirus infection by reducing inflammation without causing immunosuppression. Of importance, these pro-resolving effects are also observed for Ang-(1–7) in the settings of bacterial infection [ 26 , 38 ]. Ang-(1–7) treatment rescued the number of leukocytes in MHV-infected mice in the blood, and thus maintaining the host's ability to fight infections [ 11 ]. Here, no direct anti-viral Ang-(1–7) actions in human airway epithelial cells were observed in vitro , suggesting that the reduction of virus titers in vivo are most likely associated with an overall better host resilience to infection. Of note, Ang-(1–7) inhibits the phosphorylation of JAK/STAT proteins [ 39 ], which are essential regulators of local and systemic response to viral infections [ 40 ]. Indeed, JAK inhibitors such as baricitinib has antiviral properties against SARS-CoV-2 infection [ 41 ] suggesting that adjunctive anti-viral and anti-inflammatory/pro-resolving based strategies as potential therapies in the settings of infectious diseases [ 11 , 12 ]. Whether Ang-(1–7) is regulating the JAK/STAT pathway and contributing for the observed effect in viral load remain to be investigated. Further studies will fully establish the role of Ang-(1–7) in promoting pathogen control during coronavirus infection. Transgenic mice expressing human ACE2 (K18-hACE2) are highly susceptible to SARS-CoV-2 infection [ 1 ]. In these animals, the infection results in intense production of cytokines locally and systemically, increased weight loss, leukopenia, and sustained viral replication in the nasal turbinates, lungs, and brains resulting in a viral sepsis-like disease [ 2 ]. Similarly to the results obtained during MHV-3 infection, significant improvements were observed after Ang-(1–7) treatment in SARS-CoV-2 infection. Ang-(1–7) notably attenuated weight loss, rescue lymphopenia reduced lung inflammation/injury and viral loads and improved the overall clinical score when compared with the vehicle-treated mice. The role of the ACE2/Ang-(1–7)/MasR axis in balancing actions that attenuate inflammation, oxidative stress, apoptosis, and fibrosis are well described [ 10 ]. Of interest, the membrane-bound ACE2 was shown to serve as a viral receptor for SARS-CoV-2 and other coronaviruses in the host cells. After virus binding and adsorption, ACE2 is internalized and degraded [ 18 ]. Thus, the enzymatic production of Ang-(1–7) decreases, preventing inflammation resolution and perpetuating tissue damage, and coagulopathy, features of severe COVID-19 [ 42 ]. In this regard, administration of an ACE2 recombinant protein is effective in protecting mice from ALI induced by the systemic injection of SARS-CoV-2 spike protein [ 43 ]. Increased levels of proinflammatory cytokines in serum have been associated with pulmonary inflammation and severe lung damage in patients with COVID-19 [ 6 , 44 ]. Of note, SARS-CoV-2 infection leads to an over activation of macrophages with exacerbated secretion of pro-inflammatory mediators and associated damage [ 45 ]. Here, high levels of pro-inflammatory cytokines such as IL-6, TNF, CXCL1, and IFN-γ were found in the plasma and lungs of coronavirus infected animals, associated to increased infiltration of macrophages. Ang-(1–7) reduced infiltration of these leukocytes to the airways, tamed production of pro-inflammatory cytokines and prevented lung damage and loss of lung function. Similarly, we have shown that in a pre-clinical model of IAV infection [ 14 ] and allergic inflammation [ 46 ], Ang-1-7 reduced cytokine production that was associated with the attenuation of the overall lung damage. In addition, Ang-(1–7) shifts macrophage phenotype towards anti-inflammatory and pro-resolving profiles [ 38 , 47 ]. Of note, it has been shown that Ang-(1–7) decrease the levels of IL-6 and IL-8 in alveolar epithelial cells stimulated with SARS-CoV-2 spike protein [ 48 ]. Lymphocyte count is a biomarker for monitoring the severity and mortality of COVID-19 disease [ 49 ]. Indeed, a notable reduction in T lymphocytes is positively associated with in-hospital mortality and the severity of illness [ 9 ]. In our study, we have observed reduced leukocyte levels in the peripheral blood of animals infected with coronavirus, alongside heightened number of these cells in bronchoalveolar lavage. This suggests an accumulation of inflammatory cells in lung tissue, indicative of leukocyte trafficking from the bloodstream to the viral-infected respiratory tract [ 50 ]. Our findings demonstrate that treatment with Ang-(1–7) regulates leukocyte numbers in the airways and attenuates lymphopenia induced by coronavirus infection. Curiously, analog drugs of Ang-(1–7) are widely recognized for their capacity to stimulate hematopoiesis and accelerate the replenishment of circulating cells [ 51 ]. Thus, Ang-(1–7) could be acting in bone marrow to restore lymphopenia caused by coronavirus infection [ 52 , 53 ]. Yet, further studies will clarify the associated mechanism. In conclusion, this study offers evidence supporting Ang-(1–7) as a crucial regulator of the immune response during coronavirus infection. Our findings demonstrate for the first time that Ang-(1–7) regulates both local and systemic immune responses, mitigates pulmonary inflammation, improves clinical outcomes, reduces viral load, and enhances survival in mice infected with MHV-3 or SARS-CoV-2. Altogether, these findings support the hypothesis that angiotensin-(1–7) could serve as an interesting therapeutic strategy for critical illnesses such as COVID-19 [ 54 ], and reinforce the need for new clinical trial using Ang-(1–7) based peptides or other MasR agonists. Declarations ACKNOWLEDGEMENTS The authors are thankful to Ilma Marçal and Tânia Colina for technical assistance. Thanks to the animal biosafety level 3 laboratory at UFMG (Laboratório Institucional de Pesquisa, LIPq); Centro de Laboratórios Multiusuários, CELAM; Laboratório de Biossegurança Nível 3, NB3-ICB. AUTHOR CONTRIBUTIONS L.P.S., and E.B.S.L. analyzed data and wrote the paper. E.B.S.L., A.F.S.C, I.Z., A.H.A.M., C.C, E.S.L., F.S.C., L.C.O, F.R., F.R.S.S., C.M.Q-J., performed the experiments and analyzed data. R.A.S.S. provided expertise. R.C.R. performed the lung mechanical assessment. M.M.T., V.V.C., L.P.S and L.P.T conception and design, analysis, and interpretation of data, drafting, editing and revising manuscript and project funding. All authors approved the final version of the manuscript. FUNDING This study was was supported by grants from Fundação de Amparo à Pesquisa do Estado de Minas Gerais, grant numbers: BPD-01010-22 and APQ-03221-18 to L.P.S., APQ02281-18, APQ02618-23 and RED-00202-22; Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, Brazill) by the grant PQ- 310799/2022-8, 408482/2022-2; and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior –CAPES/Brazil (Projeto: CAPES - Programa: 9951 - Programa Estratégico Emergencial de Prevenção e Combate a Surtos, Endemias, Epidemias e Pandemias AUX 0641/2020 - Processo 88881.507175/2020-01). This work also received support from the National Institute of Science and Technology in Dengue and Host-Microorganism Interaction (INCT em Dengue), sponsored by the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq; Brazil) (Processo CNPQ: 465425 /2014-3) and the Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG; Brazil) (Processo FAPEMIG: 25036). And by FINEP - Financiadora de Estudos e Projetos under MCTI/FINEP – MS/SCTIE/DGITIS/CGITS (6205283B-BB28-4F9C-AA65-808FE4450542) grant. Conflict of interest The authors declare no conflict of interest. References Oladunni FS, Park J-G, Pino PA, Gonzalez O, Akhter A, Allué-Guardia A, et al. Lethality of SARS-CoV-2 infection in K18 human angiotensin-converting enzyme 2 transgenic mice. Nat Commun. 2020;11:6122. Souza TML, Pinho VD, Setim CF, Sacramento CQ, Marcon R, Fintelman-Rodrigues N, et al. Preclinical development of kinetin as a safe error-prone SARS-CoV-2 antiviral able to attenuate virus-induced inflammation. Nat Commun. 2023;14:199. Yinda CK, Port JR, Bushmaker T, Offei Owusu I, Purushotham JN, Avanzato VA, et al. K18-hACE2 mice develop respiratory disease resembling severe COVID-19. PLoS Pathog. 2021;17:e1009195. Janiuk K, Jabłońska E, Garley M. Significance of NETs Formation in COVID-19. Cells. 2021;10:151. 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Angiotensin-(1-7) synergizes with colony-stimulating factors in hematopoietic recovery. Cancer Chemother Pharmacol. 2013;72:1235–45. Rodgers KE, diZerega GS. Contribution of the Local RAS to Hematopoietic Function: A Novel Therapeutic Target. Front Endocrinol (Lausanne). 2013;4:157 Garcia B, Zarbock A, Bellomo R, Legrand M. The alternative renin–angiotensin system in critically ill patients: pathophysiology and therapeutic implications. Critical Care. 2023;27:453. Additional Declarations No competing interests reported. Supplementary Files Graphicalabstract.tif Supplementaryfig1.tif Supplementary Fig. 1 Effect of Ang-(1-7) on SARS-CoV-2 replication in CALU-3 cells. CALU-3 cells were infected with SARS-CoV-2 at a multiplicity of infection (MOI) of 0.1 for 1 hour. After the infection period, the inoculum was removed. The cells then received treatment with Ang-(1-7) at different concentrations (50 nM, 100 nM, 200 nM) or vehicle. The cell supernatant was collected and analyzed at 24 and 48 hours post-infection. A Experimental design; BSARS-CoV-2 viable viral loads was determined in cell supernatants by plaque assay and results expressed as P.F.U per mL. Data were analyzed by one-way ANOVA, followed by the Tukey’s post hoc test. 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Sousa","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA3UlEQVRIiWNgGAWjYBACPmYow/548wEgJSFDUAsbTAvDmWMJIC08hLXAWTdyDEAUEVrYeQ9/+NhWJ8/YkPP51Y0aCx4G9sNHN+B3GF+a5My2w4bNDGe3WeccAzqMJy3tBn4tPGbMvG0HGNsYe7cZ57ABtUjwmBHSYvyZt63OvoeZ55lxzj/itBhI87YxJ85g42F+nNtGlBagX2acO5y8gYfNjDm3T4KHjZBf+PnPHv7woazOdoP848efc77VyfGzHz6GVwtyRLBJgEn8ylG1MH8grHoUjIJRMApGIgAAwwE/GQhT/HUAAAAASUVORK5CYII=","orcid":"","institution":"Programa de Pós-graduação em Análises Clínicas e Toxicológicas, Faculdade de Farmácia, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil.","correspondingAuthor":true,"prefix":"","firstName":"Lirlândia","middleName":"P.","lastName":"Sousa","suffix":""}],"badges":[],"createdAt":"2024-06-04 16:59:20","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4529565/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4529565/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s00011-024-01948-8","type":"published","date":"2024-09-18T15:57:21+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":58561584,"identity":"db615678-85f9-40f0-9ffa-8b822f456c5b","added_by":"auto","created_at":"2024-06-18 09:11:16","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":201582,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of Ang-(1-7) treatment in features of MHV-induced infection. \u003cstrong\u003eA \u003c/strong\u003eC57BL/6J mice were intranasally infected with MHV-3 (1x10³ PFU/animal), followed by Ang-(1-7) treatments; \u003cstrong\u003eB\u003c/strong\u003e Blood counts of total leukocytes; \u003cstrong\u003eC\u003c/strong\u003e Lymphocytes; \u003cstrong\u003eD \u003c/strong\u003eGranulocytes and\u003cstrong\u003eE\u003c/strong\u003e Thrombocytes. BAL were harvested and evaluated for total and differential counting. \u003cstrong\u003eF\u003c/strong\u003e Total leukocytes; \u003cstrong\u003eG\u003c/strong\u003e Monocytes/macrophages; \u003cstrong\u003eH\u003c/strong\u003eLymphocytes and \u003cstrong\u003eI \u003c/strong\u003eGranulocytes. \u003cstrong\u003eJ \u003c/strong\u003eHeatmap of levels of cytokines and chemokines measured by enzyme-linked immunosorbent assay (ELISA) in lungs of mock controls, vehicle-treated MHV-3-infected mice, and Ang-(1-7)-treated MHV-3-infected mice. Differences between infection groups and the mock control were assessed by one-way ANOVA plus Tukey multiple-comparison test (SEM; n=6-7). * For \u003cem\u003ep \u003c/em\u003e\u0026lt; 0.05 vs Mock and # for \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 vs Vehicle. The difference between the vehicle and treated groups is indicated in the graphs by the \u003cem\u003ep\u003c/em\u003e-value\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-4529565/v1/c9d0a4aa5e75cb81289e7ed8.png"},{"id":58560948,"identity":"6ed2a2b9-6c26-4cf9-b504-b255051ef297","added_by":"auto","created_at":"2024-06-18 09:03:16","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":238216,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of delayed Ang-(1-7) treatment in features of MHV-induced infection. \u003cstrong\u003eA\u003c/strong\u003e C57BL/6J mice were intranasally infected with MHV-3 (1x10³ PFU/animal), followed by Ang-(1-7) treatments. \u003cstrong\u003eB\u003c/strong\u003e Blood counts of total leukocytes; \u003cstrong\u003eC\u003c/strong\u003e Lymphocytes; \u003cstrong\u003eD\u003c/strong\u003e Granulocytes and \u003cstrong\u003eE\u003c/strong\u003eThrombocytes. BAL were harvested and evaluated for total and differential counting. \u003cstrong\u003eF\u003c/strong\u003e Total leukocyte; \u003cstrong\u003eG\u003c/strong\u003e Monocytes/macrophages, \u003cstrong\u003eH\u003c/strong\u003eLymphocytes and \u003cstrong\u003eI\u003c/strong\u003e Granulocytes. Heatmap of levels of cytokines and chemokines measured by enzyme-linked immunosorbent assay (ELISA) in \u003cstrong\u003eK\u003c/strong\u003eLungs and \u003cstrong\u003eL\u003c/strong\u003e Plasma of mock controls, vehicle-treated MHV-3-infected mice, and Ang-(1-7)-treated MHV-3-infected mice. Differences between infection groups and the mock control were assessed by one-way ANOVA plus Tukey multiple-comparison test (SEM; n=6-7). * For \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 vs Mock and # for \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 vs Vehicle. The difference between the vehicle and treated groups is indicated in the graphs by the \u003cem\u003ep\u003c/em\u003e-value\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-4529565/v1/42f8e401e1882fed8a61bdb5.png"},{"id":58560951,"identity":"949eccbf-5ebd-45ee-8be4-2cdeb3a24c53","added_by":"auto","created_at":"2024-06-18 09:03:17","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1999879,"visible":true,"origin":"","legend":"\u003cp\u003eImpact of the Ang-(1-7) treatment in viral loads of MHV-infected mice.\u003cstrong\u003e \u003c/strong\u003eC57BL/6 mice were infected with MHV-3 (1x10³ PFU/animal), followed by Ang-(1-7) treatments. Mice were euthanized at 3 dpi or 5 dpi and samples were collected for virus detection. Immunohistochemistry (IHC) analysis of viral double-stranded RNA (dsRNA). \u003cstrong\u003eA\u003c/strong\u003e Scale = 200 μm (lower magnification) and 50 μm (higher magnification) at 3 dpi; \u003cstrong\u003eB-E\u003c/strong\u003eViral load determined in organs (lung e liver) and plasma of MHV-3-infected mice by plaque assay. Differences between infection groups and the mock control were assessed by one-way ANOVA plus Tukey multiple-comparison test (SEM; n=6-7). * For \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 vs Mock and # for \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 vs Vehicle. The difference between the vehicle and treated groups is indicated in the graphs by the \u003cem\u003ep\u003c/em\u003e-value\u003c/p\u003e","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-4529565/v1/9d4bb31818e429b74bb6ef07.png"},{"id":58560954,"identity":"0de7bd5e-aaaf-4fbd-bd88-bcce932b8b66","added_by":"auto","created_at":"2024-06-18 09:03:17","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2873678,"visible":true,"origin":"","legend":"\u003cp\u003eImpact of Ang-(1-7) treatment on MHV-3-induced lung damage. \u003cstrong\u003eA\u003c/strong\u003e C57/Bl 6 mice were infected with MHV-3 (1x10³ PFU/animal/intranasally, treated with Ang-(1-7) or vehicle at 24, 36 and 48h p.i. with Ang-(1-7) (30 μg intraperitoneally) and euthanized at 72 hpi for lung mechanics and histopathological analysis. Hematoxylin and eosin (H\u0026amp;E) staining of lung sections; Bars, 50 μm (high magnification); \u003cstrong\u003eB\u003c/strong\u003e The histopathological score evaluated airway, vascular and parenchymal inflammation, neutrophilic infiltration, and epithelial lesion. \u003cstrong\u003eC\u003c/strong\u003e Total protein in BALF; \u003cstrong\u003eD-F\u003c/strong\u003e Assessment of respiratory mechanic function by analyses of flow, forced expiratory volume, and resistance. Differences between infection groups and the mock control were assessed by one-way ANOVA plus Tukey multiple-comparison test (SEM; n=6-7). * For \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 vs Mock and # for \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 vs Vehicle. The difference between the vehicle and treated groups is indicated in the graphs by the \u003cem\u003ep\u003c/em\u003e-value\u003c/p\u003e","description":"","filename":"Fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-4529565/v1/44ad810cef8a3b8c572a6e53.png"},{"id":58560957,"identity":"b101d2c3-4df7-4b84-8cf0-127ded7e405d","added_by":"auto","created_at":"2024-06-18 09:03:17","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":144645,"visible":true,"origin":"","legend":"\u003cp\u003eAng-(1-7) rescue mice from MHV-induced lethality.\u003cstrong\u003e A \u003c/strong\u003eC57BL/6J mice were intranasally infected with MHV-3 (1x10² PFUs/animal), followed by Ang-(1-7) treatments. \u003cstrong\u003eB\u003c/strong\u003e Percentage of weight of infected mice treated or not with Ang-(1-7); \u003cstrong\u003eC\u003c/strong\u003e Weight Loss in polled mice from Day 5-8 in % (C); \u003cstrong\u003eD\u003c/strong\u003e Survival curve of infected mice treated or not with Ang-(1-7). Survival rates were monitored for 10 days and those animals that reached the criteria were humane euthanasia (n=10). The difference between the vehicle and treated groups is indicated in the graphs by # for \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 or the \u003cem\u003ep\u003c/em\u003e-value by t-test. Log-rank test was used to compare survival curves\u003c/p\u003e","description":"","filename":"Fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-4529565/v1/fcfece86e40afc08ed0e7110.png"},{"id":58560953,"identity":"4c7920dc-c07d-449f-8e01-52319830ceed","added_by":"auto","created_at":"2024-06-18 09:03:17","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":237488,"visible":true,"origin":"","legend":"\u003cp\u003eAng-(1-7) is protective in a model of SARS-CoV-2 infection. \u003cstrong\u003eA\u003c/strong\u003e hK18ACE2 Mice infected with (2x10\u003csup\u003e4\u003c/sup\u003e PFUs/animal i.n), followed by Ang-(1-7) treatments. \u003cstrong\u003eB-C\u003c/strong\u003e Lymphocytes 3dpi and 5dpi; \u003cstrong\u003eC\u003c/strong\u003e % body weight curve of infected mice treated or not with Ang-(1-7); \u003cstrong\u003eD\u003c/strong\u003e Weight loss in polled mice from day 4-5 in %; \u003cstrong\u003eE\u003c/strong\u003e Clinical score (day 5); \u003cstrong\u003eF\u003c/strong\u003e Viral load, and \u003cstrong\u003eG\u003c/strong\u003e cytokines/chemokines in the lungs of Mock, vehicle-treated SARS-COV-2 infected mice, and Ang-(1-7)-treated SARS-COV-2 infected mice. Differences between infection groups and the mock control were assessed by one-way ANOVA plus Tukey multiple-comparison test (SEM; n=5-7). * For \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 vs Mock and # for \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 vs Vehicle. The difference between the vehicle and treated groups is indicated in the graphs by the \u003cem\u003ep\u003c/em\u003e-value\u003c/p\u003e","description":"","filename":"Fig6.png","url":"https://assets-eu.researchsquare.com/files/rs-4529565/v1/6ea1121c298a3ac068c38d48.png"},{"id":58560955,"identity":"313a2599-f8a5-4345-aa4c-f0ff897ab477","added_by":"auto","created_at":"2024-06-18 09:03:17","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":3026011,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of Ang-(1-7) treatment on lung damage triggered by SARS-COV-2. hK18ACE2 Mice infected with (2x10\u003csup\u003e4\u003c/sup\u003e PFUs/animal i.n, n=7), were euthanized after 120h for lung histological analysis. \u003cstrong\u003eA\u003c/strong\u003e Hematoxylin and eosin (H\u0026amp;E) staining of lung sections; Bars, 50 μm (high magnification); \u003cstrong\u003eB\u003c/strong\u003e The histopathological score evaluated airway, vascular and parenchymal inflammation, neutrophilic infiltration, and epithelial lesion. * For \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 vs Mock and # for \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 vs Vehicle. The difference between the vehicle and treated groups is indicated in the graphs by the \u003cem\u003ep\u003c/em\u003e-value\u003c/p\u003e","description":"","filename":"Fig7.png","url":"https://assets-eu.researchsquare.com/files/rs-4529565/v1/573db6ee187a6619e71b44af.png"},{"id":65103987,"identity":"745117f3-47ec-4ef9-9e2e-01a5e3933cbf","added_by":"auto","created_at":"2024-09-23 16:10:31","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":13853937,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4529565/v1/c0b3aada-9549-4270-9241-b260d30b070b.pdf"},{"id":58560956,"identity":"6e932a37-b3db-4a88-ad75-a88672aab809","added_by":"auto","created_at":"2024-06-18 09:03:17","extension":"tif","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":7179842,"visible":true,"origin":"","legend":"","description":"","filename":"Graphicalabstract.tif","url":"https://assets-eu.researchsquare.com/files/rs-4529565/v1/8f51351d83eade4a53e11d7c.tif"},{"id":58560952,"identity":"af922cdf-2a71-498f-9172-eef1475c9011","added_by":"auto","created_at":"2024-06-18 09:03:17","extension":"tif","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":872120,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Fig. 1 \u003c/strong\u003eEffect of\u003cstrong\u003e \u003c/strong\u003eAng-(1-7) on SARS-CoV-2 replication in CALU-3 cells. CALU-3 cells were infected with SARS-CoV-2 at a multiplicity of infection (MOI) of 0.1 for 1 hour. After the infection period, the inoculum was removed. The cells then received treatment with Ang-(1-7) at different concentrations (50 nM, 100 nM, 200 nM) or vehicle. The cell supernatant was collected and analyzed at 24 and 48 hours post-infection. \u003cstrong\u003eA \u003c/strong\u003eExperimental design; \u003cstrong\u003eB\u003c/strong\u003eSARS-CoV-2 viable viral loads was determined in cell supernatants by plaque assay and results expressed as P.F.U per mL. Data were analyzed by one-way ANOVA, followed by the Tukey’s post hoc test. Created with BioRender\u003c/p\u003e","description":"","filename":"Supplementaryfig1.tif","url":"https://assets-eu.researchsquare.com/files/rs-4529565/v1/a02ef3e6583efb12b0166dec.tif"}],"financialInterests":"No competing interests reported.","formattedTitle":"Angiotensin-(1-7) decreases inflammation and lung damage caused by betacoronavirus infection in mice","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eThe global pandemic of coronavirus disease 2019 (COVID-19) caused by the extremely contagious severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has led to nearly 700\u0026nbsp;million cases and 7\u0026nbsp;million deaths worldwide as of May of 2024 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.worldometers.info/coronavirus/\u003c/span\u003e\u003cspan address=\"https://www.worldometers.info/coronavirus/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). In most cases, respiratory infection by SARS-CoV-2 are asymptomatic or cause mild respiratory symptoms. Yet, COVID-19 can cause acute respiratory distress syndrome (ARDS) and severe systemic inflammation leading to death [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Comprehensive studies using clinical and experimental approaches, have been employed to investigate the pathogenesis of betacoronavirus infection, which is associated with a systemic dysregulated host immune response with bystander tissue damage, increased morbidity and mortality [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Indeed, infection with SARS-CoV-2 can lead to compromise of alveolar structure, and local and systemic pro-inflammatory cytokines production [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. In addition, severe COVID-19 patients display a lower blood lymphocyte count compared to non-severe patients [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e] indicating weakened immunity, and potentially worsening the prognosis [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe main receptor for SARS-CoV-2 and other coronaviruses is the angiotensin converting enzyme 2. This enzyme is an important component of the renin-angiotensin system (RAS). More specifically, ACE-2 is a key enzyme for angiotensin-(1\u0026ndash;7) [(Ang-(1\u0026ndash;7)] production, a mediator involved in the RAS non-classical pathway [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e] and possesses both anti-inflammatory and pro-resolving actions in the lungs [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. The biological effects of Ang-(1\u0026ndash;7) are mainly mediated by Mas receptor (MasR) and have been shown to antagonize the pro-inflammatory actions of Ang II [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. RAS dysregulation has been linked to inflammatory response exacerbation and development of severe acute respiratory syndrome, seen during severe pulmonary infections [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], such as influenza A virus (IAV) infection [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. The impact of dysbalanced RAS responses in COVID-19 pathogenesis is yet to be explored [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eManagement of COVID-19 severe disease include antiviral, anticoagulant and anti-inflammatory steroids [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Indeed, regulation of the disbalanced inflammation evoked by SARS-CoV-2 has proved to be effective in protection of severe disease [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Over the years, the Ang-(1\u0026ndash;7) hormone peptide has been associated with a number of pharmacological mechanisms in modulation of the inflammatory response by decreasing secretion of proinflammatory cytokine and leukocyte influx to the inflammatory sites and inducing key steps of resolution of inflammation, including apoptosis, efferocytosis and clearance of pathogen [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Despite the growing evidence supporting the anti-inflammatory and pro-resolving bioactions of Ang-(1\u0026ndash;7), studies addressing the effect of the peptide during viral infection, especially in coronavirus infection in mice are lacking. Here, we have investigated the effect of Ang-(1\u0026ndash;7) in inflammatory response triggered by murine and human betacoronaviruses and found that Ang-(1\u0026ndash;7) modulates the inflammatory response during infection, while restores lymphopenia and decrease virus loads and lung damage, resulting in improvement of lung function. These finds have a translational potential and reinforce clinical studies in SARS-CoV-2-infected patients suggesting that Ang-(1\u0026ndash;7) could serve as an anti-inflammatory/pro-resolving molecule to be used as an adjunctive therapy along with antivirals in the treatment of COVID-19.\u003c/p\u003e"},{"header":"MATERIAL AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eCell, Virus, and Plaque Assay\u003c/h2\u003e \u003cp\u003eL929 (ATCC CCL-1) and Calu-3 (ATCC HTB-55) cells were cultured at 37\u0026deg;C with 5% CO2 in high-glucose Dulbecco's Modified Eagle Medium (DMEM) for L929 cells or Minimal Essential Medium (MEM) for Calu-3 cells. Both media were supplemented with 7% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 mg/ml streptomycin. The MHV-3 strain was provided and sequenced (GenBank accession number MW620427.1.) by Dr. Clarice Arns and Dr. Ricardo Dur\u0026atilde;es-Carvalho from the State University of Campinas (UNICAMP, Brazil), and propagated in L929 cells. The SARS-CoV-2 gamma variant (P1 lineage; #EPI_ISL_1060902, hCoV-19/Brazil/AM-L70-71-CD1739/2020) was isolated from nasopharyngeal swabs of COVID-19 confirmed cases on Vero E6 cells. For viral titration, 100 \u0026micro;L of serially diluted virus suspensions, plasma samples, and lung and liver tissue homogenates were inoculated onto confluent monolayers of L929 cells (for MHV-3) or Calu-3 cells (for SARS-CoV-2) grown in 24-well plates. After gentle agitation for 1 h (4 \u0026times; 15 min), the samples were harvested, and the culture medium was replaced by DMEM containing 1.6% carboxymethylcellulose, 2% FBS, and 1% penicillin-streptomycin-glutamine and maintained at 37\u0026ordm;C and 5% CO\u003csub\u003e2\u003c/sub\u003e for 2 days (for MHV-3) or 3 days (for SARS-CoV-2). The cells were fixed with 10% neutral-buffered formalin for 1 h and stained with 0.1% crystal violet. Viral titers were determined as plaque-forming units (PFU) expressed as Log/PFU per g of tissue.\u003c/p\u003e \u003cp\u003e \u003cb\u003eEthical statement and Mouse models\u003c/b\u003e \u003c/p\u003e \u003cp\u003eFor the experiments using MHV-3, male and female C57BL/6 mice aged six to eight weeks were obtained from the Central Animal House at UFMG and housed in the animal facilities of the Biochemistry and Immunology Department at the same institution. In the SARS-CoV-2 experiments, we used male and female transgenic mice expressing the human ACE-2 receptor (K18-hACE2 mice from Jackson Laboratories) aged 10\u0026ndash;12 weeks. These experiments were conducted in the Biosafety Level 3 (BSL-3) multiuser facility at the Institute of Biological Sciences, UFMG. All mice were kept under controlled conditions, with free access to food and water, at a temperature of 29\u0026ndash;30\u0026deg;C, following a 12-hour light/dark cycle, and maintained at 50\u0026ndash;58% humidity. All the procedures performed in this study adhered to the Brazilian Guideline for the Care and Use of Animals in Teaching or Scientific Research Activities, with approval from the Animal Ethics Committees of UFMG (protocol number 159/2021, 191/2021 and CTNBio No 8.842/2023).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eMHV-3 infection\u003c/h2\u003e \u003cp\u003eMice were anesthetized intraperitoneally with ketamine (50 mg/kg, Syntec, S\u0026atilde;o Paulo, Brazil) and xylazine (5 mg/kg, Syntec). To induce a SARS-like disease, the mice received an intranasal inoculation of 10\u0026sup3; PFU of MHV-3 in 30 \u0026micro;L of 0.9% sterile saline solution (Equiplex, S\u0026atilde;o Paulo, Brazil) [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Control group mice were administered intranasally with the same volume of 0.9% sterile saline. If any mouse experienced a weight loss exceeding 25%, euthanasia was performed to prevent suffering. Three and five days post-infection, blood samples were collected from the abdominal vena cava of anesthetized mice using heparinized tubes. Euthanasia was then performed using an overdose of anesthetics (ketamine 240 mg/kg and xylazine 45 mg/kg, i.p.). Bronchoalveolar lavage fluid (BAL) was collected by instilling 1 mL of phosphate-buffered saline (PBS) through a tracheal catheter, withdrawing and re-instilling the fluid twice more. This process was repeated, and the lavages were pooled. The BAL was centrifuged (5 minutes, 300 \u0026times; g, 4\u0026deg;C), and the supernatant was collected for protein level analysis using the Bradford assay. Part of the resuspended cell pellet was used for total and differential leukocyte counting. Following the BAL collection, the lungs were harvested for subsequent viral titration, cytokine assays, and histopathological examination. To determination of survival curves mice were infected with 10\u003csup\u003e2\u003c/sup\u003e PFU of MHV-3 and continuously monitoring of body weight loss and clinical signs of disease for up to 10 days.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eSARS-CoV-2 infection\u003c/h2\u003e \u003cp\u003eMice were anesthetized intraperitoneally using a mixture of ketamine (50 mg/kg, Syntec) and xylazine (5 mg/kg, Syntec) before being intranasally inoculated with 2x10\u003csup\u003e4\u003c/sup\u003e PFU of the SARS-CoV-2 gamma strain in 30 \u0026micro;L of 0.9% sterile saline solution [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Control group mice were administered the same volume of 0.9% sterile saline intranasally. Over the next five days, mice were observed daily for changes in body weight and clinical score, encompassing ruffled fur, back arching, weight loss and activity level. Euthanasia was performed on any mice that experienced more than 25% weight loss to prevent undue suffering. Three and five days after inoculation, mice were anesthetized for blood cell collection from the vena cava. Afterwards, mice were euthanized using an overdose of anesthetics (ketamine 240 mg/kg and xylazine 45 mg/kg, i.p.). Lungs were then harvested and subjected to different analyses: routine histology, ELISA and virus titration.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eAngiotensin-(1\u0026ndash;7) treatment\u003c/h2\u003e \u003cp\u003eAng-(1\u0026ndash;7) was sourced as a synthetic peptide from Bachem Inc., with its purity exceeding 99% as confirmed by high-performance liquid chromatography (HPLC). The peptide was diluted (saline\u0026thinsp;+\u0026thinsp;0.02% DMSO) and administered intraperitoneally (i.p) at a dose of 30 \u0026micro;g/mouse, with the administration protocol varying based on the specific experiment, as detailed in the figures. The dose of Ang-(1\u0026ndash;7) was based on previous studies in murine models of infection [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. For the 3-day post-inoculation (3dpi) treatment regimen, Ang-(1\u0026ndash;7) administration started 24 hours after MHV-3 or SARS-CoV-2 inoculation and continued at 36- and 48-hours later. In the 5-dpi treatment protocol, the peptide was administered at 24, 36, 48, 72, and 96 hours post-inoculation. In survival curve studies, mice received the peptide at 24, 36, 48, 72, 96, and 120 hours post-inoculation, with treatments continuing until death was observed. The vehicle group received saline\u0026thinsp;+\u0026thinsp;0.02% DMSO. For Sars-CoV-2 a group of infected mice was treated with Remdesivir (25mg/kg, ip., 2x/day - GILEAD Sciences, S\u0026atilde;o Paulo, Brazil), starting 6 hours after virus inoculation, as a positive antiviral control group of the experiment.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eHematological Evaluation\u003c/h2\u003e \u003cp\u003eThe numbers of total leucocytes, monocytes, granulocytes, lymphocytes, and circulating platelets were determined in blood samples using the Celltac MEK-6500K hemocytometer (Nihon Kohden, Indaiatuba, S\u0026atilde;o Paulo, Brazil).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eBAL protein measurement\u003c/h2\u003e \u003cp\u003eTo evaluate edema formation and lung tissue damage, protein concentration in the bronchoalveolar lavage fluid (BAL) was quantified using the Bradford assay (Bio-Rad, Hercules, California, USA). In this procedure, the working reagent was diluted fivefold and incubated with BSA standards or BAL samples. Following incubation at room temperature, absorbance was measured at 595 nm.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eCytokine Assay\u003c/h2\u003e \u003cp\u003eLung homogenates were prepared by homogenizing lung samples in cold cytokine extraction buffer, containing 100 mM Tris (pH 7.4), 150 mM NaCl, 1 mM EGTA, 1 mM EDTA, 1 mM Triton X-100, 1% sodium deoxycholate, 0.5% NP-40, and a protease inhibitor cocktail (1%). The homogenate was centrifuged at 3,000\u0026times; g for 10 minutes at 4\u0026deg;C, and the supernatant was collected for further analysis. The concentrations of TNF, IFN-γ, IL-10, IL-6, CCL2, and CXCL1 in the lung homogenate supernatant and in plasma samples were measured using mouse ELISA kits (DuoSet ELISA System, R\u0026amp;D Systems Inc., Minneapolis, MN, USA) according to the manufacturer's instructions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eHistopathology\u003c/h2\u003e \u003cp\u003eLung samples were fixed in 4% neutral buffered formalin for 48 hours, followed by dehydration in ethanol and embedding in paraffin. Sections of 5 \u0026micro;m thickness were cut and stained with hematoxylin and eosin (H\u0026amp;E) for examination under light microscopy. A pathologist, blinded to the experimental groups, performed a histopathological assessment based on the criteria described by Andrade et al. (2021). The evaluation included scoring of airway (0\u0026ndash;4), vascular (0\u0026ndash;4) and parenchymal inflammation (0\u0026ndash;5), and polymorphonuclear (PMN) infiltrate (0\u0026ndash;5), culminating in a total possible score of 18 points.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eImmunohistochemistry\u003c/h2\u003e \u003cp\u003eParaffin-embedded MHV-3 lung samples were sectioned (5\u0026micro;m thickness) and used for detecting viral double strand RNA (dsRNA) by immunohistochemistry. Sections were dewaxed, hydrated and submitted to antigen retrieval with boiling (95\u0026ordm;C) EDTA solution (EDTA 0,37M, pH 8,0) for 20 minutes. The endogenous peroxidase activity was blocked with a 0.3% hydrogen peroxide solution, and then a protein block solution (Abcam ab64226) was used to inhibit nonspecific reactions. Slides were then treated with Fc-blocking reagents from the Mouse-on-Mouse kit (Vector labs, California, USA), and incubated with the primary anti-dsRNA antibody (1:50, Merck MABE1134, Clone rJ2) overnight. Then, the secondary antibody (biotinylated goat anti-mouse IgG from the Mouse-on-Mouse kit) was applied for 30 minutes, followed by avidin-peroxidase treatment. The reaction was visualized using a DAB chromogenic solution and counterstained with hematoxylin.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eAssessment of Pulmonary Function in MHV-3 Infected Mice\u003c/h2\u003e \u003cp\u003eTo evaluate lung function in mice infected with MHV-3, invasive forced spirometry was utilized. Mice were anesthetized with ketamine and xylazine and then tracheostomized. They were placed in a body plethysmograph and connected to a computer-controlled ventilator (Forced Pulmonary Maneuver System; Buxco Research Systems, Wilmington, NC, USA). This setup allowed for the measurement of various pulmonary parameters under mechanical ventilation, including flow respiratory, forced Expiratory volume at 20 milliseconds and lung resistance (Rl), as determined by resistance and compliance tests. To ensure the accuracy of the results, suboptimal maneuvers were discarded. Each mouse underwent at least three acceptable maneuvers for each test, providing reliable mean values for all the numerical parameters.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eCalu-3 cells infection\u003c/h2\u003e \u003cp\u003eCalu-3 cells, purchased from Banco de C\u0026eacute;lulas do Rio de Janeiro (BCRJ), code 0264, were infected with SARS-CoV-2 (Wuhan strain) at an MOI of 0.1. Cells were infected at densities of 2 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells/well in 24-well plates for 1 h at 37\u0026deg;C. After 1 h, cell monolayers were washed, supernatant removed and 1 mL RPMI supplemented with Angio 1\u0026ndash;7 at different concentrations (50, 100 and 200 nM) was added. Calu-3 cells infected and untreated or treated with vehicle only (saline\u0026thinsp;+\u0026thinsp;0.02% DMSO) were kept as controls. After 48 h, the supernatants were collected, and viable virus titers determined via plaque assay in permissive Vero cells. Results are expressed as Log10 P.F.U per mL of supernatant.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eAll results are presented as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM. Data were analyzed by one-way ANOVA, and differences between groups were assessed using the Tukey post-test. When only two groups were evaluated Student\u0026rsquo;s \u003cem\u003et-test\u003c/em\u003e was used. A \u003cem\u003eP\u003c/em\u003e value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered significant. Calculations were performed using the Prism 8.0 software for Windows (GraphPad Software, San Diego, CA).\u003c/p\u003e \u003c/div\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eAng-(1\u0026ndash;7) modulates inflammation and rescued lymphopenia induced by MHV-infection\u003c/h2\u003e \u003cp\u003eTo evaluate the therapeutic effect of Ang-(1\u0026ndash;7) during coronavirus infection we first employed a model of murine coronavirus (MHV-3) infection [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e] that resembles relevant clinical aspects of severe COVID-19, including lymphopenia and features of macrophage activation syndrome and cytokine storm [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Thus, mice were treated intraperitoneally as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea, and euthanized at 3 dpi, at the peak of lung disease in the model [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Intranasal infection of mice with MHV-3 led to leukopenia (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb) primarily due to a decreased number of lymphocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec) in blood of infected mice. Of interest, Ang-(1\u0026ndash;7) treatment restored leukocyte blood numbers, by increasing lymphocytes and granulocyte counts in the blood at 3 dpi (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed). Thrombocytopenia was a feature of the infection and Ang-(1\u0026ndash;7) did not modify the blood platelets counts in infected mice at this timepoint evaluated (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee). Notably, treatment with Ang-(1\u0026ndash;7) reduced leukocyte infiltration to the alveoli of infected mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ef-h), which was mainly characterized by monocytes/macrophages (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eg). Neutrophils were barely detected in the alveolar space of MHV-infected mice at 3 dpi (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ei). Akin to the anti-inflammatory effect of Ang-(1\u0026ndash;7) in other models of viral infection [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], treatment of MHV-infected mice decrease the lung levels IL-6, TNF, CXCL-1 and IFN-γ (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ej).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo test whether serial administration of Ang-(1\u0026ndash;7) could affect systemic signs seen at 5-day post-MHV infection, we applied a longer therapeutical protocol (scheme in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). In keeping with our previous findings, longer Ang-(1\u0026ndash;7) treatment restored blood leukocytes/lymphocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb-c) and increased neutrophils (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed) numbers [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Notably, longer Ang-(1\u0026ndash;7) treatment partially restored the virus-induced thrombocytopenia (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee) and decreased the numbers of monocytes/macrophages numbers into the airways (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ef-h). Ang-(1\u0026ndash;7) decreased the lung and systemic levels of inflammatory cytokines/chemokines (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ek-l). Collectively, these results demonstrate that treatment with Ang-(1\u0026ndash;7) modulates the local and systemic inflammatory response and rescued lymphopenia and thrombocytopenia throughout the MHV infection in mice.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eAng-(1\u0026ndash;7) decreases viral load and damage/mechanical dysfunction in lungs of MHV-3 infected mice\u003c/h2\u003e \u003cp\u003eNext, we addressed whether Ang-(1\u0026ndash;7) treatment could affect viral load in MHV-infected mice. Viral double-strand RNA (dsRNA) was detected by immunostaining of lung sections at 3 days post-infection (dpi) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). Importantly, in lung slices from Ang-(1\u0026ndash;7)-treated mice the staining was lower (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). In agreement with the immunohistochemistry analysis, plate unit forming (PFU) assay showed reduced viral titers in the lungs, liver, and plasma of Ang-(1\u0026ndash;7)-treated mice at 3 dpi and 5 dpi (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb-e) when compared to vehicle-treated infected-animals.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eHistopathological scores of lungs of infected mice evaluated at 3 dpi showed pronounced tissue damage that was significantly reduced after Ang-(1\u0026ndash;7) treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea-b). In addition, total protein levels in bronchoalveolar lavage fluid, an indirect measurement of edema, were reduced after Ang-(1\u0026ndash;7) treatment compared to the vehicle group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec). Moreover, Ang-(1\u0026ndash;7) improved lung function of infected mice as evaluated by flow parameters, forced expiratory volume, and pulmonary resistance (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed-f). In summary, these data suggest that Ang-(1\u0026ndash;7) treatment reduced viral loads while protecting mice from inflammation-related lung damage and dysfunction caused by MHV-infection.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eAng-(1\u0026ndash;7) decreases lethality of MHV-infected mice\u003c/h2\u003e \u003cp\u003eGiven the protective effect of Ang-(1\u0026ndash;7) during MHV infection, we questioned if Ang-(1\u0026ndash;7)-mediated immunomodulation and viral control could result in improvement of lethality in mice. To test that, infected mice were treated for 5 days with Ang-(1\u0026ndash;7) (scheme in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea) and were monitored daily for weight loss and lethality. Infected animals began to lose weight progressively after 2\u0026ndash;3 days post-infection (dpi) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb) and succumbed to the infection between 6 and 8 dpi (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). Treatment with Ang-(1\u0026ndash;7) prevented the weight loss observed from the 5 dpi (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec). Of note, treated animals exhibited progressive weight gain until the end of the experimental analysis. Notably, Ang-(1\u0026ndash;7) partially rescued mice from MHV-induced lethality (100% \u003cem\u003eversus\u003c/em\u003e 40%, vehicle to Ang-(1\u0026ndash;7) respectively - Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eAng-(1\u0026ndash;7) restores lymphopenia, improves clinical parameters, reduces viral loads, and protects mice from lung injury induced by SARS-CoV-2 in hK18ACE2 mice\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo validate our findings in animals actually infected with SARS-CoV-2, we infected transgenic mice expressing the human angiotensin I-converting enzyme 2 (ACE2) receptor (K18-hACE2 mice) with SARS-CoV-2 and treated infected-mice with Ang-(1\u0026ndash;7) (scheme in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea). In agreement with previous studies [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], SARS-CoV-2 infection in K18-hACE2 mice led to intense weight loss of mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec). In addition, as for the MHV infection, blood lymphocyte counts were significantly reduced with SARS-CoV-2 infection (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb). Ang-(1\u0026ndash;7) treatment promoted a modest recovery in lymphocyte circulation starting at 3 dpi (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb) and continued treatment led to a significant recovery of lymphocytes in the blood at 5 dpi (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb). Notably, treatment with Ang-(1\u0026ndash;7) promoted significant recovery of the weight of infected mice on days 4\u0026ndash;5 after infection compared to the vehicle-treated group (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec-d). In addition, SARS-CoV-2 infected mice treated with Ang-(1\u0026ndash;7) showed improved clinical scores at 5 dpi (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ee). Importantly, pulmonary viral loads in the lungs were significantly reduced after Ang-(1\u0026ndash;7) treatment as evidenced by plaque-forming assays (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e.f). Lung levels of IL-6, TNF, and CXCL1 were significantly reduced in Ang-(1\u0026ndash;7) treated mice in comparison to the vehicle treated mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eg). Lungs histopathological analyses of SARS-CoV-2 infected mice showed tissue damage that was decreased by Ang-(1\u0026ndash;7) treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea-b). Lastly, we questioned whether Ang-(1\u0026ndash;7) could impact viral replication directly by employing an in vitro infection with SARS-CoV-2. Of importance, no direct antiviral effect was observed after treating lung epithelial Calu-3 cells infected by SARSCOV-2 with Ang-(1\u0026ndash;7) (supplementary Fig.\u0026nbsp;1). Taken together, our results indicate that treatment with Ang-(1\u0026ndash;7) protects mice against SARS-CoV-2 by taming inflammation and damage at the same time it reduces viral titers.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eRAS activation results in the production of angiotensin II, which upon binding to AT1 receptor evoke effects as vasoconstriction, inflammation, and oxidative stress [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Conversely, angiotensin-converting enzyme (ACE) 2 counteracts these effects by degrading angiotensin II into Ang-(1\u0026ndash;7), which, upon activation of the MAS receptor, elicits anti-inflammatory, antioxidative, vasodilatory, and pro-resolving responses [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Early in COVID-19 pandemic several reviews claimed a putative benefic effect of Ang-(1\u0026ndash;7) [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e], given the known actions of this peptide. However, while higher systemic levels of Ang II were consistently documented in COVID-19 patients [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e] compared to healthy donors, divergent findings of Ang-(1\u0026ndash;7) measurement were reported in severely ill hospitalized COVID-19 patients, with some exhibiting elevated Ang-(1\u0026ndash;7) concentrations while others show decreased levels [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. In addition, two randomized clinical trials using synthetic Ang-(1\u0026ndash;7) (TXA-127) and an angiotensin II type 1 receptor-biased ligand (TRV-027) showed no clinical benefit for patients with severe COVID-19 [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e], as initially hypothesized. Nevertheless, some concerns regarding the administration protocol have been raised by experts in the field [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. In this regard, recent results from a clinical trial using a Mas-receptor activation by 20-hydroxyecdysone (BIO101) has shown positive results in severe COVID-19 by significantly reducing the risk of death or respiratory failure and supporting the use of MasR activators/agonists during disease [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. The protective bioactions of the ACE2/Ang (1\u0026ndash;7)/MasR axis in pre-clinical models of lung injury, including influenza virus infection [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] and the lack of specific pre-clinical date regarding the role Ang-(1\u0026ndash;7) in coronavirus diseases, emphasize the urgent need to understand the effects of Ang-(1\u0026ndash;7) during betacoronavirus infection for better planning of novel clinical studies in humans. Here, we have investigated the effect of the delayed administration of Ang-(1\u0026ndash;7) on the immunopathology evoked by two betacoronavirus, MHV-3 and SARS-CoV-2, in mice. Importantly, this pro-resolving peptide afforded significant protection in infected mice by taming the inflammatory response, restoring lymphopenia, and decreasing viral loads and lung damage. Notably, Ang-(1\u0026ndash;7) improved lung function and partially rescued mice from morbidity and lethality.\u003c/p\u003e \u003cp\u003eMHV-3 is a betacoronavirus that infects mice and can be used in BSL-2 safety conditions. Infection with MHV-3 caused severe acute respiratory syndrome in C57BL/6 mice with efficient viral replication in the lungs, tissue damage associated with inflammation, and compromised respiratory function [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. SARS-CoV-2 triggered similar host inflammatory responses, characterized by an overabundance of pro-inflammatory cytokines, leading to the infiltration of immune cells into both the bronchioalveolar space and lung interstitial compartment [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. These events contribute to lung tissue damage, ultimately leading to reduced lung compliance, and impaired lung function [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. In this context, mice infected with MHV-3 exhibited elevated edema and heightened infiltration of inflammatory cells to the airways, along with hyperplasia and disruption of tissue architecture, characteristics associated with acute respiratory distress syndrome [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. In the present study, the treatment with Ang-(1\u0026ndash;7) improved clinical parameters and protected mice from death, resulting in higher percentage of survival in Ang-(1\u0026ndash;7)-treated mice. Mechanistically, Ang-(1\u0026ndash;7) treatment decreased monocyte/macrophage infiltration to the airways coupled to decreased levels of cytokines, viral RNA and PFUs in the lungs of MHV-infected mice compared to vehicle-treated group. The effects of Ang-(1\u0026ndash;7) taming inflammation and promoting viral clearance were associated with improvement of lung function and respiratory mechanics, in agreement with the data obtained from Ang-(1\u0026ndash;7)-treated IAV-infected mice [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn addition to its beneficial role on lung inflammation and function, Ang-(1\u0026ndash;7) reduced virus titers in plasma, when evaluated at the time point of viremia (5dpi) in this model [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Therefore, our findings suggest that administration of Ang-(1\u0026ndash;7) is protective during coronavirus infection by reducing inflammation without causing immunosuppression. Of importance, these pro-resolving effects are also observed for Ang-(1\u0026ndash;7) in the settings of bacterial infection [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Ang-(1\u0026ndash;7) treatment rescued the number of leukocytes in MHV-infected mice in the blood, and thus maintaining the host's ability to fight infections [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Here, no direct anti-viral Ang-(1\u0026ndash;7) actions in human airway epithelial cells were observed \u003cem\u003ein vitro\u003c/em\u003e, suggesting that the reduction of virus titers \u003cem\u003ein vivo\u003c/em\u003e are most likely associated with an overall better host resilience to infection. Of note, Ang-(1\u0026ndash;7) inhibits the phosphorylation of JAK/STAT proteins [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e], which are essential regulators of local and systemic response to viral infections [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Indeed, JAK inhibitors such as baricitinib has antiviral properties against SARS-CoV-2 infection [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e] suggesting that adjunctive anti-viral and anti-inflammatory/pro-resolving based strategies as potential therapies in the settings of infectious diseases [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Whether Ang-(1\u0026ndash;7) is regulating the JAK/STAT pathway and contributing for the observed effect in viral load remain to be investigated. Further studies will fully establish the role of Ang-(1\u0026ndash;7) in promoting pathogen control during coronavirus infection.\u003c/p\u003e \u003cp\u003eTransgenic mice expressing human ACE2 (K18-hACE2) are highly susceptible to SARS-CoV-2 infection [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. In these animals, the infection results in intense production of cytokines locally and systemically, increased weight loss, leukopenia, and sustained viral replication in the nasal turbinates, lungs, and brains resulting in a viral sepsis-like disease [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Similarly to the results obtained during MHV-3 infection, significant improvements were observed after Ang-(1\u0026ndash;7) treatment in SARS-CoV-2 infection. Ang-(1\u0026ndash;7) notably attenuated weight loss, rescue lymphopenia reduced lung inflammation/injury and viral loads and improved the overall clinical score when compared with the vehicle-treated mice.\u003c/p\u003e \u003cp\u003eThe role of the ACE2/Ang-(1\u0026ndash;7)/MasR axis in balancing actions that attenuate inflammation, oxidative stress, apoptosis, and fibrosis are well described [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Of interest, the membrane-bound ACE2 was shown to serve as a viral receptor for SARS-CoV-2 and other coronaviruses in the host cells. After virus binding and adsorption, ACE2 is internalized and degraded [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Thus, the enzymatic production of Ang-(1\u0026ndash;7) decreases, preventing inflammation resolution and perpetuating tissue damage, and coagulopathy, features of severe COVID-19 [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. In this regard, administration of an ACE2 recombinant protein is effective in protecting mice from ALI induced by the systemic injection of SARS-CoV-2 spike protein [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIncreased levels of proinflammatory cytokines in serum have been associated with pulmonary inflammation and severe lung damage in patients with COVID-19 [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Of note, SARS-CoV-2 infection leads to an over activation of macrophages with exacerbated secretion of pro-inflammatory mediators and associated damage [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. Here, high levels of pro-inflammatory cytokines such as IL-6, TNF, CXCL1, and IFN-γ were found in the plasma and lungs of coronavirus infected animals, associated to increased infiltration of macrophages. Ang-(1\u0026ndash;7) reduced infiltration of these leukocytes to the airways, tamed production of pro-inflammatory cytokines and prevented lung damage and loss of lung function. Similarly, we have shown that in a pre-clinical model of IAV infection [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e] and allergic inflammation [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e], Ang-1-7 reduced cytokine production that was associated with the attenuation of the overall lung damage. In addition, Ang-(1\u0026ndash;7) shifts macrophage phenotype towards anti-inflammatory and pro-resolving profiles [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. Of note, it has been shown that Ang-(1\u0026ndash;7) decrease the levels of IL-6 and IL-8 in alveolar epithelial cells stimulated with SARS-CoV-2 spike protein [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eLymphocyte count is a biomarker for monitoring the severity and mortality of COVID-19 disease [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. Indeed, a notable reduction in T lymphocytes is positively associated with in-hospital mortality and the severity of illness [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. In our study, we have observed reduced leukocyte levels in the peripheral blood of animals infected with coronavirus, alongside heightened number of these cells in bronchoalveolar lavage. This suggests an accumulation of inflammatory cells in lung tissue, indicative of leukocyte trafficking from the bloodstream to the viral-infected respiratory tract [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. Our findings demonstrate that treatment with Ang-(1\u0026ndash;7) regulates leukocyte numbers in the airways and attenuates lymphopenia induced by coronavirus infection. Curiously, analog drugs of Ang-(1\u0026ndash;7) are widely recognized for their capacity to stimulate hematopoiesis and accelerate the replenishment of circulating cells [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. Thus, Ang-(1\u0026ndash;7) could be acting in bone marrow to restore lymphopenia caused by coronavirus infection [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. Yet, further studies will clarify the associated mechanism.\u003c/p\u003e \u003cp\u003eIn conclusion, this study offers evidence supporting Ang-(1\u0026ndash;7) as a crucial regulator of the immune response during coronavirus infection. Our findings demonstrate for the first time that Ang-(1\u0026ndash;7) regulates both local and systemic immune responses, mitigates pulmonary inflammation, improves clinical outcomes, reduces viral load, and enhances survival in mice infected with MHV-3 or SARS-CoV-2. Altogether, these findings support the hypothesis that angiotensin-(1\u0026ndash;7) could serve as an interesting therapeutic strategy for critical illnesses such as COVID-19 [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e], and reinforce the need for new clinical trial using Ang-(1\u0026ndash;7) based peptides or other MasR agonists.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eACKNOWLEDGEMENTS\u0026nbsp;\u003c/strong\u003eThe authors are thankful to Ilma Mar\u0026ccedil;al and T\u0026acirc;nia Colina for technical assistance.\u0026nbsp;Thanks to the animal biosafety level 3 laboratory at UFMG (Laborat\u0026oacute;rio Institucional de Pesquisa, LIPq); Centro de Laborat\u0026oacute;rios Multiusu\u0026aacute;rios, CELAM; Laborat\u0026oacute;rio de Biosseguran\u0026ccedil;a N\u0026iacute;vel 3, NB3-ICB.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAUTHOR CONTRIBUTIONS\u0026nbsp;\u003c/strong\u003eL.P.S., and E.B.S.L. analyzed data and wrote the paper. E.B.S.L., A.F.S.C, I.Z., A.H.A.M., C.C, E.S.L., F.S.C., L.C.O, F.R., F.R.S.S., C.M.Q-J., performed the experiments and analyzed data. R.A.S.S. provided expertise. R.C.R. performed the lung mechanical assessment. M.M.T., V.V.C., L.P.S and L.P.T conception and design, analysis, and interpretation of data, drafting, editing and revising manuscript and project funding.\u0026nbsp;All authors approved the final version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFUNDING\u0026nbsp;\u003c/strong\u003eThis study was was supported by grants from Funda\u0026ccedil;\u0026atilde;o de Amparo \u0026agrave; Pesquisa do Estado de Minas Gerais, grant numbers: BPD-01010-22 and APQ-03221-18 to L.P.S., APQ02281-18, APQ02618-23 and RED-00202-22; Conselho Nacional de Desenvolvimento Cient\u0026iacute;fico e Tecnol\u0026oacute;gico (CNPq, Brazill) by the grant PQ- 310799/2022-8, \u0026nbsp;408482/2022-2; and Coordena\u0026ccedil;\u0026atilde;o de Aperfei\u0026ccedil;oamento de Pessoal de N\u0026iacute;vel Superior \u0026ndash;CAPES/Brazil (Projeto: CAPES - Programa: 9951 - Programa Estrat\u0026eacute;gico Emergencial de Preven\u0026ccedil;\u0026atilde;o e Combate a Surtos, Endemias, Epidemias e Pandemias AUX 0641/2020 - Processo 88881.507175/2020-01). This work also received support from the National Institute of Science and Technology in Dengue and Host-Microorganism Interaction (INCT em Dengue), sponsored by the Conselho Nacional de Desenvolvimento Cient\u0026iacute;fico e Tecnol\u0026oacute;gico (CNPq; Brazil) (Processo CNPQ: 465425 /2014-3) and the Funda\u0026ccedil;\u0026atilde;o de Amparo \u0026agrave; Pesquisa do Estado de Minas Gerais (FAPEMIG; Brazil) (Processo FAPEMIG: 25036). And by FINEP - Financiadora de Estudos e Projetos under MCTI/FINEP \u0026ndash; MS/SCTIE/DGITIS/CGITS (6205283B-BB28-4F9C-AA65-808FE4450542) grant.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eOladunni FS, Park J-G, Pino PA, Gonzalez O, Akhter A, Allu\u0026eacute;-Guardia A, et al. 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Increased circulating levels of angiotensin-(1-7) in severely ill COVID-19 patients. ERJ Open Res. 2021;7:00114\u0026ndash;2021. \u003c/li\u003e\n\u003cli\u003eSelf WH, Shotwell MS, Gibbs KW, de Wit M, Files DC, Harkins M, et al. Renin-Angiotensin System Modulation With Synthetic Angiotensin (1-7) and Angiotensin II Type 1 Receptor-Biased Ligand in Adults With COVID-19: Two Randomized Clinical Trials. JAMA. 2023;329:1170\u0026ndash;82. \u003c/li\u003e\n\u003cli\u003eDos Santos RAS, Taccone FS, Annoni F. Renin-Angiotensin System Modulation in Adults With COVID-19. JAMA. 2023;330:663\u0026ndash;4. \u003c/li\u003e\n\u003cli\u003eLobo SM, Plantef\u0026egrave;ve G, Nair G, Joaquim Cavalcante A, Franzin de Moraes N, Nunes E, et al. Efficacy of oral 20-hydroxyecdysone (BIO101), a MAS receptor activator, in adults with severe COVID-19 (COVA): a randomized, placebo-controlled, phase 2/3 trial. EClinicalMedicine. 2024;68:102383. \u003c/li\u003e\n\u003cli\u003eMyatra SN, Alhazzani W, Belley-Cote E, M\u0026oslash;ller MH, Arabi YM, Chawla R, et al. Awake proning in patients with COVID-19-related hypoxemic acute respiratory failure: A rapid practice guideline. Acta Anaesthesiol Scand. 2023;67:569\u0026ndash;75.\u003c/li\u003e\n\u003cli\u003eZaidan I, Tavares LP, Sugimoto MA, Lima KM, Negreiros-Lima GL, Teixeira LC, et al. Angiotensin-(1-7)/MasR axis promotes migration of monocytes/macrophages with a regulatory phenotype to perform phagocytosis and efferocytosis. JCI Insight. 2022;7:e147819. \u003c/li\u003e\n\u003cli\u003eItcho K, Oki K, Kobuke K, Ohno H, Yoneda M, Hattori N. Angiotensin 1-7 suppresses angiotensin II mediated aldosterone production via JAK/STAT signaling inhibition. The Journal of Steroid Biochemistry and Molecular Biology. 2019;185:137\u0026ndash;41. \u003c/li\u003e\n\u003cli\u003eEzeonwumelu IJ, Garcia-Vidal E, Ballana E. JAK-STAT Pathway: A Novel Target to Tackle Viral Infections. Viruses. 2021;13:2379. \u003c/li\u003e\n\u003cli\u003eRichardson P, Griffin I, Tucker C, Smith D, Oechsle O, Phelan A, et al. Baricitinib as potential treatment for 2019-nCoV acute respiratory disease. Lancet. 2020;395:e30\u0026ndash;1.\u003c/li\u003e\n\u003cli\u003eHeyman SN, Walther T, Abassi Z. Angiotensin-(1-7)-A Potential Remedy for AKI: Insights Derived from the COVID-19 Pandemic. J Clin Med. 2021;10:1200.\u003c/li\u003e\n\u003cli\u003eZhang L, Zhang Y, Qin X, Jiang X, Zhang J, Mao L, et al. Recombinant ACE2 protein protects against acute lung injury induced by SARS-CoV-2 spike RBD protein. Crit Care. 2022;26:171. \u003c/li\u003e\n\u003cli\u003eHuang C, Wang Y, Li X, Ren L, Zhao J, Hu Y, et al. Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China. Lancet. 2020;395:497\u0026ndash;506. \u003c/li\u003e\n\u003cli\u003eFelkle D, Zięba K, Kaleta K, Czaja J, Zyzdorf A, Sobocińska W, et al. Overreactive macrophages in SARS-CoV-2 infection: The effects of ACEI. International Immunopharmacology. 2023;124:110858. \u003c/li\u003e\n\u003cli\u003eMagalhaes GS, Gregorio JF, Beltrami VA, Felix FB, Oliveira-Campos L, Bonilha CS, et al. A single dose of angiotensin-(1\u0026ndash;7) resolves eosinophilic inflammation and protects the lungs from a secondary inflammatory challenge. Inflamm Res [Internet]. 2024 [cited 2024 May 11];\u003c/li\u003e\n\u003cli\u003ede Carvalho Santuchi M, Dutra MF, Vago JP, Lima KM, Galv\u0026atilde;o I, de Souza-Neto FP, et al. Angiotensin-(1-7) and Alamandine Promote Anti-inflammatory Response in Macrophages In Vitro and In Vivo. Mediators Inflamm. 2019;2019:2401081. \u003c/li\u003e\n\u003cli\u003eShen Y-L, Hsieh Y-A, Hu P-W, Lo P-C, Hsiao Y-H, Ko H-K, et al. Angiotensin-(1\u0026ndash;7) attenuates SARS-CoV2 spike protein-induced interleukin-6 and interleukin-8 production in alveolar epithelial cells through activation of Mas receptor. Journal of Microbiology, Immunology and Infection. 2023;56:1147\u0026ndash;57.\u003c/li\u003e\n\u003cli\u003eZhang P, Du W, Yang T, Zhao L, Xiong R, Li Y, et al. Lymphocyte subsets as a predictor of severity and prognosis in COVID-19 patients. Int J Immunopathol Pharmacol. 2021;35:20587384211048567. \u003c/li\u003e\n\u003cli\u003eAlon R, Sportiello M, Kozlovski S, Kumar A, Reilly EC, Zarbock A, et al. Leukocyte trafficking to the lungs and beyond: lessons from influenza for COVID-19. Nat Rev Immunol. 2021;21:49\u0026ndash;64.\u003c/li\u003e\n\u003cli\u003eGaffney K, Weinberg M, Soto M, Louie S, Rodgers K. Development of angiotensin II (1-7) analog as an oral therapeutic for the treatment of chemotherapy-induced myelosuppression. Haematologica. 2018;103:e567\u0026ndash;70.\u003c/li\u003e\n\u003cli\u003eRodgers KE, Espinoza TB, Roda N, Meeks CJ, diZerega GS. Angiotensin-(1-7) synergizes with colony-stimulating factors in hematopoietic recovery. Cancer Chemother Pharmacol. 2013;72:1235\u0026ndash;45.\u003c/li\u003e\n\u003cli\u003eRodgers KE, diZerega GS. Contribution of the Local RAS to Hematopoietic Function: A Novel Therapeutic Target. Front Endocrinol (Lausanne). 2013;4:157\u003c/li\u003e\n\u003cli\u003eGarcia B, Zarbock A, Bellomo R, Legrand M. The alternative renin\u0026ndash;angiotensin system in critically ill patients: pathophysiology and therapeutic implications. Critical Care. 2023;27:453. \u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"inflammation-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"inre","sideBox":"Learn more about [Inflammation Research](http://link.springer.com/journal/11)","snPcode":"11","submissionUrl":"https://submission.nature.com/new-submission/11/3","title":"Inflammation Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Covid-19, Coronavirus, MHV-3, SARS-CoV-2, Angiotensin-(1-7)","lastPublishedDoi":"10.21203/rs.3.rs-4529565/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4529565/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eObjective: \u003c/strong\u003ePro-resolving molecules, including the peptide Angiotensin-(1-7) [Ang-(1-7)], have potential adjunctive therapy for infections. Here we evaluate the actions of Ang-(1-7) in betacoronavirus infection in mice.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods: \u003c/strong\u003eC57BL/6 mice were infected intranasally with the murine betacoronavirus MHV-3 and K18-hACE2 mice were infected with SARS-CoV-2. Mice were treated with Ang-(1-7) (30 μg/mouse, i.p.) at 24-, 36-, and 48-hours post-infection (hpi) or at 24, 36, 48, 72, and 96 h. For lethality evaluation, one additional dose of Ang-(1-7) was given at 120 hpi. At 3- and 5-days post- infection (dpi) blood cell, inflammatory mediators, viral loads, and lung histopathology were evaluated.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults: \u003c/strong\u003eAng-(1-7) rescued lymphopenia in MHV-infected mice, and decreased airways leukocyte infiltration and lung damage at 3- and 5-dpi. The levels of pro-inflammatory cytokines and virus titers in lung and plasma were decreased by Ang-(1-7) during MHV infection. Ang-(1-7) improved lung function and increased survival rates in MHV-infected mice. Notably, Ang-(1-7) treatment during SARS-CoV-2 infection restored blood lymphocytes to baseline, decreased weight loss, virus titters and levels of inflammatory cytokines, resulting in improvement of pulmonary damage and clinical scores.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion: \u003c/strong\u003eAng-(1-7) protected mice from lung damage and death during betacoronavirus infections by modulating inflammation, hematological parameters and enhancing viral clearance.\u003c/p\u003e","manuscriptTitle":"Angiotensin-(1-7) decreases inflammation and lung damage caused by betacoronavirus infection in mice","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-06-18 09:03:11","doi":"10.21203/rs.3.rs-4529565/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-07-08T07:19:34+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-07-07T21:59:42+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-07-07T15:02:06+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"182153743421478822732070692631059901735","date":"2024-06-25T02:25:08+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"47257784463882192758189042832472041838","date":"2024-06-23T04:08:51+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"193295077178776128182782814064478300947","date":"2024-06-22T15:52:52+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-06-20T19:53:16+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-06-05T18:32:53+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-06-05T18:32:38+00:00","index":"","fulltext":""},{"type":"submitted","content":"Inflammation Research","date":"2024-06-04T16:57:25+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"inflammation-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"inre","sideBox":"Learn more about [Inflammation Research](http://link.springer.com/journal/11)","snPcode":"11","submissionUrl":"https://submission.nature.com/new-submission/11/3","title":"Inflammation Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"6b9e82d6-eec5-4a34-83da-8d3c1cd9f2fe","owner":[],"postedDate":"June 18th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-09-23T16:01:04+00:00","versionOfRecord":{"articleIdentity":"rs-4529565","link":"https://doi.org/10.1007/s00011-024-01948-8","journal":{"identity":"inflammation-research","isVorOnly":false,"title":"Inflammation Research"},"publishedOn":"2024-09-18 15:57:21","publishedOnDateReadable":"September 18th, 2024"},"versionCreatedAt":"2024-06-18 09:03:11","video":"","vorDoi":"10.1007/s00011-024-01948-8","vorDoiUrl":"https://doi.org/10.1007/s00011-024-01948-8","workflowStages":[]},"version":"v1","identity":"rs-4529565","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4529565","identity":"rs-4529565","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}