Cutting Edge
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Cutting Edge: Severe SARS-CoV-2 Infection in Humans Is Defined by a Shift in the Serum Lipidome, Resulting in Dysregulation of Eicosanoid Immune Mediators Benjamin Schwarz,*,1 Lokesh Sharma,†,1 Lydia Roberts,*,1 Xiaohua Peng,† † ‡ † Santos Bermejo, x Ian Leighton,* Arnau Casanovas-Massana, Maksym Minasyan, Shelli Farhadian, Albert I. Ko,‡ Yale IMPACT Team,2 Charles S. Dela Cruz,†,1 and Catharine M. Bosio*,1 The COVID-19 pandemic has affected more than and converted via enzymatic hydroxylation to immune regulating 20 million people worldwide, with mortality exceeding lipid mediators (LMs) (1–7). LMs function as inflammatory, 800,000 patients. Risk factors associated with severe immune-regulatory, or proresolving immune signals during disease and mortality include advanced age, hyperten- chronic and acute immune responses (1, 8). Previous studies have sion, diabetes, and obesity. Each of these risk factors demonstrated that comorbidities associated with severe COVID- pathologically disrupts the lipidome, including immu- 19 including obesity, hypertension, diabetes and heart disease nomodulatory eicosanoid and docosanoid lipid media- feature pathological disruption of the lipidome including altered tors (LMs). We hypothesized that dysregulation of LMs baseline levels of LMs and the PUFA-containing LM precursors may be a defining feature of the severity of COVID-19. in absence of infection (9–13). Several studies have examined By examining LMs and polyunsaturated fatty acid pre- systemic metabolic correlates of COVID-19, including shifts in cursor lipids in serum from hospitalized COVID-19 pa- the lipidome (14–17) (J. Troisi et al., Research Square, 2020, https://doi.org/10.21203/rs.3.rs-34085/v1). Collectively, these tients, we demonstrate that moderate and severe disease studies demonstrated that severe COVID-19 is marked by a are separated by specific differences in abundance of systemic dysregulation of metabolism and widespread changes in immune-regulatory and proinflammatory LMs. This thelipidomesuggestingthepotential for dysregulation of LMs. difference in LM balance corresponded with decreased However, the specific behavior of LMs and their implications for LM products of ALOX12 and COX2 and an increase disease severity remains unexplored in the context of COVID-19. LMs products of ALOX5 and cytochrome p450. Given Based on these previous results, we hypothesized that severe the important immune-regulatory role of LMs, these COVID-19 is associated with a disrupted balance of LMs po- data provide mechanistic insight into an immuno- tentially as a result of the associated metabolic comorbidities. In lipidomic imbalance in severe COVID-19. The this study, we characterize a dysregulation of the LM lipidome, Journal of Immunology, 2021, 206: 329–334. including the PUFA-containing LM precursors, in severe COVID-19 and identify new potential targets for modulating the ipids function in disease to rearrange cellular signaling aberrant immune response that results in morbidity. structures, modify metabolic processes, absorb reactive L species, and act directly as both autocrine and endo- Materials and Methods crine ligands in the regulation of the immune system. In the Ethics statement context of an immune insult, eicosanoid and docosanoid poly- This study was approved by Yale Human Research Protection Program unsaturated fatty acids (PUFA) are liberated from glycerolipids Institutional Review Boards (FWA00002571, Protocol ID. 2000027690). *Laboratory of Bacteriology, National Institute of Allergy and Infectious Diseases, Na- of Internal Medicine at the Yale School of Medicine, the Yale School of Public Health, tional Institutes of Health, Hamilton, MT 59840; †Section of Pulmonary and Critical and the Beatrice Kleinberg Neuwirth Fund. L.S. is supported by Parker B. Francis Care and Sleep Medicine, Yale University School of Medicine, New Haven, CT 06520; Fellowship 0000. C.S.D.C. is supported by Veterans Affairs Merit Grant BX004661 ‡Department of Epidemiology of Microbial Diseases, Yale School of Public Health, New and U.S. Department of Defense Grant PR181442. x Haven, CT 06520; and Section of Infectious Diseases, Department of Medicine, Yale Address correspondence and reprint requests to Dr. Charles S. Dela Cruz or Dr. Catharine University School of Medicine, New Haven, CT 06520 M. Bosio, Section of Pulmonary and Critical Care and Sleep Medicine, Yale Univer- 1B.S., L.S., L.R., C.S.D.C., and C.M.B. contributed equally. sity School of Medicine, 300 Cedar Street, TACS441D, New Haven, CT 06520 2 All authors appear at the end of the article. (C.S.D.C.) or National Institute of Allergy and Infectious Diseases, National Institutes of Health, 903 South 4th Street, Hamilton, MT 59840 (C.M.B.). E-mail addresses: ORCIDs: 0000-0002-2973-2390 (X.P.); 0000-0003-0078-1989 (S.B.); 0000-0002-3301- [email protected] (C.S.D.C.) or [email protected] (C.M.B.) 6143 (A.C.-M.); 0000-0001-9023-2339 (A.I.K.); 0000-0002-5258-1797 (C.S.D.C.). The online version of this article contains supplemental material. Received for publication September 10, 2020. Accepted for publication November 11, Abbreviations used in this article: BMI, body mass index; COX, cyclooxygenase; CYP, 2020. cytochrome p450; LC-MS/MS, liquid chromatography tandem mass spectrometry; LM, This work was supported by the Intramural Research Program of the National Institute lipid mediator; PUFA, polyunsaturated fatty acid; Rv, resolvin. of Allergy and Infectious Diseases, National Institutes of Health (U19 supplement AI089992-09S2 to A.I.K. and C.S.D.C.). This work was supported by the Department Copyright Ó 2021 by The American Association of Immunologists, Inc. 0022-1767/21/$37.50 www.jimmunol.org/cgi/doi/10.4049/jimmunol.2001025 330 CUTTING EDGE: DYSREGULATION OF EICOSANOIDS IN COVID-19 Informed consents were obtained from all enrolled patients. Samples from (severe). Serum draws from healthy patients without known healthy patients were from frozen banked donations which were obtained comorbidities were collected prior to the COVID-19 pan- under the protocol (HIC 0901004619; CPIRT) before the onset of COVID-19 outbreak. demic, to avoid any contribution of asymptomatic or con- valescent cases in this dataset. The demographics, preexisting Patient cohort and serum collection conditions, and treatment details of these patients are indicated COVID-19 patients were recruited among those who were admitted to the in Supplemental Table I. To ensure recovery of both lipids Yale-New Haven Hospital between March 18, 2020, and May 9, 2020 and and polar metabolites a modified chloroform extraction was were positive for SARS-CoV-2 by RT-PCR from nasopharyngeal and/or used and measurements were made using a series of targeted oropharyngeal swabs and displayed respiratory symptoms. Patients in this study were enrolled through the IMPACT biorepository study after obtaining liquid chromatography tandem mass spectrometry (LC-MS/ informed consent. Samples were collected an average of 3 6 1 and 4 6 2d MS) methods providing high-confidence feature identification postadmission for moderate and severe groups, respectively (Supplemental (20, 22, 23). Table I). Control biospecimens are from healthy subjects without known inflammatory or lung disease or comorbidities from our Yale CPIRT bio- Changes in primarily polar metabolites, including methanol- repository. Basic demographics and clinical information of study participants soluble polar lipids, among COVID-19 patient cohorts from were obtained and shown in Supplemental Table I. Following collection, all China, Italy, and France have been reported (14–17). In 2 samples were immediately frozen at 80˚C. Prior to thawing, all samples agreement with those studies, we observed a dysregulation of including healthy controls were g-irradiated (2 Mrad) to inactivate potential infectious virus. amino acids and lactate and dysregulation of nucleotide cata- bolic products such as xanthine, hypoxanthine, and urate Sample processing for aqueous, organic, and LM extraction (Supplemental Fig. 1A) (14). Among lipid species, separation Liquid chromatography mass spectrometry grade solvents were used for all liquid of disease from healthy and severe from moderate disease was chromatography mass spectrometry experiments. Sample order was randomized marked by changes in sphingomyelins, lysophospholipids, for each extraction. For aqueous and organic metabolites samples were extracted using a modified Bligh and Dyer extractions with details in B. Schwarz et al., cholesterol esters and multiple classes of glycerophospholipids medRxiv, 2020 (https://doi.org/10.1101/2020.07.09.20149849) (18). in agreement with previous studies (Fig. 1A, 1B, Supplemental Fig. 1B–M) (14–17). LMs sample processing and extraction To pursue our hypothesis that dysregulation of PUFA- LMs were extracted from patient serum as previously described with modifi- containing lipids and immune-regulatory LMs may contrib- cations detailed in B. Schwarz et al., medRxiv, 2020 (https://doi.org/10.1101/ ute to the inflammatory progression of severe COVID-19, 2020.07.09.20149849) (19). we specifically interrogated patterns of LM precursors Liquid chromatography tandem mass spectrometry analysis among the portion of lipids that significantly varied be- Aqueous metabolite, lipid, and LM samples were analyzed using a series of tween any two groups in the cohort. Disease groups contained targeted multiple-reaction monitoring methods. All samples were separated lowered levels of PUFA-containing phosphatidylcholine, using a Sciex