The hygiene hypothesis : immunological mechanisms of airway tolerance

The

hygiene

hypothesis:

immunological

mechanisms

of

airway

tolerance

Eline

Haspeslagh

,

Ines

Heyndrickx

,

Hamida

Hammad

and

Bart

N

Lambrecht

Thehygienehypothesiswasinitiallyproposedasan

explanationforthealarmingriseinallergyprevalenceinthelast century.Theimmunologicalideabehindthishypothesiswasa lackofinfectionsassociatedwithaWesternlifestyleanda consequentialreductionintype1immuneresponses.Itisnow understoodthatthedevelopmentoftolerancetoallergens dependsonmicrobialcolonizationandimmunostimulatory environmentalsignalsduringearly-lifeorpassedonbythe mother.Theseenvironmentalcuesaresensedandintegrated bybarrierepithelialcellsofthelungsandpossiblyskin,whichin turninstructdendriticcellstoregulateorimpedeadaptiveTcell responses.Recentreportsalsoimplicateimmunoregulatory macrophagesaspowerfulsuppressorsofallergybythe microbiome.Weproposethatlossofadequatemicrobial stimulationduetoaWesternlifestylemayresultin hypersensitivebarriertissuesandtheobservedriseintype 2allergicdisease.

Addresses

1

LaboratoryofImmunoregulationandMucosalImmunology,VIBCenter forInflammationResearch,Technologiepark927,B-9052Ghent (Zwijnaarde),Belgium

2_{DepartmentofBiomedicalMolecularBiology,GhentUniversity,}

Technologiepark927,B-9052Ghent(Zwijnaarde),Belgium

3

DepartmentofInternalMedicine,GhentUniversity,DePintelaan 185K12,B-9000Ghent,Belgium

4_{DepartmentofPulmonaryMedicine,ErasmusMC,’s-Gravendijkwal}

230,3015CERotterdam,TheNetherlands

Correspondingauthors:Hammad,Hamida(Hamida.Hammad@UGent. be),Lambrecht,BartN([email protected])

CurrentOpinioninImmunology2018,54:102–108 ThisreviewcomesfromathemedissueonAllergyand hypersensitivity

EditedbyOnurBoyman,AlexanderEggelandMarioNoti

https://doi.org/10.1016/j.coi.2018.06.007

0952-7915/ã2018TheAuthors.PublishedbyElsevierLtd.Thisisan openaccessarticleundertheCCBY-NC-NDlicense( http://creative-commons.org/licenses/by-nc-nd/4.0/).

Introduction

Allergicsensitizationischaracterizedbythepresenceof

allergen-specific immunoglobulin E (IgE) in serum.

Exposuretoallergensviainhalation,ingestionorcontact

with the skin can lead to diseases such as asthma, hay

fever,eczemaand,insomecases,tosystemicanaphylaxis.

Duringthelast150years,allergieshaveemergedinavery

rapidwayandtheirprevalenceisstillontherise.

Nowa-days,morethan30%ofchildrenareallergic,upto10%of

childrensufferfromasthmaandallergicrhinitis,and5–

7%ofchildrenhavedevelopedfoodallergy.Itisstillnot

entirelyclearwhy asthmaprevalenceissohigh,butthe

rapidtimeframeofitsoriginationandexpansionsuggests

that environmental or behavioral changes in Western

lifestyleare involved.

A

modern

lifestyle

is

associated

with

dysbiosis

Animportantevolutionofthelast150yearsisasuccessful

decreaseofinfectiousdiseaseburden,duetothemassive

introduction of hygiene measures, antibiotics, and

vac-cines.In1989,Strachanobservedthatgrowingupinlarge

familieswithmoreoldersiblingsdecreasedthechancesof

developinghayfever or eczema [1].Hepostulated that

therecent increase in allergy incidencewas a result of

‘decliningfamilysize,improvementsinhousehold

ame-nities, and higher standards of personal cleanliness’,

which had reduced ‘the opportunity for crossinfection

inyoungfamilies’.Theoriginal‘hygienehypothesis’was

thus introduced. Since then, this hypothesis has been

supported by numerous studies, especially in murine

models,showing thatexposure to bacteria,viruses,

hel-minthsor microbe-derivedproducts couldprotect from

allergy(reviewedin[2],[3

]).However,itshouldbekept

inmindthatnotallpathogensareprotective;forinstance,

respiratorysyncytialvirus(RSV)orrhinovirusare

associ-atedwithahigherrisktodevelopwheezeandasthmaup

toadulthood[4].

Changesin lifestylecanalsoheavilyinfluencethe

com-positionanddiversityofthemicrobiomeatseveral

muco-sal surfaces. These microbial communities have

co-evolved with and within the human body for millions

ofyears,and,consequently,thehumanimmunesystem

has been calibrated and fine-tuned so to maintain and

shape symbiotic relationships with them (reviewed in

[5]).Twotheories,the‘Oldfriends’andthe‘Biodiversity’

hypotheses,havebeenproposedbyRookandby

Haah-telaasamoreaccurate,oratleastcomplementary,

expla-nationfortherecentallergypandemic[6,7].They

stipu-latethatthereasonfortheincreasedincidenceinallergic

disordersisareducedexposuretosuchbeneficial

reported thatalterationsinthecomposition of theskin,

the nose or the gut microbiome are associated with

eczema, asthmaandfoodallergy[8–10].Thesechanges

do not affect a single commensal, but rather reflect a

reduced total microbial diversity[11],and they maybe

caused byseveral factors,including sibling orderin the

family[12],exposuretoanimals[13],andotherearly-life

events[14].Theimportanceofahealthymicrobiomein

controlling allergies was further substantiated in mice,

with germ-free micebeing especiallyprone to develop

overt allergic (airway)disease, aphenotypereverted by

microbial recolonization[15,16].However,otherstudies

showed that germ-free mice are not universally more

susceptible to house dustmite driven asthma,and that

onlyselectedstrainsoflungmicrobiotaseemtosuppress

asthma[17].Duringthelast30years,thebodyof

correla-tiveepidemiologicalstudieshasexpandedvastly, andis

the subject of many excellent reviews. Here, we will

zoominonrecentadvancesinthesearchforthe

under-lying immunological mechanisms explaining the

observed effects.

Microbes

induce

protective

regulatory

DCs

and

T

cells

Allergies are generally aberrant immune reactions to

innocuous antigens, orchestrated by T helper 2 (Th2)

cellsandtype2innatelymphoidcells(ILC2s).Inthecase

ofasthma,thistype2cellactivityleadstomucus

hyper-secretion, goblet cell hyperplasia, smooth muscle cell

hyperreactivity, and the infiltration and/or activationof

eosinophils, mast cells and basophils,ultimately

culmi-nating in breathing difficulties and airway remodeling

[18].Dendriticcells(DCs)arealwaysfoundatthebody’s

barriers,andbecausetheyexpressawiderangeofpattern

recognition receptors (PRRs), they can sense the

envi-ronment for the presence of danger signals [19]. Our

group hasshownthatTh2responsesto housedustmite

(HDM) allergens were induced by IRF4-dependent

cDC2s in thelungsand in theskin [20,21
] (Figure 1).

ThesecDC2scapturetheHDMallergensintheairways

andmigratetothedraininglymphnodes,requiring

ILC2-derivedIL-13,wheretheypresenttheallergenstonaı¨ve

T cells [22]. It is easy to imagine that environmental

changessensedatthelevelofthelungs,theskinbutalso

ofthegutwillmodifythecontextofallergenrecognition

by DCs, and either protect against or enhance allergic

responses.

Chronic Helicobacter pylori infection has been inversely

linkedto asthma inhumansand caneffectivelyprotect

mice from OVA-induced asthma [23,24]. In mice, H.

pylori infection induced the accumulation of CD103+

cDCsin thelungs,which wererequiredfor the

protec-tion,aswastheirIL-10production[24].Inarecentstudy,

semi-therapeuticH.pyloriextracttreatmentalsoreduced

airwayallergy,shiftedtheCD11b+/CD103+DCratioin

thelungs,andreducedtheantigenprocessingbylungand

lymphnode DCs[25].Other studiesdemonstrated

pro-tective modulation ofinvitro bone-marrowderivedDC

cultures (BMDCs). A synthetic TLR1/TLR2-agonist

inducedLPS-toleranceandIL-10productioninBMDCs,

whereas thecowshedLactococcuslactisinstigated a

Th1-polarizingprogram,bothrenderingtheBMDCsunableto

sensitize mice to OVA-allergen upon adoptive transfer

[26,27
].

Trompetteetal.recentlyfoundthatfeedingmicea

fiber-rich diet changed the composition of the lung and gut

microbiome, thelattermetabolizing thefiberinto

circu-lating short-chain fatty acids (SCFA’s) [28]. The

increasedSCFA levelsprotectedthemicefrom allergic

lung inflammation.Mechanistically,theSCFA’s altered

DC precursor generation in the bone marrow, and the

DCssubsequentlyseedingthelungshadahigher

phago-cyticcapacityandwereimpairedinpolarizingTh2cells.

Additionalstudieshavesupportedtheprotectiveeffectof

dietary fiber supplementationon allergicasthma

devel-opment in mice [29], and onwheeze in humaninfants

whenthefiberwasgiventothepregnantmother[30].

One mechanismbywhichtheDCsin microbe-exposed

animalscanconferprotection,isbyinducingthe

genera-tionofregulatoryTcells(Tregs).Microbialcolonization

in 2-week old mice was shown to be necessaryfor the

transientupregulationofPD-L1onlungCD11b+DCs,

and the expansionof a specific pulmonary Tregsubset

[31].PD-L1blockadeinneonatesresultedinexaggerated

responsivenesstoHDMthroughadulthood,suggestinga

crucialroleforthismicrobial-inducedDC–Tregaxisfor

immunologicaltolerance.Inanothermouse modelofH.

pylori-mediatedasthmaprotection,theHelicobacter

infec-tion inhibited TLR-inducedDC maturation and

repro-grammed the DCs towards a FoxP3+ Treg-polarizing

phenotype [32]. The bacterial component flagellin B,

givensemi-therapeuticallytogetherwithallergen, could

alsoinhibit murineallergicasthmasymptomsinaDC

andCD25+Treg-dependent manner[33
].

Although helminths are prototypical inducers of type

2 immunity, they have been correlated with reduced

allergen skin prick test reactivity, and to some degree

with asthma protection (reviewed in [34]). A general

explanationfor thisnon-intuitiveassociationisthat

hel-minthsinduceaso-called‘modifiedTh20response,with

immunoregulatory cells such as Tregs complementing

theTh2-armofimmunity,andregulatingtheresponseto

bystanderantigenssuchasaeroallergens.Therefore,

sev-eralgroupshavetriedtofindhelminth-derivedproducts

withimmunomodulatorypropertiesthatcouldbeusedto

suppressTh2immunity.Forinstance,an

anti-inflamma-toryprotein(-2;AIP-2)fromtheparasitichookwormwas

identified to suppress murine airway allergy in a

DC-dependentandTreg-dependentmanner[35].Inanother

AvCystatin was demonstrated to induce regulatory

macrophagesthatprotectedagainstexperimentalasthma

upon adoptivetransfer [36].Regulatory alveolar

macro-phagesfrombonemarroworiginwererecentlyalso

impli-cated in long-lasting protection conferred by a latent

murinegammaherpesvirusinfection,amodelfor

Epstein-–Barrvirusinfectioninmice[37
].Theregulatory

macro-phagesinducedbytheinfection replacedthelong-lived

andself-replenishingalveolarmacrophagesthatare

gen-eratedshortlyafterbirth,andbecamelong-livedaswell.

Figure1 Treg Th2 TLR4 IL-25 IL-33 GM-CSF CCL20 IL-1α Th0 IRF4+ CD11b+ OX40L Notch L IL-12 Allergens (HDM) Endotoxin Farming environment Microbial colonization RSV

Second hand smoke Neonatal lungs H. polygyrus HES Microbial colonization H. pylori L. lactis SCFAs Flagellin B AIP-2 Th1 Th0 IL-1R IL-33 TLR4 TLR4 rM AvCystatin Herpes

Current Opinion in Immunology

Proposedmodelofairwaytolerance.Intheabsenceofimmunoregulatorypathways,epithelialbarriercellsreadilyrespondtoallergenbindingon theirpatternrecognitionreceptors,amongwhichTLR4,bythesecretionofinflammatorymediators(IL-1a,IL-25,IL-33,GM-CSF,CCL20,and others).Thesemediatorslicenseantigen-bearingIRF4+CD11b+conventionaldendriticcells(cDC2s)topolarizenaı¨ve TcellstoThelper2(Th2) cellsinthelung-draininglymphnodes.Neonatalandgerm-freemiceareespeciallypronetodevelopsuchTh2responses.Respiratorysyncytial virus(RSV)infectionandsecondhandcigarettesmoke,twoknownasthmariskfactors,increaseIL-33secretionandmaytherebystimulatethis pathway.Exposuretoendotoxin,farmdustormicrobialcolonizationbluntstheepithelialresponsebyincreasingtheexpressionofnegative regulators.EpithelialIL-33releaseisalsoinhibitedbyhelminth-derivedexcretedandsecretedproducts(HES).DCsdevoidofepithelialactivation signalsdonotinduceTcellactivation(Th0).OtherprotectivefactorsimpedeTcellactivitybyinfluencingthematuration,antigenpresentation,or phagocyticcapacityofDCs.SomeprotectivefactorsinduceDCsthatprovokeregulatoryTcell(Treg)activityorThelper1(Th1)activity.

Regulatorymacrophages(rM)canalsoinduceTregs,orblockDC-mediatedTh2-polarization.Abbreviations:HDM,housedustmite;TLR4,Tolllike receptor4;IL-1R,IL-1receptor;H.pylori,Helicobacterpylori;L.lactis,Lactococcuslactis;SCFA,shortchainfattyacids;AIP-2,anti-inflammatory protein2.

This potentially explains the long-lasting effects of

microbial stimuliinthelungsonallergysuppression.

Allergic

asthma

is

initiated

by

aberrant

immune

responses

at

barrier

tissues

To initiate an allergen-specific Th2 response, cDC2s

need to be instructed by barrier epithelial cells (ECs)

liningtheairways.BarrierECsarepermanentlyexposed

to environmental insults or innocuous signals and, like

DCs,theyarewell-equippedtointegratethesesignalsvia

arangeofPRRs(reviewedin[38]).ActivationofPRRson

ECs by allergens induces NF-kB activation and ROS

production,resultinginthesecretionofawiderangeof

inflammatorymediators,amongwhichthecytokines

IL-33, IL-25 and TSLP. DCs react to these cytokines by

OX40LandNotchligandupregulation,and

downregula-tion of IL-12 production,an activationstate thatfavors

Th2polarizationinthelung-draininglymphnode[39,40].

Interestingly,thebarriertissueoftheskinalsoconstitutes

a possible entry route for aeroallergens [21
]. Thus,

barrier cellsact very upstream in theinflammatory

cas-cadeofeventsleadingtoallergicsensitization(Figure1).

OurgrouphaspreviouslyreportedthatthePRRtoll-like

receptor4(TLR4)onairwayECswascriticallynecessary

to mount aTh2-mediated asthmaticresponseto HDM

[41].Strikingly,severalHDMallergenshavetheintrinsic

capacitytofacilitateoramplifyTLR4signalingby

bind-ingdirectlytoproteinsoftheTLR4signalingcomplexor

to itsligands[42,43].However,TLR4is bestknownas

thereceptorforLPS,alsotermedendotoxin,acomponent

ofgram-negativebacteria.Itisdifficulttoreconcilehowa

receptorspecializedinbacterialsensingcancontributeto

Th2immunityandallergy,especiallygiventhefactthat

highendotoxinlevelsinchildren’smattressesare

protec-tive against atopic sensitization and asthma in humans

and mice [44–46]. Another study also associated house

dustendotoxinlevelswithasignificantlyreducedriskof

allergic sensitization or eczema, specifically in children

with apolymorphism in the CD14gene [47].Infact, a

bodyofepidemiologicalstudieshaveconvincingly

corre-latedatraditionalfarmingenvironment,whereendotoxin

levelsarehigh,withprotectionagainsthayfever,allergic

sensitization,andasthma(reviewedin[48]).Inahallmark

study,childrengrowinguponagriculturalHutteriteand

Amish farms in the US were compared, and the latter

werefound to havesixtimes lesschanceof developing

atopyandasthma[49

].Thesetwofarmingpopulations

share a similar genetic ancestry and lifestyle. Farming

practices, however, differ, and the Amish house dust

containedalmost7timesmoreendotoxinthanthehouse

dustfromHutteritefarms.OnlythetransferoftheAmish

dust intranasally to mice inhibited subsequent

experi-mentalasthmadevelopment.Wehaverecentlyconfirmed

that farm dust collectedfrom Bavarian farms(in which

farmingpracticesresembletheAmish’sones),andLPS,

conferred protectionagainst experimental asthma.This

protection was mediated by an increased epithelial

expression ofTNFAIP3(betterknownasA20),a

nega-tiveregulatoroftheNF-kBpathway,whichbluntedthe

epithelial cell response to HDM and downstream DC

activity[50].AsimilartolerancetoLPSmediatedbyA20

inductionwasdemonstratedinintestinalECs[51].

Inter-estingly, A20 expression was very low in neonatal rats,

spontaneously increased shortly after birth coinciding

withmicrobialcolonization,andcouldbedownregulated

by treatment with antibiotics. It remains to be

investi-gatedifthegutorlungmicrobiotacansimilarlyinfluence

expressionofA20andothernegativeregulatorsinairway

ECs.ECmodulationhasrecentlybeendemonstratedfor

Heligmosomoidespolygyrus,ahelminthoftenconfirmedto

protectagainstmurineallergy[52].Secretedandexcreted

products(HES)ofthisparasiteinhibitedIL-33releaseby

ECs and thereby suppressed Alternaria-induced airway

allergy [3

]). Mechanistically, the H. polygyrus alarmin

release inhibitor (HpARI), a 26kDa protein, binds to

activated IL-33 and at the same time tethers IL-33 to

theDNAofnecroticcells,thusinhibitingIL-33actionin

adualmanner andinhibitinginnateeosinophilic airway

inflammation [53

]. Other molecules secreted by the

parasite are more related to TGFb and can induce a

Foxp3+Tregpopulationwithimmunoregulatory

poten-tial[54].

A dysregulated immune responseattheskin can cause

atopic dermatitis(AD),which initselfisariskfactorto

accumulate more allergies later in life, among which

asthma, aprocess known as ‘the atopic march’. In fact,

ADandasthmashareseveralriskfactors.ADisstrongly

correlatedwithchangesintheskinmicrobiome,themost

well-knownbeingpertinentStaphylococcusaureus

coloni-zation of allergic skin. In a recent study, IL-17Ra/

micespontaneouslydevelopedADwithnaturally

occur-ringskin dysbiosisand acompromisedskin barrier,and

antibiotictreatmentamelioratedskininflammation[55].

Asimilardysbiosis–ADaxishasalsobeendemonstrated

inADAM17/mice[56].Topicaltreatmentwith

non-pathogenic bacteria, on the other hand, can alleviate

cutaneous inflammation in murine AD [57]. It has

become clear in recent years that tonic sensing of skin

commensalsheavilyshapeshostDCandTcellfunctions

[58–60].Itremains to beinvestigated whattherelative

importance isof passivebarrierintegrityandactive

sig-nalingthroughkeratinocytePRRs,alsopoisedtorapidly

respond to innate immune ligands, in this

microbe-immunecellcross-talk[61].Itwillalsobeofgreatinterest

tostudyhowenvironmentalexposuresinfluencetheskin

microbiome and/or the immune threshold of skin

epithelium.

One intriguing observation is that allergies tend to

developearlyinlife.Inthesametimewindow,andeven

in utero,protective effects of environmental factorsand

recently demonstrated that the lung environment in

neonatalmiceisstronglytype2-skewed,with agradual

increase in IL-33 release by lung ECs, and with the

spontaneous recruitment of several Th2-associated

innate immune cells, peaking 2 weeks after birth

[64

].Thisspontaneouswaveofearlytype2immunity

islikelytobecausedbythemechanicalstressinducedby

the breathing patterns [65], but also by the constant

remodeling necessary to build up new lung structures.

Interestingly,this periodisproneto favorstronger Th2

sensitizationtoinhaledallergens[64

],butalsotofavor

lower immunity to bacteria [65]. Many environmental

factors, likesecond handsmokingor RSV infectionare

knowntofacilitateTh2sensitizationinchildren.These

triggershavein commonthattheyinducehighlevelsof

IL-33[66,67].Itistemptingto speculatethattheserisk

factors act by prolonging or amplifying the epithelial

cytokineresponsetoallergensduringearly-life,andthat

combined early-life exposures thus define the final

thresholdforECactivation.

Conclusion

EffectsofmicrobesoninducingTregcells,Th1cellsand

allergen cross-reactive antibody responses, are

well-observed. In addition, we propose a model in which

environmentalandmicrobialstimuliaresensedand

inte-gratedbybarriertissuesofthelung,theskinandthegut,

resultinginatonicDCactivationstatuspromotingeither

inflammatory or tolerogenic immunity. Together with

directeffectsonDCsandTcells,mostprotectivestimuli

thus seem to converge in the same central tolerogenic

immunepathways.Fullyunderstandingthefundamental

immunological pathways underlying these protective

triggers,theirrelativecontribution,andhowtheyinteract,

should hopefullyallow usto pinpoint,modify or newly

develop prophylactic or therapeutic therapies to cure

asthma.

Conflict

of

interest

statement

Nothingdeclared.

Acknowledgements

ResearchintheLambrechtandHammadlabissupportedbyanAdvanced EuropeanResearchCouncil(ERC)grant,anFWOFlandersExcellenceof Sciencegrant,andtheWorldWithoutAsthma(AWWA)programofthe DutchLungFoundation.Figure1includesderivativematerialfromServier MedicalArt(https://smart.servier.com/)byServier,availableundera CreativeCommonsAttribution3.0UnportedLicense(https:// creativecommons.org/licenses/by/3.0/).

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