{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,22]],"date-time":"2026-09-22T18:40:22Z","timestamp":1790102422268,"version":"4.0.1"},"reference-count":54,"publisher":"Public Library of Science (PLoS)","issue":"12","license":[{"start":{"date-parts":[[2020,12,10]],"date-time":"2020-12-10T00:00:00Z","timestamp":1607558400000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":["www.plosone.org"],"crossmark-restriction":false},"short-container-title":["PLoS ONE"],"abstract":"<jats:p>Retinoic acid (RA) has been shown to improve epithelial and endothelial barrier function and development and even suppress damage inflicted by inflammation on these barriers through regulating immune cell activity. This paper thus sought to determine whether RA could improve baseline barrier function and attenuate TNF-\u03b1-induced barrier leak in the human bronchial epithelial cell culture model, 16HBE14o- (16HBE). We show for the first time that RA increases baseline barrier function of these cell layers indicated by an 89% increase in transepithelial electrical resistance (TER) and 22% decrease in<jats:sup>14<\/jats:sup>C-mannitol flux. A simultaneous, RA-induced 70% increase in claudin-4 attests to RA affecting the tight junctional (TJ) complex itself. RA was also effective in alleviating TNF-\u03b1-induced 16HBE barrier leak, attenuating 60% of the TNF-\u03b1-induced leak to<jats:sup>14<\/jats:sup>C-mannitol and 80% of the leak to<jats:sup>14<\/jats:sup>C-inulin. Interleukin-6-induced barrier leak was also reduced by RA. Treatment of 16HBE cell layers with TNF-\u03b1 resulted in dramatic decrease in immunostaining for occludin and claudin-4, as well as a downward \u201cband-shift\u201d in occludin Western immunoblots. The presence of RA partially reversed TNF-\u03b1\u2019s effects on these select TJ proteins. Lastly, RA completely abrogated the TNF-\u03b1-induced increase in ERK-1,2 phosphorylation without significantly decreasing the TNF-driven increase in total ERK-1,2. This study suggests RA could be effective as a prophylactic agent in minimizing airway barrier leak and as a therapeutic in preventing leak triggered by inflammatory cascades. Given the growing literature suggesting a \u201ccytokine storm\u201d may be related to COVID-19 morbidity, RA may be a useful adjuvant for use with anti-viral therapies.<\/jats:p>","DOI":"10.1371\/journal.pone.0242536","type":"journal-article","created":{"date-parts":[[2020,12,10]],"date-time":"2020-12-10T19:10:39Z","timestamp":1607627439000},"page":"e0242536","update-policy":"https:\/\/doi.org\/10.1371\/journal.pone.corrections_policy","source":"Crossref","is-referenced-by-count":21,"title":["Retinoic acid improves baseline barrier function and attenuates TNF-\u03b1-induced barrier leak in human bronchial epithelial cell culture model, 16HBE 14o-"],"prefix":"10.1371","volume":"15","author":[{"given":"Patrick J.","family":"Callaghan","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Elizabeth","family":"Rybakovsky","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Bryan","family":"Ferrick","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sunil","family":"Thomas","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5285-7530","authenticated-orcid":true,"given":"James M.","family":"Mullin","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"340","published-online":{"date-parts":[[2020,12,10]]},"reference":[{"issue":"6","key":"pone.0242536.ref001","doi-asserted-by":"crossref","first-page":"395","DOI":"10.1016\/S1359-6446(05)03379-9","article-title":"Keynote review: epithelial and endothelial barriers in human disease","volume":"10","author":"JM Mullin","year":"2005","journal-title":"Drug Discovery Today"},{"key":"pone.0242536.ref002","doi-asserted-by":"crossref","first-page":"207","DOI":"10.1146\/annurev.cellbio.22.010305.104219","article-title":"Tight junctions and cell polarity","volume":"22","author":"K Shin","year":"2006","journal-title":"Annual Review of Cell and Developmental Biology"},{"key":"pone.0242536.ref003","first-page":"1","article-title":"Tight junction structure and regulation: role in health and disease","volume":"4","author":"H Clarke","year":"2001","journal-title":"Current Topics in Biochemical Research"},{"key":"pone.0242536.ref004","doi-asserted-by":"crossref","first-page":"47","DOI":"10.1016\/j.semcdb.2015.04.009","article-title":"Claudins: gatekeepers of lung epithelial function","volume":"42","author":"B Schlingmann","year":"2015","journal-title":"Seminars in cell and developmental biology"},{"issue":"1","key":"pone.0242536.ref005","doi-asserted-by":"crossref","first-page":"135","DOI":"10.1007\/s00424-016-1917-3","article-title":"Tight junctions in pulmonary epithelia during lung inflammation","volume":"469","author":"OH Wittekindt","year":"2017","journal-title":"Pflugers archive: European journal of physiology"},{"issue":"5","key":"pone.0242536.ref006","doi-asserted-by":"crossref","first-page":"461","DOI":"10.1007\/BF01487039","article-title":"Tumor necrosis factor induced acute lung leak in rats: less than with interleukin-1","volume":"20","author":"Y Koh","year":"1996","journal-title":"Inflammation"},{"key":"pone.0242536.ref007","doi-asserted-by":"crossref","first-page":"772","DOI":"10.1513\/pats.200805-041HR","article-title":"Airway epithelial cells current concepts and challenges","volume":"7","author":"GR Crystal","year":"2008","journal-title":"American thoracic society"},{"key":"pone.0242536.ref008","doi-asserted-by":"crossref","first-page":"70","DOI":"10.1186\/1465-9921-12-70","article-title":"Claudins in lung diseases","volume":"12","author":"Y Soini","year":"2011","journal-title":"Respiratory research"},{"issue":"1","key":"pone.0242536.ref009","doi-asserted-by":"crossref","first-page":"72","DOI":"10.1016\/j.jaci.2016.02.035","article-title":"Claudin-18 deficiency is associated with airway epithelial barrier dysfunction and asthma","volume":"139","author":"K Sweerus","year":"2017","journal-title":"The Journal of allergy and clinical immunology"},{"key":"pone.0242536.ref010","doi-asserted-by":"crossref","first-page":"213","DOI":"10.1016\/0091-6749(75)90139-6","article-title":"Nasal mucosal hyperpermeability to macromolecules in atopic rhinitis and extrinsic asthma","volume":"4","author":"GF Buckle","year":"1975","journal-title":"The Journal of allergy and clinical immunology"},{"key":"pone.0242536.ref011","doi-asserted-by":"crossref","first-page":"38","DOI":"10.1165\/ajrcmb.10.1.7507342","article-title":"CFTR expression and chloride secretion in polarized immortal human bronchial epithelial cells","volume":"10","author":"AL Cozens","year":"1994","journal-title":"American journal of respiratory cell and molecular biology"},{"key":"pone.0242536.ref012","doi-asserted-by":"crossref","first-page":"46","DOI":"10.1016\/j.yexcr.2016.08.025","article-title":"Human airway epithelial cells investigated by atomic force microscopy: a hint to cystic fibrosis epithelial pathology","volume":"348","author":"M Lasalvia","year":"2016","journal-title":"Experimental cell research"},{"key":"pone.0242536.ref013","doi-asserted-by":"crossref","first-page":"333","DOI":"10.1177\/0394632017734479","article-title":"HSP60 activity on human bronchial epithelial cells","volume":"30","author":"C Sangiorgi","year":"2017","journal-title":"International journal of immunopathology and pharmacology"},{"key":"pone.0242536.ref014","first-page":"15267","article-title":"Human normal bronchial cells: a novel in vitro cell model for toxicity evaluation","volume":"10","author":"W Feng","year":"2015","journal-title":"PLos one"},{"key":"pone.0242536.ref015","doi-asserted-by":"crossref","first-page":"350","DOI":"10.1016\/j.intimp.2011.12.006","article-title":"Glucocorticoids enhance airway epithelial barrier integrity","volume":"12","author":"A Sekiyama","year":"2012","journal-title":"International immunopharmacology"},{"key":"pone.0242536.ref016","doi-asserted-by":"crossref","first-page":"161","DOI":"10.1016\/S0378-5173(03)00129-7","article-title":"The human bronchial epithelial cell line 16HBE14o- as a model system of the airways for studying drug transport","volume":"257","author":"B Forbes","year":"2003","journal-title":"International journal of pharmaceutics"},{"issue":"50","key":"pone.0242536.ref017","doi-asserted-by":"crossref","first-page":"10950","DOI":"10.1021\/acs.jafc.7b04203","article-title":"Improvement of human-oral-epithelial-barrier function and of tight junctions by micronutrients","volume":"65","author":"E Rybakovsky","year":"2017","journal-title":"Journal of agricultural and food Chemistry"},{"issue":"10","key":"pone.0242536.ref018","doi-asserted-by":"crossref","first-page":"5674","DOI":"10.1096\/fj.201701469R","article-title":"Retinoic acid signaling is essential for maintenance of the blood-retinal barrier","volume":"32","author":"LM Pollock","year":"2018","journal-title":"The Journal of the federation of American societies for experimental biology"},{"issue":"4","key":"pone.0242536.ref019","doi-asserted-by":"crossref","first-page":"1660","DOI":"10.1523\/JNEUROSCI.1338-12.2013","article-title":"Retinoic acid induces blood-brain barrier development","volume":"33","author":"MR Mizee","year":"2013","journal-title":"Journal of neuroscience"},{"issue":"4","key":"pone.0242536.ref020","doi-asserted-by":"crossref","first-page":"337","DOI":"10.1016\/j.jphs.2019.06.012","article-title":"Retinoic acid promotes barrier functions in human iPSC-derived intestinal epithelial monolayers","volume":"140","author":"S Yamada","year":"2019","journal-title":"Journal of pharmacological sciences"},{"issue":"3","key":"pone.0242536.ref021","doi-asserted-by":"crossref","first-page":"72","DOI":"10.1007\/s40629-018-0054-2","article-title":"Vitamin A and D in allergy: from experimental animal models and cellular studies to human disease","volume":"27","author":"K Hufnagl","year":"2018","journal-title":"Allergo journal international"},{"issue":"2","key":"pone.0242536.ref022","first-page":"171","article-title":"Effects of bacterial lysates and all trans-retinoic acid on airway inflammation in asthmatic mice","volume":"22","author":"Y Lei","year":"2020","journal-title":"Chinese journal of contemporary pediatrics"},{"key":"pone.0242536.ref023","doi-asserted-by":"crossref","first-page":"28","DOI":"10.1186\/1471-2172-14-28","article-title":"All-trans retinoic acid attenuates airway inflammation by inhibiting Th2 and Th17 response in experimental allergic asthma","volume":"14","author":"J Wu","year":"2013","journal-title":"BMC immunology"},{"issue":"1","key":"pone.0242536.ref024","doi-asserted-by":"crossref","first-page":"12","DOI":"10.1006\/excr.1996.0244","article-title":"Chronic exposure of LLC-PK1 epithelia to the phorbol ester TPA produces poly-like foci with leaky tight junctions and altered Protein Kinase C-\u03b1 expression and localization","volume":"227","author":"JM Mullin","year":"1996","journal-title":"Experimental cell research"},{"issue":"2","key":"pone.0242536.ref025","doi-asserted-by":"crossref","first-page":"276","DOI":"10.1016\/j.ab.2004.02.049","article-title":"A high-affinity reversible protein stain for Western blots","volume":"329","author":"BS Antharavally","year":"2004","journal-title":"Anal Biochem"},{"issue":"3","key":"pone.0242536.ref026","doi-asserted-by":"crossref","first-page":"665","DOI":"10.1016\/j.jaci.2013.03.005","article-title":"TNF-\u03b1-mediated bronchial barrier disruption and regulation by src-family kinase activation","volume":"132","author":"MA Hardyman","year":"2013","journal-title":"The Journal of allergy and clinical immunology"},{"key":"pone.0242536.ref027","doi-asserted-by":"crossref","first-page":"137","DOI":"10.1242\/jcs.112.1.137","article-title":"Tumor necrosis factor-alpha (TNF-\u03b1) regulates the epithelial barrier in the human intestinal cell line HT-29\/B6","volume":"112","author":"H Schmitz","year":"1999","journal-title":"Journal of cell science"},{"key":"pone.0242536.ref028","doi-asserted-by":"crossref","first-page":"1140","DOI":"10.1038\/labinvest.2012.67","article-title":"Cytokines induce tight junction disassembly in airway cells via an EGFR-dependent MAPK\/ERK1\/2-pathway","volume":"92","author":"L Petecchia","year":"2012","journal-title":"Laboratory investigation"},{"key":"pone.0242536.ref029","doi-asserted-by":"crossref","first-page":"e0170346","DOI":"10.1371\/journal.pone.0170346","article-title":"TNF\u03b1 alters occludin and cerebral endothelial permeability: Role of p38MAPK","volume":"12","author":"Y Ni","year":"2017","journal-title":"PLoS ONE"},{"key":"pone.0242536.ref030","doi-asserted-by":"crossref","first-page":"3187","DOI":"10.1242\/jcs.113.18.3187","article-title":"Protein Kinase C activation leads to dephosphorylation of occludin and tight junction permeability increase in LLC-PK1 epithelial cell sheets","volume":"113","author":"H Clarke","year":"2000","journal-title":"Journal of cell science"},{"issue":"3","key":"pone.0242536.ref031","doi-asserted-by":"crossref","first-page":"379","DOI":"10.33549\/physiolres.934492","article-title":"COVID-19 and the immune system","volume":"69","author":"J Paces","year":"2020","journal-title":"Physiol Res"},{"key":"pone.0242536.ref032","doi-asserted-by":"crossref","first-page":"1446","DOI":"10.3389\/fimmu.2020.01446","article-title":"The COVID-19 Cytokine Storm; What We Know So Far","volume":"11","author":"D Ragab","year":"2020","journal-title":"Front Immunol"},{"key":"pone.0242536.ref033","doi-asserted-by":"crossref","first-page":"105808","DOI":"10.1016\/j.intimp.2019.105808","article-title":"Retinoic acid modulates IL-4, IL-10 and MCP-1 pathways in immune mediated hepatitis and interrupts CD4+ T cells infiltration","volume":"75","author":"M Elshal","year":"2019","journal-title":"International immunopharmacology"},{"issue":"2","key":"pone.0242536.ref034","first-page":"172","article-title":"Prophylactic administration of all-trans retinoic acid alleviates inflammation in rats with collagen-induced arthritis","volume":"37","author":"X Zeng","year":"2016","journal-title":"Journal of Southern Medical University"},{"key":"pone.0242536.ref035","doi-asserted-by":"crossref","first-page":"101","DOI":"10.1016\/j.jnutbio.2017.01.004","article-title":"All-trans-retinoic acid represses chemokine expression in adipocytes and adipose tissue by inhibiting NF-kB signaling","volume":"42","author":"E Karkeni","year":"2017","journal-title":"The Journal of nutritional biochemistry"},{"issue":"4","key":"pone.0242536.ref036","first-page":"479","article-title":"The effects of all-trans retinoic acid on the expression of inflammatory cytokines and cartilage damage related proteases in rats with collagen-induced arthritis","volume":"47","author":"XJ Xie","year":"2016","journal-title":"Journal of Sichuan University"},{"key":"pone.0242536.ref037","doi-asserted-by":"crossref","first-page":"47","DOI":"10.1016\/j.jnutbio.2018.06.002","article-title":"All-trans retinoic acid ameliorates inflammatory response mediated by TLR4\/NFkB during initiation of diabetic nephropathy","volume":"60","author":"E Sierra-Mondragon","year":"2018","journal-title":"The Journal of nutritional biochemistry"},{"issue":"5","key":"pone.0242536.ref038","doi-asserted-by":"crossref","first-page":"869","DOI":"10.1152\/ajprenal.1996.270.5.F869","article-title":"Tissue remodeling during tumor necrosis factor-induced apoptosis in LLC-PK1 renal epithelial cells","volume":"270","author":"AP Soler","year":"1996","journal-title":"The American journal of physiology\u2013renal physiology"},{"issue":"2","key":"pone.0242536.ref039","doi-asserted-by":"crossref","first-page":"223","DOI":"10.4291\/wjgp.v7.i2.223","article-title":"Sieving characteristics of cytokine- and peroxide-induced epithelial barrier leak: Inhibition by berberine","volume":"7","author":"KM DiGuilio","year":"2016","journal-title":"World J Gastrointest Pathophysiol"},{"key":"pone.0242536.ref040","doi-asserted-by":"crossref","first-page":"226","DOI":"10.1002\/(SICI)1097-4652(199705)171:2<226::AID-JCP14>3.0.CO;2-B","article-title":"Different size limiations for increased transepithelial paracellular solute flux across phorbol ester and tumor necrosis factor-treated epithelial cell sheets","volume":"171","author":"JM Mullin","year":"1996","journal-title":"Journal of cellular physiology"},{"key":"pone.0242536.ref041","doi-asserted-by":"crossref","first-page":"1749","DOI":"10.1096\/fj.99-0898com","article-title":"Leaks in the epithelial barrier caused by spontaneous and TNF-\u03b1-induced single-cell apoptosis","volume":"14","author":"AH Gitter","year":"2000","journal-title":"The FASEB journal"},{"issue":"9","key":"pone.0242536.ref042","doi-asserted-by":"crossref","first-page":"3218","DOI":"10.1091\/mbc.e02-03-0134","article-title":"Regulation of airway tight junctions by proinflammatory cytokines","volume":"13","author":"CB Coyne","year":"2002","journal-title":"Molecular biology of the cell"},{"key":"pone.0242536.ref043","doi-asserted-by":"crossref","first-page":"56","DOI":"10.1016\/S0171-9335(99)80007-7","article-title":"Activation of NF-kB is necessary for restoration of the barrier function of an epithelium undergoing TNF-\u03b1-induced apoptosis","volume":"78","author":"AP Soler","year":"1999","journal-title":"European journal of cell biology"},{"key":"pone.0242536.ref044","doi-asserted-by":"crossref","first-page":"85","DOI":"10.1002\/(SICI)1097-4644(20000701)78:1<85::AID-JCB8>3.0.CO;2-F","article-title":"Multiple domains of occludin are involved in the regulation of paracellular permeability","volume":"78","author":"SM Balda","year":"2000","journal-title":"Journal of cellular biochemistry"},{"key":"pone.0242536.ref045","doi-asserted-by":"crossref","first-page":"1393","DOI":"10.1083\/jcb.137.6.1393","article-title":"Possible involvement of phosphorylation of occludin in tight junction formation","volume":"137","author":"A Sakakibara","year":"1997","journal-title":"Journal of cell biology"},{"issue":"3","key":"pone.0242536.ref046","doi-asserted-by":"crossref","first-page":"993","DOI":"10.3390\/ijms21030993","article-title":"Contributions of myosin light chain kinase to regulation of epithelial paracellular permeability and mucosal homeostasis","volume":"21","author":"W. Q. He","year":"2020","journal-title":"International journal of molecular sciences"},{"key":"pone.0242536.ref047","doi-asserted-by":"crossref","first-page":"107","DOI":"10.1023\/A:1011086426652","article-title":"Tumor necrosis factor \u03b1 activates the phosphorylation of ERK, SAPK\/JNK, and P38 kinase in primary cultures of neurons","volume":"26","author":"G Barbin","year":"2001","journal-title":"Neurochemical research"},{"issue":"12","key":"pone.0242536.ref048","doi-asserted-by":"crossref","first-page":"5538","DOI":"10.1091\/mbc.e05-04-0294","article-title":"Ras mutation impairs epithelial barrier function to a wide range of nonelectrolytes","volume":"16","author":"JM Mullin","year":"2005","journal-title":"Mol Biol Cell"},{"issue":"1","key":"pone.0242536.ref049","doi-asserted-by":"crossref","first-page":"51","DOI":"10.1042\/BJ20100249","article-title":"Contrasting effects of ERK on tight junction integrity in differentiated and under-differentiated Caco-2 cell monolayers","volume":"433","author":"S Aggarwal","year":"2011","journal-title":"Journal of biochemistry"},{"key":"pone.0242536.ref050","doi-asserted-by":"crossref","first-page":"108464","DOI":"10.1016\/j.clim.2020.108464","article-title":"Targeting T-cell senescence and cytokine storm with rapamycin to prevent severe progression in COVID-19","author":"L Omarjee","year":"2020","journal-title":"Clinical immunology"},{"key":"pone.0242536.ref051","doi-asserted-by":"crossref","first-page":"102567","DOI":"10.1016\/j.autrev.2020.102567","article-title":"Should we stimulate or suppress immune responses in COVID-19? Cytokine and anti-cytokine interventions","volume":"4","author":"Y Jamilloux","year":"2020","journal-title":"Autoimmunity reviews"},{"key":"pone.0242536.ref052","doi-asserted-by":"crossref","first-page":"102562","DOI":"10.1016\/j.autrev.2020.102562","article-title":"Cytokine storm syndrome in severe COVID-19","author":"P Ruscitti","year":"2020","journal-title":"Autoimmunity reviews"},{"key":"pone.0242536.ref053","doi-asserted-by":"crossref","first-page":"100044","DOI":"10.1016\/j.medidd.2020.100044","article-title":"Melatonin inhibits COVID-19-induced cytokine storm by reversing aerobic clycolysis in immune cells: a mechanistic analysis","author":"RJ Reiter","year":"2020","journal-title":"Medicine in drug discovery"},{"key":"pone.0242536.ref054","article-title":"The imperfect cytokine storm: severe COVID-19 with ARDS in patient on durable LVAD support","author":"VQ Chau","year":"2020","journal-title":"JACC: Case reports."}],"container-title":["PLOS ONE"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/dx.plos.org\/10.1371\/journal.pone.0242536","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2022,12,4]],"date-time":"2022-12-04T01:31:21Z","timestamp":1670117481000},"score":1,"resource":{"primary":{"URL":"https:\/\/dx.plos.org\/10.1371\/journal.pone.0242536"}},"subtitle":[],"editor":[{"given":"Michael","family":"Koval","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"editor"}]}],"short-title":[],"issued":{"date-parts":[[2020,12,10]]},"references-count":54,"journal-issue":{"issue":"12","published-online":{"date-parts":[[2020,12,10]]}},"URL":"https:\/\/doi.org\/10.1371\/journal.pone.0242536","relation":{},"ISSN":["1932-6203"],"issn-type":[{"value":"1932-6203","type":"electronic"}],"subject":[],"published":{"date-parts":[[2020,12,10]]}}}