Modulators Of Purinergic Receptors and Related Immune Checkpoint For Treating Acute Respiratory Distress Syndrome
20230302031 · 2023-09-28
Assignee
Inventors
- Jean-Luc PERFETTINI (Meaux, FR)
- Deborah LECUYER (Guyancourt, FR)
- Desiree TANNOUS (Paris, FR)
- Awatef Allouch (Bry-sur-Marne, FR)
- Oliver DELELIS (Viroflay, FR)
- Frederic SUBRA (Chantilly, FR)
Cpc classification
A61K31/7048
HUMAN NECESSITIES
A61K31/191
HUMAN NECESSITIES
A61K45/06
HUMAN NECESSITIES
A61K31/196
HUMAN NECESSITIES
International classification
A61K31/7048
HUMAN NECESSITIES
A61K45/06
HUMAN NECESSITIES
A61K31/196
HUMAN NECESSITIES
A61K31/64
HUMAN NECESSITIES
Abstract
The inventors herein show that purinergic receptors regulate the conversion of macrophage pro-inflammatory reprogramming into anti-inflammatory phenotype in patients suffering from COVID-19 disease. Moreover, they show that P2Y receptor agonists repress NLRP3 inflammasome-dependent IL-1b secretion, but also impair the replication and the cytopathogenic effects of SARS-CoV-2. These results therefore suggest that some purinergic receptors agonists can treat acute lung injury and respiratory disease that are associated with SARS-CoV-2 infection. In addition, their results show that antagonists of the purinergic receptors P2X impair the replication of said virus. The present invention therefore proposes to use purinergic receptors modulators and NLR3-P2Y2R immune checkpoint modulators to treat patients suffering from a virus-induced acute respiratory distress syndrome.
Claims
1.-25. (canceled)
26. Method for treating a subject suffering from an inflammatory disease associated with an accumulation of pro-inflammatory macrophages and/or associated with an over-activation of the NRLP3 inflammasome, said method comprising the step of administering to said subject an agonist of a purinergic P2Y receptor.
27. The method of claim 26, wherein said inflammatory disease is Acute Respiratory Distress Syndrome (ARDS), the cryopyrin associated periodic syndrome, rheumatoid arthritis, obesity, or Alzheimer's disease.
28. The method of claim 26, wherein said inflammatory disease is an ARDS caused by sepsis, pneumonia, pancreatitis, surgery, radiation, virus, or a chemotherapeutic drug.
29. The method of claim 26, wherein said inflammatory disease is a virus-induced ARDS.
30. The method of claim 26, wherein said inflammatory disease is the COVID19 disease associated with the infection of the Severe Acute Respiratory Syndrome coronavirus 2.
31. The method of claim 26, wherein said agonist is an agonist of the P2Y2 receptor.
32. The method of claim 26, wherein said agonist is MRS2698, Uridine triphosphate (UTP), 4-thio-UTP, 2-thioUTP, Diquafosol, PSB1114, ATP, Denufosol, Ap4A, UTPγS, 5BrUTP, or MRS2768.
33. The method of claim 26, wherein said agonist is used to impair the replication of the Severe Acute Respiratory Syndrome coronavirus 2 in epithelial cells and for treating a subject suffering from the COVID19 disease.
34. The method of claim 26, wherein said agonist is administered to a subject suffering from an ARDS caused by an influenza virus, a respiratory virus, or an herpesvirus.
35. The method of claim 26, wherein said agonist is administered to a subject suffering from a coronavirus.
36. The method of claim 26, wherein said agonist is administered to a subject suffering from a Betacoronavirus.
37. The method of claim 26, wherein said agonist is administered to a subject suffering from a SARS-CoV-2 virus.
38. A method for impairing the replication of the Severe Acute Respiratory Syndrome coronavirus 2 in epithelial cells, said method comprising the step of contacting said cells with an agonist of a purinergic P2Y receptor.
39. The method of claim 38, wherein said agonist is an agonist of the P2Y2 receptor.
40. The method of claim 38, wherein said agonist is MRS2698, Uridine triphosphate (UTP), 4-thio-UTP, 2-thioUTP, Diquafosol, PSB1114, ATP, Denufosol, Ap4A, UTPγS, 5BrUTP, or MRS2768.
41. The method of claim 38, wherein said agonist is used to impair the replication of the Severe Acute Respiratory Syndrome coronavirus 2 in epithelial cells and for treating a subject suffering from the COVID19 disease.
42. The method of claim 38, wherein said agonist is administered to a subject suffering from an ARDS caused by an influenza virus, a respiratory virus, or an herpesvirus.
43. The method of claim 38, wherein said agonist is administered to a subject suffering from a coronavirus.
44. The method of claim 38, wherein said agonist is administered to a subject suffering from a Betacoronavirus.
45. The method of claim 38, wherein said agonist is administered to a subject suffering from a SARS-CoV-2 virus.
Description
DESCRIPTION OF THE FIGURES
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EXAMPLES
1. Materials and Methods
[0167] 1.1. Cell lines and SARS-COV-2
[0168] Monocytic THP1 cells were obtained from ATCC and were maintained in RMPI-1640-Glutamax medium supplemented with 10% heat inactivated fetal bovine serum (FBS) and 100 UI/mL penicillin-streptomycin (Life technology). THP1 macrophages were obtained by treatment for 3 hours with 100 nM phorbol-12-myristate-13-acetate (PMA, Invivogen) of THP1 monocytes and after extensive washings were let to differentiate for 72 hours before experimentation. The African green monkey kidney epithelial (Vero E6) cells were purchased from ATCC (ATCC CRL-1587) and cultured in Dulbecco's modified Eagle's medium (DMEM, Gibco, USA) with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 μg/mL streptomycin at 37° C. All cell lines used were mycoplasma-free. SARS-CoV-2 was propagated on Vero E6 cells in a biosafety level-3 (BLS-3) laboratory. After 72 hours of infection with a multiplicity of infection of 0.2, the supernatant was collected and centrifuged during 5 minutes at 1500 rpm at 4° C. to remove cellular debris. Then, supernatant was centrifuged during 20 minutes at 3000 rpm at 4° C. and stored at −20° C. Viral titration was performed by determining cytopathogenic effects associated with viral infection. Cell lysis was determined using agarose-containing semi-solid medium (Björn Meyer; Institut Pasteur). LPS, ATP, UTP, Diquafosol and Denufosol, pyridoxal phosphate-6-azophenyl-2′,4′-disulfonic acid (PPDAS) and P2X7 receptor agonist 2′,3′-O-(4-benzoyl-benzoyl)ATP (BzATP) were obtained from Sigma, Diquafosol from Clinisciences and Denufosol from Carbosynth.
1.2. Western Blots
[0169] Human THP-1 cells were cultured in RPMI 1640 media, supplemented with 10% FBS and differentiated by treatment for 3 hours with 100 nM phorbol-12-myristate-13-acetate (PMA, Invivogen). After 2 days, macrophage THP-1 cells were stimulated first 3 hours with ultrapure LPS from E. coli (10 ng/ml, LPS) and then stimulated for 6 hours with ATP (5 mM, Sigma) or treated with 50 ng or 20 ng of IFNg during 24 or 12 hours as indicated. Then, supernatants and cells were collected for western blot analysis.
[0170] Cells or supernatant were lysed in appropriated buffer (250 mM NaCl, 0.1% NP-40, 5 mM EDTA, 10 mM Na3VO4, 10 mM NaF, 5 mM DTT, 3 mM Na4P2O7, 1 mM EGTA, 10 mM Glycerol phosphate, 10 mM Tris-Hcl (pH=7.5) and the protease and phosphatase inhibitors (Roche)). Equal amount of supernatant or 10-40 μg of protein extracts were run on 4-12% or 10% SDS-PAGE and transferred at 4° C. onto a nitrocellulose membrane (0.2 Micron). After incubation for 2 hours at room temperature with 5% nonfat milk or BSA (Bovine Serum Albumine) in Tris-buffered saline and 0.1% Tween 20 (TBS-Tween), membranes were incubated with primary antibody at 4° C. overnight. Horseradish peroxidase-conjugated goat anti-mouse or anti-rabbit (SouthernBiotech) antibodies were then incubated for 1 hour 30 minutes and revealed with the enhanced ECL detection system (GE Healthcare). The primary antibodies against. IL-1β, IRF5 and GAPDH were from Abcam and Horseradish peroxidase-conjugated goat anti-rabbit (SouthernBiotech) antibodies were incubated for 1 hours and revealed with the enhanced ECL detection system (GE Healthcare). Western blots shown are representative of at least of three independent experiments.
1.3. Cytopathogenic assay
[0171] The cytotoxic tests were performed using Vero E6 cells. Twenty-four hours before infection, 4×10.sup.3 cells were seeded per well on 96 well plates. Cells were pretreated with indicated concentrations of UTP, Dequifosol, Denufosol and BzATP during 4 hours before infection and infected with a multiplicity of infection between 1 and 2. Viability of cells was then determined after 72 hours of infection using (bromure de 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium) (MTT) assay following manufacter's instructions.
1.4. Quantitative RT-PCR
[0172] After infection, total cell RNA was extracted with the RNeasy minikit (QIAGEN) according to the manufacturer's instructions. RNA was recovered according to the manufacturer's instructions for total cell RNA extraction. Quantifications were performed by real-time PCR on a Light Cycler instrument (Roche Diagnostics, Meylan, France) using the second-derivative-maximum method provided by the Light Cycler quantification software, version 3.5 (Roche Diagnostics). Standard curves for Sars-Cov2 RNA quantifications were provided in the quantification kit and were generated by amplification of serial dilutions of the provided positive control. Sequences of the oligonucleotides and probes used to quantify the RdRp and E genes are given below.
[0173] For the RdRp gene the LightMix®Modular Wuhan CoV RdRP-gene530 (Cat.-No. 53-0777-96, Tib Mol biol) was used with the following primers and probe: [0174] RdRp SARSr-F 5′: gTgARATggTCATgTgTggCgg (SEQ. ID NO:3); [0175] RdRp SAR5r-R 5′: CARATgTTAAASACACTATTAgCATA (SEQ ID NO :4); [0176] RdRp SARSr-P2 5′: 6FAM-CAggTggAACCTCATCAggAgATgC-BBQ (6FAM-SEQ ID NO:5-BBQ).
[0177] For the E-gene: [0178] E Sarbeco P1 5′: 6FAM-ACACTAgCCATCCTTACTgCgCTTCg-BBQ (6FAM-SEQ ID NO:6-BBQ): [0179] E Sarbeco F 5′:ACAggTACgTTAATAgTTAATAgCgT (SEQ ID NO:7); [0180] E Sarbeco R 5′: ATATTgCAgCAgTACgCACACA (SEQ ID NO:8).
[0181] Conditions used for the amplification of RdRp and E genes are the same and are as follow: Reverse transcription (5 minutes), denaturation (5 minutes) and 45 cyles with the following steps (95° C., 5 s−60° C., 15 s−72° C. 15 s). Results were normalized by the total amount of RNA in the sample and also reported to the condition without any compound (wild-type condition).
1.4. In Vitro Infection with SARS-CoV-2
[0182] ACE2-A549 and Caco2 cells were infected for 48 hours with SARS-CoV-2 (BetaCoV/France/IDF0372/2020) at multiplicity of infection (MOI) ranging from 0.1 to 6 in absence or in presence of 5 UI/mL Apyrase (Sigma), 500 μM of c(Sigma) or 100 μM of Tranilast (Sigma).
1.5. Immunofluorescence
[0183] After viral infection with a MOI of 6, cells were fixed at room temperature during 20 min with 4% paraformaldehyde (PFA), permeabilized with 0.3% Triton-X100 and blocked with 10% FBS for 1 hour at room temperature, Then, cells were stained for 2 hours with mouse anti-Spike S antibody (#GTX632604, Genetex) and 1 hour with goat anti-mouse IgG conjuguated to Alexa Fluor 546 (#A11030, Invitrogen). Nuclei were also stained with Hoechst 33342 (#H3570, Invitrogen) as previously described. Cells were analyzed by fluorescent microscopy on Leica DMI8.
1.6. Flow Cytometry
[0184] Mice were sacrificed and the right lungs were removed, washed in cold PBS, minced, and digested using the lung dissociation kit (Mittenyi, #130-095-927) for 30 min under agitation at 37° C. After enzyme digestion, lung tissue was passed through a 70 μm filter and red blood cells were lysed with ACK lysing buffer (#A10492-01, Gibco) for 10 min on ice. Then, cells were washed once in DMEM medium than in PBS. Cells were incubated with purified anti-mouse CD16/32 (#101302, BioLegend) for 10 minutes at 4° C. For membrane staining, anti-CD45APC-Vio770 (#130-110-662, miltenyi Biotec), anti-Ly6G PerCP-Vio 700 (#130-117-500, Miltenyi Biotec), anti-CD169 PE (#130-104-953, Miltenyi Biotec), anti-CD11c PE-Vio 770 (#130-110-703, Miltenyi Biotec), anti-CD11b BUV395 (#563553, B D Horizon), anti-Ly6C AF700 (#128024, BioLegend), anti-CD64BV605 (139323, BioLegend) and anti-Siglec-F PE-CF594 (#562757, B D Horizon) antibodies were incubated for 20 min at 4° C. Cells were fixed with 4% PFA. Samples were acquired on an LSR Fortessa II (B D, Franklin Lakes, NJ) with FACSDiva software, and data were analyzed with FlowJo 10.0.7 software (Tree Star, Inc., Ashland, OR).
1.7. Celt Viability Assay
[0185] Cell viability was determined after 48 hours using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (#M5655, Sigma) following manufacturer's instructions.
1.8. Western Blotting
[0186] After infection with a MOI of 2, total cellular proteins were extracted lysed in appropriated buffer containing 1% NP40, 20 mM HEPES, 10 mM KCl, 0.5 M EDTA, 10% Glycerol, protease and phosphatase inhibitors for 30 min at 4′C. After centrifugation, supernatants were collected, subjected to protein quantification and 15 to 20 μg of protein were loaded. A classical western blotting was then performed.
1.9. Quantitative RT-PCR for SARS-COV-2
[0187] After infection with a MOI of 0.1, total cell RNA was extracted using QIAshredder kit (#79654, Qiagen) and the RNeasy kit plus Mini kit (#74136, Qiagen) according to the manufacturer's instructions. RNA was recovered according to the manufacturer's instructions for total cell RNA extraction. Quantifications of SARS-COV-2 E RNA were performed by real-time PCR on a Light Cycler instrument (Roche Diagnostics, Meylan, France) using the second-derivative-maximum method provided by the Light Cycler quantification software (version 3.5 (Roche Diagnostics)). Standard curves for SARS-CoV-2 RNA quantifications were provided in the quantification kit and were generated by amplification of serial dilutions of the provided positive control.
1.10. Mouse Model of Bleomycin-Induced Lung Inflammation
[0188] Seven weeks old C57BL/6 mice were obtained from Janvier laboratories, maintained on 12 h dark/light cycles and provided with water and standard rodent diet ad libitum. Pulmonary inflammation was induced in C57BL/6 mice by intratracheal instillation of 50 UI of bleomycin. Every 3 days, 300 μg of Diquafosol were administrated intraperitoneally in mice. Mice were sacrificed at 21 days after bleomycin instillation.
1.11. Computerized Tomography (CT) Scan
[0189] Lung CT scan was acquired 1 day before the sacrifice using Bioimaging IVIS Spectrum-CT (PerkinElmer) and the Living Image Software 4.3 (PerkinElmer).
1.12. Quantification and Statistical Analysis
[0190] All values were expressed as the mean±SEM of cell individual samples.
[0191] Student t-test and one-way ANOVA test were used for statistical analysis. Statistical data were analyzed with Graphpad prism 8 software. Statistical significance was given as *P≤0.05, **P≤0.001 and ****P<0.0001.
2. Results
2.1. P2Y2 Agonists Inhibit NLRP3-Dependent IL-1β Secretion and Macrophage Pro-Inflammatory Reprogramming
[0192] To study the effects of P2Y2 agonists on NLRP3 inflammasome activation, PMA-treated THP1 macrophages were analyzed for IL-1β secretion after pretreatment with 50 μM of P2Y2 agonist MRS2768 during 1 hour and stimulation with 10 ng/ml LPS during 3 hours and 5 mM ATP during 6 hours (LPS+ATP). As previously described in many studies, stimulation of PMA-treated THP1 macrophages with LPS+ATP led to a robust secretion of IL-1 β in the supernatant of treated cells (
[0193] Then, PMA-treated THP1 macrophages were simulated with LPS+ATP in presence of indicated concentrations of Diquafosol, which is a P2Y2 agonist used for the treatment of cystic fibrosis lung disease (Kellerman et al., 2002) and supernatants were analyzed for IL-1β release. As shown with MRS2768 (
[0194] Considering that NLRP3 was also shown to contribute to macrophage pro-inflammatory reprograming (Camell et al., 2015), PMA-treated THP1 macrophages were also stimulated with IFNγ during 24 hours in presence or in absence of different concentrations of the natural P2Y2 agonist uridine-5′-triphosphate (UTP) and the expression of Interferon regulatory factor 5 (IRF5), which is a central transcription factors of macrophage pro-inflammatory reprogramming (Krausgruher et al., 2011.sup.35) was determined. A decrease of IRFS expression was detected in presence of 10 and 100 μM of UTP (
2.2. Purinergic Receptors Control Viral Replication of SARS-CoV-2 and Associated Cytopathogenic Effects
[0195] Considering that purinergic receptor P2Y2 and other purinergic receptors (such as purinergic receptor P2X7) may regulate permissivity to viral infection (Séror et al., 2011.sup.22; Paoletti et al., 2019.sup.20), the impact of P2Y2 agonists (UTP, Diquafosol and Denufosol), of P2X receptor antagonist pyridoxal phosphate-6-azophenyl-2′,4′-disulfonic acid (PPDAS) and of P2X7 receptor agonist 2′,3′-O-(4-benzoyl-benzoyl)ATP (BzATP) was next determined on the replication of SARS-CoV-2.
[0196] African green monkey kidney epithelial (Vero E6) cells were infected with SARS-CoV-2 during 2 hours (
[0197] To study viral replication, mRNA expression of RNA-dependent RNA polymerase (RdRp) and E genes were analyzed using quantitative RT-PCR. The activation of the P2Y2 by UTP, Diquafosol and Denufosol strongly reduced the amount of RdRp and E mRNA in Vero cells after 16 hours (
[0198] Interestingly, PPDAS also repressed RdRp and E mRNA expression after 16 (
[0199] In parallel, the impact of purinergic modulators on cell damages elicited by SARS-CoV-2 infection was evaluated. Vero cells were infected with SARS-COV-2 during 72 hours in presence of indicated concentrations of UTP (
[0200] These results indicate that the modulation of purinergic receptor activity during SARS-CoV-2 infection affects SARS-CoV-2 pathogenicity. Altogether, these results demonstrate that purinergic receptors and extracellular nucleotides play a central rote during SARS-CoV-2 infection.
2.3. Mechanisms of Action of the Modulators of the Invention
[0201] The rapid worldwide spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) poses a global health emergency that has not been resolved. Even though over 33,282,969 people are infected and more than 1,000,867 people have died from SARS-CoV-2 infection (https://coronavirus.jhu.edu/map.html), no cure or prophylactic treatment is currently available. With the exception of the antiviral remdesivir, which was authorized by the US Food and Drug administration for emergency use for the treatment of coronavirus 19 (COVID-19) (https://www.fda.gov), and steroid treatment which partially reduces the mortality of COVID-19 patients with severe disease (1), the majority of preclinical studies and clinical trials evaluating the repurposing of anti-malarial compounds (such as hydroxychloroquine and chloroquine), nonspecific anti-viral agents (such as lopinavir and ritonavir) or anti-inflammatory drugs (such as the IL-6 receptor antagonist tocilizumab) for the treatment of COVID-19 failed to identify highly effective treatment. A better understanding of mechanisms whereby SARS-COV-2 hijacks host cells for viral entry and dysregulates the anti-viral immune response is urgently needed to develop efficient therapeutic strategies for COVID-19.
[0202] The entry of SARS-CoV-2 into host cells starts with binding of viral spike (S) glycoprotein to angiotensin converting enzyme 2 (ACE-2) and with subsequent priming of S glycoprotein by the serine protease TMPRSS2 or cathepsin B/L (2, 3). The viral membrane then fuses with host cellular membranes (2, 4), leading to the release of viral RNA into host cytosol and replication using specialized proteins (such as RNA-dependent RNA polymerase (RdRp) (5)), intracellular expression of viral structural proteins (such as E and S proteins) and finally to the assembly and release of viral progeny (6). Host factors (such as p38MAPK, CK2, AXL and kinase PIFFWE kinases) are involved in the regulation of early and late steps of SARS-CoV-2 infection (7), but the host cellular pathways used by SARS-COV-2 to establish a viral infection are still poorly understood. Even though SARS-CoV-2-infected people are mainly asymptomatic or exhibit mild to moderate symptoms, approximately 15% of patients experience severe disease with atypical pneumonia and 5% develop an acute respiratory distress syndrome (ARDS) and/or multiple organ failure that is associated with a high mortality rate (around 50%) (8). Studies of COVID-19 patients with severe disease revealed a high level of plasma pro-inflammatory cytokines (including IL-1β, IL-6, IL-10, IL-18 and TNF) (9) and lactate dehydrogenase (LDH) (10), indicating overt hyper-inflammation during COVID-19, also known as “cytokine storm” or “cytokine release syndrome”. Until now, few molecular mechanisms driving COVID-19 associated hyper-inflammation have been identified and proposed to explain the pathogenesis of COVID-19.
[0203] Nucleotide-binding domain leucine-rich repeat-containing receptor (NLR) proteins and purinergic (P2) receptors are the main germline-encoded pattern recognition receptors regulating the secretion of IL-1 family members in response to microbial infection, inflammation, and inflammatory diseases. Upon activation, NLR protein 3 (NLRP3), which is the most studied NLR protein (11), forms large complexes, called inflammasomes, which activate caspase-1, induce the release of mature cytokines IL-1β and IL-18 (11, 12) and can lead to the inflammatory cell death of stimulated, stressed or infected host cells, which is also known as pyroptosis (13), SARS-CoV-2 viral proteins such as viral spike (S) glycoprotein (14), SARS-Cov open reading frame-8b (15) and the transmembrane pore-forming viral Viroporin 3a (also known as SARS-COV 3a) (16) were recently shown to activate the NLRP3 inflammasome, thus indicating that the NLRP3 inflammasome could represent a novel molecular target for the treatment of COVID-19. Purinergic receptors are membrane-bound innate receptors that bind extracellular nucleotides (such as adenosine triphosphate (ATP) and uridine triphosphate (UTP)), and control numerous cellular functions (such as cytokine secretion and migration) mainly on immune cells, but also on other cell types that are involved in SARS-CoV-2 pathogenesis such as type 1 and 2 pneumocytes, endothelial cells, platelets, cardiomyocytes and kidney cells (17, 18).
[0204] Purinergic receptors are divided into two families, the tonotropic P2X receptors and the metabotropic P2Y receptors, which can regulate the NLRP3 inflammasome (18-20). P2X7 activation was extensively shown to control NLRP3 inflammasome activation and cytokine release in response to danger signals (21). We recently demonstrated that the purinergic receptor P2Y2 interacts with NLRP3 and induces its ubiquitination and degradation (20), indicating that P2Y2 may potentially regulate negatively NLRP3 inflammasome activation. We previously revealed that purinergic receptors P2Y2 and P2X7 also control viral entry through the modulation of the fusogenic activity of HIV-1 envelope (22). The contribution of purinergic receptors to viral infection has been confirmed with other purinergic receptors and with several viruses (such as human cytomegalovirus and hepatitis B, C and D viruses) (23). Recently, we demonstrated that NLRP3 represses P2Y2-dependent viral entry during the early steps of HIV-1 infection (20). In addition, the P2Y2-NLRP3 interaction is enhanced during the inflammation, which is associated with chronic infections with HIV-1 and SIV (20). In this context, we hypothesized that the P2Y2-NLRP3 interaction could be enhanced during COVID-19 and that the therapeutic modulation of P2Y2 and NLRP3 could regulate both susceptibility of host cells to SARS-CoV-2 infection and hyper-inflammation associated with COVID-19. The contribution of P2X7 to SARS-CoV-2 pathogenesis was also addressed in this study.
2.3.1. Material and Methods
2.3.1.1. Viruses, Cells and Chemical Compounds
[0205] The BetaCoV/France/IDF0372/2020 SARS-CoV-2 strain was provided by Dr. Benoit Visseaux from the group of Prof. Diane Descamps (UMR 5 1135, Hôpital Bichat, Paris) and by the National Reference Center For Respiratory Viruses (Institut Pasteur, Paris, France). For in vitro studies, viral stocks were prepared by propagation in African green monkey kidney epithelial (Vero E6) cells in a biosafety level-3 (BLS-3) laboratory and titrated using lysis plaque assay as previously described (53). SARS-CoV-2 stock titer was 2×10.sup.6 PFU/mL. The supernatant was aliquoted and stored at −80° C. For in vivo studies, hCOV-19/France/IDF0372/2020 strain was amplified and virus stock produced as previously-described (54). Vero E6 cells were purchased from ATCC (ATCC CRL-1587) and cultured in Dulbecco's modified Eagle's medium (DMEM, Gibco, USA) with 10% heat inactivated fetal bovine serum (FBS), 100 UI/mL penicillin (Life technology), and 100 μg/mL streptomycin (Life technology) at 37° C. ACE2-overexpressing 1549 (ACE2-A549) cells were a gift from Dr. Olivier Schwartz (Institut Pasteur, Paris). Monocyte THP1 cells (ATCC TIB2002) were obtained from ATCC and were maintained in RMPI-1640-Glutamax medium supplemented with 10% heat inactivated FBS, 100 UI/mL penicillin, and 100 μg/mL streptomycin. Buffy coats from healthy donors were obtained from the French blood bank (Etablissement Français du Sang (EFS)). Informed written consent from each donor was obtained accordingly to French law. To generate Monocytes Derived Macrophages (MDMs), human monocytes were separated from peripheral blood mononuclear cells (PBMCs) by adherence to the plastic, detached and cultured for 6 days in hydrophobic Teflon dishes (Lumox Duthsher) in macrophage medium (RPMI 1640 supplemented with 200 mM L-glutamine, 100 UI of penicillin, 100 μg streptomycin, 10 mM HEPES, 10 mM sodium pyruvate, 50 μM β-mercaptoethanol, 1% minimum essential medium vitamins, 1% non-essential amino acids (Life technology)) supplemented with 15% of heat inactivated human serum AB (Life technology). Then, monocytes-derived macrophages (MDMs) were harvested and resuspended in macrophage medium containing 10% of FBS, as previously described (20, 55 , 56). Control THP1 cells (sh.Co.) and THP1 cells depleted for NLRP3 (sh-1NLRP3 and sh-2NLRP3) or P2Y2 (shP2Y2) were previously published (20). THP1 cells depleted for CASP1 (sh-1CASP1 and sh-2CASP1) were produced following the same experimental procedure and simultaneously to control and NLRP3- or P2Y2-depleted THP1 cells as indicated below. Uridine 5′-triphosphate (UTP) (#U6625), 2′(3′)-O-(4-Benzoylbenzoyl) adenosine 5′-triphosphate (BzATP) (#B6396), Kaempferol (#K0133), Suramin (#S2671), oxidized ATP (OxATP) (#A6779), pyridoxal-phosphate-6-azopheny-2′,4′-disulfonate (PPADS) (#P178) were purchased from Sigma-Aldrich. AR-C118925XX (#4890), Diquafosol (#HY-B0606) and Denufosol (#ND45968) were respectively from Tocris, MedChemExpress and Biosynth Carbosynth. Phorbol myristate acetate (PMA) (#tlrl-pma) was from Invivogen and recombinant human interferon g (IFNγ) was from R&D systems (#285-IF).
2.3.1.2. Patients' Samples
[0206] This non-interventional study received approval by the institutional review boards of Gustave Roussy hospital (Villejuif, France) and the National Institute for Infectious Disease <<Lazzaro Spallanzani>> and following the principles stated in the Declaration of Helsinski. Peripheral blood samples (Gustave Roussy) were obtained with informed consent of patients. The collection of blood samples from SARS-CoV-2 (n=7) and positive (n-18) patients was obtained from patients that were detected either negative or positive for SARS-CoV-2 by real time PCR (RT-PCR) at days before and at the same day of the blood sampling. The SARS-CoV-2 detection was performed on nasopharyngeal samples by RT-PCR using the GeneFinder COVID-19 PLUS RealAmp Kit (ELITECH), which detects SARS-CoV-2 by negative amplification of RdRp gene, E gene and N gene according to WHO recommended protocol. Moderate SARS-CoV-2 cases were defined as WHO Ordinal Scale for Clinical Improvement (OSCI) scale 3 and 4 and ≤5L/min of oxygen flow to maintain oxygen saturation (SpO2)>94%. Severe SARS-CoV-2 cases were defined as OCSI scale 4-8 and prolonged need of a ≥6 L/min of oxygen flow to maintain SpO2>94%. In this study all the five severe SARS-CoV-2 patients were admitted to the intensive care unit and needed mechanical ventilation. Fresh blood samples (250 μl) of SARS-CoV2 positive (n=18) or negative (n=7) patients were dropped on the microscopy slides that were pre-treated with Poly-L-lysine solution (0.1%) to let peripheral blood cells adhere for 1 hour at room temperature. Adherent cells were then washed with phosphate buffered saline (PBS) solution, dried for 5 minutes and fixed by 2% paraformaldehyde (PFA) during 20 minutes at room temperature. After washing with PBS, the slides were conserved at 4° C. in PBS. Human autopsies were performed at the National Institute for Infectious Diseases Lazzaro Spallanzani-IRCCS Hospital (Rome, Italy) according to guidance for post-mortem collection and submission of specimens and biosafety practices (CDC March 2020, Interim Guidance and (57)) to reduce the risk of transmission of infectious pathogens during and after the post-mortem examination. Autopsies were performed in accordance with the law owing to the unknown cause of death, and to both scientific and public interest in a pandemic novel disease. All performed procedures and investigations were in accordance with the ethical standards of the Institutional Review Board of Lazzaro Spallanzani National Institute for Infectious Disease (Ethics Committee Approval number: n° 9/2020). Post-mortem lung sections were obtained from 3 non-COVID-19 patients and 7 COVID-19 patients with severe disease. Controls (n=3) were patients deceased after hemorragia, cardiorespiratory failure or interstitial pneumonia associated with pulmonary capillaritis. All COVID-19 patients (n=7) deceased after cardiorespiratory failure and exhibited in their vast majority diffuse alveolar damage (n=6) or fibrosis (n=1). Detection of SARS-CoV-2 was performed by RT-PCR on all patients using ocular, nasopharyngeal, oropharyngeal and rectal swabs.
2.3.1.3. Samples from Non-Human Primates
[0207] Cynomolgus macaques (Macaca fascicularis) aged from 4 to 7 years originating from Mauritius Island and housed in Infectious Disease Models for Innovative Therapies (IDMIT) infrastructure of Commissariat à l'Energie Atomique et aux Energies Alternatives (CEA, Fontenay-aux-roses, France) were used. The protocols were approved by the ethical committee of animal experimentations of CEA under the protocol number CEA #44. Challenged animals were exposed to a total dose of 10.sup.6 PFU of SARS-CoV-2 (BetaCoV/France/IDF0372/2020 SARS-CoV-2 strain) via the combination of intranasal and intra-tracheal routes. The bronchoalveolar lavage fluids (BALFs) from SARS-CoV2 negative (n=4) and positive (n=5) Cynomoglus macaques were obtained three days after infections. Viral loads (0.76-2.4 (copies/mL)) were assessed in bronchoalveolar lavages by RT-PCR with a plasmid standard concentration range containing an RdRp gene fragment including the RdRp-IP4 RT-PCR target sequence. The protocol describing the procedure for the detection of SARS-CoV-2 is available on the WHO website (https://www.who.int/docs/default-source/coronaviruse/real-time-rt-pcr-assays-for-the-detection-of-sars-cov-2-institut-pasteur-paris. pdf?sfvrsn=3662fcb6_2). All BALFs were chilled in the ice up during handling, well mixed by low vortexing and filtered using cell strainers of 100 μm pore membranes (Falcon #352360) to eliminate debris. Cellular suspension was then centrifuged at 1000 rpm for 10 minutes at 4 ° C. and washed twice with 50 mL cold PBS containing 20% FBS. Viable cells (0.5×10.sup.6) were suspended in 200-μl cold PBS containing 20% FBS and dropped on poly-L-Lysine coated slides by using cytospin centrifuge (Cytospin 2 Shandon, Block scientific) at 800 rpm for 3 minutes. BALF cells were then air dried on slides for 30 minutes, fixed with 4% PFA solution for 20 minutes, washed twice with PBS and conserved at 4° C.
2.3.1.4. Samples from P2y2.sup.+/+ and P2y2.sup.−/− Mice
[0208] Two weeks old P2y2.sup.+/+ and P2y2.sup.−/− transgenic mice were obtained from Dr. Isabelle Couillin (58) and sacrificed upon arrival following the Federation of European Laboratory Animal Science Association guidelines and in accordance with the Ethical Committee of the Gustave Roussy Cancer Campus (CEE A26) (Villejuif, France). After sacrifice, plasmatic serum was aliquoted and stored at −80° C., lung biopsies were either fixed or digested for biological analysis as previously described (59).
2.3.1.5. In vitro infection with SARS-CoV-2
[0209] Vero (E6) cells and ACE2-4549 cells were infected during indicated times with SARS-CoV-2 (BetaCoV/France/IDF0372/2020) at multiplicity of infection (MOI) from 0.1 to 0.2, for infectivity analysis and from MOI 1 to 2 for cytopathogenicity analyses, in absence or in presence of 500 μM UTP, 100 μM Diquafosol, 5 μM Denufosol, 50 μM PPADS, 100 μM OxATP or 100 μM BzATP unless stated otherwise.
2.3.1.6. RNA Interference
[0210] Plasmids coding for the gag-pot HIV-1 genes (pCMV GAG-POL HIV University of Michigan), for the vector genome carrying shRNA of interest (pLKO.1 shRNA, Thermo Scientific) and for the plasmid coding for an envelope of VSVG (pMDG-VSV-G) were transfected into HEK293T cells using calcium phosphate reagent (Promega) to obtain lentiviral vector particles. After 48 hours, supernatants were filtered using 0.45-μm cellulose acetate filters (Sartorius stedim), aliquoted and stored at −80° C. Monocytic THP1 cells (4×10.sup.6) were then transduced during 24 hours and grown in medium containing 1 μg/mL puromycin (Invivogen). Control THP1 (PLKO.1) cells and P2Y2- (shP2Y2), NLRP3- (sh-1NLRP3 and sh-2NLRP3) and CASP1- (sh-1CASP1 and sh-2CASP1)-depleted THP1 cells were thus obtained. Validation of shCo., shP2Y2, sh-1NLRP3, sh-2NLRP3-containing THP1 cells were previously described and validated (20). MDMs were transfected using smart pools of siGenome non-targeting control and P2Y2 specific siRNAs from Dharmacon as previously described (20, 55, 56). After 48 hours of transfection, cell lysates and supernatants were analyzed for protein expression by western blot, flow cytometry and ELISA. Sequences of Human siRNA, siGENOME 5MARTpool and shRNA used in this study are shown in the following table:
TABLE-US-00002 Human target sg Target sequence SEQ ID NO siGENOME Non Targeting siRNA SMARTpool 1 UAGCGACUAAACACAUCAA SEQ ID NO: 9 2 UAAGGCUAUGAAGAGAUAC SEQ ID NO: 10 3 AUGUAUUGGCCUGUAUUAG SEQ ID NO: 11 4 AUGAACGUGAAUUGCUCAA SEQ ID NO: 12 siGENOME SMARTpool P2Y2 1 UGCCUAGGGCCAAGCGCAA SEQ ID NO: 13 2 UAACUGGAGCUCCGAUUUA SEQ ID NO: 14 3 UCUCAGGAGUAGUCUCAUA SEQ ID NO: 15 4 AGUCAUCGUUUGUGUGUAU SEQ ID NO: 16 pLKO.1 shP2Y2 1 ATGTTCCACCTGGCTGTGT SEQ ID NO: 17 CTGATGCACT pLKO.1 1 ATAATGAGAGCAAGACGTG SEQ ID NO: 18 sh-1Caspase-1 TG pLKO.1 2 AGCATCATCCTCAAACTCT SEQ ID NO: 19 sh-2Caspase-1 TC
2.3.1.7. Quantitative RT-PCR for SARS-CoV-2
[0211] After infection, total cell RNA was extracted using QIAshredder kit (#79654, Qiagen) and the RNeasy kit plus Mini kit (#74134, Qiagen) according to the manufacturer's instructions. RNA was recovered according to the manufacturer's instructions for total cell RNA extraction. Quantifications were performed by real-time PCR on a Light Cycler instrument (Roche Diagnostics, Meylan, France) using the second-derivative-maximum method provided by the Light Cycler quantification software (version 3.5 (Roche Diagnostics)). Standard curves for SARS-CoV-2 RNA quantifications were provided in the quantification kit and were generated by amplification of serial dilutions of the provided positive control. Sequences of the oligonucleotides and probes used to quantify the RdRp and E genes are given in the following Table.
TABLE-US-00003 Gene Primers and Probe sequences (SEQ ID NO: target 3-5 and 6-8) SARS- RdRp SARSr-F 5′: gTgARATggTCATgTgTggCggRdRp Cov-2 RdRp SARSr-R 5′: CARATgTTAAASACACTATTAgCATA RdRp SARSr-P2 5′: 6FAMCAggTggAACCTCATCAggAgATgC-BBQ SARS- E Sarbeco F 5′: ACAggTACgTTAATAgTTAATAgCgT Cov-2 E Sarbeco R 5′: ATATTgCAgCAgTACgCACACA. E-gene E Sarbeco P1 5′: 6FAM- CAggTggAACCTCATCAggAgATgC-BBQ
[0212] The amplification of RdRp and E genes was obtained after 5 minutes of reverse transcription, 5 minutes of denaturation and 45 cycles with the following steps (95° C. during 5 seconds, 60° C. during 15 seconds and 72° C. during 15 seconds). Results were normalized by the total amount of RNA in the sample and also reported to the condition without any compound (control condition). Data are presented as fold changes and were calculated with relative quantification of ΔΔCT obtained from quantitative RT-PCR.
2.3.1.8. Immunofluorescence and Flow Cytometry
[0213] Peripheral human blood cells, non-human primates cells, ACE2-A549 and THP1 cells were permeabilized with 0.3% Triton-X100, blocked with 10% FBS for 1 hour at room temperature before overnight staining at 4° C. with anti-NLRP3 (#ab4207, Abcam) and anti-P2Y2 (#APR-010, Alomone) antibodies at 1/50 dilutions. After washings with PBS, the proximity ligation assay was performed according to manufacture's instructions. Briefly, the primary antibodies were hybridized with the Duolink In Situ PLA Probes anti Rabbit PLUS (#DU092002, Sigma) and anti-Goat Minus PLUS (#DU092006, Sigma) for 1 hour at 37° C. followed by a ligation step of 30 minutes at 37° C. and an amplification step of 1 hour and 40 minutes at 37° C. The ligase and the polymerase enzymes catalyzing these reactions were included in the Duolink® In Situ Detection Reagents Green (#DUO92014, Sigma). Additional immunostaining step was done at room temperature in humid chamber. For THP1 and ACE2-A549 cells, nuclei were stained with Hoechst 33342 (#1874027, Invitrogen) (1/1000) for 30 minutes. Peripheral human blood cells were incubated with Alexa Fluor 647 anti-CD3 (#300416, BioLegend) (1/50) and Alexa Fluor 594 anti-human CD14 (#325630, BioLegend) (1/50) and Hoechst 33342 (1/500) for 2 hours. Bronchoalveolar lavage fluid (BALF) cells obtained from non-human primates were incubated with Alexa Fluor 647 anti-human CD68 (#562111, BD Pharmigen) (1/50) for two hours. After washings with PBS, cells were air dried at room temperature for 1 hour on slides and protected from the light. Then, slides were mounted with Duolink In situ Mounting Medium with DAPI (#DU082040, Sigma) and conserved at 4° C. For the detection of SARS-CoV-2-infected cells in vitro, Vero E6 and ACE2-A549 cells were fixed in 4% paraformaldehyde in PBS for 5 minutes, permeabilized with 0.3% Triton (Sigma) in PBS, and incubated in PBS containing 10% FBS for 1 hour. SARS-CoV-2-infected ACE2-A549 cells were subjected to P2Y2-NLRP3 PLA staining as described above, but SARS-CoV-2-infected Vero E6 and ACE2-4549 cells also were incubated during 2 hours with mouse anti-Spike S antibody (#GTX632604, Genetex) and then during 1 hour with goat anti-mouse IgG conjugated to Alexa Fluor 546 (#A11030, Invitrogen). Nuclei were also stained with Hoechst 33342 (#H3570, Invitrogen) as previously described. PMA-differentiated THP1 macrophages and MDMs that were treated during 72 hours with 100 or 1000 ng/mL IFNγ or 10 ng/mL LPS were also analyzed for P2Y2-NLRP3 PLA staining following the same procedure. The visualization and quantification of PLA assays were performed in blind with confocal microscopy (SP8, Leica), which is equipped with two PMT and two high sensitivity hybrid detectors using a 63× oil objective. The representative PLA cells were imaged by confocal microscopy (SP8, Leica) using hybrid detectors (pinhole airy: 1; pixel size: 180 nm, magnification zoom: 3.5×) at optimal optical sectioning (OOS) of 0.8 μm. The confocal PLA images were then analyzed by Image J software in the best focal plan for the construction of z projection images on maximum intensity. LPS+ATP-stimulated, PMA-THP1 macrophages that were depleted (or not) for NLRP3 were analyzed for PYK2Y402* (#3291, Cell Signaling) and F-actin polymerization (using Alexa Fluor 488 Phalloïdin (#A12379, Invitrogen)) by confocal microscopy as previously published (20). For immunofluorescence analysis of mouse lungs, 4-82 m sections were cut from the paraffin blocks of the paraformaldehyde fixed tissues from mice. After paraffin removal, slides were subjected to antigen retrieval by microwave boiling in 1 mmol/L EDTA pH 9.0. After permeabilization with 0,3% Triton during 5 minutes and saturation in PBS containing 20% FBS during 1 hour, slides were first stained with green TUNEL assay (#Roche, #11684809910) during 1 hour at 37° C. according to the manufacturer's instructions and then incubated with anti-CD40 (#14-0401, ebioscience) or anti-CASP1 p10 (#sc-22164, Santa Cruz) overnight at 4° C. Then, cells were incubated with anti-rat IgG conjugated to Alexa Fluor 546 (#A11081, Invitrogen) or anti-goat IgG conjugated to Alexa Fluor 647 (#A21447, Invitrogen) fluorochromes at room temperature during 1 hour and 30 minutes. Cells were analyzed by fluorescent confocal microscopy on Leica SPE (using a 63× objective). Z series of optical sections at 0.4-μm increments were acquired. For flow-cytometry analysis, MDMs (10.sup.6 cells/mL) were harvested after indicated treatments in RPM) complete medium, washed twice with PBS, saturated at 4° C. for 20 minutes in PBS containing 10% FBS and incubated with anti-CD163 Alexa Fluor 647 (#562669, B D Pharmingen)) antibodies during 1 hour and 30 minutes. Membrane expression of CD163 was then analyzed using LSRFortessa (B D) flow cytometer. ACE2 positive Vero and ACE2-A549 cells were determined after two hours blocking with 20% FBS and overnight incubation with anti-Angiotensin converting enzyme (CD143) (#557928, B D Biosciences) for at 4° C. Mice alveolar macrophages were dissociated from lung (using Lung Dissociation kit from Miltenyi Biotech) and analyzed using anti-CD11b (APC-Cy7) (#557657, B D Pharmingen), anti-CD11c (PE-Cy7) (#117318, BioLegend), anti-CD40 (eFluo710) (#46-0401, ebioscience), anti-F4/80 (RTC) (#11-4801, ebioscience) and anti-Ly-6G (GR-1-PE) (#12-5931, ebioscience) using LSRFortessa (BD) flow cytometer as previously reported (59).
2.3.1.9. Western Blots
[0214] Total cellular proteins were extracted and lysed in appropriated buffer containing 250 mM NaCl, 0.1% NP-40, 5 mM EDTA. 10 mM Na3VO4, 10 mM NaF, 5 mM DTT, 3 mM Na4P2O7, 1 mM EGTA, 10 mM Glycerol phosphate, 10 mM Tris-HCl (pH=7.5) and the protease and phosphatase inhibitors (Roche)). Then, 10-40 μg of protein extracts were run on 4-12% or 12% SDS-PAGE and transferred at 4° C. onto a nitrocellulose membrane (0.2 Micron). After 2 hours saturation at room temperature with 5% nonfat milk or BSA (Bovine Serum Albumin) in Tris-buffered saline and 0.1% Tween 20 (TBS-Tween), nitrocellulose membranes were incubated with primary antibody at 4° C. overnight. Horseradish peroxidase (HRP)-conjugated goat anti-mouse or anti-rabbit antibodies (SouthernBiotech) or rabbit anti-goat antibodies (SouthernBiotech) were then incubated for 1 hour and revealed with the enhanced ECL detection system (GE Healthcare). The primary antibodies against P2Y2 (#APR-010) and P2X7 (#APR-004) were obtained from Alomone laboratories. Primary antibodies against ACE2 (#AF933) and Spike S (#GTX632604) were from Biotechene and GeneTex. Primary antibodies against IL-1β (#ab2105), IRF5 (#ab21689) and β-Actin-HRP (#ab49900) were purchased from Abcam. Anti-NLRP3 (Cryo-2), anti-CASP1 (#2225), anti-α-Tubulin (#T9026) and GAPDH (#MAB374) were from Adipogen, Cell signaling, Sigma and Millipore, respectively.
2.3.1.10. Detection of Cytokines
[0215] Plasma serum from P2y2.sup.+/+ and P2y2.sup.−/− mice and supernatants harvested from PMA-stimulated THP1 macrophages that were depleted or not for P2Y2 or NLRP3, and/or stimulated with 10 ng/mL LPS during 3 hours and 5 mM ATP during 6 hours or with 20 ng/mL IFNγ, in presence or in absence of 100 μM Suramin, 100 μM OxATP, 100 μM Kaempferol or 100 μM Diquafosol at indicated times were analyzed using western blot or ELISA for IL-1β (Ebioscience) and IL-10 (BD) according to the manufacturers' instructions.
2.3.1.11. Cell Viability Assays
[0216] Human THP1 cells were cultured in RPMI 1640 media, supplemented with 10% FBS. THP1 cells were differentiated by treatment for 3 hours with 100 nM phorbol-12-myristate-13-acetate (PMA, Invivogen). After 2 days, control or THP1 cells depleted or not for P2Y2, NLRP3 or CASP1 were stimulated for 3 hours with ultrapure LPS from E. coli (10 ng/mL, Sigma) and for 6 hours with ATP (5 mM, Sigma), and analyzed for LDH release using LDH kit (Roche). Cell viability in drug-treated cells was also measured. Vero E6 cells and ACE2-A549 cells were pretreated with indicated concentrations of UTP, Diquafosol, Denufosol, PPADS, OxATP and BzATP during 4 hours before infection and infected or not with SARS-CoV-2 BetaCoV/France/IDF0372/2020 strain with a multiplicity of infection between 1 and 2. Cell viability was determined after 72 hours using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (#M5655, Sigma) following manufacturer's instructions. Cell viability was also performed in uninfected Vero E6 cells and ACE2-A549 cells with the same compound dilutions.
2.3.1.12. Macrophage Migration Assay
[0217] The migration of treated-PMA-primed THP1 cells was determined using a Boyden chamber system (Roche CIM16 plate XCELLigence DP) 9 hours after LPS (10 ng/mL) and ATP (5 mM) stimulation.
2.3.1.13. Microarray Assay
[0218] MDMS were treated with 100 μM Kaempferol during 72 hours. Then, mRNAs were isolated using RNeasy kit (#74104, Quiagen) and gene expression analyses were performed with Agilent® SurePrint G3 Human GE 8x60K Microarray (Agilent Technologies, AMADID 39494) with the following single-color design. RNAs were labeled with Cy3 using the one-color Agilent labeling kit (Low Input Quick Amp Labeling Kit 5190-2306) adapted for small amount of total RNA (100 ng total RNA per reaction). Hybridization were then performed on microarray using 800 ng of linearly amplified cRNA labeled, following the manufacturer protocol (Agilent SureHyb Chamber; 800 ng of labeled extract; duration of hybridization of 17 hours; 40 μL per array; Temperature of 65° C.). After washing in acetonitrile, slides were scanned by using an Agilent G2565 C DNA microarray scanner with defaults parameters (100° PMT, 3 μm resolution, at 20° C. in free ozone concentration environment). Microarray images were analyzed by means of Feature Extraction software version (10.7.3.1) from Agitent technologies. Defaults settings were used.
2.3.1.14. Microarray Data Processing and Analysis
[0219] Raw data files from Feature Extraction were imported into R with LIMMA (Smyth, 2004, Statistical applications in Genetics and molecular biology, vol 3, N°1, article 3), an R package from the Bioconductor project, and processed as follow: gMedianSignal data were imported, controls probes were systematically removed, and flagged probes (gisSaturated, glsFeatpopnOL, glsFeatNonUnifOL) were set to NA. Inter-array normalization was performed by quantile normalization. To get a single value for each transcript, taking the median of each replicated probes summarized data. Missing values were inferred using KNN algorithm from the package ‘impute’ from R bioconductor. Normalized data were then analyzed. To assess differentially expressed genes between two groups, we start by fitting a linear model to the data. Then, we used an empirical Bayes method to moderate the standard errors of the estimated log-fold changes. The top-ranked genes were selected with the following criteria: an absolute fold-change>2 and an adjusted p-value (FDR)<0.05.
2.3.1.15. Immunochemistry
[0220] Post-mortem lung specimens were fixed in formalin and embedded in paraffin. Tissue sections were deparaffinized, rehydrated, incubated in 10 mM sodium citrate, pH 6.0, microwaved for antigen retrieval and treated with 3% H2O2 to block endogenous peroxidase activity. Then, mouse antibodies against NLRP3/NALP3 (#AG-20B-0014, AdipoGen) (1:100), CD68 (#KP-1, Ventana) (prediluted), or double-stranded RNA (#J2-2004, Scicons J2) (1:500) and biotinylated goat anti-mouse IgG (#BA-9200, Vector) were incubated with lung sections. Immune-reactivities were vizualized using avidin-biotin complex-based peroxidase system (#PK-7100, Vector) and 3,3′-diaminobenzidine (DAB) peroxidase (HRP) substrate Kit (#SK-4100, Vector). Lung sections were also stained with hematoxylin and eosin, as previously described (60) and assessed by two independent observers without the knowledge of clinical diagnosis, using a Leica DM2500 LED Optical microscope and a 63× objective.
2,3.1.16. Statistical Analysis
[0221] For the pairwise comparison of two groups within the same cell line (THP1 monocytes, PMA-differentiated THP1 macrophages, Vero E6 or ACE2-A549), where the cell populations have normal distribution, we used the unpaired two-tailed t-test to compare the means±SEM (standard error of the mean). For the comparison of two groups of samples from independent individuals (blood cells of healthy donors or patients, bronchoalveolar lavage (BALF) cells of non-human primates and serum or organ cells of mice) where the sample populations did not assume the same distributions, we used the non-parametric unpaired two-tailed or one-tailed Mann-Whitney test to compare the means of the ranks of two groups. When the control sample values from independent individuals were normalized to the value of 1 to compare the fold changes in the treated group, we used the non-parametric test Wilcoxon matched-pairs signed rank test. For the multiple comparisons of more than two groups, we used the one-way ANOVA test when the sample groups have one variable parameter (such as gene knockdown or stimulus or drug treatments or infections). When the sample groups have two variable parameters (such as gene knockdowns and stimulus or drug treatments and/or infections) we used the two-way ANOVA test to compare the means of the absolute values, of the frequencies or of the fold changes. The p-values of the multiple comparisons of ANOVA tests were adjusted with the Tukey's Honestly Significant Difference (HSD) test when the means are compared with every other mean or with Dunnett's correction test when every mean is compared to the control mean. Statistical data were analyzed with Graphpad prism 6 and 8 software. Statistical significance was given as *P<0.05, **P<0.01, ***P<0.001, and ****P<0.0001.
2.3,2. Results
[0222] 2.3.2.1. Expression of NLRP3 on Lung Tissue Samples Obtained from Uninfected and SARS-CoV-2-Infected Carriers To study the potential contribution of the P2Y2 and NLRP3 interaction to pathogenesis of SARS-CoV-2 infection, we first analyzed the expression of NLRP3 on lung tissue samples obtained from three uninfected carriers and seven SARS-CoV-2-infected carriers who died from COVID-19. Autopsies were stained with hematoxylin and eosin, or incubated with antibody against NLRP3 and analyzed. As recently described (24), parenchymal multifocal damages with intra-alveolar inflammation, fibrin and hyaline membrane formation that are consistent with a diagnosis of diffuse alveolar damage were observed in all SARS-CoV-2-infected carriers. The pattern of organizing pneumonia (with fibrotic organization and type 2 pneumocyte hyperplasia) and fibroblastic foci formed by loose organizing connective tissue consistent with alveolar duct fibrosis were also detected (
2.3.2.2. Modulation of the P2Y2-NLRP3 interaction in Peripheral Blood Cells During SARS-CoV-2 Infection.
[0223] Based on our previous work showing that the purinergic receptor P2Y2 interacts with and regulates NLRP3 protein expression during acute and chronic infection with HIV-1 (20), we then determined the ability of the P2Y2-NLRP3 interaction to be modulated in peripheral blood cells during SARS-CoV-2 infection. By means of a proximity ligation assay (PLA), we revealed that the P2Y2-NLRP3 interaction is enhanced in neutrophils (
[0224] The enhancement of the P2Y2-NLRP3 interaction was also confirmed in macrophages when analyzing bronchoalveolar fluid lavages (BALFs) obtained from non-human primate (NHP) Macaca fascicularis that were infected with SARS-CoV-2, as compared with uninfected NHP (
2.3.2.3. Expression of IL1β in P2y2.sup.−/ − and P2y2.sup.+/ + Mice
[0225] Since NLRP3 and P2Y2 have been separately reported to be involved in macrophage activation, which is a key event during SARS-CoV-2 pathogenesis (27-30), we then assessed basal expression levels of IL-1β in P2y2.sup.−/ − and P2y2.sup.+/ + mice. Interestingly, the baseline plasma levels of IL-1β in P2y2.sup.−/ − mice were higher than in wild type controls (
2.3.2.4. Modulation of the P2Y2-NRLP3 Interaction During Macrophage Activation
[0226] Given that macrophages are key cellular targets for the treatment of COVID-19 (30), we then analyzed the modulation of the P2Y2-NLRP3 interaction during macrophage activation. Interestingly, we found that the P2Y2-NLRP3 interaction is enhanced during pro-inflammatory macrophage reprogramming detected after treatment of PMA-THP1 macrophages (
[0227] Taken together, these results indicate that P2Y2 inhibits NLRP3 inflammasome dependent IL-1β release. Since IL-1β release is a hallmark of pro-inflammatory macrophage activation, we next analyzed the impact of P2Y2 inactivation on functional reprogramming of macrophages (34). P2Y2 inhibition by kaempferol (
[0228] Altogether, these results indicate that P2Y2 acts as an endogenous negative modulator of macrophage pro-inflammatory functions and raise the possibility that P2Y2 agonists could be used as candidate drugs for the treatment of COVID-19-associated hyper-inflammation.
2.3.2.5. The P2Y2-NLRP3 Interaction Controls the Susceptibility to SARS COV2 Through the Modulation of Viral Entry.
[0229] Since the P2Y2-NLRP3 interaction also increased in A549 type II pneumocytes ((
[0230] We next examined the impact of a non-selective antagonist of purinergic receptors P2X, the pyridoxal-phosphate-6-azopheny-2′,4′-disulfonate (PPADS) and OxATP, on SARS-COV-2 viral replication and related cellular damage, to evaluate the possibility that P2X7 might also affect viral replication. The inhibition of P2X and P2X7 activities by PPADS and OxATP reduced the frequency of Spike-positive cells (
[0231] We then examined whether purinergic receptors control SARS-CoV-2 replication through the modulation of viral entry. Since ACE2 down-regulation detected during the early steps of SARS-CoV-2 infection follows viral entry (40), Vero E6 cells were infected during 24 hours in presence of modulators of purinergic receptors and assessed for ACE2 expression. P2Y2 agonists (UTP and Diquafosol) and P2X7 antagonist (OxATP) repressed ACE2 down regulation detected in response to SARS-CoV-2 infection as a consequence of viral entry (
2.3.3. Conclusions
[0232] With the goal of studying the potential contribution of the interaction between P2Y2 and NLRP3 to the pathogenesis of SARS-CoV-2 infection, our study revealed that two major sensors of danger signals, namely, P2Y2 and NLRP3, increased their interaction during COVID-19. The enhanced P2Y2-NLRP3 interaction is detected on immune cells, namely neutrophils, monocytes/macrophages and lymphocytes, whose differentiation or specific functions were found to be altered during COVID-19, contributing to organ dysfunction and disease severity. In the majority of these processes, dysregulated immune functions can be regulated by NLRP3. NLRP3-dependent inflammasome activation and uncontrolled extracellular traps (NET) production by neutrophils have been proposed to contribute to hyper-inflammation and dysregulated coagulation in COVID-19 patients with severe disease (41, 42). Activation of the NLRP3 inflammasome and pyroptosis have been detected during SARS-CoV-2 infection (14) and COVID-19 (43). In addition, danger signals such as calprotectin recently associated with COVID-19 disease severity (44), are known to activate the NLRP3 inflammasome (45) or to be released as a consequence of its activation (46) and may contribute to pyroptosis (46). Recent studies showed that numerous patients with severe COVID-19 exhibit a higher plasma concentration of inflammasome-induced cytokines (such as IL-1β and IL-18) than patients with moderate disease (9, 47). Our study reveals that the detection of the P2Y2-NLRP3 interaction positively correlated l with disease severity and increased with viral infection. Accordingly, the detection of P2Y2-NLRP3 interaction on circulating blood cells could be a prognostic marker for the transition between moderate to severe disease during COVID-19.
[0233] By deciphering the unexpected relationship between P2Y2 and NLRP3 during COVID-19, our study showed that P2Y2 regulates macrophage functions and represents an endogenous repressor of macrophage pro-inflammatory functions through the negative modulation of NLRP3 inflammasome activation, pro-inflammatory reprogramming and pyroptosis. Our study also demonstrated that purinergic receptors P2Y2 and P2X7 control the susceptibility to SARS-CoV-2. P2Y2 acts as a restriction factor while P2X7 promotes viral entry, without interfering with ACE2 membrane expression. Since we previously observed that the purinergic receptors P2Y2 and P2X7 control HIV-1 envelope-elicited fusion ((20, 22), it is conceivable that P2Y2 and P2X7 may modulate the entry of SARS-CoV-2 into host cells through modulation of the Spike S fusogenic activity. Interestingly, the FDA-approved drug Ivermectin, which is known to potentiate the activity of the purinergic receptor P2X4 (48), which can form heterotrimeric receptors with P2X7 (49), was recently shown to inhibit the replication of SARS-CoV-2 in vitro (50).
[0234] According to a recent retrospective study revealing that the early administration of IL-1 receptor antagonist anakinra significantly improved prognosis of COVID-19 patients with severe inflammatory failure (51), our work confirms that targeting inflammasome-dependent IL-1β/IL-18 and/or purinergic signaling pathways may offer a novel opportunity for the treatment of viral infection and hyper-inflammation associated with COVID-19. Our study provides the first rationale for testing P2Y2 agonists such as Diquafosol and Denufosol which have been previously approved or proposed for the treatments of dry eye (33) and cystic fibrosis (52), respectively, alone or in combination with specific antagonists for P2X7 such as CE-224,535 or JNJ-54175446, which were assessed without success for the treatment of rheumatoid arthritis (NCT00628095) or under evaluation as antidepressant for the treatment of major depressive disorders (NCT04116606), respectively. Repurposing of these existing drugs for blocking both viral replication and COVID-19-associated hyper-inflammation should rapidly improve the health of COVID-19 patients.
2.3.4. Validation data
[0235] As mentioned above, ATP is able to enhance the entry of SARS-CoV-2 into permissive cells and favor viral propagation. To confirm this, the present inventors used modulators that are able to reduce the level of circulating ATP. They show in
[0236] Moreover, to confirm that the NLRP3 inflammasome represent a novel molecular target for the treatment of COVID-19, the present inventors used antagonists of NRLP3, as Tranilast, to reduce the inflammasome activity. They demonstrated that the NLRP3 inhibitor Tranilast indeed inhibits SARS-COV-2 replication (
[0237] Finally, to confirm the role of the P2Y2R agonists in the COVID-19-associated hyper-inflammation, the present inventors demonstrated that the P2Y2 agonist Diquafosol abolishes Bleomycin-induced lung inflammation (
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