key: cord-1039525-f7jk4ooq authors: Ferreira, A. C.; Soares, V. C.; Quintanilha, I. G. d. A.; Dias, S. d. S. G.; Rodrigues, N. F.; Sacramento, C. Q.; Mattos, M.; Freitas, C. S.; Temerozo, J. R.; Teixeira, L.; Hottz, E. D.; Barreto, E.; Pao, C. R.; Palhinha, L.; Miranda, M.; Bou-Habib, D. C.; Bozza, F. A.; Bozza, P. T.; Lopes e Souza, T. M. title: SARS-CoV-2 induces inflammasome-dependent pyroptosis and downmodulation of HLA-DR in human monocytes, which can be prevented by atazanavir. date: 2020-08-31 journal: nan DOI: 10.1101/2020.08.25.20182055 sha: 37b5e6e08c8447349839dc3fa5ffea0adb1d2c4b doc_id: 1039525 cord_uid: f7jk4ooq Infection by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been associated with leukopenia and uncontrolled inflammatory response in critically ill patients. A better comprehension of SARS-CoV-2-induced monocytes death is essential for the identification of therapies capable to control the hyper-inflammation and reduce viral replication in patients with COVID-19. Here, we show that SARS-CoV-2 induces inflammasome activation and cell death by pyroptosis in human monocytes, experimentally infected and in patients under intensive care. Pyroptosis was dependent on caspase-1 engagement, prior to IL-1beta production and inflammatory cell death. Monocytes exposed to SARS-CoV-2 downregulate HLA-DR, suggesting a potential limitation to orchestrate the immune response. Our results originally describe the mechanism by which monocytes, a central cellular component recruited from peripheral blood to respiratory tract, succumb in patients with severe 2019 coronavirus disease (COVID-19), and emphasize the need for identifying anti-inflammatory and antiviral strategies to prevent SARS-CoV-2-induced pyroptosis. (VECTASHIELD). Fluorescence was analyzed by fluorescence microscopy with an x100 151 objective lens (Olympus, Tokyo, Japan) . 152 The levels of IL-1ß, TNF-α, IL-6, IL-8 and LDH were quantified in the culture 154 supernatants from infected and uninfected monocytes using ELISA kits, according to the 155 manufacturer's intructions (R&D System). 156 Extracellular lactate dehydrogenase (LDH) was quantified using Doles ® kit 157 according to manufacturer's instructions. In brief, cell culture supernatants were 158 centrifuged at 5,000 rpm for 1 minute, to remove cellular debris, and then 25 μL were 159 placed into 96-well plates and incubated with 5 μL of ferric alum and 100 μL of LDH 160 substrate for 3 minutes at 37 o C. Nicotinamide adenine dinucleotide (NAD, oxidized 161 form) was added followed by the addition of a stabilizing solution. After 10 minutes, the 162 reaction was read in a spectrophotometer at 492 nm. 163 Cellular extracts of 1x10 6 cells were homogenized in the RIPA lysis buffer in the 165 presence of proteinase inhibitor cocktail (Roche), and the protein levels were measured 166 by BCA protein assay kit. A total of 20 μg of protein was loaded onto a 10% sodium 167 dodecyl sulfate polyacrylamide gel (SDS-PAGE) for separation by electrophoresis and 168 the protein bands were then transferred to a polyvinylidene difluoride membranes 169 (ImmobilonP-SQ, Millipore). The membranes were blocked with 5 % albumin diluted in 170 Tris-buffered saline containing 0.05 % of Tween 20 for 2 hours at room temperature and 171 incubated with the specific primary antibodies (Cell Signaling Technology), to detect pro-172 caspase-1 and cleaved-caspase-1, after overnight incubation at 4 °C. After washing, We prospectively enrolled severe COVID19 RT-PCR-confirmed cases, as well as 182 SARS-CoV-2-negative healthy controls. Blood samples were obtained from 12 patients 183 with severe COVID-19 within 72 hours from intensive care unit (ICU) admission in two 184 reference centers (Instituto Estadual do Cérebro Paulo Niemeyer and Hospital Copa Star, 185 Rio de Janeiro, Brazil). Severe COVID-19 was defined as critically ill patients presenting 186 viral pneumonia on computed tomography scan and in mechanical ventilation. All 187 patients had SARSCoV-2 confirmed diagnostic through RT-PCR of nasal swab or 188 tracheal aspirates. Peripheral vein blood was also collected from 8 SARS-CoV-2-negative 189 healthy control participants as tested by RT-PCR on the day of blood sampling. The 190 characteristics of severe (n = 12), and control (n = 8) participants are presented in Table 191 1. Severe COVID-19 patients usually present older age and higher prevalence of 192 comorbidities as obesity, cardiovascular diseases and diabetes as in previously reported 193 patient cohorts. In the present study, the SARS-CoV-2-negative control group was 194 designed to include subjects of older age and chronic non-communicable diseases, so it 195 is matched with critically ill COVID-19 patients (Table 1) . Patients with acute respiratory 196 distress syndrome (ARDS) were managed with neuromuscular blockade and a protective 197 ventilation strategy that included low tidal volume (6 mL/kg of predicted body weight) 198 and limited driving pressure (less than 16 cmH2O) as well as optimal PEEP calculated 199 based on the best lung compliance and PaO2/FiO2 ratio. In those with severe ARDS and 200 PaO2/FiO2 ratio below 150 despite optimal ventilatory settings, prone position was 201 initiated. Our management protocol included antithrombotic prophylaxis with enoxaparin 202 40 to 60 mg per day. Patients did not receive routine steroids, antivirals or other anti-203 inflammatory or anti-platelet drugs. The SARS-CoV-2-negative control participants 204 were not under anti-inflammatory or anti-platelet drugs for at least two weeks. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted August 31, 2020. . study protocol (CONEP 30650420.4.1001 .0008), and informed consent was obtained 213 from all participants or patients' representatives. 214 The assays were performed in blinded way. They were performed by one 216 professional, codified and read by another fellow. All experiments were carried out at 217 least three independent times, including a minimum of two technical replicates in each 218 assay. The dose-response curves used to calculate EC50 and CC50 values were generated 219 by variable slope plot from Prism GraphPad software 8.0. The equations to fit the best 220 curve were generated based on R 2 values ≥ 0.9. Student's T-test was used to access 221 statistically significant P values <0.05. The statistical analyses specific to each software 222 program used in the bioinformatics analysis are described above. 223 To explore the mechanism by which SARS-CoV-2 triggers monocyte death, 226 human primary monocytes were infected and treated with pharmacological inhibitors of 227 pyroptosis (AC-YVAD-CMK, an inhibitor of Caspase-1 activity), apoptosis (ZVAD-228 FMK, a pan-caspase inhibitor), and necroptosis (Nec-1, an inhibitor of Receptor-229 interacting serine/threonine-protein kinase 1; RIPK1). Next, cell death was analyzed by 230 quantifying the LDH activity in the culture supernatant, and by AnnexinV/propidium 231 iodide (PI) cell labeling through for flow cytometry and fluorescence microscopy. As 232 shown in Figure 1A is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted August 31, 2020. . https://doi.org/10.1101/2020.08.25.20182055 doi: medRxiv preprint To gain insights on the mechanisms of monocyte pyroptosis in SARS-CoV-2 243 infection, we accessed caspase-1 and IL-1 activation -two key events that require 244 inflammasome's proteolytic activity. To do so, human monocytes were pretreated with 245 AC-YVAD-CMK or the IL-1 receptor antagonist (IL-1RA), and infected with SARS-246 CoV-2. Cells stimulated with LPS+ATP were used as a positive control of inflammasome 247 activation. We observed that SARS-CoV-2 induced pro-caspase-1 cleavage, similarly to 248 positive control (Figure 2A ). Caspase-1 activation was not observed in infected cells 249 pretreated with AC-YVAD-CMK ( Figure 2A ). To confirm these results, cells were 250 labeled with FAM-YVAD-FLICA (as an indicative of caspase-1 activation) and analyzed 251 by flow cytometry. We found that SARS-CoV-2 infection induced the activation of 252 caspase-1 in monocytes in the same magnitude to the positive control group ( Figure 2B ). 253 Moreover, SARS-CoV-2-induced caspase-1 activation was a specific event, since we did 254 not observe activation of the apoptotic caspases-3 and -7 in infected monocytes ( Figure 255 2C). Importantly, inhibition of IL-1R engagement reduced SARS-CoV-2-mediated 256 caspase-1 activation and LDH release, suggesting that inflammasome-dependent IL-1β 257 secretion amplify caspase-1 activation and pyroptosis in SARS-CoV-2 infection ( Figure 258 2B and 2D). 259 monocytes 261 As caspase-1 activity is critical for the production of IL-1β (15,16), we measured 262 the levels of this cytokine in our experiments. Our data show that SARS-CoV-2 infection 263 was able to increase IL-1β production ( Figure 2A ). This increment was prevented by the 264 treatment with caspase-1 pharmacological inhibitor AC-YVAD-CMK and the pan-265 caspase inhibitor ZVAD ( Figure 2A ). To confirm whether the observed effect is 266 specifically related to inflammasome activation, we evaluated the production of IL-8, in 267 which induction is independent of the inflammasomes pathway. SARS-CoV-2 infection 268 induced a higher IL-8 production, which was not prevented by caspase-1 inhibition 269 ( Figure 2B ). Importantly, caspase-1 specific inhibition, but not pan-caspase or RIPK1 270 inhibitor, also led to lower levels of the pro-inflammatory cytokines IL-6 and TNF-α, 271 highlighting the participation of the caspase-1-IL-1β axis in inflammatory amplification 272 during SARS-CoV-2 infection. ( Figure 3C and 3D). 273 . CC-BY-NC-ND 4.0 International license It is made available under a perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted August 31, 2020. . https://doi.org/10.1101/2020.08.25.20182055 doi: medRxiv preprint To investigate the impact of inflammasome activation and cell death through 276 pyroptosis on the orchestration of anti-SARS-CoV-2 immune response, human 277 monocytes were pretreated with AC-YVAD-CMK or with IL-1RA, and then infected 278 with SARS-CoV-2. Cells were then marked with HLA-DR and analyzed by flow 279 cytometry. We found that SARS-CoV-2 infection induced a decrease in HLA-DR 280 expression in monocytes ( Figure 3A and B). However, the inhibition of caspase-1 and 281 IL-1R engagement did not prevent the SARS-CoV-2-mediated decreasing of HLA 282 expression ( Figure 3C and D). 283 Given our findings that pyroptosis and decreased HLA-DR expression are 285 triggered during SARS-CoV-2 replication, we tested whether atazanavir (ATV), an HIV 286 protease inhibitor recently shown to possess antiviral activity against the new CoV in 287 monocytes (17), could prevent these events. Flow cytometry analysis of SARS-CoV-2-288 infected monocytes treated with ATV demonstrated a significant reduction in caspase-1 289 activity, observed by the decreased FAM-YVAD-FLICA labeling ( Figure 4A ). Other 290 orally available repurposed anti-SARS-CoV-2 drugs, such as lopinavir (LPV) and 291 ribavirin (RBV), did not affect SARS-CoV-2-induced pyroptosis ( Figure 4B ). Moreover, 292 treatments with ATV did not alter the activity of caspase-1, -3 and -7 in monocytes 293 exposed to LPS and ATP, used as a positive control of pyroptosis ( Figure S2 ), indicating 294 a specific antiviral activity of this drug. Consistently, treatment with ATV also reduced 295 the levels of IL-1β, IL-6 and TNF-α in SARS-CoV-2-infected monocytes, when 296 compared to the untreated cells ( Figure 4C -F). ATV did not interfere with the production 297 of IL-8, which is independent of inflammasome engagement ( Figure 4D ). 298 We then confirmed whether ATV can prevent SARS-CoV-2-induced pyroptosis. 299 Lower levels of LDH were detected in the supernatants of SARS-CoV-2-infected 300 monocytes treated with ATV when compared to infected and untreated cells ( Figure 4G ). 301 The treatment with ATV was also able to prevent the decrease of HLA-DR expression in 302 infected monocytes ( Figure 4H and I). LPV and RBV did not alter the HLA expression 303 in monocytes infected with SARS-Cov-2 ( Figure S3 ). 304 . CC-BY-NC-ND 4.0 International license It is made available under a perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted August 31, 2020. . https://doi.org/10.1101/2020.08.25.20182055 doi: medRxiv preprint patients 306 To clinically validate our findings, we evaluated if monocytes obtained from 307 critically ill patients with COVID-19 would also display signals of inflammasome 308 engagement and pyroptosis. Western blot analyzes showed that monocytes from 309 COVID-19 patients had increased levels of cleaved caspase-1 compared to monocytes 310 from healthy donors (HD) ( Figure 5A ). To confirm this result, cells were marked with 311 FAM-YVAD-FLICA and analyzed by flow cytometry. We observed that monocytes from 312 COVID-19 patients had increased caspase-1 activation ( Figure 5B ) and significantly 313 higher PI+ staining, when compared to monocytes from HD ( Figure 5C ). Corroborating 314 with our in vitro data, we also detected higher levels of IL-1β in the plasma of critically 315 ill patients ( Figure 5D ). 316 is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted August 31, 2020. . https://doi.org/10.1101/2020.08.25.20182055 doi: medRxiv preprint response mediated by monocytes/macrophages, when they should orchestrate the 335 antiviral immune response (26, 27) . In this work, we demonstrate that SARS-CoV-2 336 infection triggers inflammasome activation, increases IL-1ß secretion by monocytes, 337 resulting in pyroptotic cell death, which could be a key event for SARS-CoV-2 338 pathogenesis in critically ill patients and prevented by antiviral drugs (28). 339 Our results demonstrate that SARS-CoV-2 leads to an intense cell death in human is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted August 31, 2020. . https://doi.org/10.1101/2020.08.25.20182055 doi: medRxiv preprint peripheral monocytes isolated from patients with severe COVID-19. We found that these 367 cells present higher caspase-1 activation when compared with monocytes isolated from 368 HD. Recent clinical data reports reveal the presence of high levels of LDH and leukopenia 369 in critically ill patients (6, 8, 40, 41) . Our data also demonstrate intense monocyte death in 370 COVID-19 patients as detected through flow cytometry analyzes. Altogether, these data 371 suggest that the severity of COVID-19 may be associated with inflammasome activation 372 in monocytes that results in large amounts of IL-1ß and generates an excessive 373 inflammatory response characterized by high levels of IL-6 and TNF-α. Consistently, 374 treatment with IL-1RA has been associated with clinical and inflammatory improvements 375 in critically ill COVID-19 patients (42). These results are in line with clinical case reports 376 that demonstrate that monocytes/macrophages are key cells in the deleterious pro-377 inflammatory events that characterize the most serious cases of . 378 Effective therapies for COVID-19 should combine antiviral with anti-379 inflammatory properties to reduce viral load and to mitigate the intense inflammatory 380 response. In the present study we used ATV, an antiretroviral approved in 2003 for HIV-381 1 treatment and previously described by us as having anti-SARS-CoV-2 effects in 382 different cell types (including monocytes) and impairing virus-induced enhancement of 383 IL-6 and TNF-α levels (17). Our data reveal that ATV, besides inhibiting inflammasome 384 and pyroptosis, prevents the decreasing of HLA expression in monocytes infected with 385 SARS-CoV-2. In addition, ATV inhibited the release of LDH and the production of IL-386 1ß, f IL-6 and TNF-α by SARS-CoV-2-infected monocytes, which are key players in the 387 cytokine storm associated with 48) . 388 In this work, we originally describe that infection with SARS-CoV-2 can induce 389 pyroptotic cell death by inflammasome activation, which may be related to the intense 390 leukopenia and exacerbated inflammation seen in severe cases of the COVID-19. Since 391 there is no specific therapy for this disease, our results point out that ATV has a promising 392 therapeutic potential against SARS-CoV-2-induced cell death. 393 is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted August 31, 2020. . is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted August 31, 2020. . is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted August 31, 2020. . https://doi.org/10. 1101 /2020 for the formation of inflammasomes and pyroptosis induction. Cell culture supernatants 566 were collected for the measurement of the levels of (A) IL-1β, (B) IL-8, (C) IL-6 and (D) 567 TNF-α. Graphs data are representative of six independent experiments. Data are presented 568 as the mean ± SEM # P < 0.05 versus infected and untreated group (Nil); * P < 0.05 versus 569 positive control group (LPS+ATP). 570 is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted August 31, 2020. . https://doi.org/10.1101/2020.08.25.20182055 doi: medRxiv preprint is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted August 31, 2020. . https://doi.org/10.1101/2020.08.25.20182055 doi: medRxiv preprint . CC-BY-NC-ND 4.0 International license It is made available under a perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted August 31, 2020. Induction of 410 pro-inflammatory cytokines (IL-1 and IL-6) and lung inflammation by COVID-411 19: anti-inflammatory strategies 894 895 896 897 898 899 900 901 902 903 904 905 906 907 908 909 910 911 912 913 914 915 916 917 918 919 920 921 922