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Up-Regulated Vitamin D Receptor by Pelargonium sidoides Extract EPs® 7630 Contributes to Rhinovirus Defense in Bronchial Epithelial Cells

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EPs®7630, extracted from Pelargonium sidoides, reduces the severity of viral upper respiratory tract infections. Vitamin D also improves anti-viral host defense through similar signaling pathways. This study assessed if EPs®7630 modifies vitamin D receptor (VDR) expression and function by human bronchial epithelial cells. Bronchial epithelial cells were incubated with EPs®7630 over 48 h before calcitriol stimulation and/or infection with Rhinovirus (RV)-16. Protein expression was determined by Western-blotting. Intracellular signaling of mitogen activated protein kinases (MAPK) was studied by chemical inhibitors. The anti-viral effect was assessed by immunofluorescence for RV-16 protein. EPs®7630 upregulated VDR expression through Erk1/2 MAPK and thereby increased the cell’s sensitivity to calcitriol. Compared ton untreated cells, the shift of the VDR into the nucleus at 5.3 times lower calcitriol concentration. EPs®7630 increased Erk1/2 MAPK signaling, but reduced p38 phosphorylation, and had no effect on Jun N-terminal kinase (JNK). EPs®7630 improved the anti-viral effect of vitamin D on RV-16 infection by 2.1 folds compared to vitamin D alone or to untreated cells. Furthermore, EPs®7630 improved the differentiation of epithelial cells by upregulating E-cadherin expression through Erk1/2. In conclusion, EPs®7630 increased host defense against Rhinovirus infection by upregulating the VDR and the differentiation of epithelial cells.
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Pharmaceuticals2021,14,172.https://doi.org/10.3390/ph14020172www.mdpi.com/journal/pharmaceuticals
Article
UpRegulatedVitaminDReceptorbyPelargoniumsidoides
ExtractEPs
®
7630ContributestoRhinovirusDefensein
BronchialEpithelialCells
MichaelRoth
1,
*,QingzhuSun
1,2
andMichaelTamm
1
1
PulmonaryCellResearch&Pneumology,Dept.Biomedicine&InternalMedicine,
UniversityHospitalBasel,Petersgraben4,CH4031Basel,Switzerland;sunqingzhu@nwafu.edu.cn(Q.S.);
michael.tamm@usb.ch(M.T.)
2
CollegeofAnimalScienceandTechnology,NorthwestA&FUniversity,Yangling712100,Shaanxi,China
*Correspondence:michael.roth@usb.ch;Tel.:+41612652337
Abstract:EPs
®
7630,extractedfromPelargoniumsidoides,reducestheseverityofviralupper
respiratorytractinfections.VitaminDalsoimprovesantiviralhostdefensethroughsimilar
signalingpathways.ThisstudyassessedifEPs
®
7630modifiesvitaminDreceptor(VDR)expression
andfunctionbyhumanbronchialepithelialcells.Bronchialepithelialcellswereincubatedwith
EPs
®
7630over48hbeforecalcitriolstimulationand/orinfectionwithRhinovirus(RV)16.Protein
expressionwasdeterminedbyWesternblotting.Intracellularsignalingofmitogenactivated
proteinkinases(MAPK)wasstudiedbychemicalinhibitors.Theantiviraleffectwasassessedby
immunofluorescenceforRV16protein.EPs
®
7630upregulatedVDRexpressionthroughErk1/2
MAPKandtherebyincreasedthecell’ssensitivitytocalcitriol.Comparedtonuntreatedcells,the
shiftoftheVDRintothenucleusat5.3timeslowercalcitriolconcentration.EPs
®
7630increased
Erk1/2MAPKsignaling,butreducedp38phosphorylation,andhadnoeffectonJunNterminal
kinase(JNK).EPs
®
7630improvedtheantiviraleffectofvitaminDonRV16infectionby2.1folds
comparedtovitaminDaloneortountreatedcells.Furthermore,EPs
®
7630improvedthe
differentiationofepithelialcellsbyupregulatingEcadherinexpressionthroughErk1/2.In
conclusion,EPs
®
7630increasedhostdefenseagainstRhinovirusinfectionbyupregulatingtheVDR
andthedifferentiationofepithelialcells.
Keywords:rhinovirus;EPs
®
7630;vitaminDreceptor;viralreplication;bronchialepithelialcell
1.Introduction
Viralinfectionsarethemostfrequentcauseofrespiratoryailmentsandpresenta
severehealthproblemtochildrenandtheelderly[1,2].EPs
®
7630isanherbaldrug
preparationfromtherootsofPelargoniumsidoides,whicheffectivelyreducestheseverity
ofsymptomsinpatientswithacutebronchitis,acutesinusitismaxillaris,
tonsillopharyngitis,orcommoncold,combinedwithagoodtolerability[1–4].
ThemechanismsbywhichEPs
®
7630reducesviralinfectionwereinvestigatedin
severalnonclinicaltrials.IthasbeenreportedthatEPs
®
7630significantlyreducedthe
abilityofvariousvirusestoattachtothehostcells,ortopreventvirusreleasefrom
infectedcells.Incellcultureexperiments,EPs
®
7630inhibitedtheattachmentofHIV1to
humanimmunecells,protectingthemfromviralentry[5],whereasprotectionofcells
againstinfluenzaAwasmediatedviainhibitionofhemagglutinandneuraminidase
activity[6].Inhumanbronchialepithelialcells,EPs
®
7630reducedRhinovirus16(RV16)
replicationbydownregulatingtheexpressionofinduciblecostimulator(ICOS)andits
ligand(ICOSL),aswellasthesurfacecalreticulinreceptor,whereashostdefense
supportingproteinβ‐defensin1andsuppressorofcytokinesignaling1(SOCS1)were
Citation:Roth,M.;Sun,Q.;
Tamm,M.UpRegulatedVitaminD
ReceptorbyPelargoniumsidoides
ExtractEPs
®
7630Contributesto
RhinovirusDefenseinBronchial
EpithelialCells.Pharmaceuticals2021,
14,172.https://doi.org/10.3390/
ph14020172
AcademicEditor:DejanStojković
Received:2February2021
Accepted:19February2021
Published:22February2021
Publisher’sNote:MDPIstays
neutralwithregardtojurisdictional
claimsinpublishedmapsand
institutionalaffiliations.
Copyright:©2021bytheauthors.
LicenseeMDPI,Basel,Switzerland.
Thisarticleisanopenaccessarticle
distributedunderthetermsand
conditionsoftheCreativeCommons
Attribution(CCBY)license
(http://creativecommons.org/licenses
/by/4.0/).
Pharmaceuticals2021,14,1722of12
increased[7].However,theintracellularsignalingmechanismunderlyingtheantiviral
effectsofEPs
®
7630remainedtobedefined.
FortypeAand–BRhinoviruses,theintracellularadhesionmolecule1(ICAM1)isa
majordockingproteinofhostcells[8],requiredforviralinfectionofdifferenthostcells
[7,9].VitaminDreceptor(VDR)activationhasbeenshowntoreducetheadherenceofRV
tointracellularadhesionmolecule1(ICAM1)[10].Inthiscontext,lowserumlevelsof
vitaminDcorrelatedwithincreasedsusceptibilitytorespiratoryvirusinfection.In
children,rhinovirusinfectioninverselycorrelatedwithvitaminDlevels[11],andlow
serumvitaminDincreasedthesensitivitytoRVinfection[12].VitaminDdeficiency
correlatedwiththefrequencyofexacerbationcausedbyrhinovirusinfectioninchronic
obstructivepulmonarydisease(COPD)patients[13].Furthermore,infectionwitheither
RVorRespiratorysyncytialvirus(RSV)reducedtheexpressionoftheVDRbyhuman
epithelialcells,butthiswasreversedbyvitaminDsupplementation[14].
TheimportanceoftheVDRinhostdefenseissupportedbyreportsthattheVDR
genotypeaffectstheriskofupperrespiratorytractinfections.Inchildrenwithasthma,a
genomeanalysisindicatedthattheVDRisoneofthemostimportantfactorsthatregulates
thesusceptibilitytovirusinducedupperrespiratorytractinfections[15].Inacohortof
1462adultsfromtheU.K.,alinkbetweentheminorVDRallele,rs4334089SNPandthe
susceptibilitytoupperrespiratorytractinfectionswasconfirmed[16].Inchildrenwith
severebronchiolitis,SNPrs2228570VDRtogetherwithSNPsofTolllikereceptor4and
Tolllikereceptor2wasassociatedwithincreasedriskofdeath[17].
TheexpressionoftheVDRiscontrolledbyextracellularsignalregulatedkinase1/2
(Erk1/2)mitogenactivatedproteinkinase(MAPK)inmusclecells[18].EPs
®
7630hasbeen
showntodifferentiallyregulateErk1/2MAPKinimmunecells,whilesuppressingprotein
38)(p38)MAPK[19].Hence,itseemsfeasiblethatEPs
®
7630couldalsoaffectVDR
expression.
Inthisstudy,weassessedtheeffectofEPs
®
7630ontheexpressionandactivationof
theVDRinbronchialepithelialcellsanddetermineditsfunctionalrelevancefortheanti
viralactivity.
2.Results
2.1.AnalysisofEPs
®
7630ComponentsbyHighPressureLiquidChromatography(HPLC)
Figure1showsatypicalHPLCUVHRMSprofileofEPs
®
7630.Thechemical
structureofthedifferentbenzopyranonescontainedinEPs
®
7630areassignedbysmall
letters(d–o)andthechemicalstructureanalysisisprovidedinTable1.Moredetailsofthe
EPs
®
7630constituentswerepublishedbyothers[20,21].
Figure1.Highpressureliquidchromatography(HPLC)fingerprintdetectedbyUVat280nm.The
majorpeakswereassignedbyanalysisofhighresolutionmassspectrometry(HRMS)dataandare
givenasfollows:(a)adenosine3,5′‐cyclicmonophosphate,(b)guanosine3,5′‐cyclic
monophosphate,(c)1methylguanosine3,5′‐cyclicmonophosphate,and(d)benzopyranones.The
assignedstructuresaregiveninTable1.
Pharmaceuticals2021,14,1723of12
Table1.BenzopyranonesofEPs®7630asanalyzedbyHPLCUVHRMS(seeFigure1).
AssignedPeak
inFigure1NameR1R2R3R4
d6,8bissulfooxy7hydroxy2H1
benzopyran2one
HSO4OHSO4
e6,7dihydroxy8sulfooxy2H1
benzopyran2one
HOHOHSO4
f7,8dihydroxy6sulfooxy2H1
benzopyran2one
HSO4OHOH
g8hydroxy7methoxy6(sulfooxy)
2H1benzopyran2one
HSO4OCH3OH
h6methoxy7sulfooxy2H1
benzopyran2one
HOCH3SO4H
k5,6dimethoxy7,8dihydroxy2H1
benzopyran2one
OCH3OCH3OHOH
m7hydroxy5,6dimethoxy8
sulfooxy2H1benzopyran2one
OCH3OCH3OHSO4
n5,6dimethoxy7sulfooxy2H1
benzopyran2one(Umckalin7
sulphate)
OCH3OCH3SO4H
o7hydroxy5,6dimethoxy2H1
benzopyran2one(Umckalin)
OCH3OCH3OHH
2.2.VDRRegulationbyEPs®7630andtheUnderlyingSignalingPathway
EpithelialcellstreatedwithEPs®7630increasedtheexpressionoftheVDRina
concentrationdependentmannerover24h(Figure2A).Therewasnodifferencebetween
primaryepithelialcellsorBEAS2Bcells.Thiseffectbecamesignificantatconcentration>
5μg/mLEPs®7630after24h(Figure2A).KineticsoftheEPs®7630inducedVDR
expressionwerestudiedwithafixedconcentrationofEPs®7630(10μg/mL)over48h
whichshowedacontinuousincrease(Figure2B).
Basedonearlierreports,theinvolvementofMAPKsintheEPs®7630mediated
activationoftheVDRexpressionwasstudied.InFigure2Cthekineticofthestimulating
effectofEPs®7630onphosphorylationofErk1/2isshown.NoneoftheotherMAPKswas
significantlymodifiedbyEPs®7630(datanotshown).BlockingMAPKsignaling
pathwaysbyspecificchemicalinhibitors,thestimulatingeffectofEPs®7630(10μg/mL)
onVDRexpressionwassignificantlyreducedbyPD98059inaconcentrationdependent
manner(Figure2D).Incontrast,neithertheinhibitionofp38MAPKbySB203580,northat
ofJunNterminalkinase(JNK)bySP600125hadanysignificanteffectonEPs®7630
inducedVDRexpression(Figure2D).
OO
R
1
R
2
R
3
R
4
Pharmaceuticals2021,14,1724of12
Figure2.EPs
®
7630modifiesvitaminDreceptor(VDR)expression.(A)RepresentativeWesternblotsandimageanalyses
ofVDRexpressioninprimarybronchialepithelialcells(n=4)stimulatedwithincreasingconcentrationsofEPs
®
7630over
24h.Barsrepresentmean±SEM.(B)KineticofEPs
®
7630(10μg/mL)inducedVDRexpressionover48h(n=4).(C)The
effectofEPs
®
7630onthephosphorylationofErk1/2mitogenactivatedproteinkinases(MAPK)over120min.(D)The
effectofinhibitorsforJunNterminalkinase(JNK),p38,andErk1/2onEPs
®
7630inducedVDRexpression(n=4).
RepresentativeWesternblotsaredepictedabovethecorrespondingbars.SimilarresultswereobtainedinBEAS2Bcells.
EPs
®
7630notonlyincreasedtheoverallexpressionoftheVDR,butalsoinducedits
translocationintothenucleus(Figure3A).However,thiseffectwasmuchlower
comparedtocalcitriolinducednuclearaccumulationoftheVDR(Figure3B).Botheffects
wereconcentrationdependent.Inuntreatedcells,theVDRwaslocatedmainlyinthe
cytosol(Figure3C).Calcitriolsignificantlyincreasedthenumberofcellswithnuclear
VDR,andthiswasfurtherincreasedwhenthecellswerealsotreatedwithEPs
®
7630(10
μg/mL)(Figure3C).WhencellsweretreatedwiththecombinationofEPs
®
7630(10
μg/mL)andcalcitriol,onlyErk1/2MAPKinhibition(PD98059)suppressedthe
accumulationoftheVDRintothenucleus(Figure3D).
Figure3.EPs
®
7630andcalcitriolactivatetheVDR.(A)Concentrationdependentincreaseof
nuclearVDRbyEPs
®
7630over24hinprimaryhumanbronchialepithelialcells(n=4).(B)
Pharmaceuticals2021,14,1725of12
ConcentrationdependentincreaseofnuclearVDRbycalcitriolover24h(n=4).(C)Theeffectof
combinedEPs
®
7630withcalcitriolontheratioofcytosolicversusnuclearVDRaccumulation(n=
4)over24h.(D)TheeffectofMAPKinhibitorsforJNK(SP600125),p38(SB203580),andErk1/2
(PD98059)onEPs
®
7630andcalcitriolinducedVDRexpression(n=3).Barsrepresentmean±SEM.
SimilarresultswereobtainedinBEAS2Bcells.
2.3.CalcitriolSupportstheAntiViralEffectofEPs
®
7630
Inapreviousstudy,EPs
®
7630reducedtheinfectionofepithelialcellsbyRV16[7].
ThisobservationwasconfirmedbycellcountingoftheRVpositivestainedBEAS2Bcells
(Figure4A).CalcitriolalonealsoreducedRVinfectionofBEAS2Bcellsina
concentrationdependentmannerasshowninFigure4B.Whencombined,theinhibitory
effectofthetwodrugsonRV16infectionswassignificantlyimproved,comparedto
calcitriolalone(Figure4C).Thiseffectwasratheradditivethansynergistic.
RepresentativeimmunefluorescencephotographsoftheantiviraleffectofEPs
®
7630in
oneprimaryepithelialcellline,withandwithoutcalcitriolareshowninFigure4D.
Figure4.TheeffectofEPs
®
7630andcalcitriolonRV16infectioninBEAS2Bcells.(A)Concentrationdependenteffectof
EPs
®
7630onRV16staininginBEAS2Bcells.(B)ConcentrationdependenteffectofcalcitriolonRV16staininginBEAS
2Bcells.(C)TheeffectofcombinedEPs
®
7630(fixedconcentration)withcalcitriol(increasingconcentrations)onRV16
stainingonBEAS2Bcells.Barsrepresentmean±SEMoftriplicateexperiments.(D)Representativeimmunofluorescence
photographsofRV16positiveprimaryepithelialcellsinthepresenceandabsenceofEPs
®
7630(1μg/mL),orcalcitriol(1
μM),orthecombinationofboth(EPs
®
76301μg/mL+calcitriol1μM).
2.4.EPs
®
7630IncreasedEpithelialCellDifferentiation
Ecadherinisanindicatorofepithelialcellmaturationandisimportantforthecell
cellcontact.TreatingBEAS2BcellswithEPs
®
7630over24hsignificantlyincreasedthe
expressionofEcadherininaconcentrationdependentmanner(Figure5A).When
inhibitingMAPKs,onlyPD98059,theinhibitorofErk1/2,significantlyreducedthe
expressionofEcadherininBEAS2BcellstreatedwithEPs
®
7630(Figure5B).Similar
resultswereobtainedinoneoftheprimaryepithelialcelllines(datanotshown).
Pharmaceuticals2021,14,1726of12
Figure5.EPs
®
7630increasesEcadherinexpressionthroughErk1/2MAPKinBEAS2Bcells.(A)Concentrationeffectof
EPs
®
7630ontheexpressionofEcadherinover24hinBEAS2Bcells.(B)TheeffectofinhibitorsforJNK,p38,andErk1/2
onEPs
®
7630inducedEcadherinexpression.Barsrepresentmean±SEMoftriplicateexperiments.
3.Discussion
EPs
®
7630enhancedtheexpressionoftheVDRinhumanbronchialepithelialcells
throughErk1/2MAPKsignaling.IncreasedVDRexpressionimproveditsactivationby
calcitriolandmighttherebyenhancetheantiviraleffectofEPs
®
7630.
ThepresentstudyshowedthatEPs
®
7630activatesErk1/2MAPKsignalingpathway,
butinhibitedp38MAPKsignaling,andhadnoeffectonJNK.Thedataareinlinewith
earlierstudiesinhumanmonocytes[19].Erk1/2MAPKwasthemajorregulatingsignaling
proteinforEPs
®
7630inducedexpressionoftheVDR.Thiseffectmightoccurthrough
proanthocyanidinscontainedinEPs
®
7630[22].Inhumangranulosacells,
proanthocyanidinsincreasedtheexpressionofErk1/2MAPKandofhormonesathigher
concentrations[23].Therebyanotherproanthocyanidin,resveraterol,increasedthe
expressionoftheVDRinotherconditions[24].However,otherstudiesreportedthat
proanthocyanidinsandtheirmetabolitesinhibitedErk1/2,p38andJNKMAPKs,and
therebyreducedthereproductionofthehepatitisBvirus[25].Thesestudiesindicatethat
theeffectofproanthocyanidinsondifferentMAPKsmightbeconcentrationdependent,
orvariedwithcelltypeorvirus.
AsdisplayedinFigure1andTable1,EPs
®
7630containsdifferentformsof
benzopyranones;littleisknownabouttheeffectofthiscompoundontheregulationof
Erk1/2signaling,VDRexpression,andEcadherinexpression.Erk1/2activationis
essentialforepithelialwoundhealing,proliferationandmigration[26].Inlinewithour
results,glycitein,adifferentbenzopyranone,hasbeenreportedtoupregulatetheVDRin
columncancercellsthroughErk1/2[27].TheroleofErk1/2ontheregulationofEcadherin
hasmainlybeenstudiedinthecontextofepithelialmesenchymaltransition.Inthis
condition,Erk1/2phosphorylationwasinverselycorrelatedwithEcadherinexpression
[28].However,underdifferentconditionssuchashypoxia,epithelialcelldifferentiation
involvedtheactivationofErk1/2,followedbyEcadherinexpression[29].Furthermore,
EcadherinhasanegativefeedbackeffectonErk1/2signalinginintestinalepithelialcells
[30].
Inaddition,theexpressionoftheVDRislinkedtotheexpressionofEcadherin.In
humanbronchialepithelialcells(A549),vitaminDincreasedtheexpressionofEcadherin
andtherebyinduceddifferentiation[31].Ingingivalkeratinocytes,vitaminDimproved
intercellularjunctionsbyincreasingEcadherinexpression[32].ThelossofVDRwas
associatedwithreducedexpressionofEcadherinandepithelialcelldifferentiationin
chronickidneydisease[33].Thus,theliteratureindicatesamechanismlinkingVDRwith
Ecadherinandthereforeepithelialcelldifferentiationwithviraldefense.Oneshortfallof
thisstudyisthatEps
®
7630isacompoundofvarioussubstances,therefore,wewerenot
abletodelineatetheexactsignalingmechanism.
TheincreasedexpressionoftheVDRbyEPs
®
7630mightalsoaccountforthe
improveddifferentiationofepithelialcellsbyincreasingtheirbarrierfunctionandhost
Pharmaceuticals2021,14,1727of12
defensemechanisms.LowvitaminDlevelswereassociatedwithincreasedepithelialde
differentiationandremodelinginCOPDpatients[34].Ingingivalkeratinocytes,theVDR
actionmaintainedtheexpressionofEcadherinandtherebyreducedtissueremodeling
[35].Incancercells,VDRactivationpreventedepithelialmesenchymaltransitionand
therebymaintainedtheepithelialcellcharacter[36].However,thecontributionofthe
VDRtoepithelialcelldifferentiationmightvaryduringthecourseofmaturation[37].
Inthisstudy,EPs®7630increasedtheexpressionofEcadherininnondifferentiated
bronchialepithelialcellsbyactivatingErk1/2MAPK.Thisobservationisinlinewiththe
abovedescribedEPs®7630‐inducedactivationofErk1/2MAPKinothercelltypes[19].
FuturestudieshavetoverifyifEPs®7630inducedmaturationofepithelialcellsinvolves
theVDR.Inlinewiththeseresults,EcadherinexpressionwassuppressedbyRVinfection
inahumanbronchialepithelialcelllineandresultedinEMT[38].Itwassuggestedthat
thismechanismmightcontributetothethicknessofthebasalmembraneinasthma.
Furthermore,RVinfectiondelayedtherepairofTGF‐β‐inducedepitheliuminjury,which
correlatedwithreducedepithelialcelldifferentiation,andlowEcadherinexpression[39].
Inconclusion,anincreaseofEcadherinexpressionindicatesthatEPs®7630might
reducethethickeningoftheairwaywallinairwayinfectionorinflammation.The
upregulationoftheVDRandtheimprovedresponsetovitaminDinEPs®7630treated
epithelialcellssupportstheantiviraleffectofthecompound.However,thiseffecthasto
beconfirmedbyclinicalstudiesandinanimalmodels.
4.MaterialsandMethods
4.1.PrimaryBronchialEpithelialCellsandBEAS2BCell:
Primarybronchialepithelialwereisolatedandcharacterizedasdescribedearlier[32].
Endobronchialtissuebiopsieswereobtainedfromsixpatientsbyendobronchoscopyfor
diagnosticreasonsbythelungclinic(UniversityHospitalBasel,Switzerland).Allpatients
providedwritteninformedconsentfortheuseofoneadditionalanonymousbiopsyfor
scientificinvestigations.ThestudywasapprovedbythelocalInstitutionalEthical
Committee(EKBB05/06).
EpithelialcellswereisolatedbycelltypeselectivemediumCnTPRA(CellnTec,
Bern,Switzerland).BEAS2Bcellsweregrowninthesamecellculturemedium.Thecell
phenotypewasmonitoredbyphasecontrastmicroscopyandbystainingfor:positiveE
cadherinandcytokeratin13,aswellasfornegativefibronectinstaining(Figure6A).
4.2.Drugs
EPs®7630,aherbaldrugpreparationfromtherootsofP.sidoides(1:8–10),extraction
solvent:ethanol11%(w/w).TheconcentrationofEPs®7630is60mg/day,whichis
equivalentto1μg/kgforanaverageperson,orto1μg/mL.Theconcentrationsusedfor
thisstudyrangedbetween0.1–100μg/mL,withasingleapplicationatday0.EPs®7630
wassuppliedasdrypowderbySchwabePharmaAG(Karlsruhe,Germany)andwas
dissolvedincellculturemediumtothedesiredendconcentration.
VitaminD,calcitriol(SigmaAldrichMerck,Buchs,Switzerland),wasdissolvedin
ethanol(100μg/mL)beforebeingaddedtothecellculturesatfinalconcentrationsranging
fromconcentrationsof0.1–10μM,togetherwithEPs®7630.
4.3.HighPressureLiquidChromatography
EPs®7630highpressureliquidchromatographyUVhighresolutionmass
spectrometry(HRMS):TheHPLCUVHRMSchromatogramswererecordedonan
Thermo®VanquishUHPLCcoupledtoaDADandThermoOrbitrap®Fusionmass
detectorusingaWaters®AtlantisT3(3μM,2mm×150mm)columnwithoutprecolumn.
EluentAconsistedof2.5%(v/v)acetonitrileand0.5%(v/v)formicacidinwater.EluentB
consistedof5%(v/v)waterand0.5%(v/v)formicacidinacetonitrile.
Pharmaceuticals2021,14,1728of12
Ataflowrateof0.2mL/min,thegradientwasasfollows:from0.0–10.0minlinear
for0%to5%eluentB,from10.0–65.0minlinearfor5%to50%eluentB,from65.0to66.0
minlinearfor50%to100%eluentB,from66.0to71.0minisocratic100%eluentBcolumn
wash,from71.0–72.0minlinearfrom100%to0%eluentBfollowedby8minequilibration
periodwith0%eluentB,resultinginatotalruntimeof80.00min.UVdetection
wavelengthof280nmandacolumntemperatureof40°Cwereapplied.Theinjection
volumewas4μLofa5mg/mLP.sidoidesextractEPs
®
7630dissolvedineluentA.HRMS
basedpeakassignmentwasperformedusingACDLabsSpectrusProcessorSoftware
v2017.2.1.
4.4.CellTreatment
ConfluentepithelialcellswerestimulatedwithEPs
®
7630(0.1–10μg/mL),or
calcitriol,orthecombinationofboth,forupto48hbeforebeinginfectedwith1MOIof
RV16asdepictedinFigure6B.
Figure6.Cellcharacterizationandtreatment.(A)Celltypecharacterizationbyphasecontrastmicroscopyandpositive
immunofluorescencepancytokeratin,Ecadherin,andfibronectin(negativecontrol).(B)Treatmentschemeofcells.
ConfluentepithelialcellswerepretreatedwitheitherEPs
®
7630for24hbeforecalcitriolwasaddedforanother24hand
priortoinfectionwith1MOIRV16.RNAandproteinwereisolatedover4days.Noninfectedcellswereusedtocalculate
changesofproteinexpressionatthecorrespondingtimepoints.
ForEPs
®
7630inducedVDRexpression,cellswerepretreatedfor24or48hwith
EPs
®
7630(10μg/mL).TodeterminetheeffectofEPs
®
7630andcalcitriolonVDR
translocationpretreatedcellswereexposedtoincreasingconcentrationofvitaminD
(calcitriol0.1–10μMinDMSO).TheexpressionoftheVDRwasdeterminedbyWestern
blottingintotalproteinextracts.Insomeexperiments,theEPs
®
7630pretreatedcellswere
infectedwith1MOIRV16forupto48h.Totalproteinswerecollectedover4daysand
Pharmaceuticals2021,14,1729of12
analyzedasdescribedbelowforproteinexpression,orcellsgrownoncoverslipswere
fixedwith2%formaldehydefor2×5min.followedbystainingandfluorescence
microscopyforRV16proteinexpression(Figure6B).
4.5.RhinovirusInfectionandDetection
TheRV16strainwaspurchasedfromATCC(RV16,VR283,AmericanTypeCulture
Collection,Manassas,USA).Cellswereseededin8wellchamberslides(ThermoFisher
Scientific,Basel,Switzerland)andat80–90%confluencetheywereinfectedwithRV16(1×
multiplicityofinfection:MOI)by5mincentrifugation(200×g).Cellswerecontinued
understandardcellcultureconditionsforupto4days.RV16infectionwasmonitoredby
immunofluorescenceforantiRV16antibody(cat#18758,QEDBioscienceInc.,SanDiego,
CA,USA).Cellswerefixedbyformalin(4%inPBS,2×5min.),washedtwice(PBS),and
permeabilized(5min.,0.01%TWEEN100inPBS).Unspecificantibodybindingwas
blocked(30min.,2%bovineserumalbumininPBS)andafterwardsincubatedwiththe
antiRV16antibody(1:100dilution,overnight,(4°C).Cellswerewashed(PBS),
followedbyincubationwithantimouseFITClabelledantibody(Abcam,Switzerland,1
h,roomtemperature).NucleiwerestainedbyDAPIforcellcounting(ThermoFisher
Scientific).ThenumberofRV16positivecellswasdeterminedafter3washes(PBS)by
immunofluorescencemicroscopy(EVOSFLoidcellimagingstation,ThermoFisher
Scientific).AllexperimentswereperformedinaBioSafetyLevelIIlaboratory.
4.6.Cytosolic—NuclearProteinTranslocation
ConfluentepithelialcellsweretreatedwithEPs®7630(10μg/mL)for48hbefore
beingstimulatedwithincreasingconcentrationsofvitaminD(0.1–10mM)overvarious
timeperiods(0,3,6,and24h).ThecellcompartmentaldistributionoftheVDRwas
determinedbyimmunofluorescencestainingusingEVOScellimagingsystem(Thermo
FisherScientific).
4.7.WesternBlotting
CellswerelysedinRIPAbuffer,orascytosolicandnuclearproteins.Theprotein
contentwasquantifiedbyBCA(ThermoFisherScientific).Denaturedproteins(10μg)
weresizefractionatedbyelectrophoresis(8–16%SDS–PAGE,ThermoFisherScientific),
andtransferredontoPVDFmembranes.Unspecificbindingofantibodieswasblockedby
30minincubationofthemembraneswith2%bovineserumalbumininphosphatebuffer
saline(PBS)containing0.05%TWEEN20.Proteinsweredetectedbyincubatingthe
membraneswithoneoftheprimaryantibodiestoeithertheVDR,Erk1/2MAPK,p38(α,
β,γ,δ),JNK,Ecadherin,orGAPDH(all:AbcamPlc,Cambridge,UK)forovernightat
4oC.Followingthreewasheswithblockingbuffer,themembraneswereincubatedwith
secondaryspeciesspecificHRPconjugatedantibodies(Abcam).Proteinbandswere
visualizedbychemiluminescence,applyingSuperSignalWestDurasubstrate(Thermo
FisherScientific)anddocumentedbyc300(AzureBiosystems,Dublin,CA,USA).
4.8.Immunofluorescence
Epithelialcellswereseededon8wellPCAslides(cat94.6140.802,Sarstedt,Sevelen,
Switzerland)andallowedtoadhereovernight.Cellswerethentreatedwithmedium
alone,orbyincreasingconcentrationsofEPs®7630(0.01–10μg/mL)overvarious
durationsuptofivedays.Cellswerefixedin4%paraformaldehyde(inPBS,2×5min),
andimmunofluorescencestainingwasperformedasdescribedearlier[7].Nucleiwere
stainedbyDAPI.
Pharmaceuticals2021,14,17210of12
4.9.Statistics
TheNullhypothesiswas:nomodificationofproteinexpression,activationor
locationbyanytreatmentcomparedtountreatedcells.TheNullhypothesiswastestedby
ANOVA,Student’sttestandsubsequentMannWhitneyUtestasappropriate;pvalue<
0.05wasacceptedassignificant.
5.Conclusions
ThisstudyindicatesthattheprotectiveeffectofEPs®7630againstrhinovirus
infectionmightinvolvetheupregulationoftheVDRandtheimprovedepithelialcell
differentiation.
AuthorContributions:Conceptualization,M.R.andM.T.;methodology,M.R.andQ.S.;validation,
M.R.andM.T.;formalanalysis,M.R.andQ.S.;investigation.M.R.andQ.S.;datacuration,M.R.and
Q.S.;writing—originaldraftpreparation,M.R.andQ.S.;writing—reviewandediting,M.R.,Q.S.
andM.T.;visualization,M.R.andQ.S.;supervision,M.R.andM.T.;projectadministration,M.R.and
M.T.;fundingacquisition,M.T.Allauthorshavereadandagreedtothepublishedversionofthe
manuscript.
Funding:ThestudywasfundedbyanunrestrictedresearchgrantbyDr.WillmarSchwabeGmbH
&Co.KG(76227Karlsruhe,Germany)toMTamm.
InstitutionalReviewBoardStatement:ThestudywasapprovedbythelocalInstitutionalEthical
Committee(EKBB05/06).Allbiopsieswereanonymous.
InformedConsentStatement:Informedconsentwasobtainedfromallsubjectsinvolvedinthe
study.
DataAvailabilityStatement:Thedatasetsusedand/oranalyzedduringthecurrentstudyare
availablefromthecorrespondingauthoronreasonablerequest.
Acknowledgments:WethankA.Bell(UK)forhisassistantwithEnglishwritingandediting.We
thankHeikeSchneiderandŽarkoKulić fromtheDepartmentPreclinicalResearchand
DevelopmentofDr.WillmarSchwabeGmbH&Co.KG,Karlsruhe,Germanyforthemeasurement
andanalysisoftheHPLCUVHRMSchromatograms.
ConflictsofInterest:Theauthorsdeclarenoconflictsofinterest.Thefundershadnoroleinthe
designofthestudy;inthecollection,analyses,orinterpretationofdata;inthewritingofthe
manuscript,orinthedecisiontopublishtheresults.
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... The fractions generated by ultrafiltration were analyzed using HPLC-UV, gel permeation chromatography (GPC), and NMR spectroscopy. HPLC analysis was used to monitor the purine derivatives and benzopyranones in the fractions according to the LC-MS assignment reported by Roth et al. (Roth et al., 2021). Polymeric and oligomeric prodelphinidins appeared as broad signals in HPLC analysis, whereas carbohydrates were not detectable due to the absence of chromophores. ...
... Interestingly, whereas the 1-3 kDa fraction comprises mainly monomeric gallocatechin and prodelphinidin dimers to tetramers with proven effects in virus entry, the <1 kDa fraction contains highly substituted benzopyranones like umckalin and other small molecules. Although little is known about the antiviral effect of these compounds, umckalin and other benzopyranones were shown to modulate immune responses and affect antiviral defense mechanisms (Robertson et al., 2016;Roth et al., 2021). Considering the TMPRSS2-independent entry inhibition that we observed in Vero cells, it appears likely that umckalin and other small molecules contributed to the inhibition of SARS-CoV-2 entry in our study. ...
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Treatment options for COVID-19 are currently limited. Drugs reducing both viral loads and SARS-CoV-2-induced inflammatory responses would be ideal candidates for COVID-19 therapeutics. Previous in vitro and clinical studies suggest that the proprietary Pelargonium sidoides DC. root extract EPs 7630 has antiviral and immunomodulatory properties, limiting symptom severity and disease duration of infections with several upper respiratory viruses. Here we assessed if EPs 7630 affects SARS-CoV-2 propagation and the innate immune response in the human lung cell line Calu-3. In direct comparison to other highly pathogenic CoV (SARS-CoV, MERS-CoV), SARS-CoV-2 growth was most efficiently inhibited at a non-toxic concentration with an IC50 of 1.61 μg/ml. Particularly, the cellular entry step of SARS-CoV-2 was significantly reduced by EPs 7630 pretreatment (10–100 μg/ml) as shown by spike protein-carrying pseudovirus particles and infectious SARS-CoV-2. Using sequential ultrafiltration, EPs 7630 was separated into fractions containing either prodelphinidins of different oligomerization degrees or small molecule constituents like benzopyranones and purine derivatives. Prodelphinidins with a low oligomerization degree and small molecule constituents were most efficient in inhibiting SARS-CoV-2 entry already at 10 μg/ml and had comparable effects on immune gene regulation as EPs 7630. Downregulation of multiple pro-inflammatory genes (CCL5, IL6, IL1B) was accompanied by upregulation of anti-inflammatory TNFAIP3 at 48 h post-infection. At high concentrations (100 μg/ml) moderately oligomerized prodelphinidins reduced SARS-CoV-2 propagation most efficiently and exhibited pronounced immune gene modulation. Assessment of cytokine secretion in EPs 7630-treated and SARS-CoV-2-coinfected Calu-3 cells showed that pro-inflammatory cytokines IL-1β and IL-6 were elevated whereas multiple other COVID-19-associated cytokines (IL-8, IL-13, TNF-α), chemokines (CXCL9, CXCL10), and growth factors (PDGF, VEGF-A, CD40L) were significantly reduced by EPs 7630. SARS-CoV-2 entry inhibition and the differential immunomodulatory functions of EPs 7630 against SARS-CoV-2 encourage further in vivo studies.
... This was attributed to suppression by 25D of RV-induced expression of intercellular adhesion molecule 1 (ICAM-1), a cell surface molecule that functions as the receptor for RVs, and platelet-activating factor receptor (PAFR) [79] (Fig. 2). Most recently, EPs®7630, a herbal drug preparation from the roots of Pelargonium sidoides, which is efficacious against respiratory tract infections, was found to increase host defense against RV by upregulating the VDR in human bronchial epithelial cells [80]. ...
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