key: cord-0906167-whloioyi authors: Kaur, G.; Yogeswaran, S.; Muthumalage, T.; Rahman, I. title: Persistently increased systemic ACE2 activity and Furin levels are associated with increased inflammatory response in smokers with SARS-CoV-2 COVID-19 date: 2021-01-15 journal: nan DOI: 10.1101/2021.01.14.21249836 sha: 7c71ee9ac569ba2dfae86698790ed07b11789305 doc_id: 906167 cord_uid: whloioyi Tobacco smoking is known to be involved in the pathogenesis of several cardiopulmonary diseases, and smokers are susceptible to infectious agents. However, the progression of lung injury based on COVID-19 susceptibility and severity amongst smokers and those with pre-existing pulmonary diseases is not known. We determined the systemic expression and activity of COVID-19 related proteins, cytokine, chemokines, and lipid mediators (lipidomics) amongst COVID-19 patients with and without a history of smoking with a view to define biomarkers. We obtained serum from COVID-19 positive and COVID-19 recovered patients with and without a history of smoking. We conducted a Luminex multiplex assay (cytokine levels), LC-MS (eicosanoids or oxylipin panel) and enzymatic activity assays on the serum samples to study the systemic changes in COVID-19 patients. On comparing the cytokine profiles among COVID-19 positive and COVID-19 negative patients, we found a significant upregulation in the production of pro-inflammatory cytokines like IL-1beta, IL-8, IL-2, VEGF and IL-10 in COVID-19 positive patients as compared to the respective controls. Interestingly, smoking history resulted in further augmentation of the release of some hyper-inflammatory cytokines, like IFN-gamma, Eotaxin, MCP-1 and IL-9 amongst COVID-19 positive patients. The enzymatic activity for ACE2, the binding partner for SARS-CoV2 virus in the host cell, was found to be significantly increased in the serum of patients with a smoking history compared to the serum collected from the non-smoking controls. Similarly to our cytokine analysis, our measurement of serum Furin levels was also affected by the patients smoking history, in which we reported a substantial rise in serum Furin levels of COVID-19 patients. The analysis of lipid mediators revealed a distinct signature amongst the COVID-19 positive versus recovered subjects in PGF2alpha, HETEs, LXA4 and LTB4 levels. However, we did not find any changes in the levels of any lipid mediators based on the smoking history of the patients. Overall, our results point towards distinct systemic signatures amongst COVID-19 positive patients. We also show that smoking adversely affects the systemic levels of inflammatory markers and COVID-19 related proteins, thus suggesting that COVID-19 infection may have severe outcomes amongst smokers which is reflected systemically. The current pandemic of coronavirus disease 2019 (COVID-19) has emerged as a major 72 public health threat worldwide. Viral pneumonia and acute respiratory failure are the most 73 common clinical manifestations of severe COVID-19, featuring fever, cough, hypoxemia, 74 dyspnea, and bilateral infiltrates on chest radiography (1) . ACE2 (angiotensin converting 75 enzyme 2) receptor is the viral binding site for the SARS-CoV2 in human. ACE2 is 76 abundantly expressed in the lung epithelium, specifically type II pneumocytes, goblet 77 circumstances, ACE2 converts angiotensin 2 to metabolites, many of which exert 79 vasodilatory properties. However, since the onset of this pandemic, ACE2 is being widely 80 studied in the context of COVID-19. In this regard, it is still mostly unknown the specific 81 nature of the change in ACE2 induced by SARS-CoV-2 infection, whether it's the 82 modulation of ACE-2 activity or its expression levels that have significance in affecting 83 COVID-19 disease outcome. 84 85 With continual spreading of the virus and reports of a novel, mutant strain causing further 86 alarm and panic, questions regarding COVID19 risk-factors have become even more 87 urgent and a cause for concern. While old age, heart disease and diabetes are the 88 universally accepted risk factors for COVID19, there are many other factors that are 89 subject to debate and require empirical analysis. One such risk factor is smoking. While 90 initial reports on COVID-19 risk factors have indicated little to no risk amongst smokers, 91 recent data suggests otherwise (2, 5). A meta-analysis of 15 studies with a total of 2473 92 confirmed COVID-19 patients reported that COPD patients (63% vs 33.4 in people 93 without COPD) and current smokers (22%) are at a higher risk of severity and mortality 94 due to COVID-19 compared to disease-free and non-smoking individuals respectively 95 (95% confidence interval)(6, 7). Similar findings were reported by Patanavanich et al 96 (2020) , whose research suggested that smoking nearly doubles the rate of COVID-19 97 progression amongst patients (8). Despite these findings, the exact pathogenesis of 98 COVID-19 and the clinical features resulting in severe outcomes amongst smokers is 99 largely unexplored. 100 In consideration of this lack of research and the growing call of concern for further 102 research into COVID-19 risk-factors, we investigated whether a variation in systemic 103 markers for inflammation and COVID-19 infection existed between smokers and non-104 smokers. We also studied the gender-based differences in the expression and activity of 105 COVID-19 related proteins (ACE2 and Furin) to understand the progression of the 106 disease amongst both sexes. Evidence from previous literature has suggested 107 upregulated levels of ACE-2 in the lungs of smokers. However, there is no evidence 108 correlating this increased expression to COVID-19 disease development and severity. 109 Our study investigates the relationship between COVID-19 biomarkers and disease 110 severity and shows a marked increase in the inflammatory spillover amongst COVID-19 111 positive patients with a smoking history compared to controls. We were also able to show 112 a pronounced increase in cytokine/chemokine and Furin (S1/S2 cleavage protein) levels 113 (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted January 15, 2021. ; https://doi.org/10.1101/2021.01.14.21249836 doi: medRxiv preprint Assessment of pro-inflammatory mediators in blood sera using Luminex multiplex 140 assay 141 The levels of pro-inflammatory cytokines/chemokines like MCP-1, IL-8, IFN-, TNF- and 142 IL-7 in the sera were measured by Luminex multiplex assay using Bio-Plex Pro™ Human 143 cytokine 27-plex assay (Cat#M500KCAFOY, BIO-RAD, Hercules, CA) as per 144 manufacturer's directions. Blood plasma was diluted two-folds and the levels of 27 pro-145 inflammatory mediators (expressed as pg/ml) were measured using Luminex FlexMap3D 146 system (Luminex, Austin, TX). 147 To determine the level of Furin in sera collected from COVID-19 positive and negative samples, in addition to the appropriate standards and controls (positive, negative, and 160 background), were then added to a 96-well plate. After that, 50 ul of ACE2 substrate was 161 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted January 15, 2021. ; https://doi.org/10.1101/2021.01.14.21249836 doi: medRxiv preprint added to both sample and control wells, and subsequently, fluorescence was measured 162 at an excitation maximum of 320 nm and an emission maximum of 420 nm using Cytation 163 5 microplate reader (BioTek Instruments, Inc. Winooski, VT). Total protein content per 164 sample was determined using the Bradford protein assay kit (Thermo Fisher, Waltham, 165 MA). Sample ACE2 activity for each sample was calculated using the following formula: Lipid extraction: Lipid extraction from the serum samples was performed by protein 182 precipitation followed by solid-phase extraction (SPE). Protein precipitation was 183 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted January 15, 2021. ; https://doi.org/10.1101/2021.01.14.21249836 doi: medRxiv preprint performed by addition of 50 µL H2O: Acetonitrile solution to each sample. Thereafter, 184 SPE was performed using Strata-X cartridges (33 μm, 200 mg/10 mL; Phenomenex, PA). 185 The extracted lipids were finally dissolved in 100 µL water/acetonitrile 60:40 (v:v) solution. 186 To prepare the calibration curves, a mixture of the 20 calibration standards was prepared 187 in methanol. All statistical calculations were performed using GraphPad Prism 8.0. Data is expressed 197 as mean ± SE. Pairwise comparisons were done using unpaired Student's t-test. 198 Differences were considered statistically significant at *p < 0.05, **p < 0.01, and *** p < 199 0.001 when compared with respective controls. Since in humans, ACE2 binds to the SARS-CoV2 spike protein, we investigated changes 205 in COVID-19 positive and COVID-19 negative serum ACE2 activities. To our surprise, we 206 found a significantly higher ACE2 activity in serum from COVID-19 negative patients 207 compared to serum from COVID19 positive patients. (Fig 1a) . This could have occurred 208 as in this study the COVID-19 negative patient sera actually belong to COVID-19 209 recovered patient group. As expected, after repeating these experiments on blood plasma 210 samples from COVID-19 positive and healthy subjects, we found a significant increase in 211 ACE2 activity in the COVID-19 positive samples compared to healthy controls (data not 212 shown). 213 214 However, it was interesting to find a significant increase in ACE2 activity in serological 215 samples from COVID-19 patients (current and recovered) with a smoking history as 216 compared to non-smokers (Fig 1b) . In fact, ACE2 activity was found to be more 217 pronounced among male patients compared to females (Fig 1c) . Our results show that 218 age and smoking status play a crucial role in governing the COVID-19 related enzyme 219 activity in human subjects, thereby affecting the disease pathogenesis amongst Another key to understanding COVID-19 virulence as a function of susceptibility to viral 226 entry is analyzing changes in Furin levels. Unlike other Coronaviruses, SARS-CoV-2 has 227 a lessened dependence on target host cell proteases and depends more on proprotein 228 convertase Furin for its viral entry. Based on this, we measured Furin-levels in patient 229 sera from COVID-19-positive and COVID-19 negative (COVID-19 recovered) groups 230 using ELISA. We observed a marked increase in the serum Furin levels (p=0.083) in 231 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted January 15, 2021. ; https://doi.org/10.1101/2021.01.14.21249836 doi: medRxiv preprint COVID-19 positive patients as compared to controls (Fig 2a) . A significant upregulation 232 of Furin levels was noted among smokers compared to non-smokers among sera 233 collected from COVID-positive (current and previous) patients (Fig 2b) . Similarly, 234 although not significant, Furin levels among male patients were elevated compared to 235 females; this suggests gender-based variations in the Furin expression on SARS-CoV2 236 infection. (Fig 2c) . Our results showed significant increases in the levels of IL-8, IL-10, IL-2, VEGF and IL-246 1(p= 0.0616)in COVID-19 positive patient sera compared to healthy controls (Fig 3) . 247 Parameters such as, IL-5, GM-CSF, IL-12(p70), and IL-15 were undetected in the patient 248 sera from diseased and normal subjects. 249 250 Intriguingly, when analyzing cytokine/chemokine levels in patient sera based on smoking 251 status, a unique trend emerged. We noted a substantial increase in the production of 252 pro-inflammatory markers like, IFN- (p=0.0836), MCP-1 and Eotaxin in the COVID-19 253 positive patient sera who had a smoking history compared to the non-smoking controls. 254 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted January 15, 2021. ; https://doi.org/10.1101/2021.01.14.21249836 doi: medRxiv preprint Furthermore, we found a moderate increase in the levels of IL-9 (p=0.0991) amongst 255 smokers infected with COVID 19compared to non-smokers (Fig 4) . These results support 256 our hypothesis and further elucidate the role smoking has in exacerbating both disease 257 severity and outcome with regards to SARS-CoV-2 virulence amongst smokers. it is important to recognize the high-risk populations and the underlying differences in the 276 disease pathogenesis to limit the viral spread in an efficient manner. In this regard, we 277 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted January 15, 2021. find as it shows that ACE2 activity remains persistently elevated even after recovery and 292 might have important implications for future research. Importantly, ACE2 is expressed in 293 various tissues including the heart, kidney, lungs; and can undergo shedding into the 294 blood circulation. Reports suggest that circulating ACE2 activity is indicative of adverse 295 cardiovascular outcomes in patients with hypertension, coronary artery disease and aortic 296 stenosis (10-12). It is also known to have a negative correlation with the glomerular 297 filtration rate in type 1 diabetes (13, 14) . Considering this, our results suggest 298 dysregulated shedding of active ACE2 even after recovery amongst COVID-19 patients 299 which may have adverse effects in the long run and is subject to further investigation. 300 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted January 15, 2021. ; https://doi.org/10.1101/2021.01.14.21249836 doi: medRxiv preprint Interestingly, we found that the ACE2 activity was significantly higher for individuals with 301 a smoking history, thus suggesting that smoking can greatly affect the disease severity 302 and outcomes amongst COVID-19 patients. As has been reported previously (15), our 303 data also shows gender-based upregulation of ACE2 activity amongst males as 304 compared to females. This could correlate to the increased morbidity and mortality 305 amongst male patients and must be interesting to study in future. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted January 15, 2021. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted January 15, 2021. ; https://doi.org/10.1101/2021.01.14.21249836 doi: medRxiv preprint heterogeneous demographics to deduce better conclusions. Also, we intend to use (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted January 15, 2021. ; https://doi.org/10.1101/2021.01.14.21249836 doi: medRxiv preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted January 15, 2021. ; https://doi.org/10.1101/2021.01.14.21249836 doi: medRxiv preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted January 15, 2021. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted January 15, 2021. ; https://doi.org/10.1101/2021.01.14.21249836 doi: medRxiv preprint Cytokines not detected IL-5, GM-CSF, IL-12(p70) and IL-15 545 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted January 15, 2021. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted January 15, 2021. ; https://doi.org/10.1101/2021.01.14.21249836 doi: medRxiv preprint represents the individual sample being tested. Lipid abundance ratios are colored 557 according to the fold changes and the color key indicates the magnitude of log2 fold 558 change. 559 560 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted January 15, 2021. ; https://doi.org/10.1101/2021.01.14.21249836 doi: medRxiv preprint Respiratory System Disorders: Current Knowledge Arteriosclerosis, thrombosis, and vascular biology SARS-CoV-2 COVID-19 susceptibility and lung 389 inflammatory storm by smoking and vaping CoV-2 Receptor ACE2 Is an Interferon-Stimulated Gene in Human Airway Epithelial Cells and Is 393 Detected in Specific Cell Subsets across Tissues SARS-CoV-2 395 entry factors are highly expressed in nasal epithelial cells together with innate immune genes. 396 Nature medicine CoV-2 and COVID-19: A review of reviews considering implications for public health policy and 399 practice Severity and Mortality associated with COPD and Smoking in patients with COVID-402 19: A Rapid Systematic Review and Meta-Analysis Smoking, ACE-2 and COVID-19: ongoing controversies. The 404 European respiratory journal Smoking Is Associated With COVID-19 Progression: A Nicotine & tobacco research : official journal of the Society for Research on 407 Nicotine and Tobacco Emergence of a Highly Fit SARS-CoV-2 Variant Circulating ACE2: a novel biomarker of cardiovascular risk Circulating 421 angiotensin converting enzyme 2 activity as a biomarker of silent atherosclerosis in patients with 422 chronic kidney disease Circulating plasma concentrations of angiotensin-converting enzyme 2 in men and women with 425 heart failure and effects of renin-angiotensin-aldosterone inhibitors The role of furin cleavage site in SARS-428 CoV-2 spike protein-mediated membrane fusion in the presence or absence of trypsin. Signal 429 transduction and targeted therapy Furin Protease: From SARS CoV-2 to Anthrax, Diabetes, and 431 Hypertension. The Permanente journal Structure, Function, 433 and Antigenicity of the SARS-CoV-2 Spike Glycoprotein Clinical features of patients infected 435 with 2019 novel coronavirus in Wuhan COVID-19: A New Virus, but a Familiar Receptor and Cytokine 438 Release Syndrome We thank Dr. Shikha Sharma for technical assistance.