key: cord-0739021-kcuf5x8k
authors: Sharif-Askari, Narjes Saheb; Sharif-Askari, Fatemeh Saheb; Mdkhana, Bushra; Hussain Alsayed, Hawra Ali; Alsafar, Habiba; Alrais, Zeyad Faoor; Hamid, Qutayba; Halwani, Rabih
title: Upregulation of oxidative stress gene markers during SARS-COV-2 viral infection
date: 2021-06-26
journal: Free Radic Biol Med
DOI: 10.1016/j.freeradbiomed.2021.06.018
sha: 2183ef4fc4d347824fd7a0d9d01035cb7e417aa5
doc_id: 739021
cord_uid: kcuf5x8k

Severe viral infections, including SARS-COV-2, could trigger disruption of the balance between pro-oxidant and antioxidant mediators; the magnitude of which could reflect the severity of infection and lung injury. Using publicly available COVID-19 transcriptomic datasets, we conducted an in-silico analyses to evaluate the expression levels of 125 oxidative stress genes, including 37 pro-oxidant genes, 32 oxidative-responsive genes, and 56 antioxidant genes. Seven oxidative stress genes were found to be upregulated in whole blood and lung autopsies (MPO, S100A8, S100A9, SRXN1, GCLM, SESN2, and TXN); these genes were higher in severe versus non-severe COVID-19 leucocytes. Oxidative genes were upregulated in inflammatory cells comprising macrophages and CD8(+) T cells isolated from bronchioalveolar fluid (BALF), and neutrophils isolated from peripheral blood. MPO, S100A8, and S100A9 were t/opmost upregulated oxidative markers within COVID-19's lung autopsies, whole blood, leucocytes, BALF derived macrophages and circulating neutrophils. The calprotectin's, S100A8 and S100A9 were upregulated in SARS-COV-2 infected human lung epithelium. To validate our in-silico analysis, we conducted qRT-PCR to measure MPO and calprotectin's levels in blood and saliva samples. Relative to uninfected donor controls, MPO, S100A8 and S100A9 were significantly higher in blood and saliva of severe versus asymptomatic COVID-19 patients. Compared to other different viral respiratory infections, coronavirus infection showed a prominent upregulation in oxidative stress genes with MPO and calprotectin at the top of the list. In conclusion, SARS-COV-2 induce the expression of oxidative stress genes via both immune as well as lung structural cells. The observed correlation between oxidative stress genes dysregulation and COVID-19 disease severity deserve more attention. Mechanistical studies are required to confirm the correlation between oxidative stress gene dysregulation, COVID-19 severity, and the net oxidative stress balance.

to AT1 receptors, which in turn activates NADPH oxidase, and augments production of 139 reactive oxygen species (19, 20) . Expected Cellular response to oxidative stress is 140 mediated by Nuclear factor-erythroid 2 related factor 2 (NRF2) which activate the host 141 antioxidant defense by encoding transcription of oxidative-responsive and antioxidant 142 genes which contain antioxidant response elements (AREs) including thioredoxins, 143 sestrins, and glutathione system (21-23). The protective NRF2 antioxidant signaling was 144 found to be suppressed in severe COVID-19 lung autopsies as well as SARS-COV-2 in-145 vitro infection model (24) . These findings could suggest that SARS-COV-2 target NRF2 146 as an evasion mechanism to enhance their viral survival and replication (24) . 147

Although the contribution of oxidative stress to disease pathogenesis had been explored 149

in several viral infection (28, 29) , its relevance to COVID-19 respiratory infection 150 deserves more attention (25, 26) . This is due to the fact that immune derangement 151 during SARS-CoV-2 infection could switch on a lethal cycle of oxidative stress, 152 inflammation and lung tissue injury. Therefore, the aim of the current study is to 153 evaluate the dysregulation of oxidative balance during SARS-COV-2 infection through 154 measuring the gene expression levels of 125 oxidative stress genes known to be 155 associated with proinflammatory, antimicrobial, oxidant-scavenging and apoptosis-156 inducing activities .  157   158   159   160  161  162  163  164  165  166  167  168  169  170  171  172  173  174  175  176  177  178  179  180  181  182  183  184  185  186 Method 192 193 For this study, we first established a list of 125 oxidative stress genes including: 37 pro-194 oxidant genes, 32 oxidative-responsive genes, and 56 antioxidant genes (Table 1 -3). 195

The oxidative stress genes were derived from Gene Ontology (GO) term: 0006979 196 (response to oxidative stress), WikiPathways oxidative stress database (27), and a 197 number of previous reports (28-31). The expression of these genes was evaluated 198 using publicly available transcriptomic COVID-19 whole transcriptomic and single-cell 199 datasets of samples obtained from bronchioalveolar fluid (BALF), lung autopsies, and 200 whole blood of COVID-19 patients with different disease severity. We also compared 201 between the blood oxidative stress gene expression levels of COVID-19 and three 202 respiratory infections: SARS-CoV-1, influenza (IVA), and respiratory syncytial virus 203 (RSV). These datasets were publicly available at National Center for Biotechnology 204 while burden of co-morbidity was obtained by measuring the Charlson Comorbidity 227

Index score (35). Logistic regression analysis was then used to determine the 228 independent association between expression of oxidative stress genes and COVID-19 229 disease severity. ICU admission factor was used as the dependent factor and oxidative 230 stress gene expression as independent factor. The model was adjusted for age, gender, 231 body mass index and Charlson Comorbidity Index score (35). Statistical analyses were 232 performed using R software (v 3.0.2), SPSS 25.00 (SPSS Inc., Chicago, IL, USA), and 233

Prism (v8; GraphPad Software). P-value of <0.05 considered statistically significant. 234

We also examined how SARS-COV-2 and IAV infection may regulate the expression of 235 oxidative stress genes in whole blood and lung autopsies. We, hence, reanalyzed the 236 data deposited by Daniel Blanco-Melo (GEO: GSE147507) (6) to compare the 237 expression of these genes in viral-infected lung epithelial cells compared to Mock-238 infected controls. For leucocyte datasets (GEO: GSE157103) and Daniel Blanco-Melo 239 (GEO: GSE147507)), we processed the RNAseq raw count using the Bioconductor 240 package limma-voom (36), and presented the results as log2 counts per million (log 241 CPM). Log-transformed normalized intensities were also used in Linear Models for 242

MicroArray data (LIMMA) analyses to identify differentially expressed genes between 243 diseased and control groups. Expression of oxidative stress genes is increased in blood and lung tissue during 311 SARS-CoV-2 infection relative to disease severity. 312 313

Using publicly available transcriptomic datasets, we have determined the expression 314 levels of 125 oxidative stress genes, including 37 pro-oxidant genes, 32 oxidative-315 responsive genes, and 56 antioxidant genes. The lists of these genes are presented in 316

Tables 1-3. The datasets used in this study are presented in Table 4 . Expression levels 317 of the oxidative stress genes were determined in lung autopsies and whole blood of 318 COVID-19 patients ( Figure 1 ). For whole blood, RNA-sequencing data was extracted 319 from 20 severe COVID-19 patients and 10 controls ( Figure 1A ). For lung, RNA-320 sequencing data was obtained from 9 deceased COVID-19 patients and 10 negative 321 controls (PRJNA646224) ( Figure 1B ). Twenty-six oxidative stress genes were 322 upregulated in whole blood, while only 10 genes in lung autopsies (Figure 1C ). Seven of 323 these genes were commonly upregulated in both whole blood and lung autopsies 324 ( Figure 1C ), including the following five pro-oxidants/oxidative responsive genes: 325

Myeloperoxidase (MPO), Calprotectin (S100A8/S100A9), Sulfiredoxin-1 (SRXN1), 326

Glutamate-cysteine ligase modifier (GCLM), and two antioxidant genes: Sestrin 2 327 (SESN2) and Thioredoxin (TXN) (Figure 1C) . A significant increase in lung tissue 328 expression of S100A8 (4.20.3 log-fold vs 2.70.3 log-fold; p-value=0.001) and SRXN1 329 (2.10.2 log-fold vs 1.040.4 log-fold; p-value=0.03) compared to whole blood was 330 observed ( Figure 1C ). We then determined the expression of these seven shared genes 331 in NHBE infected with IAV or SARS-COV-2 using data deposited by Daniel Blanco-Melo 332 (GEO: GSE147507) (6). Of these seven genes, SRXN1 and SESN2 were slightly 333 increased in IAV infected NHBE (n=4 IAV NHBE vs n=4 mock infected NHBE), while 334 calprotectin genes of S100A8 (log-fold of 1.90.15; p-value < 0.0001) and S100A9 (log-335 fold of 1.10.07; p-value < 0.0001) were noticeably increased in SARS-COV-2 infected 336

The in-vitro infected results are displayed in the Figure 1D . 338

To confirm this increase in expression of oxidative genes during COVID-19 infection, we 340 also determined the expression of these seven genes in a transcriptome dataset of 341 leucocyte isolated from 37 severe and 51 non-severe COVID-19 patients (GSE157103). 342

Confirming the previous results, this analysis showed a significant upregulation of the 343 selected seven oxidative stress genes in severe versus non-severe COVID-19 (Figure 344 2). To further characterize the association between expression of these genes and 345 COVID-19 severity we carried a logistic regression analysis. After adjustment with age, 346 gender, body mass index, and Charlson Comorbidity Index score, the expression of 347 these seven genes were significantly associated with COVID-19 severity and ICU 348 admission. (Table S2) . increase in antioxidants genes. Among oxidative genes, the expression of S100A8 (1.8 367 log-fold; p-value <0.0001) and S100A9 (1.5 log-fold; p-value <0.0001) was significantly 368 increased ( Figure 4A ). Further, a distinct upregulation of oxidative stress genes in BALF 369 CD8+ T cells isolated from the same severe COVID-19 patients, while these markers 370

were not changed in BALF CD8+ T cells isolated from non-severe COVID-19 patients 371 ( Figure 4B ). All fold changes presented in Figure 4A and 4B were significant with a p 372 In this study overall neutrophil counts were increased in severe COVID-19, while 379 presence of low-density neutrophils was associated with severe COVID-19 phenotype 380 and development of ARDS (40). Low-density neutrophils showed upregulation of MPO 381

(1.6 log-fold; p-value <0.0001), CYBB (1.1 log-fold; p-value <0.0001), S100A8 (0.8 log-382 fold; p-value <0.0001) and S100A9 (0.34 log-fold; p-value <0.0001) calprotectin genes. patients relative to disease severity.

We next examined whether the observed increase in these oxidative stress genes can 394 be detected in saliva of COVID-19 patients. This may hence suggest the usage of these 395 genes as non-invasive biomarkers for disease severity. To do that, we first validated the 396 increase of these markers in blood of asymptomatic and severe COVID-19 patients 397 using qRT-PCR. A significant increase in blood levels of MPO and calprotectin in severe 398 versus asymptomatic COVID-19 patients was observed ( Figure 4A ). MPO was 399 increased one log-fold more in severe versus asymptomatic (p-value= 0.0033), while 400 log-fold difference in S100A8 and S100A9 were 0.87 log-fold (p-value 0.004) and log-401 fold 0.9 (p-value=0.006), respectively. We then determined the level of these genes in 402 saliva from the same COVID19 patients ( Figure 4B ). This increase was comparable in 403 saliva compared to blood samples which may suggest that saliva level of expression of 404 these genes could reflect the level of COVID-19 severity. In severe versus 405 asymptomatic saliva, the log-fold difference was one log-fold (p-value=0.0001) for MPO, 406 2.7 log-fold (p-value<0.0001) for S100A8, and 0.3 log-fold (p-value=0001) for S100A9. We next compared the profile of enhanced oxidative stress gene expression observed 415 during SARS-CoV-2 to that detected during other respiratory viral infections. To do that, 416 we used transcriptomic microarrays and RNA-sequencing data of PBMCs isolated from 417 SARS-CoV-1, IAV, and RSV infected patients at the peak of disease. For each 418 condition, differential gene expression was obtained by comparing the normalized gene 419 expression of the infected group to those of healthy donors ( Figure 5A ). For IAV and 420 RSV infections, none of the oxidative stress genes were increased more than one log 421 fold-change (FC), while 7 genes for SARS-CoV-1 and 27 genes for SARS-CoV-2 422 infections were upregulated to a than one log FC ( Figure 5A ). TXN, QSOX1, MAPK14, 423 MPO, S100A9, and S100A8 were the top shared oxidative stress genes appearing in 424 both coronavirus respiratory infections, with an increase in expression of more than 1.5 425 folds following infection ( Figure 5B) . Herein, the dysregulation in the expression levels of 125 oxidative stress genes during 432 severe COVID-19 viral infection was explored using bioinformatic analysis of publicly 433 available transcriptomic datasets of lung autopsies, bronchioalveolar fluid, and blood 434 from SARS-CoV-2 infected individuals. Seven oxidative stress genes were found to be 435 upregulated in whole blood and lung autopsies of severe COVID-19 (MPO, S100A8, 436 S100A9, SRXN1, GCLM, SESN2, and TXN) ( Figure 1C ). Of these genes, calprotectin 437 genes, S100A8 and S100A9, were distinctly elevated in NHBE infected with SARS-438 COV-2 as compared to cells infected with IAV ( Figure 1D ). We then examined if the 439 increase in these genes was relative to disease severity. These genes were significantly 440 increased in blood leucocytes of severe compared to non-severe COVID-19 (Figure 441 S1). Using logistic regression, this association remained significant even after 442 adjustment with cofounding factors of age, gender, body mass index, and Charlson 443 Comorbidity Index score (Table S1) . Here we observed an overall dysregulation of oxidative stress genes expression in 472 circulating blood and lung tissue during severe COVID-19 disease. MPO and S100A8/9 473 calprotectin's were the top upregulated oxidative markers in lung autopsies, whole 474 blood, leucocytes, BALF derived macrophages and PBMC derived neutrophils. The 475 three top upregulated oxidative genes were validated in blood of severe COVID-19 476 patients using qRT-PCR. Relative to uninfected donor controls, MPO, S100A8 and 477 S100A9 were significantly higher in blood of severe versus asymptomatic COVID-19 478 patients. Interestingly, these three oxidative genes were also significantly upregulated in 479 saliva of severe relative to asymptomatic COVID-19 patients (Figure 4 ). This suggest 480 that the saliva level of these oxidative genes can be used as non-invasive markers for 481 COVID-19 disease severity. 482 483 S100A8 and S100A9 genes encode calcium binding proteins also known as Migration 484

Inhibitory Factor-Related Protein 8 and 14 (MPR8 and MRP14), respectively. They form 485 heterodimers known as calprotectin that binds to toll-like receptor 4 (TLR4) and function 486 as alarmin to stimulate the innate immune system pathways, namely MAP-kinase and 487 -kappa-B signaling (49, 50) . Given that, S100A8 and S100A9 have extensive effects 488 on the net inflammation, redox balance, and cell death via autophagy and apoptosis 489 (51, 52). They are also expressed abundantly in cells of myeloid origin such as 490 neutrophils and monocytes. These genes are not expressed in normal tissue resident 491 macrophages, however, S100A9 (MRP14) was found to be expressed in macrophage 492 during acute inflammation, while macrophage infiltrate during chronic inflammation 493 expressed both S100A9 and S100A8 (53). Interestingly, we found both S100A8 and 494 S100A9 to be upregulated in BALF derived macrophages of severe COVID-19 in 495 contrast to mild COVID-19 ( Figure 3A) . Recently, Silvin et al showed that elevated 496 calprotectin levels in peripheral blood cells could be used to discriminate severe from 497 mild COVID-19 infection (54). In this study, they used high-dimensional flow cytometry 498 and single-cell RNA sequencing of COVID-19 peripheral blood and suggested that high 499 calprotectin production is mediated by abnormal myeloid subsets (54). Calprotectin 500 genes are also expressed in lungs, particularly in lung epithelial and alveolar type II 501 pneumocytes (55). Likewise, the expression of these genes is increased with viral 502 infection (56) and lipopolysaccharide stimulation (57). In our study, through 503 bioinformatic analyses, we showed that S100A8 and S100A9 were expressed in HBE 504 and lung autopsies, and their expression was upregulated post SARS-COV-2 infection 505 ( Figure 1D ). The observed increase of these markers, especially in lung autopsies, 506 could be attributed to the increase in expression of these genes in both inflammatory as 507 well as structural lung cells. We then associated elevated calprotectin level to severe 508 COVID-19 (Figure 2, Figure S1 , Figure S2 , Figure 3 and Table S2 ) and validated this in 509 whole blood and saliva of COVID-19 patients (Figure 4) . 510

In both blood and lung autopsies, myeloperoxidase enzyme gene, MPO, was among 512 the top three oxidative stress genes. This gene is mainly expressed in neutrophil, and it 513 mediates catalysis of reactive oxygen intermediates such as hypohalous acids (58). 514

Oxidative stress stimulates neutrophil extracellular traps (NETs) formation by 515 neutrophils, NETosis, and lead to burst of neutrophil granules containing 516 myeloperoxidase enzyme and calprotectin which in turn boost the cellular oxidative 517 levels further. Similar to S100A8 and S100A9, myeloperoxidase expression level was 518 associated with disease severity (Figure 2 ). Supporting these findings, a recent 519 investigation by FP Veras et. al showed viable SARS-COV-2 ability to directly induce 520 the release of NETs by healthy neutrophils (2). This group observed that NETs 521 concentration was increased in plasma, tracheal aspirate and lung autopsies during 522

and COVID-19 severity (3, 4) . 524 In conclusion, SARS-COV-2 induce the expression of oxidative stress genes via both 577 immune as well as lung structural cells. Myeloperoxidase and calprotectin gene levels 578 are increased in blood, lung tissue, and inflammatory cells. In our study, these genes 579 were detected in salivary samples and hence they could potentially be used as a non-580 invasive severe COVID-19 marker. The observed correlation between oxidative stress 581 genes dysregulation and COVID-19 disease severity deserve more attention. These 582 changes in oxidative stress gene expression may or may not reflect alteration in the net 583 oxidative stress balance. It is warranted that further mechanistical studies are performed 584 to confirm this association. 585

The authors declare no competing interests. were shared between whole blood and lung autopsies. MPO, S100A8, and S100A9 were among 868 the top upregulated oxidative genes, while S100A8 and SRXN1 were higher in lung autopsies. 869

Results are presented as fold change of gene expression between cases and controls. Unpaired 870 student t-test was used to compare between fold changes in mild and severe COVID-19. * p < 871 0.05, * * p < 0.01, * * * p < 0.001, * * * * p < 0.0001. (D) The seven shared oxidative genes were 872 analyzed in SAR-COV-2 and influenza A virus infected human lung epithelial cells. Independent 873 biological replicates of primary human lung epithelium (NHBE) were mock treated or infected 874 with SARS-CoV-2 (USA-WA1/2020), or IAV (A/Puerto Rico/8/1934 (H1N1)). Of these seven 875 genes, SRXN1 and SESN2 were slightly increased in IAV infected NHBE (n=4 IAV NHBE vs 876 n=4 mock infected NHBE, GEO: GSE147507), while calprotectin genes of S100A8 and S100A9 877 were noticeably increased in SARS-COV-2 infected NHBE (n=3 SARS-CO-V-2 NHBE vs n=3 878 patients relative to disease severity. (A) Gene expression level of MPO, S100A8 and S100A9 902 was higher in blood from severe COVID-19 (n=7) as compared to asymptomatic COVID-19 903 (n=9). (B) Gene expression level of MPO, S100A8 and S100A9 was higher in saliva from severe 904 COVID-19 (n=10) as compared to asymptomatic . Results are presented as log2 905 fold change. Unpaired student t-test was used to compare between the independent groups. * p < 906 0.05, * * p < 0.01, * * * p < 0.001, * * * * p < 0.0001. 907 

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Correlates With Poor Prognosis of Severe Influenza A Infection

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The expression of MPO, S100A8, and S100A9 were higher in severe compared to mild

Results are presented as fold change of gene expression between cases and 925 controls

The sample size presented in A-C belonged to EGAS00001004503 928 dataset and were as following

Single-cell gene expression from PBMCs of severe COVID-19 patients

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Model-based analysis of single cell transcriptomics (MAST) algorithm in Seurat v3 was used by 937 authors to identify differentially expressed genes and to determine the fold change

Prominent upregulation of S100A8 and S100A9 in M1 macrophage group. Macrophages were 891 clustered into four groups; M1 macrophages were presented with group 1 and 2, while M2 892 macrophages were presented with group 3. Both M1 and M2 like macrophages were enriched in 893 severe COVID-19. Group 4 macrophages were predominant in moderate and healthy controls 894 and presented the less severe COVID-19. Fold changes were generated for each group of 895 macrophages relative to total macrophages. (B) Specific upregulation of oxidative stress genes in 896 CD8+ T cells from severe COVID-19 patients, while none of the oxidative genes appeared in the 897 non-severe COVID-19 cluster. All fold changes presented in the figure were significant with a p 898 value <0.05. 899 900