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Lipid storm within the lungs of severe COVID-19 patients: Extensive levels of cyclooxygenase 2

and lipoxygenase-derived inflammatory metabolites 3

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*Anne-Sophie Archambault1,2, *Younes Zaid3,4, Volatiana Rakotoarivelo1,2, Étienne Doré5,6, Isabelle 5

Dubuc5, Cyril Martin1,2, Youssef Amar7, Amine Cheikh4, Hakima Fares4, Amine El Hassani4, Youssef 6

Tijani8, Michel Laviolette1, Éric Boilard5,6,9, #Louis Flamand5,9 and #Nicolas Flamand1,2. 7

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*ASA and YZ equally contributed to this work. 9

#LF and NF equally contributed to this work 10 11 1Centre de recherche de l’Institut universitaire de cardiologie et de pneumologie de Québec, Faculté de 12 médecine, Département de médecine, Université Laval, Québec City, QC, Canada 13 2Canada Research Excellence Chair in the Microbiome-Endocannabinoidome Axis in Metabolic Health, 14 Université Laval, Québec, QC, Canada. 15 3Biology Department, Faculty of Sciences, Mohammed V University, Rabat, Morocco 16 4Cheikh Zaïd Hospital, Abulcasis University of Health Sciences, Rabat, Morocco 17 5Centre de Recherche du Centre Hospitalier Universitaire de Québec- Université Laval, Canada; 18 6Centre de Recherche Arthrite, Université Laval, Québec, Canada 19 7Moroccan Foundation for Advanced Science, Innovation & Research (MAScIR), Rabat, Morocco 20 8Faculty of Medicine, Mohammed VI University of Health Sciences, Casablanca, Morocco 21 9Département de microbiologie-infectiologie et d'immunologie, Université Laval, QC, Canada 22

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Corresponding authors: 25 Nicolas Flamand, PhD Louis Flamand, PhD 26 Centre de recherche de l’IUCPQ Centre de recherche du CHU-CHUL 27 2725 chemin Ste-Foy, Office A3140 2705 Laurier boulevard, Office T1-64 28 Québec City, QC G1V 4G5, Canada Québec City, QC G1V 4G2, Canada 29 Tel: 1-418-656-8711 ext.3733 Tel: 1-418-525-4444 ext.46164 30 Email: [email protected] Email: [email protected] 31

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Funding: This work was supported by the Cheikh Zaid Foundation awarded to YZ and grants from the 34 Canadian New Frontier Research Fund (NFRN-2019-00004) awarded to LF (PI), EB (co-PI) and NF (co-35 PI) and the Natural Sciences and Engineering Research Council of Canada awarded to NF. ASA received 36 a doctoral award from the Canadian Institutes of Health Research. ED and EB are respectfully recipient 37 of doctoral and senior awards from the Fonds de Recherche du Québec en Santé (FRQS). 38

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.

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https://doi.org/10.1101/2020.12.04.20242115

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ABSTRACT 39

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BACKGROUND. Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) is the infectious 41

agent responsible for Coronavirus disease 2019 (COVID-19). While SARS-CoV-2 infections are often 42

benign, there are also severe COVID-19 cases, characterized by severe bilobar pneumonia that can 43

decompensate to an acute respiratory distress syndrome, notably characterized by increased 44

inflammation and a cytokine storm. While there is no cure against severe COVID-19 cases, some 45

treatments significantly decrease the severity of the disease, notably aspirin and dexamethasone, which 46

both directly or indirectly target the biosynthesis (and effects) of numerous bioactive lipids. 47

48

OBJECTIVE. Our working hypothesis was that severe COVID-19 cases necessitating mechanical 49

ventilation were characterized by increased bioactive lipid levels modulating lung inflammation. We thus 50

quantitated several lung bioactive lipids using liquid chromatography combined to tandem mass 51

spectrometry. 52

53

RESULTS. We performed an exhaustive assessment of the lipid content of bronchoalveolar lavages 54

from 25 healthy controls and 33 COVID-19 patients necessitating mechanical ventilation. Severe 55

COVID-19 patients were characterized by increased fatty acid levels as well as an accompanying 56

inflammatory lipid storm. As such, most quantified bioactive lipids were heavily increased. There was a 57

predominance of cyclooxygenase metabolites, notably TXB2 >> PGE2 ~ 12-HHTrE > PGD2. 58

Leukotrienes were also increased, notably LTB4, 20-COOH-LTB4, LTE4, and eoxin E4. 15-lipoxygenase 59

metabolites derived from linoleic, arachidonic, eicosapentaenoic and docosahexaenoic acids were also 60

increased. Finally, yet importantly, specialized pro-resolving mediators, notably lipoxin A4 and the D-61

series resolvins, were also found at important levels, underscoring that the lipid storm occurring in severe 62

SARS-CoV-2 infections involves pro- and anti-inflammatory lipids. 63

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CONCLUSIONS. Our data unmask the important lipid storm occurring in the lungs of patients afflicted 65

with severe COVID-19. We discuss which clinically available drugs could be helpful at modulating the 66

lipidome we observed in the hope of minimizing the deleterious effects of pro-inflammatory lipids and 67

enhancing the effects of anti-inflammatory and/or pro-resolving lipids. 68

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Abbreviations 70 2-AG, 2-arachidonoyl-glycerol; AA, arachidonic acid; AEA, N-arachidonoyl-ethanolamine; ALT, 71 alanine transaminase; AST, aspartate transaminase;BAL, bronchoalveolar lavage; COVID-19, 72 coronavirus disease 2019; COX, cyclooxygenase; CRP, C-reactive protein; DGLA, dihomo-γ-linolenic 73 acid; DHA, docosahexaenoic acid; DPA, docosapentaenoic acid; EPA, eicosapentaenoic acid; EX, 74 eoxin; HETE, hydroxyeicosatetraenoic acid; ICU, intensive care unit; IL, interleukin; LA, linoleic acid; 75 LDH, lactate dehydrogenase; LOX, lipoxygenase; LT, leukotriene; PG, prostaglandin; Rv, resolvin; 76 SARS-CoV-2, severe acute respiratory syndrome coronavirus 2; SPM, specialized proresolving 77 mediator; TX, thromboxane. 78 79



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INTRODUCTION 80

Coronavirus disease 2019 (COVID-19) is an infectious disease caused by the Severe Acute Respiratory 81

Syndrome coronavirus 2 (SARS-CoV-2) (1). The clinical manifestations of the disease are often mild 82

but severe cases can lead to bilobar pneumonia with hyperactivation of the inflammatory cascade and 83

progression to acute respiratory distress syndrome (ARDS), necessitating mechanical ventilation (2, 3). 84

SARS-CoV-2 spreads predominantly from respiratory droplets of infected individuals to mucosal 85

epithelial cells in the upper airways and oral cavity (4). It infects cells via its homotrimeric spike protein 86

binding to host-cell expressing angiotensin-converting enzyme-2 (ACE2) receptor in a protease-87

dependent manner (5). 88

89

Autopsies from severe COVID-19 patients unmasked a heterogeneous disease that can be characterized 90

by diffuse alveolar damage, endothelial damage, thrombosis of small and medium vessels, pulmonary 91

embolism, and inflammatory cell infiltration, sometimes more lymphocytic, sometimes more 92

granulocytic (3, 6-8). An uncontrolled systemic inflammatory response known as the cytokine storm also 93

occurs. This cytokine storm, which is the consequence of an important release of pro-inflammatory 94

cytokines, such as tumor necrosis factor-α, interleukin (IL)-1β, IL-6, IL-7 and granulocyte-colony 95

stimulating factor, is suggested as an important contributor of SARS-CoV-2 lethality (8, 9). However, 96

the heterogeneity of the disease indicates that, aside cytokines (and chemokines), other immunological 97

effectors contribute to the sustained inflammatory responses observed in the most severe forms of 98

COVID-19. 99

100

Among the numerous soluble effectors involved in the infectious/inflammatory response, bioactive lipids 101

such as eicosanoids likely promote their fair share of deleterious effects, notably by enhancing leukocyte 102

recruitment and activation, by participating in the exudate formation and by stimulating platelet 103

aggregation and thrombus formation. In contrast, specialized pro-resolving mediators (SPMs), which 104

mainly consist of docosanoids, could dampen the inflammatory response and even promote its resolution 105

(10). While therapeutic approaches targeting the biosynthesis or effects of inflammatory lipids are readily 106

available, the levels of those lipids in the lungs during severe COVID-19 remain unknown. 107

108

Herein, we performed a detailed lipidomic analysis of bronchoalveolar lavage (BAL) fluids from healthy 109

volunteers and severe COVID-19 patients. We provide evidence that severe COVID-19 patients are 110

characterized by robust levels of lipids arising from the cyclooxygenase (COX) and lipoxygenase (LOX) 111



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pathways as well increased SPM levels. Our study further highlights that few associations exist between 112

clinical parameters and bioactive lipids present in the primary site of disease, the lungs. 113



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MATERIAL AND METHODS 114

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Materials 116

All lipids were obtained from Cayman Chemical (Ann Arbor, MI, USA) unless indicated otherwise. 117

DMSO and C17:1-LPA were purchased from Sigma-Aldrich (St-Louis, MO, USA). DMSO, ammonium 118

acetate, acetic acid, LC-MS-grade MeOH, MeCN and H2O were purchased from Fisher Scientific 119

(Ottawa, Canada). Cytokine/chemokine bead arrays were purchased from Becton Dickinson (BD). 120

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Ethics 122

This study was approved by the Local Ethics Committee of Cheikh Zaid Hospital (Project: 123

CEFCZ/PR/2020- PR04), Rabat, Morocco and complies with the Declaration of Helsinki and all subjects 124

signed a consent form. The analysis of bioactive lipids in the BAL of healthy donors and severe COVID-125

19 patients was approved by the Comité d’éthique de la recherche de l’Institut universitaire de cardiologie 126

et de pneumologie de Québec – Université Laval and the Comité d’éthique de la recherche du CHU de 127

Québec-Université Laval. 128

129 Clinical criteria for subject selection. Non-smoking healthy subjects taking no other medication than 130

anovulants, without any documented acute or chronic inflammatory disease and without recent (8 weeks) 131

airway infection were recruited for bronchoalveolar lavages at Institut universitaire de cardiologie et 132

pneumologie de Québec (Québec City, Canada). Only BALs in which the cell content in alveolar 133

macrophages was greater than 95%, with lymphocytes as the other main cell type, were kept for further 134

analyses. Severe COVID-19 patients were enrolled based on the inclusion criteria for intubation and the 135

need of mechanical ventilation support at Cheikh Zaid Hospital (Rabat, Morocco). Following intubation 136

and the initiation of mechanical ventilation, blood sampling was done to assess the clinical variables 137

shown in Table 1. Patients were ventilated in the prone decubitus position with a tidal volume of 6 ml/kg 138

and a PEEP varying between 8 – 14 cm H2O. The FiO2 was adjusted between 0.6 – 1.0 to obtain a SpO2 139

≥ 92%. 140

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Bronchoalveolar lavages. BAL fluids from healthy volunteers were obtained as follows: One 50 ml 142

bolus of sterile 0.9% saline was injected in a sub-segmental bronchi of the right middle lobe. The obtained 143

lavages were centrifuged (350 × g, 10 minutes) to pellet cells and the supernatants were immediately 144

frozen (-80º Celsius) until further processing. A 5 ml aliquot of the samples was thawed then reduced to 145

~500 µl using a stream of nitrogen. BAL fluids from severe COVID-19 patients were obtained less than 146



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2 hrs following intubation and as follows: a total of 100 ml of sterile 0.9% saline (2 boli of 50 ml) was 147

injected in a sub-segmental bronchi of the right middle lobe. The obtained lavages were pooled and 148

centrifuged (320 × g, 15 minutes) to pellet cells. Supernatants were next concentrated as for the BALs of 149

healthy donors using a stream of nitrogen and immediately frozen (-80º Celsius) until further processing. 150

All of the evaporated samples were processed for LC-MS/MS analysis as described below. 151

152

LC-MS/MS analyses. Samples (500 µl) were denatured by adding 500 µl of LC-MS grade MeOH 153

containing the internal standards then warmed at 60º Celsius for 30 minutes to inactivate (or not) SARS-154

CoV-2. Samples were then denatured overnight (-20º Celsius). The morning after, samples were warmed 155

to room temperature and centrifuged at 10,000 × g to remove the denaturated proteins. The obtained 156

supernatants were diluted with water containing 0.01% acetic acid to obtain a MeOH concentration of 157

10%, then acidified to pH 3 with acetic acid. Lipids were extracted by solid phase extraction (SPE) using 158

Strata-X cartridges (Polymeric Reversed Phase, 60 mg/1 ml, Phenomenex, Torrance, CA, USA) as 159

described before (11). In brief, columns were preconditioned with 2 ml MeOH containing 0.01% acetic 160

acid followed by 2 ml LC-MS grade H2O containing 0.01% acetic acid. After sample loading, the 161

columns were washed with 2 ml LC-MS grade H2O containing 0.01% acetic acid. Lipids were then eluted 162

from the cartridges with 1 ml MeOH containing 0.01% acetic acid. Eluates were dried under a stream of 163

nitrogen and reconstituted with 25 µl of solvent A (H2O + 1 mM NH4 + 0.05% acetic acid) and 25 µl of 164

solvent B (MeCN/H2O, 95/5, v/v + 1 mM NH4 + 0.05% acetic acid). A volume of 40 µl of the resulting 165

mixture was injected onto a RP‐HPLC column (Kinetex C8, 150 × 2.1 mm, 2.6 µm, Phenomenex). 166

Samples were eluted at a flow rate of 400 µl/min with a linear gradient of 10% solvent B that increased 167

to 35% in 2 min, up to 75% in 10 min, from 75% to 95% in 0.1 min, and held at 98% for 5 min before 168

re‐equilibration to 10% solvent B for 2 min. The HPLC system was directly interfaced into the 169

electrospray source of a Shimadzu 8050 triple quadrupole mass spectrometer and mass spectrometric 170

analyses were performed in the positive (+) or the negative (−) ion mode using multiple reaction 171

monitoring for the specific mass transitions of each lipid (Table 2). Quantification of each compound 172

was done using internal standards and calibration curves that were extracted by solid-phase extraction as 173

described above. 174

175

Statistical analysis 176

Statistical analyses were done using the GraphPad Prism 9 software. P values < 0.05 were considered 177

significant. 178

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RESULTS 180

While the circulating (plasmatic) cytokines’ profile observed in severe COVID-19 patients is relatively 181

well-defined (8, 12-14), a much more limited number of studies characterizing the cytokine storm in the 182

lungs of severe COVID-19 patients were available (13, 15, 16). In fact, to the best of our knowledge, no 183

study simultaneously quantitated numerous soluble mediators in BAL fluids. We thus performed their 184

lipidomic analysis in order to define whether an inflammatory lipid storm was present in the lungs of 185

severe COVID-19 patients. Samples were compared to those obtained from healthy subjects. As a 186

starting point, we investigated eicosanoids derived from the cyclooxygenase (COX) pathway. Our 187

working hypothesis was that some COX metabolites would be increased, considering that IL-1β levels 188

are elevated in the BALs of some COVID-19 patients (16) and that IL-1β is a potent inducer of 189

cyclooxygenase (COX)-2 expression (17-19). The only COX metabolites that were detected in the BALs 190

of healthy subjects were PGD2 (16 donors out of 25) and 12-HHTrE (2 donors out of 25). In contrast, 191

BALs from severe COVID-19 patients had a significant increase of all COX metabolites investigated 192

(figure 1). Most samples had detectable levels of PGD2 (27 of 33 donors) and the PGI2 metabolite 6-193

keto-PGF1α (16 of 33 donors), while all samples contained PGF2α, important levels of PGE2, the inactive 194

TXA2 metabolite TXB2, and the thromboxane synthase metabolite 12-HHTrE (figure 1A). Although 195

there was a significant increase in all metabolites, the most increased COX-derived metabolites were 196

TXB2 >> PGE2 ~ 12-HHTrE > PGD2. We next addressed if the different COX metabolites correlated 197

with each other. The levels of TXB2 strongly correlated with those of 12-HHTrE, PGD2, PGE2 and PGF2α 198

(figure 1B-E). Of note, few correlations were found between COX metabolites and the clinical 199

parameters shown in Table1. To that end, PGF2α and 12-HHTrE weakly but significantly correlated 200

negatively with AST (figure 7), 201

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Leukotrienes are also potent bioactive lipids previously documented to regulate the inflammatory 203

responses in the airway/lung in diseases, notably ARDS (20, 21). As such, their involvement in the 204

pathogenesis of COVID-19 has recently been postulated even though their levels were unknown (22). 205

We thus quantitated the levels of leukotrienes derived from the 5-lipoxygenase pathway as well as those 206

derived from the 15-lipoxygenase pathway, the 14,15-leukotrienes, which are now classified as eoxins 207

(23). Leukotrienes were not detected in the BAL fluids of healthy volunteers, with the exception of LTB4, 208

which was detected in 4 of 25 healthy subjects. In contrast, COVID-19 patients were characterized by 209

increased levels of LTB4 and its CYP4F3A metabolite 20-COOH-LTB4 (figure 2A) as well as other 210

minor metabolites (20-OH-LTB4 and 12-oxo-LTB4). Of note, the levels of LTB4 found in the BAL fluids 211

of severe COVID-19 patients are comparable to those found in BAL fluids of ARDS patients (16). As 212



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expected, there was a strong correlation between the levels of LTB4 and those of 20-COOH-LTB4 (figure 213

2B). Importantly, the ratio between the sum of 20-OH- and 20-COOH-LTB4 to LTB4 ((20-OH- + 20-214

COOH-LTB4)/LTB4) was 2.843 +/- 0.532 (mean +/- SEM), indicating an increased omega oxidation of 215

LTB4. The levels of LTB4 strongly correlated with those of TXB2 (figure 2C). The cysteinyl-leukotrienes 216

LTE4 and EXE4 were also found in most severe COVID-19 patients (figure 2D). Surprisingly, the levels 217

of EXE4 trended to be higher than those of LTE4. Furthermore, there was a trend toward a negative 218

correlation between LTE4 and EXE4 levels although it did not reach statistical significance (figure 2E). 219

This suggests that some COVID-19 patients are more prone to biosynthesize the 15-lipoxygnease-220

derived EXC4 while others are more prone to biosynthesize the 5-lipoxygenase-derived LTC4. Of note, 221

LTB4 levels correlated with those of EXE4 but not with those of LTE4 (figure 2F,G). Again, a limited 222

number of correlations between the different leukotrienes and the clinical parameters from Table 1 were 223

found (figure 7), supporting the concept that the levels of leukotrienes in the lungs are not reflected in 224

the blood by the most recognized COVID-19-related biomarkers. 225

226

The prostaglandins and leukotrienes data (figures 1 and 2) support the idea that an increase in both 227

arachidonic acid (AA) release and metabolism occurs in severe COVID-19 patients. As such, we 228

investigated the levels of some fatty acids, which are the precursors of oxidized linoleic acid (LA) 229

mediators (OXLAMs), eicosanoids and docosanoids. To that end, the levels of LA, AA, 230

docosapentaenoic acid n-3 (DPAn-3) and docosahexaenoic acid (DHA) were all significantly increased 231

in the BALs of severe COVID-19 patients. In contrast, the levels of eicosapentaenoic acid (EPA) did not 232

change (figure 3). Of note, all tested BAL fatty acids negatively correlated with AST and ALT but 233

positively correlated with the circulating platelet-to-lymphocyte ratio (figure 7). 234

235

The increased fatty acid levels (figure 3) and the increased in the 15-lipoxygenase-derived eoxins (figure 236

2D) led us to investigate if other fatty acid-related biosynthetic pathways were also enhanced in SARS-237

CoV-2-infected patients. To that end, we also quantitated additional AA-derived metabolites derived 238

from the 12- and 15-lipoxygenases. The levels of 12-HETE and 15-HETE were also significantly 239

increased in the BAL fluids of severe COVID-19 patients, although not to the same extent than the 5-240

lipoxygenase metabolite 5-HETE (figure 4A), indicating that AA metabolism via the 15-lipoxygenase 241

(and possibly the 12-lipoxygenase) pathway is increased. In addition to AA, the 15-lipoxygenase 242

pathway can metabolize numerous polyunsaturated fatty acids having a 1Z,4Z-pentadiene motif near 243

their omega end, notably LA, EPA, DPAn-3 and DHA. We thus also assessed the levels of their most 244

documented 15-lipoxygenase metabolites and found significant increases in 13-HODE (derived from 245



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LA), 15-HETrE (derived from dihomo-γ-linolenic acid, DGLA), 12-, 15-, and 18-HEPE, 17-HDPA(n-246

3) as well as 14- and 17-HDHA (figure 4B,C). Dual lipoxygenases metabolites from AA were also 247

significantly increased (figure 4D). Of note, the levels of 15-HETE strongly correlated with those of 13-248

HODE (figure 4E) supporting an increased 15-lipoxygenase activity in the lungs of severe COVID-19 249

patients. Furthermore, 15-HETE levels also correlated with those of TXB2 (figure 4F). Finally yet 250

importantly, we also detected significant increases in 9-HODE, 8-HETE, and 11-HETE, which arise from 251

the non-enzymatic oxidation of LA and AA, pointing to an increased oxidative bust in the lungs of severe 252

COVID-19 patients (figure 4A,B). 253

254

14- and 17-HDHA, which are increased in the BALs of severe COVID-19 patients (figure 4C), are often 255

regarded as good indicators of D-series resolvin and maresin levels, which are part of the ever-expanding 256

class of specialized pro-resolving mediators (SPMs). SPMs are usually more complicated to quantitate 257

due to their very low concentrations (10). Given their documented pro-resolving actions and anti-258

microbial activities, SPMs were recently postulated as being a potential approach for diminishing the 259

inflammatory burden of COVID-19 (24). We thus investigated the levels of some commercially available 260

SPMs. All SPMs were below detection limit in the BAL samples of healthy volunteers we analyzed. In 261

contrast, most investigated SPMs, aside from RvD3, were detected in the BAL fluids of severe COVID-262

19 patients. LXA4 was the most prominent one, followed by RvD4, RvD5, RvD2, RVD1 and PDX 263

(figure 5A). RvD5 levels correlated with those of RvD4 (figure 5B) but not those of DHA (figure 5C) 264

indicating that the same patients are responsible for the increased D-series resolvins, which is mostly due 265

to increased 15-lipoxygenase activity rather than an increase in DHA availability. The levels of RvD5 266

also correlated with those of TXB2 (figure 5D). Again, there was no correlation between RvD5 with the 267

clinical parameters from table 1. Unsurprisingly, the levels of LXA4 correlated with those of 5-HETE 268

and 15-HETE (figure 5E,F), given that LXA4 biosynthesis requires both the 5- and the 15-lipoxygenase 269

pathways. As for RvD5, the levels of LXA4 also correlated with those of TXB2 and the other 270

prostaglandins (figure 6). Despite our good sensitivity (see table 2), we did not detect the presence or 271

RvE1, Maresin-1 and -2. 272

273

When assessing the lipidome for which we could quantitate a given lipid mediator in at least 50% of 274

severe COVID-19 patients, we can appreciate that most lipids consistently correlated with each other 275

with few exceptions (figure 6). Indeed, Fatty acids levels were not a key determinant for the levels of 276

their metabolites, indicating that downstream enzymes (cyclooxygenases, lipoxygenases) are likely more 277

important in that respect. Furthermore, and in agreement with their almost significant negative 278



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association (figure 2E), EXE4 and LTE4 correlated with different bioactive lipid subsets, LTE4 positively 279

correlating with fatty acid and 15-HETrE levels while EXE4 mostly correlating with other 15-280

lipoxygenase metabolites. 281



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DISCUSSION 282

283

Bioactive lipids are recognized pharmaceutical targets for the treatment of numerous inflammatory 284

diseases and lipid modulators have been utilized as such, in the last fifty years, with great success. It was 285

thus imperative, at least to us, to define whether bioactive lipid levels were modulated in the context of 286

severe COVID-19. 287

288

Herein, we provide clear evidences that the BALs of severe COVID-19 patients contain large amounts 289

of bioactive lipids, which likely participate in the deleterious inflammatory response found in severe 290

cases. More specifically, our data indicate that the BALs of severe COVID-19 patients are characterized 291

by 1) an increase in COX metabolites, notably the TXA2 metabolite TXB2; 2) an increase in LTB4 and 292

its metabolites; 3) an increase in CysLT metabolites; 4) an increase of 15-lipoxygenases metabolites 293

derived from LA, AA, EPA, DPA and DHA; 5) an increase in SPMs derived from AA and DHA; and 6) 294

a limited number of correlations between BAL lipids vs. blood markers and/or clinical parameters. 295

296

Cyclooxygenase metabolites were heavily increased in the BALs of severe COVID-19 patients (figure 297

1). This was somewhat expected, notably because of the increased levels of IL-1β described before (16), 298

the latter being a good COX-2 inducer. In the lungs, COX metabolites play multiple roles. TXA2 299

promotes bronchoconstriction and activate inflammatory cells and platelets, which might be consistent 300

with the reported enhanced platelet activation and increased thrombosis in COVID-19 (14, 25, 26). On 301

the other hand, PGD2 acting via the DP2 receptor promotes the recruitment and activation of eosinophils, 302

basophils, mastocytes and innate lymphoid cells (27-30). In contrast, PGE2 is usually regarded as anti-303

inflammatory in the lungs, notably by downregulating the pro-inflammatory functions of myeloid 304

leukocytes such as neutrophils and macrophages, notably by activating the EP2 and EP4 receptors, 305

ultimately leading to increased cyclic AMP levels (31). Therapies targeting the prostaglandin pathway 306

are numerous and usually effective at controlling inflammation. Dexamethasone, a steroid improving 307

COVID-19 symptoms and diminishing mortality (32-34), limits COX-2 expression (35-37) and might 308

thus improve COVID-19 severity, at least in part, by diminishing the biosynthesis of prostaglandins and 309

possibly TXA2. Furthermore, nonsteroidal anti-inflammatory drugs (NSAIDs) such as aspirin and 310

ibuprofen are recognized inhibitor of cyclooxygenase activity. Early at the beginning of the pandemic, it 311

was postulated that NSAIDs could worsen the severity of COVID-19 (38). However, a Danish study 312

indicated that ibuprofen was not linked to severe adverse outcome (39). Furthermore, aspirin was recently 313

shown to significantly diminish ICU admissions and the need of mechanical ventilation in an 314



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observational, retrospective study (40). Altogether, our data, combined with the recent studies on their 315

safety in a context of COVID-19, support the hypothesis that dexamethasone and NSAIDs might 316

diminish the inflammatory status of severe COVID-19 patients by diminishing the 317

prostaglandins/thromboxane storm in the lungs. While TXA2 and PGD2 might have deleterious effects 318

in the lungs, PGE2 and PGI2 might have the opposite effect, notably by inhibiting leukocyte functions 319

(PGE2) and/or promoting the vasodilation of the microvasculature (PGI2). To that end, testing whether 320

blocking the deleterious effects of PGD2 and TXA2 with the dual DP2/TP antagonist Ramotraban might 321

be beneficial in this particular context, as proposed recently (41). 322

323

Prostaglandins and TXA2, as assessed by quantitating TXB2 levels, were not the only eicosanoids that 324

were increased in the BALs of severe COVID-19 patients. Indeed, leukotrienes were also increased, 325

notably LTB4, LTE4, and EXE4 (figure 2). This indicates that in addition of the deleterious effects that 326

some COX metabolites might have, those linked to LTB4 and CysLTs might also impair lung functions 327

and participate in the inflammatory burden in the lungs of severe COVID-19 patients (22). While LTB4 328

has previously been shown to stimulate host defense (41), notably during viral infections, the 329

w-LTB4/LTB4 ratio of 2.84 we observed is more reminiscent of what is found in cystic fibrosis and 330

severely burned patients, two conditions in which infections are not well controlled (42-45). 331

Conceptually, this might diminish the effectiveness of LTB4 at promoting its host-defense related 332

functions, as we recently published (46). Interestingly, there was a trend toward an inverse correlation 333

between LTE4 and EXE4, indicating that these cysteinyl-LTs are likely coming from different cellular 334

sources. This was supported by the differential and significant correlations between the different lipids 335

we observed, LTE4 and EXE4 differentially correlating with distinct lipids (figure 6). To that end, EXE4 336

levels in the BALs positively and significantly correlated with blood eosinophils counts. While there was 337

a strong trend between EXE4 and BAL eosinophils counts the latter correlation had a p value of 0.0536. 338

Altogether, this suggests that the presence and/or biosynthesis of eoxins is, in part, linked to eosinophils. 339

A therapeutic approach to target LTs from the 5-lipoxygenase pathway could be the use of Zileuton, 340

which is commercially available for the management of asthma and has a good safety profile (47). 341

342

Another pharmaceutical approach that could simultaneously diminish the levels of COX and 343

5-lipoxygenase-derived eicosanoids could be the use of phosphodiesterase 4 inhibitors such as 344

Roflumilast. While there is no evidence of phosphodiesterase 4 inhibitors’ efficacy in the management 345

of SARS-CoV-2-infected people (48), they might diminish inflammation by preventing the degradation 346

of cyclic AMP, increasing intracellular cAMP concentrations and decreasing the release of AA, the 347



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precursor of prostaglandins and leukotrienes. Furthermore, cAMP-elevating agents such as adenosine 348

and PGE2 are recognized anti-inflammatory autacoids that inhibit numerous leukocyte functions such as 349

eicosanoid biosynthesis, oxidative burst, calcium mobilization and migration (49-53). While 350

phosphodiesterase 4 inhibitors might diminish the levels of PGE2 by diminishing AA levels, they would 351

nonetheless amplify the inhibitory effects of the remaining PGE2, as recently demonstrated in cellulo 352

(54). 353

354

While it is largely accepted that the group IVA cytosolic phospholipase A2 mediates most of AA release 355

in activated human leukocytes (e.g. in (55)), the increased levels of LA, EPA and DHA support the idea 356

that other phospholipases A2 are also heavily involved in the lipidome we have quantitated. Those 357

additional phospholipases likely belong to the superfamily of secreted phospholipases A2, which can 358

hydrolyze phospholipids containing fatty acids other than AA (56, 57) and are reported to contribute to 359

lung inflammation (58-61). However, and assuming that a large part of eicosanoids is the consequence 360

of the group IVA cytosolic phospholipase A2, our findings do not allow us to pinpoint which other 361

phospholipases are involved in the release of LA, AA, EPA, DPA and DHA. 362

363

The metabolism of EPA, DPA and DHA, which are omega-3 fatty acids, often results in the biosynthesis 364

of SPMs, most of which are anti-inflammatory and pro-resolving lipids. Their presence in high levels 365

was somewhat surprising as they are generally thought to appear during the resolution of inflammation, 366

after the biosynthesis of prostaglandins and leukotrienes has occurred and following a class-switch from 367

pro-inflammatory to pro-resolving lipids (62). However, our data indicate that prostaglandins, 368

leukotrienes and SPMs can certainly co-exist and participate in the inflammatory cascade during the 369

acute phase of inflammation/infection in which resolution has not been fully engaged. Moreover, given 370

their strong documented anti-inflammatory effects and their host-defense boosting functions, they could 371

be viewed as potential mediators to enhance to limit the infection. To that end, it was recently proposed 372

that dexamethasone could participate in the upregulation of SPM biosynthesis and effects in moderate to 373

severe COVID-19 patients, although this needs to be confirmed (63). While omega-3 fatty acids, notably 374

purified preparation enriched in EPA and DHA are commercially available as dietary supplements, we 375

did not see a correlation between SPMs and the levels of EPA or DHA, indicating that SPMs levels are 376

likely more dependent on biosynthetic enzymes other than phospholipases such as lipoxygenases than 377

EPA or DHA availability. As of today, no resolution pharmacology treatment has been approved by 378

governmental health agencies yet. However, some putative E- and D-series resolvin precursors, notably 379

18-HEPE and 17-HDHA, are available as dietary supplements. 380



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381

In addition to cyclooxygenase and lipoxygenase derivatives, other bioactive lipids are recognized, at least 382

in mice, to downregulate the immune response. This is notably the case of the endocannabinoids N-383

arachidonoyl-ethanolamine (AEA) and 2-arachidonoyl-glycerol (2-AG), which are AA-derived lipids 384

(64). The anti-inflammatory effects of AEA and 2-AG are mostly related to the activation of the CB2 385

receptor, which is heavily expressed by leukocytes (65). In addition to 2-AG and AEA, their congeners 386

from the monacyl-glycerols and N-acyl-ethanolamines and N-acyl-aminoacids families, now viewed as 387

the endocannabinoidome, could potentially regulate the inflammatory response during SARS-CoV-2 388

infections, notably by activating other anti-inflammatory receptors (66). It will thus be crucial to decipher 389

whether the endocannabinoidome is modulated during COVID-19 and if a potential target such as CB2 390

receptor agonists, or endocannabinoid hydrolase inhibitors could be helpful in the treatment of COVID-391

19 or other coronavirus-mediated infections. 392

393

Nonetheless the expanded definition of a dysregulated lipidome we unravel in SARS-CoV-2 infections 394

requiring mechanical ventilation, our study has limitations. 1) BALs from healthy controls are from 395

younger individuals than those from severe COVID-19 patients. This could have an impact on some 396

mediators, notably PGD2 levels, which are increased in aged mice and worsen SARS-CoV infections 397

(67); 2) The procedure to obtain BAL fluids is slightly different between healthy controls and severe 398

COVID-19 patients. Indeed, while the volume of saline injected to healthy people was 50 ml, that of 399

severe COVID-19 patients was 100 ml. The possible outcome of this discrepancy could be and 400

underestimation of lipid levels in one group vs. the other. While some might argue that the first saline 401

bolus contains more lipids than the following one(s), other might argue that lipid levels are consistent in 402

each bolus. Given that, in our hands, the number of BAL cells is usually greater in the first bolus than in 403

the subsequent ones (when multiple boli are utilized), we are confident that the BAL fluid from the first 404

bolus likely contains more lipids than the second one. This would thus lead to a slight underestimation 405

of lipids in the COVID-19 group. 3) Unfortunately, the leukocyte counts in the BALs of severe COVID-406

19 patients did not include the number of alveolar macrophages and it is thus impossible to determine a 407

clear differential count of leukocytes in those samples. Furthermore, the eosinophil counts were 408

somewhat limited by the apparatus as it could estimate the counts to +/- 105/ml cells, which likely has an 409

impact on the different correlations involving eosinophils, notably with EXE4; 4) We could not compare 410

the levels of lipids in the BALs of severe COVID-19 patients to those found in the blood. This appears 411

important given the limited number of correlations found between BAL lipids and other circulating 412

markers such as CRP and D-dimers. To that end, a recent study indicated differences in some of the 413



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herein investigated bioactive lipids in the circulation (68). It will thus be important to pursue our 414

investigations and determine whether there is a transposition of the BAL lipidome to the circulation. 415

416

In conclusion, our data unmask that in addition to the recognized cytokine storm previously documented, 417

lung fluids of SARS-CoV-2-infected patients indicate that a lipid storm also occurs. As highlighted 418

above, the rich library of governmental health agencies-approved lipid modulators might provide 419

beneficial and usually low-cost add-ons to the current therapeutic arsenal utilized to diminish the severity 420

of COVID-19 and possibly additional coronavirus-mediated infections. 421

422

423

424

425

ACKNOWLEDGEMENTS 426

ASA, AC, VR, ML, and NF are members of the Quebec Respiratory Health research Network. 427



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Table 1. Clinical characteristics of BAL donors 660 661

Severe COVID-19 Healthy controls N 33 25

Women/Men 16/17 16/9 Death (Y/N) 2/31 N/A

SARS-CoV-2 detection in BAL (Ct) 25.303 ± 0.906 N/A Age 58.455 ± 3.175 26.130 ± 1.030

Weight (kg) 73.097 ± 2.873 N/A Hospital stay (days) 20.545 ± 2.014 N/A

Days before BAL 3.485 ± 0.763 N/A D-Dimers (mg/ml) 1.210 ± 0.129 N/A

CRP (mg/l) 20.652 ± 2.525 N/A ALT (U/l) 32.582 ± 2.144 N/A AST (U/l) 35.145 ± 2.978 N/A LDH (U/l) 616.152 ± 41.175 N/A

Hemoglobin (g/l) 123.152 ± 2.365 N/A Blood Monocytes (million/ml) 0.327 ± 0.034 N/A Blood Neutrophils (million/ml) 2.855 ± 0.194 N/A Blood Eosinophils (million/ml) 0.026 ± 0.003 N/A

Blood Platelets (million/ml) 181.848 ± 13.397 N/A Blood Lymphocytes (million/ml) 1.127 ± 0.127 N/A

Platelet to Lymphocyte ratio 223.893 ± 34.934 N/A BAL Neutrophils (million/ml) 24.694 ± 2.152 N/A BAL Eosinophils (million/ml) 0.197 ± 0.077 N/A

BAL Lymphocytes (million/ml) 21.830 ± 2.393 N/A Data are presented as the mean ± SEM. ALT, alanine transaminase; AST, aspartate transaminase; 662 CRP, C-reactive protein; LDH, Lactate dehydrogenase; N/A, not available; 663 664



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Table 2. Mass transition of the investigated lipid mediators. 665 666

Compounds ISTD used Q1 > Q3 Retention time (min) LLOQ (fmol)

TXB2-D4 373.10>199.35 4,083 TXB2 TXB2-D4 369.20>195.30 4,090 250

PGE2-D4 355.20>275.35 4,627 6-keto PGF1α PGE2-D4 369.30>163.10 3,493 250

PGF3α PGE2-D4 351.30>193.30 3,954 50 PGE3 PGE2-D4 349.30>269.35 4,160 25 PGF2α PGE2-D4 353.40>309.30 4,412 250 PGE1 PGE2-D4 353.30>273.40 4,770 50 PGE2 PGE2-D4 351.20>271.15 4,627 50 PGD2 PGE2-D4 351.30>271.20 4,847 50

1a,1b-dihomo-PGF2α PGE2-D4 381.20>337.40 5,428 50 RvD2-D5 380.40>175.20 4,685

RvE1 RvD2-D5 349.30>195.20 3,529 250 RvD3 RvD2-D5 375.40>147.20 4,689 250 RvD2 RvD2-D5 375.40>175.20 4,780 500 RvD1 RvD2-D5 375.40>141.10 5,126 500 RvD4 RvD2-D5 375.40>101.05 5,588 250

Maresin 1 RvD2-D5 359.40>177.25 6,684 500 PDX RvD2-D5 359.30>153.15 6,877 25 RvD5 RvD2-D5 359.40>199.25 6,899 250

Maresin 2 RvD2-D5 359.40>221.05 7,433 250 LTC4-D5 629.30>272.20 4,999

EXD4 LTC4-D5 495.30>176.90 4,918 250 EXC4 LTC4-D5 624.30>272.25 5,025 500 LTC4 LTC4-D5 624.30>272.20 5,065 500 LXA4 LTB4-D4 351.50>115.05 5,051 250 EXE4 LTC4-D5 438.30>351.10 5,263 700 LTD4 LTC4-D5 495.30>176.85 5,359 250 LTE4 LTC4-D5 438.30>333.20 5,677 500

LTB4-D4 339.30>197.20 6,742 20-COOH-LTB4 LTB4-D4 365.00>347.25 3,549 250

20-OH-LTB4 LTB4-D4 351.10>195.15 3,670 50 LTB5 LTB4-D4 333.40>195.25 5,992 250 LTB4 LTB4-D4 335.30>195.25 6,765 25

12-oxo-LTB4 LTB4-D4 333.40>179.20 7,399 250 LTB3 LTB4-D4 337.40>195.20 7,523 250

13-HODE-D4 299.10>198.15 9,196 13(S)-HOTrE 13-HODE-D4 292.90>195.25 8,474 250

9-HODE 13-HODE-D4 295.30>171.35 9,237 25



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13-HODE 13-HODE-D4 295.50>195.30 9,238 25 13-KODE 13-HODE-D4 295.20>277.30 9,584 25

15-HETE-D8 327.20>226.20 9,503 8,15-DiHETE 15-HETE-D8 335.40>317.30 6,480 250

5(S),15(S)-DiHETE 15-HETE-D8 335.20>255.30 6,630 250 5(S),12(S)-DiHETE 15-HETE-D8 334.99>195.10 7,023 250

12(S)-HHTrE 15-HETE-D8 279.20>179.25 7,699 250 14(15)-DIHET 15-HETE-D8 337.40>207.30 7,712 25

5(S),6(R)-DiHETE 15-HETE-D8 335.00>145.10 8,020 250 18-HEPE 15-HETE-D8 317.30>259.40 8,497 250 15-HEPE 15-HETE-D8 317.40>219.25 8,840 250 12-HEPE 15-HETE-D8 317.30>179.35 9,015 250 15-HETE 15-HETE-D8 319.40>219.30 9,574 250 15-KETE 15-HETE-D8 317.20>113.00 9,707 25 17-HDHA 15-HETE-D8 343.50>281.30 9,719 500 14-HDHA 15-HETE-D8 343.50>281.30 9,878 250 11-HETE 15-HETE-D8 319.30>167.35 9,770 25 8-HETE 15-HETE-D8 319.30>155.25 9,841 50 12-HETE 15-HETE-D8 319.10>179.25 9,924 250 5-HETE 15-HETE-D8 319.30>115.05 10,008 250

17-HDPA 15-HETE-D8 345.20>327.10 10,086 250 15-HETrE 15-HETE-D8 321.50>303.40 10,095 25

14(15)-EET 15-HETE-D8 319.40>219.25 10,623 500 EPA-D5 306.20>262.20 12,128

EPA EPA-D5 301.20>257.30 12,156 25 DHA-D5 332.20>288.20 12,889

DHA DHA-D5 327.50>283.30 12,919 50 DPA(n-3)-D5 334.40>290.35 13,178

DPA(n-3) DPA(n-3)-D5 329.50>285.15 13,191 25 AA-D8 311.20>267.25 13,006

AA AA-D8 303.20>259.30 13,052 250 LA AA-D8 279.40>261.35 13,079 2500

667 668 669



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670

671 Figure 1. Levels of cyclooxygenase-derived metabolites in the BAL fluids of healthy and severe 672 COVID-19 patients. BALs were obtained and processed as described in Methods. A) Results are from 673 the BALs from 25 healthy subjects and 33 severe COVID-19 patients. P values were obtained by 674 performing a Mann-Whitney test: **** = p < 0.0001. B-E) Correlation tests were performed with 675 COVID-19 patients by using the non-parametric Spearman’s rank. P

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684

685 Figure 2. Comparison of leukotriene and eoxin levels in the BAL fluids of healthy subjects and 686 severe COVID-19 patients. A,D) BALs were obtained and processed as described in methods. Lipids 687 then were extracted and quantitated by LC-MS/MS. Results are from the BALs from 25 healthy subjects 688 and 33 severe COVID19 patients. P values were obtained by performing a Mann-Whitney test: * p < 689 0.05; *** p < 0.001; **** p < 0.0001 B,C,E-G) Correlation tests were performed with severe COVID-690 19 patients by using the non-parametric Spearman’s rank. P

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Figure 3. Fatty acid levels in the BAL fluids of healthy subjects and severe COVID-19 patients. 696 BALs were obtained and processed as described in Methods. Lipids then were extracted and quantitated 697 by LC-MS/MS. Results are from the BALs from 25 healthy subjects and 33 severe COVID19 patients. 698 P values were obtained by performing a Mann-Whitney test: **** p < 0.0001. 699 700



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701 Figure 4. Comparison of 12- and 15-lipoxygenase-derived oxylipins and diHETEs levels in the 702 BALs of healthy subjects and severe COVID-19 patients. A-D) BALs were obtained and processed 703 as described in Methods. Lipids then were extracted and quantitated by LC-MS/MS. Results are from the 704 BALs from 25 healthy subjects and 33 Severe COVID-19 patients. P values were obtained by performing 705 a Mann-Whitney test: **** p < 0.0001. 706 707 708 709



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710 711 Figure 5. Comparison of specialized pro-resolving lipid mediator levels in the BAL fluids of healthy 712 subjects and severe COVID19 patients. A) BALs were obtained and processed as described in 713 Methods. Lipids then were extracted and quantitated by LC-MS/MS. Results are from the BALs from 25 714 healthy subjects and 33 severe COVID-19 patients. . P values were obtained by performing a Mann-715 Whitney test: * p < 0.05; ** p < 0.01; **** p < 0.0001. B-G) Correlation tests were performed with 716 severe COVID-19 patients by using the non-parametric Spearman’s rank. P

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Figure 6. Correlation between selected bioactive lipids in the BALs of severe COVID-19 patients. Correlation tests were performed by 720 using the non-parametric Spearman’s rank using the Graphpad Prism Software. P

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723

Figure 7. Correlation between bioactive lipids in the BALs of severe COVID-19 patients and 724 clinical parameters. Correlation tests were performed by using the non-parametric Spearman’s rank 725 using the Graphpad Prism Software. P

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