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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
4
*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
8
*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
23
24
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
32 33
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
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ABSTRACT 39
40
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
64
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
69
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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
115
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
121
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
141
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
179
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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
202
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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REFERENCES 428 429 430 431 1. Wu F, Zhao S, Yu B, Chen YM, Wang W, Song ZG, Hu Y, Tao ZW, Tian JH, Pei YY, Yuan ML, 432
Zhang YL, Dai FH, Liu Y, Wang QM, Zheng JJ, Xu L, Holmes EC, Zhang YZ. A new 433 coronavirus associated with human respiratory disease in China. Nature 2020; 579: 265-269. 434
2. Verdoni L, Mazza A, Gervasoni A, Martelli L, Ruggeri M, Ciuffreda M, Bonanomi E, D'Antiga L. 435 An outbreak of severe Kawasaki-like disease at the Italian epicentre of the SARS-CoV-2 436 epidemic: an observational cohort study. Lancet 2020; 395: 1771-1778. 437
3. Huang C, Wang Y, Li X, Ren L, Zhao J, Hu Y, Zhang L, Fan G, Xu J, Gu X, Cheng Z, Yu T, Xia J, 438 Wei Y, Wu W, Xie X, Yin W, Li H, Liu M, Xiao Y, Gao H, Guo L, Xie J, Wang G, Jiang R, 439 Gao Z, Jin Q, Wang J, Cao B. Clinical features of patients infected with 2019 novel coronavirus 440 in Wuhan, China. Lancet 2020; 395: 497-506. 441
4. Zhang XSD, C. SARS-CoV-2 and Health Care Worker Protection in Low-Risk Settings: a Review of 442 Modes of Transmission and a Novel Airborne Model Involving Inhalable Particles. Clinical 443 Microbiology Reviews 2020; 34. 444
5. Oberfeld B, Achanta A, Carpenter K, Chen P, Gilette NM, Langat P, Said JT, Schiff AE, Zhou AS, 445 Barczak AK, Pillai S. SnapShot: COVID-19. Cell 2020; 181: 954-954 e951. 446
6. Damiani S, Fiorentino M, De Palma A, Foschini MP, Lazzarotto T, Gabrielli L, Viale PL, Attard L, 447 Riefolo M, D'Errico A. Pathological Post Mortem Findings in Lungs Infected With Sars-Cov 2. 448 J Pathol 2020. 449
7. Barton LM, Duval EJ, Stroberg E, Ghosh S, Mukhopadhyay S. COVID-19 Autopsies, Oklahoma, 450 USA. Am J Clin Pathol 2020; 153: 725-733. 451
8. Zhang H, Zhou P, Wei Y, Yue H, Wang Y, Hu M, Zhang S, Cao T, Yang C, Li M, Guo G, Chen X, 452 Chen Y, Lei M, Liu H, Zhao J, Peng P, Wang CY, Du R. Histopathologic Changes and SARS-453 CoV-2 Immunostaining in the Lung of a Patient With COVID-19. Ann Intern Med 2020; 172: 454 629-632. 455
9. Amigues I, Pearlman AH, Patel A, Reid P, Robinson PC, Sinha R, Kim AH, Youngstein T, 456 Jayatilleke A, Konig MF. Coronavirus disease 2019: investigational therapies in the prevention 457 and treatment of hyperinflammation. Expert Rev Clin Immunol 2020. 458
10. Serhan CN, Levy BD. Resolvins in inflammation: emergence of the pro-resolving superfamily of 459 mediators. J Clin Invest 2018; 128: 2657-2669. 460
11. Archambault AS, Turcotte C, Martin C, Provost V, Larose MC, Laprise C, Chakir J, Bissonnette E, 461 Laviolette M, Bosse Y, Flamand N. Comparison of eight 15-lipoxygenase (LO) inhibitors on 462 the biosynthesis of 15-LO metabolites by human neutrophils and eosinophils. PLoS One 2018; 463 13: e0202424. 464
12. Del Valle DM, Kim-Schulze S, Huang HH, Beckmann ND, Nirenberg S, Wang B, Lavin Y, Swartz 465 TH, Madduri D, Stock A, Marron TU, Xie H, Patel M, Tuballes K, Van Oekelen O, Rahman A, 466 Kovatch P, Aberg JA, Schadt E, Jagannath S, Mazumdar M, Charney AW, Firpo-Betancourt A, 467 Mendu DR, Jhang J, Reich D, Sigel K, Cordon-Cardo C, Feldmann M, Parekh S, Merad M, 468 Gnjatic S. An inflammatory cytokine signature predicts COVID-19 severity and survival. Nat 469 Med 2020. 470
13. Tang Y, Liu J, Zhang D, Xu Z, Ji J, Wen C. Cytokine Storm in COVID-19: The Current Evidence 471 and Treatment Strategies. Front Immunol 2020; 11: 1708. 472
14. Zaid Y, Puhm F, Allaeys I, Naya A, Oudghiri M, Khalki L, Limami Y, Zaid N, Sadki K, Ben El 473 Haj R, Mahir W, Belayachi L, Belefquih B, Benouda A, Cheikh A, Langlois MA, Cherrah Y, 474 Flamand L, Guessous F, Boilard E. Platelets Can Associate with SARS-Cov-2 RNA and Are 475 Hyperactivated in COVID-19. Circ Res 2020. 476
All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprintthis version posted December 7, 2020. ; https://doi.org/10.1101/2020.12.04.20242115doi: medRxiv preprint
https://doi.org/10.1101/2020.12.04.20242115
18
15. Xiong Y, Liu Y, Cao L, Wang D, Guo M, Jiang A, Guo D, Hu W, Yang J, Tang Z, Wu H, Lin Y, 477 Zhang M, Zhang Q, Shi M, Liu Y, Zhou Y, Lan K, Chen Y. Transcriptomic characteristics of 478 bronchoalveolar lavage fluid and peripheral blood mononuclear cells in COVID-19 patients. 479 Emerg Microbes Infect 2020; 9: 761-770. 480
16. Liao M, Liu Y, Yuan J, Wen Y, Xu G, Zhao J, Cheng L, Li J, Wang X, Wang F, Liu L, Amit I, 481 Zhang S, Zhang Z. Single-cell landscape of bronchoalveolar immune cells in patients with 482 COVID-19. Nat Med 2020; 26: 842-844. 483
17. Miller DB, Munster D, Wasvary JS, Simke JP, Peppard JV, Bowen BR, Marshall PJ. The 484 heterologous expression and characterization of human prostaglandin G/H synthase-2 (COX-2). 485 Biochem Biophys Res Commun 1994; 201: 356-362. 486
18. Newman SP, Flower RJ, Croxtall JD. Dexamethasone suppression of IL-1b-induced 487 cyclooxygenase 2 expression is not mediated by lipocortin-1 in A549 cells. Biochem Biophys 488 Res Commun 1994; 202: 931-939. 489
19. Mitchell JA, Belvisi MG, Akarasereenont P, Robbins RA, Kwon OJ, Croxtall J, Barnes PJ, Vane 490 JR. Induction of cyclo-oxygenase-2 by cytokines in human pulmonary epithelial cells: 491 regulation by dexamethasone. Br J Pharmacol 1994; 113: 1008-1014. 492
20. Stephenson AH, Lonigro AJ, Hyers TM, Webster RO, Fowler AA. Increased concentrations of 493 leukotrienes in bronchoalveolar lavage fluid of patients with ARDS or at risk for ARDS. Am 494 Rev Respir Dis 1988; 138: 714-719. 495
21. Ratnoff WD, Matthay MA, Wong MY, Ito Y, Vu KH, Wiener-Kronish J, Goetzl EJ. 496 Sulfidopeptide-leukotriene peptidases in pulmonary edema fluid from patients with the adult 497 respiratory distress syndrome. J Clin Immunol 1988; 8: 250-258. 498
22. Funk CD, Ardakani A. A Novel Strategy to Mitigate the Hyperinflammatory Response to COVID-499 19 by Targeting Leukotrienes. Front Pharmacol 2020; 11: 1214. 500
23. Feltenmark S, Gautam N, Brunnstrom A, Griffiths W, Backman L, Edenius C, Lindbom L, 501 Bjorkholm M, Claesson HE. Eoxins are proinflammatory arachidonic acid metabolites 502 produced via the 15-lipoxygenase-1 pathway in human eosinophils and mast cells. Proc Natl 503 Acad Sci U S A 2008; 105: 680-685. 504
24. Panigrahy D, Gilligan MM, Huang S, Gartung A, Cortes-Puch I, Sime PJ, Phipps RP, Serhan CN, 505 Hammock BD. Inflammation resolution: a dual-pronged approach to averting cytokine storms 506 in COVID-19? Cancer Metastasis Rev 2020; 39: 337-340. 507
25. Manne BK, Denorme F, Middleton EA, Portier I, Rowley JW, Stubben C, Petrey AC, Tolley ND, 508 Guo L, Cody M, Weyrich AS, Yost CC, Rondina MT, Campbell RA. Platelet gene expression 509 and function in patients with COVID-19. Blood 2020; 136: 1317-1329. 510
26. Hottz ED, Azevedo-Quintanilha IG, Palhinha L, Teixeira L, Barreto EA, Pao CRR, Righy C, 511 Franco S, Souza TML, Kurtz P, Bozza FA, Bozza PT. Platelet activation and platelet-monocyte 512 aggregate formation trigger tissue factor expression in patients with severe COVID-19. Blood 513 2020; 136: 1330-1341. 514
27. Nagata K, Hirai H, Tanaka K, Ogawa K, Aso T, Sugamura K, Nakamura M, Takano S. CRTH2, an 515 orphan receptor of T-helper-2-cells, is expressed on basophils and eosinophils and responds to 516 mast cell-derived factor(s). FEBS Lett 1999; 459: 195-199. 517
28. Monneret G, Gravel S, Diamond M, Rokach J, Powell WS. Prostaglandin D2 is a potent 518 chemoattractant for human eosinophils that acts via a novel DP receptor. Blood 2001; 98: 1942-519 1948. 520
29. Hirai H, Tanaka K, Yoshie O, Ogawa K, Kenmotsu K, Takamori Y, Ichimasa M, Sugamura K, 521 Nakamura M, Takano S, Nagata K. Prostaglandin D2 selectively induces chemotaxis in T helper 522 type 2 cells, eosinophils, and basophils via seven-transmembrane receptor CRTH2. J Exp Med 523 2001; 193: 255-261. 524
All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprintthis version posted December 7, 2020. ; https://doi.org/10.1101/2020.12.04.20242115doi: medRxiv preprint
https://doi.org/10.1101/2020.12.04.20242115
19
30. Chang JE, Doherty TA, Baum R, Broide D. Prostaglandin D2 regulates human type 2 innate 525 lymphoid cell chemotaxis. J Allergy Clin Immunol 2014; 133: 899-901 e893. 526
31. Medeiros A, Peres-Buzalaf C, Fortino Verdan F, Serezani CH. Prostaglandin E2 and the 527 suppression of phagocyte innate immune responses in different organs. Mediators Inflamm 528 2012; 2012: 327568. 529
32. Group RC, Horby P, Lim WS, Emberson JR, Mafham M, Bell JL, Linsell L, Staplin N, Brightling 530 C, Ustianowski A, Elmahi E, Prudon B, Green C, Felton T, Chadwick D, Rege K, Fegan C, 531 Chappell LC, Faust SN, Jaki T, Jeffery K, Montgomery A, Rowan K, Juszczak E, Baillie JK, 532 Haynes R, Landray MJ. Dexamethasone in Hospitalized Patients with Covid-19 - Preliminary 533 Report. N Engl J Med 2020. 534
33. Tomazini BM, Maia IS, Cavalcanti AB, Berwanger O, Rosa RG, Veiga VC, Avezum A, Lopes RD, 535 Bueno FR, Silva M, Baldassare FP, Costa ELV, Moura RAB, Honorato MO, Costa AN, 536 Damiani LP, Lisboa T, Kawano-Dourado L, Zampieri FG, Olivato GB, Righy C, Amendola 537 CP, Roepke RML, Freitas DHM, Forte DN, Freitas FGR, Fernandes CCF, Melro LMG, Junior 538 GFS, Morais DC, Zung S, Machado FR, Azevedo LCP, Investigators CC-BI. Effect of 539 Dexamethasone on Days Alive and Ventilator-Free in Patients With Moderate or Severe Acute 540 Respiratory Distress Syndrome and COVID-19: The CoDEX Randomized Clinical Trial. JAMA 541 2020; 324: 1307-1316. 542
34. Group WHOREAfC-TW, Sterne JAC, Murthy S, Diaz JV, Slutsky AS, Villar J, Angus DC, 543 Annane D, Azevedo LCP, Berwanger O, Cavalcanti AB, Dequin PF, Du B, Emberson J, Fisher 544 D, Giraudeau B, Gordon AC, Granholm A, Green C, Haynes R, Heming N, Higgins JPT, Horby 545 P, Juni P, Landray MJ, Le Gouge A, Leclerc M, Lim WS, Machado FR, McArthur C, Meziani 546 F, Moller MH, Perner A, Petersen MW, Savovic J, Tomazini B, Veiga VC, Webb S, Marshall 547 JC. Association Between Administration of Systemic Corticosteroids and Mortality Among 548 Critically Ill Patients With COVID-19: A Meta-analysis. JAMA 2020; 324: 1330-1341. 549
35. Crofford LJ, Wilder RL, Ristimaki AP, Sano H, Remmers EF, Epps HR, Hla T. Cyclooxygenase-1 550 and -2 expression in rheumatoid synovial tissues. Effects of interleukin-1b, phorbol ester, and 551 corticosteroids. J Clin Invest 1994; 93: 1095-1101. 552
36. Masferrer JL, Reddy ST, Zweifel BS, Seibert K, Needleman P, Gilbert RS, Herschman HR. In vivo 553 glucocorticoids regulate cyclooxygenase-2 but not cyclooxygenase-1 in peritoneal 554 macrophages. J Pharmacol Exp Ther 1994; 270: 1340-1344. 555
37. Wilborn J, Crofford LJ, Burdick MD, Kunkel SL, Strieter RM, Peters-Golden M. Cultured lung 556 fibroblasts isolated from patients with idiopathic pulmonary fibrosis have a diminished capacity 557 to synthesize prostaglandin E2 and to express cyclooxygenase-2. J Clin Invest 1995; 95: 1861-558 1868. 559
38. Little P. Non-steroidal anti-inflammatory drugs and covid-19. BMJ 2020; 368: m1185. 560 39. Lund LC, Kristensen KB, Reilev M, Christensen S, Thomsen RW, Christiansen CF, Stovring H, 561
Johansen NB, Brun NC, Hallas J, Pottegard A. Adverse outcomes and mortality in users of non-562 steroidal anti-inflammatory drugs who tested positive for SARS-CoV-2: A Danish nationwide 563 cohort study. PLoS Med 2020; 17: e1003308. 564
40. Chow JH, Khanna AK, Kethireddy S, Yamane D, Levine A, Jackson AM, McCurdy MT, Tabatabai 565 A, Kumar G, Park P, Benjenk I, Menaker J, Ahmed N, Glidewell E, Presutto E, Cain S, 566 Haridasa N, Field W, Fowler JG, Trinh D, Johnson KN, Kaur A, Lee A, Sebastian K, Ulrich A, 567 Pena S, Carpenter R, Sudhakar S, Uppal P, Fedeles BT, Sachs A, Dahbour L, Teeter W, Tanaka 568 K, Galvagno SM, Herr DL, Scalea TM, Mazzeffi MA. Aspirin Use is Associated with 569 Decreased Mechanical Ventilation, ICU Admission, and In-Hospital Mortality in Hospitalized 570 Patients with COVID-19. Anesth Analg 2020. 571
41. Gupta A, Kalantar-Zadeh K, Reddy ST. Ramatroban as a Novel Immunotherapy for COVID-19. J 572 Mol Genet Med 2020; 14. 573
All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprintthis version posted December 7, 2020. ; https://doi.org/10.1101/2020.12.04.20242115doi: medRxiv preprint
https://doi.org/10.1101/2020.12.04.20242115
20
42. Yang J, Eiserich JP, Cross CE, Morrissey BM, Hammock BD. Metabolomic profiling of regulatory 574 lipid mediators in sputum from adult cystic fibrosis patients. Free Radic Biol Med 2012; 53: 575 160-171. 576
43. Saak A, Schonfeld W, Knoller J, Steinkamp G, von der Hardt H, Konig W. Generation and 577 metabolism of leukotrienes in granulocytes of patients with cystic fibrosis. Int Arch Allergy 578 Appl Immunol 1990; 93: 227-236. 579
44. Knoller J, Schonfeld W, Koller M, Hensler T, Konig W. Arachidonic acid metabolites from 580 polymorphonuclear leukocytes of healthy donors, severely burned patients and children with 581 cystic fibrosis--routine monitoring by high-performance liquid chromatography. J Chromatogr 582 1988; 427: 199-208. 583
45. Brom J, Konig W, Koller M, Gross-Weege W, Erbs G, Muller F. Metabolism of leukotriene B4 by 584 polymorphonuclear granulocytes of severely burned patients. Prostaglandins Leukot Med 1987; 585 27: 209-225. 586
46. Archambault AS, Poirier S, Lefebvre JS, Robichaud PP, Larose MC, Turcotte C, Martin C, Provost 587 V, Boudreau LH, McDonald PP, Laviolette M, Surette ME, Flamand N. 20-Hydroxy- and 20-588 carboxy-leukotriene (LT) B4 downregulate LTB4-mediated responses of human neutrophils and 589 eosinophils. J Leukoc Biol 2019; 105: 1131-1142. 590
47. Bouchette D, Preuss CV. Zileuton. StatPearls. Treasure Island (FL); 2020. 591 48. Halpin DMG, Criner GJ, Papi A, Singh D, Anzueto A, Martinez FJ, Agusti AA, Vogelmeier CF, 592
Committee GS. Global Initiative for the Diagnosis, Management, and Prevention of Chronic 593 Obstructive Lung Disease: The 2020 GOLD Science Committee Report on COVID-19 & 594 COPD. Am J Respir Crit Care Med 2020. 595
49. Ham EA, Soderman DD, Zanetti ME, Dougherty HW, McCauley E, Kuehl FA, Jr. Inhibition by 596 prostaglandins of leukotriene B4 release from activated neutrophils. Proc Natl Acad Sci U S A 597 1983; 80: 4349-4353. 598
50. Fonteh AN, Winkler JD, Torphy TJ, Heravi J, Undem BJ, Chilton FH. Influence of isoproterenol 599 and phosphodiesterase inhibitors on platelet-activating factor biosynthesis in the human 600 neutrophil. J Immunol 1993; 151: 339-350. 601
51. Denis D, Riendeau D. Phosphodiesterase 4-dependent regulation of cyclic AMP levels and 602 leukotriene B4 biosynthesis in human polymorphonuclear leukocytes. Eur J Pharmacol 1999; 603 367: 343-350. 604
52. Flamand N, Boudreault S, Picard S, Austin M, Surette ME, Plante H, Krump E, Vallee MJ, Gilbert 605 C, Naccache P, Laviolette M, Borgeat P. Adenosine, a potent natural suppressor of arachidonic 606 acid release and leukotriene biosynthesis in human neutrophils. Am J Respir Crit Care Med 607 2000; 161: S88-94. 608
53. Flamand N, Plante H, Picard S, Laviolette M, Borgeat P. Histamine-induced inhibition of 609 leukotriene biosynthesis in human neutrophils: involvement of the H2 receptor and cAMP. Br J 610 Pharmacol 2004; 141: 552-561. 611
54. Turcotte C, Zarini S, Jean S, Martin C, Murphy RC, Marsolais D, Laviolette M, Blanchet MR, 612 Flamand N. The Endocannabinoid Metabolite Prostaglandin E2 (PGE2)-Glycerol Inhibits 613 Human Neutrophil Functions: Involvement of Its Hydrolysis into PGE2 and EP Receptors. J 614 Immunol 2017; 198: 3255-3263. 615
55. Flamand N, Picard S, Lemieux L, Pouliot M, Bourgoin SG, Borgeat P. Effects of pyrrophenone, an 616 inhibitor of group IVA phospholipase A2, on eicosanoid and PAF biosynthesis in human 617 neutrophils. Br J Pharmacol 2006; 149: 385-392. 618
56. Hanasaki K, Ono T, Saiga A, Morioka Y, Ikeda M, Kawamoto K, Higashino K, Nakano K, 619 Yamada K, Ishizaki J, Arita H. Purified group X secretory phospholipase A2 induced prominent 620 release of arachidonic acid from human myeloid leukemia cells. J Biol Chem 1999; 274: 34203-621 34211. 622
All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprintthis version posted December 7, 2020. ; https://doi.org/10.1101/2020.12.04.20242115doi: medRxiv preprint
https://doi.org/10.1101/2020.12.04.20242115
21
57. Murakami M, Masuda S, Shimbara S, Bezzine S, Lazdunski M, Lambeau G, Gelb MH, Matsukura 623 S, Kokubu F, Adachi M, Kudo I. Cellular arachidonate-releasing function of novel classes of 624 secretory phospholipase A2s (groups III and XII). J Biol Chem 2003; 278: 10657-10667. 625
58. Nolin JD, Murphy RC, Gelb MH, Altemeier WA, Henderson WR, Jr., Hallstrand TS. Function of 626 secreted phospholipase A2 group-X in asthma and allergic disease. Biochim Biophys Acta Mol 627 Cell Biol Lipids 2019; 1864: 827-837. 628
59. Hallstrand TS, Lai Y, Altemeier WA, Appel CL, Johnson B, Frevert CW, Hudkins KL, Bollinger 629 JG, Woodruff PG, Hyde DM, Henderson WR, Jr., Gelb MH. Regulation and function of 630 epithelial secreted phospholipase A2 group X in asthma. Am J Respir Crit Care Med 2013; 188: 631 42-50. 632
60. Arbibe L, Koumanov K, Vial D, Rougeot C, Faure G, Havet N, Longacre S, Vargaftig BB, Bereziat 633 G, Voelker DR, Wolf C, Touqui L. Generation of lyso-phospholipids from surfactant in acute 634 lung injury is mediated by type-II phospholipase A2 and inhibited by a direct surfactant protein 635 A-phospholipase A2 protein interaction. J Clin Invest 1998; 102: 1152-1160. 636
61. Ohtsuki M, Taketomi Y, Arata S, Masuda S, Ishikawa Y, Ishii T, Takanezawa Y, Aoki J, Arai H, 637 Yamamoto K, Kudo I, Murakami M. Transgenic expression of group V, but not group X, 638 secreted phospholipase A2 in mice leads to neonatal lethality because of lung dysfunction. J 639 Biol Chem 2006; 281: 36420-36433. 640
62. Buckley CD, Gilroy DW, Serhan CN. Proresolving lipid mediators and mechanisms in the 641 resolution of acute inflammation. Immunity 2014; 40: 315-327. 642
63. Andreakos E, Papadaki M, Serhan CN. Dexamethasone, pro-resolving lipid mediators and 643 resolution of inflammation in COVID-19. Allergy 2020. 644
64. Turcotte C, Chouinard F, Lefebvre JS, Flamand N. Regulation of inflammation by cannabinoids, 645 the endocannabinoids 2-arachidonoyl-glycerol and arachidonoyl-ethanolamide, and their 646 metabolites. J Leukoc Biol 2015; 97: 1049-1070. 647
65. Turcotte C, Blanchet MR, Laviolette M, Flamand N. The CB2 receptor and its role as a regulator of 648 inflammation. Cell Mol Life Sci 2016; 73: 4449-4470. 649
66. Di Marzo V. New approaches and challenges to targeting the endocannabinoid system. Nat Rev 650 Drug Discov 2018; 17: 623-639. 651
67. Zhao J, Zhao J, Legge K, Perlman S. Age-related increases in PGD2 expression impair respiratory 652 DC migration, resulting in diminished T cell responses upon respiratory virus infection in mice. 653 J Clin Invest 2011; 121: 4921-4930. 654
68. Schwarz B, Sharma L, Roberts L, Peng X, Bermejo S, Leighton I, Massana AC, Farhadian S, Ko A, 655 DelaCruz C, Bosio CM. Severe SARS-CoV-2 infection in humans is defined by a shift in the 656 serum lipidome resulting in dysregulation of eicosanoid immune mediators. medRxiv 2020. 657
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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