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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 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, , and . Each of these risk factors demonstrated that comorbidities associated with severe COVID- pathologically disrupts the lipidome, including immu- 19 including obesity, , 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, ; 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 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 ExionLC AC system and analyzed using a Sciex 5500 QTRAP PUFA-containing phosphatidylserine, and PUFA-containing mass spectrometer. All methods were derived from previously published phosphatidylethanolamine-plasmalogen and increased levels of methodology with modifications detailed in B. Schwarz et al., medRxiv, 2020 (https://doi.org/10.1101/2020.07.09.20149849) (19, 20). PUFA free fatty acids as well as PUFA-containing phospha- tidylethanolamine, PUFA-containing lysophospholipids, Single-cell RNA sequencing analysis and PUFA-containing triacylglycerols compared with healthy The published single-cell RNA sequencing dataset from Wilk et al. (21) was controls (Fig. 1C–F). These PUFA patterns were exacerbated interrogated using Seurat v3.0. in severe patients compared with moderate patients. Of these Statistical analysis PUFA-containing lipids, plasmalogen was of particular interest as a primary pool of PUFAs for the generation of LMs in both Demographic data are presented as either counts and percentages (for cate- immune and structural cells (24, 25). This shift in PUFA pools gorical data) or means and standard deviations (for continuous data). To investigate the difference in the control, moderate and severe groups, GraphPad strongly suggested differential mobilization of PUFAs for the Prism (version 8.4.2) was used. The results were compared using the x2 test or generation of LMs between moderate and severe COVID-19. Fisher exact test for categorical variables and one-way ANOVA or unpaired To assess LMs directly, we targeted 67 LMs species using t test was used for continuous variables with significance set at p # 0.05. Univariate and multivariate analysis was performed in MarkerView Soft- LC-MS/MS with comparison with standards and available ware 1.3.1. The aqueous dataset and the combined LMs/cytokine dataset data spectral libraries (https://serhanlab.bwh.harvard.edu/). Prin- were autoscaled prior to multivariate analysis to visualize the contribution of ciple component analysis of the LM lipidome showed sepa- low ionization efficiency species. Lipid datasets were pareto scaled to avoid ration of infected and healthy cohorts and between moderate overrepresenting low abundance signals within each lipid class. For all datasets, a missing value cut-off of 50% and a CV of 30% were used as filters. For all and severe patients (Fig. 2A). Nearly all LMs measured were univariate analyses, an unpaired t test was used, and a Benjamini–Hochberg positively correlated with infection and moderate and severe correction with a false discovery of 10% was used on the combined polar disease were characterized by unique profiles of LMs (Fig. 2B, metabolite and lipidomic datasets to correct for multiple comparisons. Ad- justed p value cutoff levels were moderate versus healthy, 0.050; severe versus Supplemental Fig. 1N–P). Of particular interest were LMs healthy, 0.032; and severe versus moderate, 0.017. present at lower levels in the severe group compared with the moderate group as these molecules behave counter to the Results and Discussion pattern of increased immune activity expected in the severe To measure differences in LM profiles between moderate and disease. Specifically, moderate disease was characterized by severe cases of COVID-19 and gain mechanistic insights into significantly higher levels of the proresolving LM resolvin how these changes may drive disease severity, we used serum (Rv) E3 and a trend toward increased presence of the PG draws from 19 healthy patients (healthy), 18 hospitalized family members, particularly PGE2 (p = 0.051), PGD2 COVID-19 patients with respiratory symptoms who did not (p = 0.220) and PGF2a (p = 0.242). In contrast, severe disease require ICU admission (moderate), and 20 patients with re- was characterized by a significant increase in free PUFAs levels, spiratory symptoms of a severity that required ICU admission monohydroxylated species, and AA-derived dihydroxylated The Journal of Immunology 331

FIGURE 1. Mobilization of plasmalogen-derived PUFAs correlates with the disease severity in COVID-19. (A) Supervised partial least-squares discriminant analysis (PLSDA) of the healthy, moderate, and severe disease groups and (B) the corresponding feature loading plot. (C–F) Heatmap of the autoscaled mean intensity of each patient group for significantly varied lipids containing C20:4 (C), C20:5 (D), C22:5 (E), and C22:6 (F) between one or more groups adjusted for a false discovery rate (FDR) of 10% because of multiple comparisons. Color scale is consistent for (C)–(F). species (Supplemental Fig. 1P). Multiple DHA- and AA- that moderate disease was characterized by higher levels of derived proresolving LMs also trend upward in severe disease LMs that require cyclooxygenase (COX) activity as well as (RvD3, p = 0.063) but do not reach significance. certain EPA products of ALOX12, whereas severe disease was LMs are generated by a single or a series of characterized by LMs that require activity of ALOX5 and mediated conversions of the parent PUFA. To examine the cytochrome p450 (CYP) . This is in good agreement potential contribution of each oxygenase to the severe with previous observations from influenza, which associated disease phenotype, we grouped LMs according to synthesis symptom severity with ALOX5 activity (26). pathway (Fig. 2C–G). Several LMs are shared between mul- Elevation of ALOX5- and CYP-dependent LMs in severe tiple enzyme groups as they require sequential stereospecific COVID-19 patient sera suggested upregulation of these hydroxylations by multiple enzymes. This grouping revealed pathways but provided no data as to the origin of these LMs.

FIGURE 2. A unique milieu of LMs defines moderate and severe COVID-19 disease. (A) Unsupervised principle component analysis (PCA) of autoscaled LMs and (B) corresponding feature loading plot. (C–G) Heatmaps of the autoscaled mean for each patient group across molecules synthesized by ALOX5 (C), ALOX12 (D), ALOX15 (E), COX (F), or CYP (G). Color scale is consistent across (C)–(G). 332 CUTTING EDGE: DYSREGULATION OF EICOSANOIDS IN COVID-19

To begin to examine potential cellular origins of these enzymes not all members reaching significance. Among comorbidities, in COVID-19 patients, we interrogated a published single- body mass index (BMI) most closely reflected the LM pattern cell RNA sequencing dataset of severe COVID-19 patient of disease severity with all but three LMs sharing a similar co- PBMCs (Fig. 3A) for expression of ALOX and CYP efficient direction and magnitude as disease severity (Fig. 4A). (21). CYP expression was not detected (data not shown), Hypertension and heart disease reflected the pattern of disease whereas ALOX5 expression was detected in multiple cell types severity but not to the degree of BMI. Of note, PGs (PGE2, (Fig. 3B). ALOX5 was significantly increased in PGD2 and PGF2a) and RvE3 were negatively correlated with and trended upward in CD14 monocytes, CD16 monocytes, disease severity, BMI, hypertension, and heart disease. Neither and developing neutrophils (a population found almost ex- age, gender, nor presence of diabetes correlated well with the clusively in diseased individuals) from severe COVID-19 pattern of LM dysregulation observed in severe cases of patients compared with healthy controls (Fig. 3C). Al- COVID-19. This correlative analysis supports the association though expression is important, ALOX enzymes are primarily of the specific LM dysregulation in severe disease with known regulated by subcellular location and pathways involving LM comorbidities, in particular high BMI. production are intercellular highlighting the importance of The downward trend of PGs in severe disease and the strong ALOX expressing cell numbers and not just expression levels negative correlation of this family of LMs with COVID-19 in these pathways (27, 28). Interestingly, severe COVID-19 is comorbidities suggested a role for these molecules in the characterized by elevated ALOX5 expressing monocyte/macrophage differential immune response between moderate and severe population and depletion of lymphocyte populations (21, 29, disease. Products of COX, including the PGs, have been shown 30). The absence of CYP in peripheral blood to play complex roles in regulating initiation and suppression is consistent with the primarily hepatic localization of these of elements of inflammation in viral infection (33). In the enzymes (31). Taken together, these data support a systemic context of COVID-19, the potentially negative effects of both increase in ALOX5 activity in severe COVID-19 and provide inhibitors of COX and the COX thromboxane a plausible foundation for the data in this study wherein (TXB2) in severe disease and death have been discussed, albeit ALOX5 products separate moderate and severe disease (32). inconclusively, further highlighting the complexity and mul- COVID-19 comorbidities are known to pathologically tifunctional nature of this pathway (34, 35). In this study, disrupt the homeostatic lipidome and may predispose patients lower levels of PGs, in the context of hospitalized COVID-19 to the different signatures of LMs seen in this study. To ex- patients, were associated with higher disease severity, but amine this potential association further, we correlated the LM further investigation is needed to understand the mechanistic dataset with the severity of disease, comorbidities, and de- role PGs play in COVID-19 progression. mographics (Fig. 4). Thirteen LMs were significantly (p # It remains unclear whether these LM patterns represent 0.05) correlated with disease severity and several families of drivers of disease, arise from preexisting conditions, or LMs group together in the positive and negative direction COVID-19 related treatment. LMs operate as part of an ex- including COX2 and ALOX12 products in the negative di- pansive immune signaling network and broadly deconvoluting rection and ALOX15 and CYP in the positive direction despite their role from other immune ligands will require controlled

FIGURE 3. Human PBMCs from COVID-19 patients are enriched for ALOX5 expressing cells and express higher levels of ALOX5.(A) UMAP dimensionality reduction plot of a published human PBMC single-cell RNA Seq dataset (21) identifying 20 cell types. (B) UMAP depicting ALOX5 expressing cells in blue. (C) Violin plots indicating ALOX5 expression levels within specific cellular populations in healthy (blue) or COVID (red) PBMCs. *p , 0.05, **p , 0.01, determined by a Mann–Whitney U test. The Journal of Immunology 333

disease severity groups is likely to be affected by different starting levels of both LMs and LM precursors. Differences in LM levels could also arise from treatment differences. Un- fortunately, in this cohort, therapies were either overrepre- sented or underrepresented to an extent that prevented correlative analysis and these therapies remain potentially confounding factors. Together, the results presented in this study provide the first, to our knowledge, detailed description of the behavior of LMs in COVID-19 disease progression (26, 36). We provide evi- dence that a systemic lipid network consisting of liberation of PUFAs from plasmalogen and their subsequent conversion to LMs, capable of modulating inflammatory responses, char- acterizes both the onset and severity of COVID-19. Specif- ically, the loss of the immune-regulatory PGs and RvE3 and the increased products of ALOX5 and CYP provides both a measure of disease severity and a potentially mechanistic understanding of the immune balance allowing for patient recovery (37). Importantly, these pathways are directly tar- getable with drugs previously approved for use in other in- flammatory conditions and, thus, provide new therapeutic opportunities to control severe COVID-19 (2, 5). Yale IMPACT Team Members Kelly Anastasio, Michael H. Askenase, Maria Batsu, Sean Bickerton, Kristina Brower, Molly L. Bucklin, Staci Cahill, Yiyun Cao, Edward Courchaine, Giuseppe DeIuliis, Bertie Geng, Laura Glick, Akiko Iwasaki, Nathan Grubaugh, Chaney Kalinich, William Khoury-Hanold, Daniel Kim, Lynda Knaggs, Maxine Kuang, Eriko Kudo, Joseph Lim, Melissa Linehan, Alice Lu-Culligan, Anjelica Martin, Irene Matos, David McDonald, Maksym Minasyan, M. Catherine Muenker, Nida Naushad, Allison Nelson, Jessica Nouws, Abeer Obaid, Camilla Odio, Saad Omer, Isabel Ott, Annsea Park, Hong-Jai Park, Mary Petrone, Sarah Prophet, Harold Rahming, Tyler Rice, Kadi-Ann Rose, Lorenzo Sewanan, Denise Shepard, Erin Silva, Michael Simonov, Mikhail Smolgovsky, Nicole Sonnert, Yvette Strong, Codruta Todeasa, Jordan Valdez, Sofia Velazquez, Pavithra Vijayakumar, Annie Watkins, Elizabeth B. White, Yexin Yang

Acknowledgments We are deeply indebted to the patients and families of patients for contribution to this study. Prof. Charles N. Serhan and his group including, K. Boyle, A. Shay, C. Jouvene, X. de la Rosa, S. Libreros, and N. Chiang generously provided methodology, consultation, and extensive training for the assessment of LMs by LC-MS/MS. AB Sciex, in particular, M. Pearson, P. Norris, and P. Baker (currently Avanti Polar Lipids), provided LC-MS/MS consultation and methods. FIGURE 4. Correlation of comorbidities with LMs and severe COVID-19. (A) Correlation plot of spearman (BMI and age) or point biserial Pearson (disease severity, hypertension, heart disease, diabetes, and gender) correlation coefficient of Disclosures each LM with disease severity or patient demographics and comorbidities. LMs The authors have no financial conflicts of interest. are ordered by the strength of correlation with disease severity. For gender, a positive correlation is correlated with male. (B) Corresponding p values of each LM patient condition correlation displayed as 2log(p). References 1. Serhan, C. N., and J. Haeggstrom. 2010. Lipid mediators in acute inflammation and resolution: eicosanoids, PAF, resolvins, and protectins. In Fundamentals of Inflammation. C. N. Serhan, P. A. Ward, D. W. Gilroy, and S. S. Ayoub, eds. experimentation. Baseline PUFA and LM levels are known to Cambridge University Press, Cambridge, p. 153–174. be dysregulated in several of the comorbidities examined in this 2. Bitto, A., L. Minutoli, A. David, N. Irrera, M. Rinaldi, F. S. Venuti, F. Squadrito, and D. Altavilla. 2012. Flavocoxid, a dual inhibitor of COX-2 and 5-LOX of study (11–13). Thus, although the LM levels in this study natural origin, attenuates the inflammatory response and protects mice from sepsis. reflect the COVID-19 disease state, the differences between Crit. Care 16: R32. 334 CUTTING EDGE: DYSREGULATION OF EICOSANOIDS IN COVID-19

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