Combinatorial Chemistry & High Throughput ScreeningCombinatorial Chemistry & High Throughput ScreeningCombinatorial Chemistry & High Throughput Screening1386-20731875-5402Bentham Science64396710.2174/0113862073256465231024075452ChemistryResearch ArticleAndrographolide Attenuates RSV-induced Inflammation by Suppressing Apoptosis and Promoting Pyroptosis after Respiratory Syncytial Virus Infection In VitroCheSiyiinfo@benthamscience.netXieXiaohonginfo@benthamscience.netLinJileiinfo@benthamscience.netLiuYinginfo@benthamscience.netXieJuninfo@benthamscience.netLiuEnmeiinfo@benthamscience.netDepartment of Respiratory Medicine, Children’s Hospital of Chongqing Medical University, National Clinical Research Center for Child Health and Disorders, Ministry of Education Key Laboratory of Child Development and Disorders,, China International Science and Technology Cooperation Base of Child Development and Critical Disorders, Chongqing Key Laboratory of PediatricsDepartment of Respiratory Medicine, Children’s Hospital of Chongqing Medical University, National Clinical Research Center for Child Health and Disorders, Ministry of Education Key Laboratory of Child Development and Disorders, China International Science and Technology Cooperation Base of Child Development and Critical Disorders, Chongqing Key Laboratory of Pediatrics,Department of Respiratory Medicine, Shanghai Children’s Medical Center, School of Medicine, Shanghai Jiao Tong UniversityState Key Laboratory of Innovative Natural Medicine and TCM Injections,, Jiangxi Qingfeng Pharmaceutical co. LTD,1506202427121776178707012025Copyright ©; 2024, Bentham Science Publishers2024Bentham Science Publishershttps://rjpbr.com/1386-2073/article/view/643967

Background::Respiratory syncytial virus (RSV), which is the predominant viral pathogen responsible for causing acute lower respiratory tract infections in children, currently lacks specific therapeutic drugs. Despite andrographolide's demonstrated effectiveness against various viral infections, its effects on RSV infection remain unclear.

Methods::In this study, RSV infection and andrographolide-intervened A549 cell lines were used. The virus load of RSV and the levels of IL-6 and IL-8 in the cell supernatant were quantified. The potential targets of andrographolide in the treatment of RSV-infected airway epithelial cells were analyzed using the Gene Expression Omnibus (GEO) database and the PharmMapper Database, and the changes in mRNA expression of these target genes were measured. To further illustrate the effect of andrographolide on the death pattern of RSV-infected airway epithelial cells, Annexin V-FITC/PI apoptosis assays and Western blotting were conducted.

Results::Andrographolide decreased the viral load and attenuated IL-6 and IL-8 levels in cell supernatant post-RSV infection. A total of 25 potential targets of andrographolide in the treatment of RSV-infected airway epithelial cells were discovered, and CASP1, CCL5, JAK2, and STAT1 were identified as significant players. Andrographolide noticeably suppressed the increased mRNA expressions of these genes post-RSV infection as well as IL-1β. The flow cytometry analysis demonstrated that andrographolide alleviated apoptosis in RSV-infected cells. Additionally, RSV infection decreased the protein levels of caspase-1, cleaved caspase-1, cleaved IL-1β, N-terminal of GSDMD, and Bcl-2. Conversely, andrographolide increased their levels.

Conclusion::These results suggest that andrographolide may reduce RSV-induced inflammation by suppressing apoptosis and promoting pyroptosis in epithelial cells, leading to effective viral clearance.

Andrographoliderespiratory syncytial virusnetwork pharmacologyapoptosispyroptosiscell supernatant post- RSV infection.Shi, T.; McAllister, D.A.; O’Brien, K.L.; Simoes, E.A.F.; Madhi, S.A.; Gessner, B.D.; Polack, F.P.; Balsells, E.; Acacio, S.; Aguayo, C.; Alassani, I.; Ali, A.; Antonio, M.; Awasthi, S.; Awori, J.O.; Azziz-Baumgartner, E.; Baggett, H.C.; Baillie, V.L.; Balmaseda, A.; Barahona, A.; Basnet, S.; Bassat, Q.; Basualdo, W.; Bigogo, G.; Bont, L.; Breiman, R.F.; Brooks, W.A.; Broor, S.; Bruce, N.; Bruden, D.; Buchy, P.; Campbell, S.; Carosone-Link, P.; Chadha, M.; Chipeta, J.; Chou, M.; Clara, W.; Cohen, C.; de Cuellar, E.; Dang, D.A.; Dash-yandag, B.; Deloria-Knoll, M.; Dherani, M.; Eap, T.; Ebruke, B.E.; Echavarria, M.; de Freitas Lázaro Emediato, C.C.; Fasce, R.A.; Feikin, D.R.; Feng, L.; Gentile, A.; Gordon, A.; Goswami, D.; Goyet, S.; Groome, M.; Halasa, N.; Hirve, S.; Homaira, N.; Howie, S.R.C.; Jara, J.; Jroundi, I.; Kartasasmita, C.B.; Khuri-Bulos, N.; Kotloff, K.L.; Krishnan, A.; Libster, R.; Lopez, O.; Lucero, M.G.; Lucion, F.; Lupisan, S.P.; Marcone, D.N.; McCracken, J.P.; Mejia, M.; Moisi, J.C.; Montgomery, J.M.; Moore, D.P.; Moraleda, C.; Moyes, J.; Munywoki, P.; Mutyara, K.; Nicol, M.P.; Nokes, D.J.; Nymadawa, P.; da Costa Oliveira, M.T.; Oshitani, H.; Pandey, N.; Paranhos-Baccalà, G.; Phillips, L.N.; Picot, V.S.; Rahman, M.; Rakoto-Andrianarivelo, M.; Rasmussen, Z.A.; Rath, B.A.; Robinson, A.; Romero, C.; Russomando, G.; Salimi, V.; Sawatwong, P.; Scheltema, N.; Schweiger, B.; Scott, J.A.G.; Seidenberg, P.; Shen, K.; Singleton, R.; Sotomayor, V.; Strand, T.A.; Sutanto, A.; Sylla, M.; Tapia, M.D.; Thamthitiwat, S.; Thomas, E.D.; Tokarz, R.; Turner, C.; Venter, M.; Waicharoen, S.; Wang, J.; Watthanaworawit, W.; Yoshida, L.M.; Yu, H.; Zar, H.J.; Campbell, H.; Nair, H. Global, regional, and national disease burden estimates of acute lower respiratory infections due to respiratory syncytial virus in young children in 2015: A systematic review and modelling study. Lancet, 2017, 390(10098), 946-958. doi: 10.1016/S0140-6736(17)30938-8 PMID: 28689664Restori, K.H.; Srinivasa, B.T.; Ward, B.J.; Fixman, E.D. Neonatal immunity, respiratory virus infections, and the development of asthma. Front. Immunol., 2018, 9, 1249. doi: 10.3389/fimmu.2018.01249 PMID: 29915592Battles, M.B.; McLellan, J.S. Respiratory syncytial virus entry and how to block it. Nat. Rev. Microbiol., 2019, 17(4), 233-245. doi: 10.1038/s41579-019-0149-x PMID: 30723301Mooney, K.; Melvin, M.; Douglas, T. Ribavirin: The need for exposure precautions. Clin. J. Oncol. Nurs., 2014, 18(5), E93-E96. doi: 10.1188/14.CJON.E93-E96 PMID: 25253120Xing, Y.; Proesmans, M. New therapies for acute RSV infections: Where are we? Eur. J. Pediatr., 2019, 178(2), 131-138. doi: 10.1007/s00431-018-03310-7 PMID: 30610420Chakravarti, R.N.; Chakravarti, D. Andrographolide, the active constituent of Andrographis paniculata Nees; a preliminary communication. Ind. Med. Gaz., 1951, 86(3), 96-97. PMID: 14860885Kumar, S.; Singh, B.; Bajpai, V. Andrographis paniculata (Burm.f.) Nees: Traditional uses, phytochemistry, pharmacological properties and quality control/quality assurance. J. Ethnopharmacol., 2021, 275, 114054. doi: 10.1016/j.jep.2021.114054 PMID: 33831465Zhang, H.; Li, S.; Si, Y.; Xu, H. Andrographolide and its derivatives: Current achievements and future perspectives. Eur. J. Med. Chem., 2021, 224, 113710. doi: 10.1016/j.ejmech.2021.113710 PMID: 34315039Banerjee, S.; Kar, A.; Mukherjee, P.K.; Haldar, P.K.; Sharma, N.; Katiyar, C.K. Immunoprotective potential of Ayurvedic herb Kalmegh (ANDROGRAPHIS PANICULATA) against respiratory viral infections – LC–MS/MS and network pharmacology analysis. Phytochem. Anal., 2021, 32(4), 629-639. doi: 10.1002/pca.3011 PMID: 33167083Wang, D.W.; Xiang, Y.J.; Wei, Z.L.; Yao, H.; Shen, T. Andrographolide and its derivatives are effective compounds for gastrointestinal protection: a review. Eur. Rev. Med. Pharmacol. Sci., 2021, 25(5), 2367-2382. PMID: 33755974Elasoru, S.E.; Rhana, P.; de Oliveira Barreto, T.; Naves de Souza, D.L.; Menezes-Filho, J.E.R.; Souza, D.S.; Loes Moreira, M.V.; Gomes Campos, M.T.; Adedosu, O.T.; Roman-Campos, D.; Melo, M.M.; Cruz, J.S. Andrographolide protects against isoproterenol-induced myocardial infarction in rats through inhibition of L-type Ca2+ and increase of cardiac transient outward K+ currents. Eur. J. Pharmacol., 2021, 906, 174194. doi: 10.1016/j.ejphar.2021.174194 PMID: 34044012Chao, W.W.; Lin, B.F. Isolation and identification of bioactive compounds in Andrographis paniculata (Chuanxinlian). Chin. Med., 2010, 5(1), 17. doi: 10.1186/1749-8546-5-17 PMID: 20465823Sareer, O.; Ahmad, S.; Umar, S. Andrographis paniculata: A critical appraisal of extraction, isolation and quantification of andrographolide and other active constituents. Nat. Prod. Res., 2014, 28(23), 2081-2101. doi: 10.1080/14786419.2014.924004 PMID: 24912126Li, B.H.; Li, Z.Y.; Liu, M.M.; Tian, J.Z.; Cui, Q.H. Progress in traditional chinese medicine against respiratory viruses: A review. Front. Pharmacol., 2021, 12, 743623. doi: 10.3389/fphar.2021.743623 PMID: 34531754Ding, Y.; Chen, L.; Wu, W.; Yang, J.; Yang, Z.; Liu, S. Andrographolide inhibits influenza A virus-induced inflammation in a murine model through NF-κB and JAK-STAT signaling pathway. Microbes Infect., 2017, 19(12), 605-615. doi: 10.1016/j.micinf.2017.08.009 PMID: 28889969Li, F.; Lee, E.M.; Sun, X.; Wang, D.; Tang, H.; Zhou, G.C. Design, synthesis and discovery of andrographolide derivatives against Zika virus infection. Eur. J. Med. Chem., 2020, 187, 111925. doi: 10.1016/j.ejmech.2019.111925 PMID: 31838328Srikanth, L.; Sarma, P.V.G.K. Andrographolide binds to spike glycoprotein and RNA-dependent RNA polymerase (NSP12) of SARS-CoV-2 by in silico approach: A probable molecule in the development of anti-coronaviral drug. J. Genet. Eng. Biotechnol., 2021, 19(1), 101. doi: 10.1186/s43141-021-00201-7 PMID: 34255214Hopkins, A.L. Network pharmacology: The next paradigm in drug discovery. Nat. Chem. Biol., 2008, 4(11), 682-690. doi: 10.1038/nchembio.118 PMID: 18936753Gias, E.; Nielsen, S.U.; Morgan, L.A.F.; Toms, G.L. Purification of human respiratory syncytial virus by ultracentrifugation in iodixanol density gradient. J. Virol. Methods, 2008, 147(2), 328-332. doi: 10.1016/j.jviromet.2007.09.013 PMID: 18029032McKimm-Breschkin, J.L. A simplified plaque assay for respiratory syncytial virus—direct visualization of plaques without immunostaining. J. Virol. Methods, 2004, 120(1), 113-117. doi: 10.1016/j.jviromet.2004.02.020 PMID: 15234816Deng, Y.; Chen, W.; Zang, N.; Li, S.; Luo, Y.; Ni, K.; Wang, L.; Xie, X.; Liu, W.; Yang, X.; Fu, Z.; Liu, E. The antiasthma effect of neonatal BCG vaccination does not depend on the Th17/Th1 but IL-17/IFN-γ balance in a BALB/c mouse asthma model. J. Clin. Immunol., 2011, 31(3), 419-429. doi: 10.1007/s10875-010-9503-5 PMID: 21340706Wang, X.; Shen, Y.; Wang, S.; Li, S.; Zhang, W.; Liu, X.; Lai, L.; Pei, J.; Li, H. PharmMapper 2017 update: A web server for potential drug target identification with a comprehensive target pharmacophore database. Nucleic Acids Res., 2017, 45(W1), W356-W360. doi: 10.1093/nar/gkx374 PMID: 28472422Vermes, I.; Haanen, C.; Steffens-Nakken, H.; Reutellingsperger, C. A novel assay for apoptosis Flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labelled Annexin V. J. Immunol. Methods, 1995, 184(1), 39-51. doi: 10.1016/0022-1759(95)00072-I PMID: 7622868Paolini, A.; Borella, R.; De Biasi, S.; Neroni, A.; Mattioli, M.; Lo Tartaro, D.; Simonini, C.; Franceschini, L.; Cicco, G.; Piparo, A.M.; Cossarizza, A.; Gibellini, L. Cell death in coronavirus infections: Uncovering its role during COVID-19. Cells, 2021, 10(7), 1585. doi: 10.3390/cells10071585 PMID: 34201847Imre, G. Cell death signalling in virus infection. Cell. Signal., 2020, 76, 109772. doi: 10.1016/j.cellsig.2020.109772 PMID: 32931899Shi, J.; Gao, W.; Shao, F. Pyroptosis: Gasdermin-mediated programmed necrotic cell death. Trends Biochem. Sci., 2017, 42(4), 245-254. doi: 10.1016/j.tibs.2016.10.004 PMID: 27932073He, Y.; Hara, H.; Núñez, G. Mechanism and regulation of NLRP3 inflammasome activation. Trends Biochem. Sci., 2016, 41(12), 1012-1021. doi: 10.1016/j.tibs.2016.09.002 PMID: 27669650Shen, C.; Zhang, Z.; Xie, T.; Ji, J.; Xu, J.; Lin, L.; Yan, J.; Kang, A.; Dai, Q.; Dong, Y.; Shan, J.; Wang, S.; Zhao, X. Rhein suppresses lung inflammatory injury induced by human respiratory syncytial virus through inhibiting NLRP3 inflammasome activation via NF-κB pathway in mice. Front. Pharmacol., 2020, 10, 1600. doi: 10.3389/fphar.2019.01600 PMID: 32047436Malinczak, C.A.; Schuler, C.F.; Duran, A.J.; Rasky, A.J.; Mire, M.M.; Núñez, G.; Lukacs, N.W.; Fonseca, W. NLRP3-inflammasome inhibition during respiratory virus infection abrogates lung immunopathology and long-term airway disease development. Viruses, 2021, 13(4), 692. doi: 10.3390/v13040692 PMID: 33923693Latif, R.; Wang, C.Y. Andrographolide as a potent and promising antiviral agent. Chin. J. Nat. Med., 2020, 18(10), 760-769. doi: 10.1016/S1875-5364(20)60016-4 PMID: 33039055Yu, B.; Dai, C.; Jiang, Z.; Li, E.; Chen, C.; Wu, X.; Chen, J.; Liu, Q.; Zhao, C.; He, J.; Ju, D.; Chen, X. Andrographolide as an Anti-H1N1 drug and the mechanism related to retinoic acid-inducible gene-I-like receptors signaling pathway. Chin. J. Integr. Med., 2014, 20(7), 540-545. doi: 10.1007/s11655-014-1860-0 PMID: 24972581Wang, D.; Guo, H.; Chang, J.; Wang, D.; Liu, B.; Gao, P.; Wei, W. Andrographolide prevents EV-D68 replication by inhibiting the acidification of virus-containing endocytic vesicles. Front. Microbiol., 2018, 9, 2407. doi: 10.3389/fmicb.2018.02407 PMID: 30349523Thomas, K.W.; Monick, M.M.; Staber, J.M.; Yarovinsky, T.; Carter, A.B.; Hunninghake, G.W. Respiratory syncytial virus inhibits apoptosis and induces NF-kappa B activity through a phosphatidylinositol 3-kinase-dependent pathway. J. Biol. Chem., 2002, 277(1), 492-501. doi: 10.1074/jbc.M108107200 PMID: 11687577Eckardt-Michel, J.; Lorek, M.; Baxmann, D.; Grunwald, T.; Keil, G.M.; Zimmer, G. The fusion protein of respiratory syncytial virus triggers p53-dependent apoptosis. J. Virol., 2008, 82(7), 3236-3249. doi: 10.1128/JVI.01887-07 PMID: 18216092Eisenhut, M. Extrapulmonary manifestations of severe respiratory syncytial virus infection--a systematic review. Crit. Care, 2006, 10(4), R107. doi: 10.1186/cc4984 PMID: 16859512Miao, E.A.; Rajan, J.V.; Aderem, A. Caspase‐1‐induced pyroptotic cell death. Immunol. Rev., 2011, 243(1), 206-214. doi: 10.1111/j.1600-065X.2011.01044.x PMID: 21884178Miao, E.A.; Leaf, I.A.; Treuting, P.M.; Mao, D.P.; Dors, M.; Sarkar, A.; Warren, S.E.; Wewers, M.D.; Aderem, A. Caspase-1-induced pyroptosis is an innate immune effector mechanism against intracellular bacteria. Nat. Immunol., 2010, 11(12), 1136-1142. doi: 10.1038/ni.1960 PMID: 21057511Maltez, V.I.; Tubbs, A.L.; Cook, K.D.; Aachoui, Y.; Falcone, E.L.; Holland, S.M.; Whitmire, J.K.; Miao, E.A. Inflammasomes coordinate pyroptosis and natural killer cell cytotoxicity to clear infection by a ubiquitous environmental bacterium. Immunity, 2015, 43(5), 987-997. doi: 10.1016/j.immuni.2015.10.010 PMID: 26572063Li, Z.; Liu, W.; Fu, J.; Cheng, S.; Xu, Y.; Wang, Z.; Liu, X.; Shi, X.; Liu, Y.; Qi, X.; Liu, X.; Ding, J.; Shao, F. Shigella evades pyroptosis by arginine ADP-riboxanation of caspase-11. Nature, 2021, 599(7884), 290-295. doi: 10.1038/s41586-021-04020-1 PMID: 34671164Xiang, Z.; Liang, Z.; Yanfeng, H.; Leitao, K. Persistence of RSV promotes proliferation and epithelial-mesenchymal transition of bronchial epithelial cells through Nodal signaling. J. Med. Microbiol., 2017, 66(10), 1499-1505. doi: 10.1099/jmm.0.000581 PMID: 28901900Lindemans, C.A.; Coffer, P.J.; Schellens, I.M.M.; de Graaff, P.M.A.; Kimpen, J.L.L.; Koenderman, L. Respiratory syncytial virus inhibits granulocyte apoptosis through a phosphatidylinositol 3-kinase and NF-kappaB-dependent mechanism. J. Immunol., 2006, 176(9), 5529-5537. doi: 10.4049/jimmunol.176.9.5529 PMID: 16622022Nakamura-López, Y.; Villegas-Sepúlveda, N.; Sarmiento-Silva, R.E.; Gómez, B. Intrinsic apoptotic pathway is subverted in mouse macrophages persistently infected by RSV. Virus Res., 2011, 158(1-2), 98-107. doi: 10.1016/j.virusres.2011.03.016 PMID: 21440589Chen, J.H.; Hsiao, G.; Lee, A.R.; Wu, C.C.; Yen, M.H. Andrographolide suppresses endothelial cell apoptosis via activation of phosphatidyl inositol-3-kinase/Akt pathway. Biochem. Pharmacol., 2004, 67(7), 1337-1345. doi: 10.1016/j.bcp.2003.12.015 PMID: 15013849Liu, W.; Liang, L.; Zhang, Q.; Li, Y.; Yan, S.; Tang, T.; Ren, Y.; Mo, J.; Liu, F.; Chen, X.; Lan, T. Effects of andrographolide on renal tubulointersticial injury and fibrosis. Evidence of its mechanism of action. Phytomedicine, 2021, 91, 153650. doi: 10.1016/j.phymed.2021.153650 PMID: 34332282Lin, K.H.; Marthandam Asokan, S.; Kuo, W.W.; Hsieh, Y.L.; Lii, C.K.; Viswanadha, V.; Lin, Y.L.; Wang, S.; Yang, C.; Huang, C.Y. Andrographolide mitigates cardiac apoptosis to provide cardio‐protection in high‐fat‐diet‐induced obese mice. Environ. Toxicol., 2020, 35(6), 707-713. doi: 10.1002/tox.22906 PMID: 32023008Li, X.; Yuan, K.; Zhu, Q.; Lu, Q.; Jiang, H.; Zhu, M.; Huang, G.; Xu, A. Andrographolide ameliorates rheumatoid arthritis by regulating the apoptosis–netosis balance of neutrophils. Int. J. Mol. Sci., 2019, 20(20), 5035. doi: 10.3390/ijms20205035 PMID: 31614480Chao, W.W.; Kuo, Y.H.; Lin, B.F. Isolation and identification of andrographis paniculata (chuanxinlian) and its biologically active constituents inhibited enterovirus 71-induced cell apoptosis. Front. Pharmacol., 2021, 12, 762285. doi: 10.3389/fphar.2021.762285 PMID: 34955832Carty, M.; Guy, C.; Bowie, A.G. Detection of viral infections by innate immunity. Biochem. Pharmacol., 2021, 183, 114316. doi: 10.1016/j.bcp.2020.114316 PMID: 33152343He, Z.; Chen, J.; Zhu, X.; An, S.; Dong, X.; Yu, J.; Zhang, S.; Wu, Y.; Li, G.; Zhang, Y.; Wu, J.; Li, M. NLRP3 inflammasome activation mediates zika virus–associated inflammation. J. Infect. Dis., 2018, 217(12), 1942-1951. doi: 10.1093/infdis/jiy129 PMID: 29518228Rodrigues, T.S.; de Sá, K.S.G.; Ishimoto, A.Y.; Becerra, A.; Oliveira, S.; Almeida, L.; Gonçalves, A.V.; Perucello, D.B.; Andrade, W.A.; Castro, R.; Veras, F.P.; Toller-Kawahisa, J.E.; Nascimento, D.C.; de Lima, M.H.F.; Silva, C.M.S.; Caetite, D.B.; Martins, R.B.; Castro, I.A.; Pontelli, M.C.; de Barros, F.C.; do Amaral, N.B.; Giannini, M.C.; Bonjorno, L.P.; Lopes, M.I.F.; Santana, R.C.; Vilar, F.C.; Auxiliadora-Martins, M.; Luppino-Assad, R.; de Almeida, S.C.L.; de Oliveira, F.R.; Batah, S.S.; Siyuan, L.; Benatti, M.N.; Cunha, T.M.; Alves-Filho, J.C.; Cunha, F.Q.; Cunha, L.D.; Frantz, F.G.; Kohlsdorf, T.; Fabro, A.T.; Arruda, E.; de Oliveira, R.D.R.; Louzada-Junior, P.; Zamboni, D.S. Inflammasomes are activated in response to SARS-CoV-2 infection and are associated with COVID-19 severity in patients. J. Exp. Med., 2021, 218(3), e20201707. doi: 10.1084/jem.20201707 PMID: 33231615Vázquez, Y.; González, L.; Noguera, L.; González, P.A.; Riedel, C.A.; Bertrand, P.; Bueno, S.M. Cytokines in the respiratory airway as biomarkers of severity and prognosis for respiratory syncytial virus infection: An update. Front. Immunol., 2019, 10, 1154. doi: 10.3389/fimmu.2019.01154 PMID: 31214165Choudhury, S.K.M.; Ma, X.; Abdullah, S.W.; Zheng, H. Activation and inhibition of the NLRP3 inflammasome by RNA viruses. J. Inflamm. Res., 2021, 14, 1145-1163. doi: 10.2147/JIR.S295706 PMID: 33814921Schuler, C.F., IV; Malinczak, C.A.; Best, S.K.K.; Morris, S.B.; Rasky, A.J.; Ptaschinski, C.; Lukacs, N.W.; Fonseca, W. Inhibition of uric acid or IL‐1β ameliorates respiratory syncytial virus immunopathology and development of asthma. Allergy, 2020, 75(9), 2279-2293. doi: 10.1111/all.14310 PMID: 32277487