key: cord-0723899-nky3azcf
authors: Solopov, Pavel A.; Luciano Colunga Biancatelli, Ruben Manuel; Catravas, John D.
title: Alcohol increases lung ACE2 expression and exacerbates SARS-CoV-2 Spike protein subunit 1-induced acute lung injury in K18-hACE2 transgenic mice.
date: 2022-04-25
journal: Am J Pathol
DOI: 10.1016/j.ajpath.2022.03.012
sha: 2fe9c8417a114eab98a83d77ca65e9012cc2e1d2
doc_id: 723899
cord_uid: nky3azcf

During the SARS-CoV-2 pandemic alcohol consumption increased markedly. Nearly 1 in 4 adults reported drinking more alcohol to cope with stress. Chronic alcohol abuse is now recognized as a factor complicating the course of acute respiratory distress syndrome (ARDS). and increasing mortality. To investigate the mechanisms behind this interaction, we developed a combined ARDS and chronic alcohol abuse mouse model by intratracheally instilling the S1 subunit of SARS-CoV-2 Spike protein (S1SP) in K18-hACE2 transgenic mice that express the human ACE2 receptor for SARS-CoV-2 and are kept on an ethanol diet. 72 hours after S1SP instillation, mice on ethanol diet exhibited a strong decline in body weight, a dramatic increase in white blood cell content of bronchoalveolar lavage fluid, and an augmented “cytokine storm”, compared to S1SP treated mice on control diet. Histologic examination of lung tissue demonstrated abnormal recruitment of immune cells in the alveolar space, abnormal parenchymal architecture, and worsening of the Ashcroft score in S1SP- and alcohol-treated animals. Along with the activation of pro-inflammatory biomarkers (NF-κB, STAT3, NLRP3 inflammasome), lung tissue homogenates from mice on alcohol diet, demonstrated overexpression of ACE2 compared to mice on control diet. This model could be useful for the development of therapeutic approaches against alcohol-exacerbated COVID-19.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) outbreak that began in December 2019 and spread rapidly across the globe causes acute lung injury, severe hypoxemia, and multiorgan failure. SARS-CoV-2 infects host cells by targeting the endothelial angiotensin-converting enzyme 2 (ACE2) in the lung, heart, kidney, and gastrointestinal tissues. The pathophysiology of acute respiratory distress syndrome (ARDS) in SARS-CoV-2 infection includes lung perfusion dysregulation and a "cytokine storm" that causes increased vascular permeability and disease severity [1] . COVID-19 can also cause psycho-social problems, including increased alcohol consumption and consequent harms [2] . Alcoholic beverage sales in the United States increased greatly immediately after the stay-at-home orders and relaxing of alcohol restrictions associated with the COVID-19 pandemic [3] . The rise in the black marketing of alcohol has also been reported [4] . Alcohol abuse increased so much that some countries even prohibited alcohol sales during the pandemic lockdown [5] . Alcohol consumption is considered an independent factor that increases the incidence of ARDS, a severe form of acute lung injury with a mortality rate up to 50 percent. This translates to tens of thousands of excess deaths in the United States each year from alcohol-associated lung injury, which is comparable to scarring of the liver (i.e., cirrhosis) in terms of alcohol-related mortality [6] [7] . Furthermore, people who drink heavily are more likely to get pneumonia [8] . Although acute alcohol exposure (less than 24 hours) favors anti-inflammatory responses, chronic alcohol consumption favors proinflammatory cytokine release [9] . Notwithstanding that alcohol consumption alone does not cause ARDS, it makes the lungs susceptible to dysfunction induced by pathologies, such as the inflammatory stresses of sepsis, trauma, etc. [10] . Studies on human monocytes reveal that several pathogens, when combined with chronic ethanol consumption, promote the production of inflammatory cytokines. In the lung, cytokine production is augmented by ethanol, exacerbating respiratory distress syndrome and greatly increasing the expression of transforming growth factor beta (TGF-β) [11] .

We have recently developed an animal model to study acute lung injury caused by subunit 1 (S1) of the SARS-CoV2 Spike protein using K18-hACE2 transgenic mice [12] . Intratracheal instillation of S1SP induced COVID-19-like lung and systemic inflammatory responses, including "cytokine storm" in bronchoalveolar lavage fluid (BALF) and serum. In this study, we have used this model to interrogate, how chronic alcohol consumption may worsen the development of COVID-19-like ARDS.

All animal studies were approved by the Old Dominion University IACUC and adhere to the principles of animal experimentation as published by the American Physiological Society. Healthy male K18-hACE2 transgenic mice (Jackson Laboratories), 8-10 weeks old, 20-25 g body weight, were placed on the Lieber-DeCarli '82 Control liquid diet for 5 days after arrival at the animal facility and then divided randomly into 4 groups ( Fig.1 ): 1) VEH groupmice, continued on Lieber-DeCarli '82 Control liquid diet for 14 days and then on day 19 instilled intratracheally (i.t.) with vehicle (sterile saline) at 2mL/kg body weight; 2) S1SP groupmice on control diet for 14 days and then -on day 19-instilled i.t. with SARS-CoV-2 S1SP at 400µg/kg in 2mL/kg body weight; 3) EtOH VEH group -mice, transferred to the Lieber-DeCarli '82 Ethanol liquid diet, consisting of 5-6 % EtOH, for two weeks and then-on day 19-instilled i.t. with vehicle (sterile saline) at 2mL/kg body weight; 4) EtOH S1SP group -mice, transferred to Lieber-DeCarli '82 Ethanol liquid diet for two weeks, then on day 19 instilled i.t. with SARS-CoV-2 S1SP at 400µg/kg in 2mL/kg body weight. The Lieber-DeCarli liquid diet, contains 36% calories from either ethanol (EtOH diet) or isocaloric maltose dextrin (Control diet), 35% calories from fat, 11% from carbohydrate and 18% from protein [13] . All animals consumed liquid food ad libitum (approximately 20-30mL per day. This treatment with the ethanol diet produces blood alcohol concentrations (BAC) of ~180 mg/dL by day 10 [14] ). Mice did not receive water during days 5 to 19. Groups 3 and 4 were transferred to the EtOH diet gradually, to minimize stress: Day 5-7: mixture of 1/3 EtOH diet and 2/3 control diet; day 8-10: mixture of 2/3 EtOH diet and 1/3 control diet; day 11-22: EtOH diet only. After i.t. instillation -day 19mice were also given free access to water. All mice were euthanized at day 22 (72 h after i.t. instillation).

Immediately after euthanasia, the chest was opened, the mouse was placed in the upright position and the lungs were instilled and inflated through the trachea with 10% formaldehyde to a pressure of 15cm H2O and then immersed in the same solution. 72 hours later, samples were embedded in paraffin. 5μm thick sections were stained with Hematoxylin & Eosin (H&E) and Masson's Trichrome stains. Twenty randomly selected fields from each slide were examined under immersion (100 x magnification). Fields from H&E stained sections were scored according to the Lung Injury Score [15] method to estimate the severity of lung inflammation; this method takes into account histological evidence of injury, including accumulation of neutrophils in the alveolar or the interstitial space, formation of hyaline membranes, presence of proteinaceous debris in the alveolar space, thickening of the alveolar walls, hemorrhage and atelectasis. In addition, fields from Masson's Trichrome-stained sections were scored according to the Ashcroft score to quantify lung architectural changes and estimate overall collagen deposition [16] .

Livers were also collected and fixed with 10% formaldehyde in the same way and paraffin sections were stained with H&E and Masson's trichrome. Twenty randomly selected fields from each slide were examined. Hepatic Steatosis Scoring were performed according to the General non-alcoholic fatty liver disease (NAFLD) Scoring System for Rodent Models [17] , that takes into account hepatocellular steatosis, hypertrophy and inflammation.

Bronchoalveolar lavage fluid (BALF) was collected by instilling and withdrawing 1mL sterile 1X PBS via the tracheal cannula. The BALF was centrifuged at 2400×g for 10 min at 4°C (Thermo Fisher Centrifuge 5417R) and the supernatant was collected and stored immediately at −80°C. The cell pellet was resuspended in 1mL sterile PBS and the total number of white blood cells was determined using a hemocytometer΄differential analysis was performed with the Wright-Giemsa stain kit.

All histopathological and morphological analyses were performed by an investigator blinded to the study groups.

BALF supernatant was collected and prepared as described above. Protein concentration was determined using the micro bicinchoninic acid (BCA) assay according to the manufacturer's protocol. BALF supernatant IL-6, KC, MCP-1, TGF-β1 and TNFα, were analyzed in triplicate via mouse/human ELISA kits.

J o u r n a l P r e -p r o o f Immediately after euthanasia, the thorax was opened, blood was drained from the heart through the right ventricle and the pulmonary circulation was flushed with sterile PBS containing EDTA. The lungs were dissected from the thorax, snap-frozen in liquid nitrogen and kept at −80°C for subsequent analysis.

Proteins in lung tissue homogenates were extracted from frozen lungs by ultrasonic homogenization (50% amplitude, 3 times for 10s) in ice-cold lysing RIPA buffer with added protease inhibitor cocktail (100:1). The protein lysates were gently mixed under rotation for 3h at 4°C, and then centrifuged twice at 14,000×g for 10 min at 4°C. The supernatants were collected, and total protein concentration was analyzed using the micro-BCA assay. Equal amounts of proteins from all samples (1000µg/mL) were used for western blot analysis. The lysates were first mixed with Tricine Sample Buffer 1:1, boiled for 5min and then separated on a 10% polyacrylamide SDS gel by electrophoresis. Separated proteins were then transferred to a nitrocellulose membrane, incubated overnight at 4°C with the appropriate primary antibody, diluted in the blocking buffer, followed by 1 hour incubation with the secondary antibody at room temperature and scanned by digital fluorescence imaging (LI-COR Odyssey CLx, Dallas, TX, USA). β-actin was used as loading control. ImageJ software v.1.8.0 was used to perform densitometry of the bands from the western blot membranes (http://imagej.nih.gov/ij/; National Institutes of Health, Bethesda, MD, USA, last access at 7/18/21). Some membranes were stripped for 5min and incubated with other primary and secondary antibodies.

Lung tissue, stored in RNAlater solution for at least 24h, was dried and homogenized in TRIzol followed by a cleaning up step using the RNeasy Mini Kit. The purified RNA was transcribed into cDNA using the SuperScriptTM IV VILO Reverse transcription Kit and analyzed by real-time qPCR with SYBR Green Master Mix on a StepOne Real-Time PCR System (Applied Biosystems v.2.3). Results were evaluated using the standard curve method and expressed as fold of control values. β-actin mRNA expression was used for the normalization of all samples.

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Statistical significance of differences among groups was determined by one way-or two wayanalysis of variance (ANOVA) followed by the Tukey post-hoc test using GraphPad Prism Software (GraphPad Software, San Diego, CA, USA). Differences among groups were considered significant at p < 0.05.

To make sure that 14 days of EtOH diet is enough for the development of chronic alcohol abuse symptoms, we first investigated morphological changes in liver samples stained with H&E and Masson's trichrome. K18-hACE2 transgenic mice on 14 days of alcohol diet, demonstrated prominent signs of severe fatty liver disease (steatosis) ( Fig.2A) , that was reflected in the profoundly increased hepatic steatosis score (Fig.2B) . Mice on control diet demonstrated healthy liver architecture.

Alcohol consumption had no effect on the body weight of transgenic mice instilled with saline. A decrease in appetite after anesthesia was reflected in a slight weight loss during the first 24 h. Both groups of transgenic mice instilled with S1SP displayed a rapid decline in body weight, unlike control groups.

However, mice on control diet started to recover 48 h after instillation, while alcohol-consuming animals continued to lose weight (Fig.3) .

Mice on regular diet instilled with S1SP exhibited significant increase in leucocyte content of BALF compared to vehicle (VEH) group. Mice on alcohol diet and treated with S1SP demonstrated a dramatic increase in white blood cell content of BALF compared to S1SP-instilled mice on normal diet (Fig.4A) . There was no difference between control and ethanol diets in the two VEH groups. A similar profile is also observed in the levels of total protein in BALF, suggesting exacerbated capillary permeability and further confirming the presence of strong acute inflammation (Fig.4B) . BALF white blood cell differential analysis demonstrated an upward shift of mononuclear cell content in EtOH-fed S1SP-instilled mice, while neutrophils primarily increased in S1SP-instilled mice on control diet (Fig.4C) .

H&E-stained lung sections from mice on control diet instilled with S1SP exhibited recruitment of neutrophils and higher lung injury score than vehicle-instilled mice on control diet. Mice on ethanol diet and instilled with saline exhibit a higher number of interstitial mononuclear cells compared to mice on control diet, altered parenchymal architecture and a higher lung injury score (Fig. 5) . S1SP-instilled mice on ethanol diet show mononuclear cell infiltration with minimal numbers of interstitial neutrophils, abnormal alveolar structure, and a lung injury score, (calculated as per the Official American Thoracic Society Workshop Report [15] ) that was 3-fold higher than mice on control diet.

Interleukin 6 (IL-6) and tumor necrosis factor-alpha (TNFα) concentrations in BALF increased in the VEH-instilled group on alcohol diet compared to the normal diet VEH group (Fig.6) . Both S1SPinstilled groups demonstrated elevated levels of IL-6 and TNFα compared to their respective controls, however, S1SP-instilled mice on ethanol diet exhibited even higher values of both cytokines. Similar results were observed with transforming growth factor β1 (TGF-β1) (Fig. 6E) . As with other cytokines, MCP-1 exacerbated increase in the EtOH S1SP group, however. significant upregulation of KC was observed in S1SP-treated mice on control diet only, in agreement with histological and BALF neutrophil concentration data that depict much lower lung and BALF neutrophil presence in EtOH-S1SP treated mice (Fig.6C) .

To explore the potential effect of increased TGF-β levels on fibroblast activation, fixed lung sections were additionally stained with Masson's trichrome to visualize collagen deposition. Significant changes in parenchymal architecture, including thickening of the alveolar walls as well as multiple segments with significant collagen deposition were observed in S1SP-instilled mice that received alcohol (Fig.7) .

Lung tissue homogenates from mice on alcohol diet, demonstrated overexpression of angiotensinconverting enzyme 2 (ACE2) compared to mice on control diet. S1SP did not further affect ACE2 expression (Fig. 8) .

As we recently published, intratracheal instillation of a single element of SARS-CoV-2, S1SP, into K18-hACE2 transgenic mice increased the expression of pro-inflammatory biomarkers in the lung [12] . This was confirmed here, where western blot analysis of lung homogenates revealed significant increases in the phosphorylation of both STAT3 and IkBα in transgenic mice on control diet instilled with S1SP. Alcohol significantly amplified the inflammatory effect of S1SP. Moreover, S1SP significantly increased the expression of inflammasome NLRP3 and even more so in mice on ethanol diet (Fig. 9 ). Profound activation of both ERK and AKT signaling was observed in mice on alcohol diet. This occurred in both VEH-and S1SP-instilled groups (Fig.10 ).

We have used a novel mouse model of SARS-CoV-2 in combination with an established model of chronic and binge ethanol feeding (the NIAAA model [18] ) to study the exacerbations of the acute respiratory distress syndrome induced by Subunit 1 of the SARS-CoV-2 Spike protein in vivo, thereby simulating the pathogenesis COVID-19 disease in alcoholics.

The NIAAA model is wide recognized and useful for the study of alcoholic liver disease (ALD) and systemic damage by alcohol consumption. This model is similar to the drinking pattern in patients with alcoholic hepatitis, who have a background of chronic alcoholism and a record of recent excessive alcohol consumption in anamnesis [18] . The model suggests using 8-to 10-week-old male C57BL/6 mice since they are an alcohol-preferring strain and have demonstrated the best survival. Other strains either refuse the alcohol diet or are too adversely affected by the 5% ethanol and as a consequence, they lose weight and exhibit high mortality rates [19] . We did not use oral ethanol gavage (binge) to avoid the possibility of compounded distress from ethanol and acute lung injury. Still, there was no doubt that mice in the present study exhibited damaged livers, as reflected in profound steatosis. As we have previously described in more detail, we used the Subunit 1 of the SARS-CoV-2 spike protein at 400 µg/kg body weight, I.T. to induce a COVID-19-like acute lung injury [12] . Similar to the previous study, transgenic mice on normal diet instilled with S1SP displayed a decline in body weight, that began recovering 48 h later. However, in alcohol-exposed mice, S1SP produced a continuously declining body weight, in agreement with a recent study where loss of body weight was significantly higher in alcohol-treated mice infected with Aspergillus fumigatus compared to similarly infected mice that did not receive alcohol [20] . This also agrees with the observation that heavy alcohol drinkers are at risk for abnormal long-term weight loss [21] .

Severe alveolar inflammation is one of the basic characteristics of ARDS associated with COVID-19. Following endothelial barrier disfunction [22] , a large number of leukocytes and plasma proteins are released into the alveolar space. S1SP could be a key factor increasing lung vascular permeability during COVID-19 [12] . The activation of pro-inflammatory transcription factors IKBα, STAT3 and NLRP3 inflammasome in the lung are likely important mediators. All these inflammatory mechanisms are enhanced by alcohol consumption. Ethanol has been reported to independently cause hyperactivation of STAT3, IKBα, and NLRP3 inflammasome both in vitro and in vivo [23] [24] [25] .

We observed monocyte, macrophage, and especially neutrophil recruitment in the BALF, and alveolar space of mice instilled with S1SP. In the intensive care units (ICU), COVID-19 patients present excessive alveolar infiltration of neutrophils [26] . Neutrophil recruitment to the focus of infection is fundamental for the fight against the invading pathogens [27] . Chronic alcohol ingestion disturbs both immunologic and nonimmunologic host defense mechanisms within the airway [28] . Neutrophil recruitment into the airways is reduced in alcohol exposed mice infected with A. fumigatus [20] . Importantly, no pathohistological differences between alcoholic and non-alcoholic groups were observed in the first two days after infection. In agreement, we observed predominantly mononuclear cell recruitment in alveoli of alcohol treated mice, receiving S1SP, in contrast to S1SP-instilled mice on normal diet who demonstrated primarily neutrophil infiltration. At the same time, spike protein-altered lung parenchymal structure was not significantly difference between mice on control and EtOH diets. Monocytes and macrophages play an important role in the pathogenesis of both alcoholic liver disease [29] and acute lung injury [30] . These cells, infected via ACE2-independent and ACE2-dependent pathways lose their ability to fight the virus and induce adaptive immune responses [31, 32] . Their impaired functions can lead to multiple organ damage, mainly due to exacerbation of ALI, provocation of a cytokine storm, and development of fibrosis [33] . Patient BALF analysis has previously shown that alcohol causes alveolar macrophage dysfunction and an alcohol-induced increase in oxidative stress [34, 35] . Here we observed hyperexpression of ACE2 in lung homogenates of K18h-ACE2 transgenic mice on alcohol diet, suggesting a yet additional mechanisms of exacerbation of COVID-19 by ethanol.

Additional evidence of the worsening of the COVID-19-related ARDS by alcohol consumption is provided by the dramatic increase of cytokine concentration in BALF. Compared to controls, mice instilled with S1SP demonstrate overexpression of cytokines, , in agreement with our previous data [12] . Here, even saline-instilled mice on ethanol diet, show significant increases in IL-6 and TNF-α compared to control mice. A few clinical studies have indicated anti-inflammatory properties of alcohol, including a reduction in IL-6, while animal studies suggest a linear relationship between alcohol drinking and IL-6 [36] . Ethanol consumption alters both IL-6 and TNF-α expression in LPS-challenged Kupffer cells [37] . Similarly, modest alcohol consumption suppresses TNF-α levels in monocytes, probably by suppressing posttranscriptional TNF-α production. However, mice who received daily 2.5 g/kg ethanol by gavage for 4 days (acute model) demonstrated increased TNF-alpha and decreased nuclear NF-kB activity in plasma, thus unleashing the apoptotic effects of TNF-alpha [38] . Here we show that the cytokine storm, associated with COVID-19-like ARDS, is more pronounced in alcohol-consuming animals. Recent meta-analysis of gene expression profiles in COVID-19 patients predicted that EtOH may augment systemic inflammation by enhancing the activity of IL-1β, IL-6, and TNF [39] . The lack of KC activation in BALF taken from S1SP mice on alcohol diet compared to S1SP mice on control diet, is consistent with the observed monocyte/neutrophil shift in BALF. This finding suggests that chronic alcohol consumption may change the immune response in ARDS. The activation of TGF-β is critical to the development of pulmonary edema in ALI and also plays an important role in the development of pulmonary fibrosis [30, [40] [41] [42] . The expression of TGF-β1, CD44v6, MMP-9, caveolin-1, and other tissue biomarkers of TGF-β signaling pathway, along with the deposition of extracellular matrix (ECM) components, collagen I, III and α-SMA, have been detected in lung sections from COVID-19 patients [43] . In the present model, alcohol did not increase the expression of TGF-β1 in mice instilled with saline but amplified it in S1SP-instilled animals.

It was previously reported that SARS-CoV-2 Spike protein leads to the induction of transcriptional regulatory molecules, such as NF-κB, and MAPK/ERK 42/44 [44] . Activation of MAPK by COVID-19 plays an important role in the survival of the virus [45] . Modulation of the MAPK pathway by alcohol is variable and depends on the organ, cell type and acute or chronic exposure, but its mechanism has been poorly studied in lungs [46] . SARS-CoV-2 endocytosis occurs through a clathrin-mediated pathway, regulated by PI3K/AKT signaling. The AKT signaling pathway was activated by the N protein of SARS-CoV in Vero E6 cells [47] . Activation of the AKT also has been linked to the induction of lung fibrosis in patients with COVID-19. Here we observed a dramatic activation of ERK 42/44 and AKT which may in part explain the associated pathologies.

In summary, our data demonstrate, that K18-hACE2 transgenic mice on an alcohol diet exhibit a more severe S1SP-induced ARDS than corresponding mice on a control diet and that overexpression of ACE2 may play a critical role in this process (Fig.11 ) It is not clear how alcohol consumption will affect the lungs in the late stages of COVID-19, especially considering that the pro-inflammatory pathways studied here are also involved in the development of pulmonary fibrosis. Thus, this model could be useful for the development of therapeutic interventions against alcohol-exacerbated COVID-19. Fig.1 . Diagram of experimental design. K18=hACE2 transgenic mice received Control or EtOH Lieber-DeCarli '82 diet 14 days before the i.t. instillation of SARS-CoV-2 S1SP or Vehicle (saline). 72 h later, mice were euthanized and lungs, liver and bronchoalveolar lavage fluid (BALF) were collected for analysis (n = 5/group). 9 . K18-hACE2 transgenic mice on alcohol diet, instilled with S1SP demonstrate activation of STAT3 (A) and IkBα (B) in lung tissue homogenates. S1SP also increased inflammasome NLRP3 expression, especially in mice on ethanol diet (C). Western blot analysis; protein band density was normalized to that of β-actin. For IkBα and STAT3 the ratio of phosphorylated to total was then calculated and all three are presented as fold of Control (VEH) (D). Means ± SEM; ***: p<0.001; **: p<0.01, *: p<0.05 with ANOVA and Tukey's, n=3-4. J o u r n a l P r e -p r o o f Fig.10 K18-hACE2 transgenic mice on alcohol diet, instilled with either vehicle or S1SP demonstrate increased phosphorylation of ERK (A) and AKT (B) in lung tissue homogenates. Western blot analysis; protein band density was normalized to that of β-actin. The ratio of phosphorylated to total was then calculated and presented as fold of Control (VEH) (C). Means ± SEM; **: p<0.01, *: p<0.05 with ANOVA and Tukey's, n=3-4. Fig. 11 . Signaling pathways in SARS-CoV-2 S1SP-(right half) and alcohol-exacerbated SARS-

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