1. 6BBYN305 Literature Based Project in Neurosciences
Cellular and Molecular Mechanisms of Itch
Camilla Siig
1207140
School of Bioscience Education
King’s College London
Guy’s Campus
Dissertation submitted in part fulfilment of a BSc (Hons)
in Biomedical Science

2. Abstract
The distinctionbetweenitchandpainhas beenamatter of debate formany years.Recentadvancementsinitch
researchhas beguntoexpose neurophysiological,cellularandmolecularmechanismsof itchthroughhumanclinical
studiesandanimal models.CurrentknowledgeaffirmsthatitchismediatedbybothunmyelinatedC-fibresandAδ
fibres,providingsupportforthe labelled-line theorywhichadvocatesitch-specificneurocircuitry.Recentresearch
has impliedthe involvementof amyriadof mediatorsincludinghistaminereceptors,TRPchannels,TLRs,Mrgprs,
inflammatorymoleculesandneuropeptides.Thisdiversityof itchmediatorsandtheircomplex interactionswithpain
and itchsuggestsa more integrative approach isneeded:populationcodingtheoryhasbeenproposedinthis
respect.Thisreviewwill coverthe developmentof anitchtransmissiontheoryandprovide evidence forits
modulationinlightof the currentliterature.The focuswillbe itchreceptorsandmediators,andtheirrole initch
signalling.

3. Table of Contents
1.0 Background 1
2.0 Itch Transmission 2
2.1 Peripheral Itch Coding and Receptors 2
2.2 Central Processing 4
3.0 Peripheral Mechanisms: Receptors and Mediators 4
3.1 Histamine 4
3.2 Mas-Related G Protein-Couple Receptors 6
3.3 Transient Receptor Potential Channels 6
3.4 Protease-activated receptors 8
3.5 Toll-like Receptors 9
3.6 Cytokines 9
3.7 Substance P 10
4.0 Modulation of the Itch Pathway 10
4.1 Peripheral Sensitisation 10
4.2 Central Sensitisation 11
5.0 Conclusion 11
6.0 References 14

4. Background
Itch, alsoreferred toas pruritus,canbe definedasan“unpleasantsensationthat elicitsthe desireorreflex to
scratch” (Patel & Dong, 2011). Its clinical classificationiscomplicated,thoughitchisbroadlyregardedaseither
peripheral (pruritoceptive)orcentral (neurogenicorneuropathic).Twycrossetal. (2003) propose fourcategories:
pruritoceptive,neuropathic,neurogenicandpsychogenic.Pruritoceptiveitchisthe mostwell-understood,
encompassingsymptomssuchasinflammation,drynessandskindamage.Commonconditionsinclude xerosis,
atopicdermatitisandscabies (Cohenetal.,2012). Neuropathicitchonthe otherhand,isan area verymuch
unexplored.Itisknowntoarise fromimpairednervesof the central nervoussystem(CNS),causingdisease anywhere
alongthe itch pathway (YosipovitchG etal., 2003). Illnessesassociatedwithneuropathicitchtendtoemerge from
infectionsalreadycausingneuropathicpainsuchasshingles,lesionsof the trigeminal nerve,stroke andmultiple
sclerosis (Oaklander,2011). Neurogenicitchischaracterisedbyalack of neural pathology, suchasin cholestasis;
and psychogenicitchevolvesfrompsychiatricorpsychological disordersincludingobsessive compulsivedisorderand
delusionalparasitophobia (YosipovitchG etal., 2003). Concerningthe alarmingprevalence of pruritusamong
patients,itisessentialthatthe pathophysiologyof abnormal itchcanbe diagnosedeffectively.Mostimportantly,
understandingthe complexityof itchmechanismsandmediatorsisthe keytotreatingclinical conditions
successfully.
Regardingthe developmentof anextensive itchtheory,earlystudiesnotedbehavioursindicatingabolishedpruritus
followingventral lateral cordotomies(surgical lesionof the ventral lateral funiculus(VLF)).Giventhatthe
spinothalamictract(STT) supportsascendingaxonsof the VLF,itwasconcludedthatitch sensationmustbe relayed
viathe STT (Davidsonet al., 2014). In 1997, Schmelzetal.firstidentifiedCfibresas“afferentnerve fibreswith
particularlythinaxons”necessaryformediatingitchsensation.Itisnow understoodthatthree typesof primary
afferentnerve exist:Aβ(fastconductingandheavilymyelinated),Aδ(slow conductingandmyelinated) andCfibres
(slowconductingandunmyelinated).Those perceivingpainanditchinvolve AδandCfibres,whichrespondto
noxiousandthermal stimuli;while those perceivingtactile sensationsrequire Aβconductionandrespondtonon-
noxiousmechanical stimuli. Sharppaincorrespondstothe fastconductionof Aδfibres,andslowerCfibresare
knownto relayprolongedachingpainoritch.Overlappingneural circuitriesassociatedwitheitherof these
modalitiesinitiallyledtothe proposal of the “intensitytheory”,whichwasacceptedbythe scientificcommunity for
a longtime (Potenzieri & Undem,2012). Itch was consideredtobe a submodalityof pain,differentiatedonlybythe
intensityorpatternof neuronal firing.Contrarytothis,recentdevelopmentsuggestsa“labelled-line theory”
wherebyitchpathwaysare distinctlymarkedbymodalityspecificmolecules,sensoryneuronsandneural pathways
(Han & Dong, 2014). The literature surroundinglabelled-line theoryisencouragingandconsensusisgravitating
towardsitsexplanation.Althoughnotall itchphenomenacanbe fullyinterpretedyet,anew andpromisingbiology
of itchisunfolding (Handwerker,2014).
The current firstline of treatmentforitchreliesonantihistamines.Thougheffectiveforminorallergiesandinsect
bites,antihistaminesare largelyinadequate atalleviatingclinical itch,especiallywhenthe conditionischronic.The

5. limitedknowledge of itchpathophysiologycombinedwiththe unavailabilityof itchanimal modelshashamperedthe
developmentof effective treatmentsforhumanitch.Currently,pruritogeninjectionsandskindehydration in vivo
modelsallowcutaneoussymptomsassociatedwithitchtobe analysed.Whilemodelsforsystemicallyderiveditch
mechanisms(suchascholestasis)have onlyrecentlybecomeapossibility (Han& Dong, 2014); anotherissue
concernsthe measurementof animal response behavioursversusdirectsensation.Forexample,certaininjection
sitesinduce aresponse where there isnodifferentiationbetweenpainanditchbehaviours (LaMotte etal., 2011). In
orderto improve the prospectsforitchtreatment,itisnecessarythatcurrentunderstandingof itchmechanismsbe
furtherinvestigated.Thisreviewwillprovideacomprehensiveandcritical accounton cellularandmolecular
mechanismsof itch;delvingintothe neurophysiologyunderlyingsensoryprocessingaswell asthe plethoraof
relevantreceptorsandmolecularmediators.Abnormalitiesof the itchpathwaywill alsobe discussed,addressingthe
histological structuresof neuronal mechanismsinchronicitchconditions.
Itch Transmission
Peripheral Itch Codingand Receptors
The interactionbetweenitchandpainhascausedgreat confusionoverthe years.The intensitytheory,originally
proposedinthe early20th
century,wasderivedfromthe observationof overlappingpainanditchresponse spotsin
humanskin (Lewiset al., 1927). However,contradictionsappeared.Tuckett (1982) demonstratedthatpaindidnot
arise fromitch withincreasingfrequencyof electrical stimulation.Neitherwaspainfoundtodull intoitchat lower
stimulationfrequencies (Ochoa& Torejbork,1989; Handwerkeret al., 1991). EnsuingSchmelz’s1997 discoveryof
histamine-responsive Cfibres,Andrew&Craig (2001) identifiedthe laminaIregionof the spinal cordas a “unique
subsetof STT neurons”acquiringitchselectivityinresponse tohistamine.Schmelzetal. (2003) supportedthis
conclusionina more thoroughinvestigation.Variouspruritogen(itchproducing)andalgogen(painproducing)
substanceswere testedonthree sub-classesof C-nociceptors.Theirmajorfindingdeterminedthathistamine-
positive Cfibres are “selective”butnot“specific”forpruriticstimuli.More recentstudiesrevealthatchemically
silencedneuronsexpressingtransientreceptorpotential vanilloid-1(TRPV1) nociceptorsexhibitseverelyimpaired
itch response (Imamachi etal., 2009), as well asreducedthermal andpainsensitivity (Cavanaugh,2009), suggesting
that TRPV1 isnecessaryforitchperception. However,the questionremains:are painanditchfacilitatedbyseparate
neuronal populations? Twomajorbreak-throughpapersfromSun& Chen (2007) andSun etal. (2009) present
strongevidence foritch-specificneural pathways.Usinggeneticallymodifiedmice knockouts,theyfoundthat
gastrin-releasingpeptide receptor(GRPR)-expressingneuronsare exclusive toandnecessaryforitchperception (Sun
et al., 2009). Similarly,Liuetal. (2009) conclude that mas-relatedg-proteincoupledreceptors(Mrgprs) playarole in
itch-specificneural transmission.Theydiscoveredthatablationof anMrgpr gene clustersignificantlyattenuateditch
response followingchloroquine(CQ) stimulation.Inaddition,the deficitcouldbe rescuedbymouse MrgprA3and
humanMrgprX1. Interestingly,in93%of cases,MrgprA3 wasfoundto be co-expressedwithGRPina subpopulation
of dorsal rootganglion(DRG) neurons:a positive findinginline withSun&Chen’spaper (2009). AnotherLiuetal.
(2010) studyinvestigatedtoll-likereceptor7(TLR7) inlightof painhypersensitivity; however,uponfindingno
evidence forabnormal painperceptioninTLR7knockoutmice,theyconsidereditseffectsonitch.“A marked

6. reductioninscratchingbehaviourinresponsetononhistaminergicpruritogens”wasobserved.TLR7,like MrgprA3,
was predominantlyexpressedinCfibresalongsideTRPV1andGRP, showingfurthersupportforitch-specific
neurophysiology.Hanetal. (2013) reportedsimilarresults:mice possessinggeneticallyablatedMrgrA3-positive
neuronsdisplayreduceditchsensationalongside unaffectedpainsensitivity.Inspite of capsaicin(analgogen)
application,MrgprA3-positive neuronssolelyexpressingTRPV1,provokeditchbehaviourandnotpain. Taken
together,these dataleantowardsa“labelled-line”theoryandprovide consistent resultsadvocatingthatitchspecific
neuronsexist.
Despite suchpositivefindings,the labelled-linetheorycannotexplaineverything.Firstly,painanditchare both
promptedbymultiple stimuliincludingmechanical,chemical,thermal andelectrical methods.Nottomention
substantial integrationof theirfunctional mechanisms (Table1) (Stander& Schmelz,2006). Secondly,their
antagonisticrelationshipispuzzling:while the stimulationof painmayeitherinhibitorenhance itchingsensation
(Stander & Schmelz,2006), its suppressionmayalsoinhibititchbutnotpainsensation (Heyeretal.,1997).
Moreover,the observationthatnonhistaminergicpruritogensmayinvolvemultiple afferentpathwayscomplicates
mattersfurther.Ringkampetal. (2011) useda selective conductionblockof myelinatedfibresonhumansubjects,
findingthata sub-groupwithA fibre-dominateditchhadsignificantlyreduceditch,prickingandburningsensation
elicitedbycowhage (aplantknowntoinduce itch).WhencomparingA fibre- withCfibre-dominateditchsubjects,
theirtime courseswere distinctlyindividualandeachsubgroupdisplayedmatchingpeakresponsesrespectiveto
theirmyelinatedorunmyelinatedcharacteristics.Similarfindingsdemonstrate cowhage asastimulusforCfibres
that are notrelatedtohistaminergicitch (Nameretal., 2008); and anotherprimate studyobservedseparateSTT
neuronsasresponsive tobothcowhage andhistamine (Davidsonetal.,2007). Itseemsthata population-coding
hypothesisexplainedbyCamperoetal. (2009) mayresolve the inconsistenciesbetweenintensityandlabelled-line
theory.Itsuggeststhat pain,itchand thermal sensationsare transmittedbylabelledafferentswhichproceedto
integrate andcrosstalk(sometimesantagonistically) inthe CNS.The response,therefore,doesnotnecessarily
correspondtothe stimulus:itmaybe an emergentsensationrelativetothe activationof multiple labelledlines (Ma,
2010).
Table 1. Commonmediators,receptors and their contributionto pain and itch. Taken from Stander & Schmelz,
2006.
Mediator Receptorspresentin skin Pruritus Pain
Histamine H1, (H2),H4 (?) receptors Inductionof itchviareceptor
stimulation
Inductionof painat high
concentrations
Tryptase Proteinase-activated
receptor-2
Inductionof itchviareceptor
stimulation
In animal experiments
Endothelin EndothelinA-receptors Inductionof itchviareceptor
stimulation
Inductionof painviareceptor
stimulation
Interleukins(IL-2,
IL-4, and IL-6)
Receptoronnerve fibers:
IL-2R, IL-6R
Delayeditch(secondary?) Sensitization
Substance P Neurokininreceptors(NKR
1–3) on mastcellsand
nerve endings
Inductionof itchviamast cell
degranulation,histamine,and
tryptase release
Neurogenicinflammation,
central sensitization
Capsaicin,heat, Transientreceptor Inductionof burningpruritus Inductionof burningpainvia

7. lowpH potential (TRP):TRPV1 viareceptorstimulation receptorstimulation
Bradykinin Bradykininreceptors(B1,
B2)
Sensitizationof nerve fibersfor
otherchemical stimuli
Sensitization/activationof
nerve fibers
Prostaglandins Prostaglandinreceptors Sensitizationof nerve fibers,
potentiationof histamine-
induceditch
Sensitizationof nerve fibers
Nerve growth
factor
TRK-A receptor Sensitizationof nerve fibers
speculated
Peripheral andcentral
sensitization
Opioidpeptides μ-, δ-receptors Inhibitionof pruritusvia
peripheral receptors-induction
of pruritusspinally
Inhibitionof painviacentral
and peripheral receptors
Cannabinoids Cannabinoid receptors
(CB1, CB2)
Suppressionof histamine-
induceditch
Peripheral andcentral
analgesiceffects
Central Processing
Althoughthere isgoodindicationforapopulationcodingtheoryintermsof peripheral processingof itch,few
explicitdetailscanbe specifiedpertainingtothe central componentof the theory.Nonetheless,acombinationof
neurophysiological,geneticandbrainimagingstudiesbegintobuildapicture.Itiscommonknowledge thatpain(i.e.
scratching) maytemporarilyinhibititchsensation.Additionally,heatandcoldhave similaractions (Ross,2011). A
2010 paperby Zhengetal. presentsanexample whereby“inhibitorylaminaIIconnectionsappeararrangedto
modulate activityfromdifferentsetsof peripheral unmyelinatedfibres”,enablingreciprocal inhibitionbetweenthe
two.Evidence forpainas a repressive mechanismof itchwasdiscoveredinexperimentsinvestigatingthe effectsof
vesicularglutamate transporter(VGLUT) 2 deletioninTRIPV1-expressingmouse afferents (Lagerstrometal., 2010).
Theyfoundsubstantiallyincreaseditchbehaviouralongsidereducedthermalpainresponse.Itchwasalleviatedupon
antihistamergicdrugadministrationandgeneticdeletionof GRPR.Collectively,itcanbe deducedthatVGLUT2 is
necessaryforTRPV1 thermal nociceptionandregulationof normal itchperception.fMRIstudiesreveal thatactive
areas usuallyassociatedwithitchinclude the prefrontal cortex(PFC),anteriorcingulate cortex (ACC),insula,
somatosensorycortex (S2),andthe cerebellum(Mochizuki etal.,2014). Importantly,the majorityof subjects
showedregional activitycorrelatingwithitchintensity (Darsowetal., 2000). In particular,areasof the
periaqueductalgreymatter(PAG) were especiallydynamic,suggestingthe regionhasamore primaryrole in itch
processing(Mochizuki etal.,2003). They alsofoundthatcorrelationsbetweenPAGactivityanditchintensitywere
resultantof painstimuli,suggestingthere maybe descendingmodulatorypathwaysforitch.Thoughthe datais
limitedanddiscrepanciesare dottedthroughoutthe literature,recentgeneticapproachescombinedwithmolecular
analysisandbrainimagingprovide asolidgroundingforrapiddevelopmentinthisfield.
Peripheral Mechanisms:Receptors andMediators
Histamine
Histamine holdsahistoricplace inthe developmentof itchunderstanding,becomingthe moststudiedpruritogenin
the literature todate (Han & Dong, 2014). It is now knowntobe an endogenousligandof histamine receptors
(HisRs),synthesised fromhistidine andpredominantlyreleasedbymastcells.Itintimatelylinkswiththe immune
system,triggeringthe familiarised“tripleresponse of local vasodilation,local edema,andflare”andhasfunctional

8. associationswithallergy,anaphylaxis,gastricacidsecretionandneural transmission (Thurmondetal., 2014). Today,
fourHisRs are recognised:H1 receptor(H1R),H2 receptor(H2R),H3 receptor(H3R) and H4 receptor(H4R).Theyare all
G-proteincoupledreceptors(GPCRS),however,are expressedindifferentcellsandactivate separate signalling
pathways (Table2) (Bongerset al., 2011). This overview of HisRsissomewhatsimplified.Mosthave beendiscovered
viapharmacological observationanddetailedsignallingcascadeshave yettobe fullydocumented.Promiscuityin
bindingaffinities,multiplefunctionsof ligands(i.e.agonistandantagonist) andcomplex transductionpathways
make it particularlychallengingtocharacterise individual receptors (Thurmondetal., 2014).
Table 2. Histamine receptors:signal transduction. Basedon Bongerset al., 2011.
HisR
subtype
Cell expression G-protein
coupling
cAMP
production
Transduction cascade
H1R CNSneurons,smoothmuscle
cells,endothelial cells
Gq Increased PLC activation,IP3 &DAG
production
H2R CNSneurons,gastricparietal cells,
cardiac muscle cells
Gs Increased PKA activation, CREB
phosphorylation
H3R FoundmainlyinCNSneuronsasa
presynapticautoreceptor
Gi/o Decreased PKA inhibition,MAPK
phosphorylation
H4R Immune cellsi.e.peripheral blood
leukocytesandmastcells
Gi/o Decreased PKA inhibition,MAPK
phosphorylation,AP-1activation
Pertainingtoitch,there isrobustevidence forhistamineasa mediator.Of the foursubtypes, H1RandH4R are the
mostrelevant(Thurmondet al.,2008). AlthoughithasbeensuggestedthatH3Rplaysa role inH1R andH4R
activation(Rossbachet al., 2011), the literature surroundingthisreceptorisunresolved (Jeffryetal.,2011).
RegardingH2R,itseffectsare minimal atbest:bothH2R agonistsand antagonistsfail toactivate orinhibititch
symptomsrespectively (Bell etal.,2004). Havingreachedsomewhatof adead endinantihistaminetreatmentfor
chronicitch, hope hasbeenrekindledinview of recentdata.Researchisnow pushingtore-evaluate the role of H1Rs
and considerH4Ras a potential targetfornovel therapeutics.
FocusingfirstonH1Rs, ithas become increasinglyevidentthatTRPV1isan importanthistaminesensorinthe signal
transductionof itch.A 2006 paperby Han etal. presentsthe phospholipase C(PLC) βisoenzymeasa critical
mediatorfoundina subpopulationof Cfibre nociceptors.UsingCa2+
imagingtechniques,theynotedhighlevelsof
PLCβ3 expressioninDRGneuronsinducedbyhistamine stimuli.Furthermore,subjecttoPLCβ3 deletion,mice
showedsignificantlyimpairedscratchingbehaviour,aresultconsistentwiththeir primaryfindings.Wooetal. (2008)
laterlinkPLCand TRPV1, assertingthatdiacylglycerol (DAG),aproductof PLC hydrolysis,activatesTRPV1directly.
Alternatively,phospholipase A2 (PLA2) mayalsoactivate TRPV1inhistaminergicitch (Kimetal., 2004; Shimet al.,
2007). Several linesof evidence indicate H4Risinvolvedinitchsignallingandisindependentfromthe H1Rpathway
(Davidson& Giesler,2010). In an assessmentof the effectsof H4Rantagonist,JNJ7777120, two modelsof
dermatitiswere usedinRossbachetal.’s2008 publication.Hapten-inducedscratchingwassignificantlyreduced
uponJNJ7777120 administration;however,the presence of inflammationremainedunaffectedinbothmodels.
Combinedadministrationof anH1R and H4R antagonistprovedmore effectivethanindividual application,suggesting
H1R and H4R are attributable toseparate aspectsof itch.IntracellularpathwaysrelevanttoH4Rare still unclear.

9. Nonetheless,mitogen-activatedproteinkinase(MAPK) (Morse etal.2001) and AP-1activation (Gutzmeretal.,
2009) have beenimplied.
Mas-RelatedG Protein-CoupledReceptors
Mrgprs were firstdiscoveredin2001 byDong etal.,beingalmostexclusivelyobservedintropomyosinreceptor
kinase (Trk) A- expressingneuronsinmice.Theirclassificationhasbeensplitintothree mainfamilies:MrgprA,
MrgprB andMrgprC, thougha more distantlyrelatedgroupalsoexist(MrgprD-H) (McNeil & Dong,2014). Onlyafter
the generationof Mrgpr knockoutmice didtheirsignificance initchsensationbecome clear (Sun& Chen,2007). The
closesthumanorthologtoMrgprA3 wasrevealedwhilstexperimentingwithitsagonist,CQ:Liuetal. (2009) found
MrgprX1 (receptorsrestrictedtoDRGneurons) inHEK293 cellsalsorespondedtoCQ.Bovine adrenal medulla
peptide (BAM8-22),apotentpruritogen,showedspecificityforMrgprC11 while MrgprA3respondedonlytoCQ (Liu
et al., 2009). Thoughthese receptorsare activatedbydistinctligands,Wilsonetal. (2011) demonstratesthat
transientreceptorpotentional ankyrin-1(TRPA1) isa commondownstreamtargetof Mrgpr-mediated
nonhistaminergicitch.Moreover,BAM8-22 has beenshowntoproduce itchand painsensationsinhumans,making
it attractive asa possible endogenousitchmediatorfornonhistaminergicitch (Siklandetal., 2011). Basedonthe
knowledge thatPLCcouplestoMrgprC11, Wilsonetal. (2011) alsoinvestigatedif gallein(a Gβγ inhibitor) affectsitch
behaviourviaMrgprA3.SignificantlyattenuatedresponsesinCa2+
signalsandCQ-induceditchsuggestthat MrgprA3
requires Gβγ forTRPA1 coupling.The same experimentwascarriedoutregarding MrgprC11, howevernosignificant
change in BAM-evokedresponsesoccurred.The dataimpliesthatPLCsignallingmust,byelimination,occurviaGq.
Unfortunately,nosupportingobservationshave beendocumentedyet.Finally,arecentpaperbyLiuet al.(2012)
showsthat the aminoacid β-alanine elicitsitchmediatedbyMrgprD.MrgprD knockoutmice were notresponsiveto
β-alanine;however,histamine-induceditchresponse wasunchangedcomparedtowildtype mice.These results
propose MrgprD is specificto β-alanine.Interestingly,Zylkaetal. (2005) reportedthatMrgprD-expressingneurons
projectedsolelytothe stratumgranulosumlayerof the epidermis.Thoughthisseemsreminiscentof labelled-line
theory,there isstill confusioninthatmultiplestimuli (noxious,thermal andmechanical)andsensations(painand
itch) are associatedwiththese neurons (Jeffryetal.,2011). Nonetheless,the fieldisimmature andthe literature on
Mrgprs is incomplete.The future lookstowardexposingthe intracellularpathwayslinkedwithitch,perhaps
beginningwithaspecificPLCisoform.
Transient ReceptorPotential Channels
The role of TRP channelsisbecomingincreasinglyrecognisedasakeycomponenttoitchtransductioninthe
periphery.Theyare partof the ion channel family,maintainingatetramericstructure formedof six transmembrane
helices.There are asmanyas 27 memberstodate; however,those thoughttoparticipate initchperceptioninclude:
TRPV1,TRPA1, and TRPV3 (Wilson& Bautista, 2014).
TRPV1,initiallydiscoveredusingcapsaicin(the active ingredientinhotchilli peppersthatcausesa burning
sensation),hasnow beenestablishedasa thermoreceptoractivatedattemperaturesabove 40°C.The relationship
betweenitchandpainhaslongbeenknown;especiallyinconsiderationof the relievingeffectspainhasonitch.
Moderntreatmentusingtopical capsaicininduces“localisedlossof nociceptivenerve fibre terminalsinthe

10. epidermisand dermis”,whichimplies the presence of TRPV1or TRPV1-expressingprimaryafferentsnecessaryfor
pruriception (Wilson& Bautista,2014). Imamachi etal.confirmedthisin2009 uponfindingthatTRPV1knockouts
had significantlyreducedscratchresponse to histamineinjection.Otherlocationsof TRPV1expressioninclude the
trigeminal ganglia,brainandDRG neurons.Pertainingtoanintracellularsignallingmechanism, Imamachietal.
(2009) replicatedHanetal.’s (2006) results,demonstratingthatPLCβisactivatedina Gq dependentmanner.
Althoughthisevidence advocatesthatTRPV1isa specificlabelledline forhistaminergicitch,recentpaperssuggest
otherwise.Pateletal. (2011) usedPirt(a molecule essential inTRPV1modulationforpainsensation) knockoutmice
to investigatewhetherithasa role initch sensation.Theyfoundthatbothhistamine-induceditchandCQ-induced
itch issubstantiallyattenuatedincomparisontowildtype mice counterparts,indicatingthatPirtisa novel mediator
of itchactingcruciallyviaTRPV1 inboth histaminergicandnonhistaminergicitch.Likewise,Thanetal. (2013) found
that CQ activatesTRPV1-expressingDRGneurons,accountingfor“43.3% of the total CQ-excitedneurons”;however,
TRPV1 wasnot a directmediatorinthiscase.In summary,TRPV1is undeniablyimportantforitchtransductionand
may be more broadlyinvolvedthanpreviouslythought.Furtherexperimentationshouldclarifymediatorsin
signallingpathwaysinordertobuilda betterpicture of itchmechanisms.
TRPA1 islocalisedinasubsetof TRPV1-expressingneuronsandisactivatedbya diverse arrayof endogenousand
exogenousirritants.Suchendogenousinflammatorysubstancesinclude 15dGJ2,PGA2 andΔ12-PGJ2, whichmay be
regulatedbyPLC-coupled receptors.Exogenousirritantsinclude allyl isothiocyanate(AITC),cinnamaldehydeand
allicin–compoundsof mustard,cinnamonandgarlic extractsrespectively (Wilson& Bautista,2014). Wilsonetal.
(2011) was firstto identifythatTRPA1is requiredforhistamine-independentitchdownstreamof MrgprA3 and
MrgprC11 in mice.Followingthis,LiuandJi (2012) determinedthatbothgeneticandpharmacological blockage of
TRPA1 profoundlyreducesoxidativeitch.Furthermore,scratchingcouldbe attenuatedin wildtype mice with
systemicadministrationof antioxidants.In2013, Wilsonetal. elaboratedontheir2011 findings,experimentingwith
a mouse model of chronicitch.TheyconfirmedthatTRPA1 isrequiredfortransducinghistamine-independentitch
elicitedbyCQandBAM8-22. Remarkably,TRPA1wasfoundto induce pathophysiologicalmarkersinchronicitch,
includingincreasesinepidermal thickness,epidermal hyperplasia,andupregulatedgene expressionassociatedwith
MgpA3 receptors,protease activated receptor-2(PAR2) andinflammatorybradykininreceptor(Bdkr2) (Wilsonetal.,
2013). Another2013 paperby Ohet al.usedan interleukin-13(IL-13- a critical cytokine forallergicinflammation)
mouse model toinhibitTRPA1andgeneticallydeletemastcells.Inbothcasesitchwas significantlydiminished,
reaffirmingTRPA1’srole initchtransduction.Interestingly,highexpressionof TRPA1wasobservedinthe “dermal
afferentnerves,mastcells,andthe epidermisinthe lesionalskinbiopsiesfrompatientswithatopicdermatitis”
whencomparedwithskinfromhealthysubjects. TakingintoaccountthatIL-13 isproducedbymast cells,itisin
accordance withthe biopsiesthatIL-13was foundtorobustlystimulate TRPA1expression.A novel findingfromLieu
et al. (2014) indicatesthatTRPA1 isnecessarilyactivatedbythe G protein-coupledbileacidreceptor,TGR5,viaa
Gβγ and PKC-mediatedmechanism.Table3 summarisesthe interactionsbetweenpruritogens,receptorsandtheir
relationshipwithTRPV1and/orTRPA1.

11. Table 3. TRPV1 and TRPA1 in itch transduction.Adapted from Zhang, 2014.
Pruritogens Pruritic receptor Excitedion channels
Histaminergic Histamine H1R, H3R, H4R TRPV1,others?
Nonhistaminergic
CQ MrgprA3 TRPA1, TRPC3
SLIGRL, BAM8-22 Mrgpr C11 TRPA1
Cowhage PAR2,PAR4 TRPA1?
IL-13 ? TRPA1
IL-31 IL-31RA TRPV1,TRPA1
TSLP TSLPR TRPA1
LTB4 BLT1 TRPV1,TRPA1
TRPV3 iswidelyexpressedinskinkeratinocytesandisactivatedbybothinnocuousandnoxioustemperaturesabove
33° (Steinhoff& Biro, 2009). Yoshiokaetal. (2009) suspectedarole forTRPV3 inpruritus,however,noevidence
couldsufficientlysupportthisidea.A 2012 paperby Yamamoto-Kasai etal.discoveredthatTRPV3knockoutmice
exhibitedsignificantlylessscratchingbehaviourafteracetone etherwatertreatment(AEW- atreatmentthatcauses
skindehydration,usedtoreplicatedchronicitchinmice),incomparisontoTRPV3-positive counterparts.This
suggestsTRPV3doesindeedcontributetoitchperception.Inadditiontothis,atopicdermatitispatientsshowed
increasedlevelsof TRPV3mRNA expression,betterlinkingthe findingsinmurinepruritustohumanpruritus.
Althoughthere isthe indicationthatprostaglandinE2(a pruritogen) isreleasedfromkeratinocytesdisplayingTRPV3
overexpression, the dataneedstobe reproducedinlightof itchratherthan pain (Huang et al.,2008). TRPV3 may
holdpromise foritchtherapeutics,however,future effortsshouldbe made toelucidate itch-promotingmechanisms
withinkeratinocytes.
Protease-activatedreceptors
Protease-activatedreceptors(PARs) are partof the GPCR family,featuringfoursubtypesaltogether:PAR1-4.Of
those four,PAR2 isbelievedtobe itchrelated,specificallyasamediatorof nonhistaminergicitch.While PAR4may
alsoregulate itch,the fieldisverymuchpremature andwhatlimiteddatathere is,isgenerallyinconclusive.PARsare
unusuallyactivatedbyproteolyticcleavage of theirN terminus.Once the N terminusisfreed,atetheredligand
sequence (TLS) becomesexposed,causingactivationof the receptoruponbinding (Kempkesetal.,2014).
Endogenousitch-inducingproteasesinclude serine proteasessuchastrypsin,tryptase,matriptaseorprostasin;while
exogenousitch-inducingproteases include the likesof mucunain (acomponentof cowhage),cathepsinS,kallikreins
(KLK) andtryptase (Han & Dong, 2014; Kempkeset al., 2014).
PAR2 receptors,inparticular,are essentialforhistamine-independentitch.Steinhoff etal.firstconfirmedtheir
identityin2003, where theyappearedtobe restrictedtocutaneoussensorynerves.The same studysaw that
patientswithatopicdermatitisexpressedupregulatedlevelsof PAR2whichwere correlatedwithitchseverity,
suggestingastronglinkbetweenthe two.Dugas-Breitetal. (2005) noticeda similarrelationshipindialysispatients
whoexhibitedraisedserumlevelsof mastcell tryptase,andassertedthe possibilitythatimmune cellsmaybe
directlyinvolvedinthe mediationof pruritus.Several recentpapershave reinforcedthisnotionuponfinding
contributionof otherproteasestoitchresponse (Tsujii etal.,2009; Andon etal., 2012). Althoughmuchremains
unknownaboutPARsignallingpathways,the relevanceof TRPchannelshasbeenrecognised. The mostrobust

12. findingsassociate PAR2withTRPA1:firstly,TRPA1andPAR2were foundtobe frequentlycolocalisedinDRGneurons
(Dai et al., 2007). Secondly,TRPA1currentswere increasedbyactivationof PAR2;andthirdly,applicationof PLC
inhibitors,butnotPKCinhibitors,suppressedsaidTRPA1currents (Dai et al., 2007). Grant etal. (2007) suggestsa
pathwayinvolvingPLCβ, actingviathe TRPV4 channel,however,conflictingresultssee thatboth“PLCand PKC
mediate PAR2-inducedsensitisationof TRPV1”(Amadesi etal.,2004). It maybe that PAR2is mediatedbymultiple
signallingpathwaysand/orthatPAR2activationislimitedtosensorynervesof the skin.Atpresent,itisdifficultto
reasonwhichisthe case. Still,itiscertainthatPARsare clinicallyrelevantinhumanpruriticskindiseasesandthus,
are worthyof furtherstudy.
Toll-like Receptors
Toll-likereceptors(TLRs) are widespreadthroughoutthe CNSandperipheral nervoussystem(PNS),beingexpressed
incellssuchas “microglia,astrocytes,oligodendrocytes,Schwanncellsandneurons” (Li & Ji,2014). Theyare known
to participate inautoimmune function,recognisingendogenousligandscalleddanger-associatedmolecularpatterns
(DAMPS);andto regulate adaptive immunity,detectingcharacteristicmolecularstructuresof pathogensnamed
pathogen-associatedmolecularpatterns(PAMPS)(Liuetal., 2012). Classedassingle transmembraneproteins,nine
TLRs existintotal.Withrespectto itchsensation,TLR3and TLR 7 are most pertinent.
In 2010, Liu et al.recordedTLR7 as a novel mediatorof itchsensation.TheirexpressionwaslimitedtoTRPV1-
positive nociceptorsalongwithcoexpressionof GRPand MrgprA3. Activationof TLR7 by imiquimodindicatedthe
itch response wasinduceddirectlybyanonhistaminergicpathway;aconclusionthatwasreproducedbyKimetal.in
2011. Interestingly,Liuet al.(2010) alsosaw significantreductioninitchbehaviourinCQ,endothelin-1andSLIGRL-
HN2,suggestingthatTLR7 has a broaderimpacton itchsignallingthaninitiallyperceived.Moreover,theyfoundthat
usinghistaminergicpruritogenshadnoeffectonscratchingbehaviourinTLR7 knockouts,comparedwithwildtype
mice,reinforcingthe ideathatTLR7 may be quite general initsaction.A laterstudybyLiu et al. (2012) findsthat
TLR3 evokesitchina similarwaytoTLR7, however,bothhistaminergic andnonhistaminergicpruritiswere markedly
decreasedinTLR3 deficientmice.Unlike TLR7,TLR3 may be indirectlyregulatingitchtransmissionthroughthe CNS.
Thiscouldexplainthe differencesbetweenLiuetal.’s2010 and 2012 studies.Consideringthese data,TLRsmay play
a vital role initch sensation,bothdirectlyandindirectly.The challengeremainstoidentifywhatsignalling
specificitiesare distincttoTLRs comparedwithotherimmune cells,andtouncoverthe role of molecular
mechanismsunderlyingTLRtransduction.
Cytokines
Cytokinesare secretedproteinsthatare releasedfromactivatedimmuneandskincellssuchasmast cellsand
keratinocytesrespectively (Cevikbasetal.,2014). Althoughtheymake upa large family,onlyafew have been
identifiedasitchmediatorsthusfar.Interleukin-31(IL-31),a memberof the IL-6 family,isthe moststudieditch
cytokine.ItispreferentiallysecretedfromThelpertype 2(TH2) cellsandsignalsthrougha heterodimericreceptor
complex formedof IL-31receptorA (IL-31RA) andoncostatinM receptor(OSMR) (Dillonetal., 2004).
Consequentially,JAKtyrosinekinasesare recruitedcausingstimulationof STAT,phosphatidylinositol 3-kinase and
variousMAPKsignallingpathways (Cornelissenetal.,2012). In2006, Bando etal. locatedIL-31 mRNA alongside

13. OSMRβ ina small subsetof DRGs andtrigeminal ganglia,howeverfailstospecifywhethersensitisationof DRGs
occurs directlyorindirectly (2006).Transgenicmice acquiringIL-31 overexpression“developed severe pruritus,
alopeciaandskinlesions”,showingsolidevidence forarole initch sensation (Dillonetal.,2004). Notto mention,
these resultshave beenreplicatedinbothhumansandNC/Ngamice (Takaoka et al., 2005; Szegedi etal., 2012).
A novel itchmediatorisThymicStromal Lymphopoietin(TSLP).In2013, Wilsonetal.foundthat TSLP directly
activatescutaneoussensoryneuronsthatcoexpressTSLPreceptors(TSLPRs) andTRPA1andcommunicate with
epithelial cells.Thisproducedrobustscratchinginamouse model of atopicdermatitis.Additionally,epithelial cells
utilised“ORAI1-mediatedCa2+
influx toregulate cytokine expressionandrelease”downstreamof nonhistaminergic
signallingviaPAR2activationinkeratinocytes.Itisnotclear yet whetherlymphocytesare necessaryforTSLP-evoked
itch.Future studiesshouldinvestigate tissue specificTSLPRknockoutmice todetermine itscontributionstoitch
throughsensoryneuronsandimmune cellsrespectively.Overall,novel therapeuticsmaytargetthe immune system
for itchrelief,however,itisessential tounderstandhow the immuneandnervoussystemscommunicate inorderto
implementsuchtreatments.
Substance P
Substance P(SP) isa neuropeptide,dominantlyexpressedincutaneousnociceptivenerve endingsandcauses
neurogenicinflammationonce released.Otherwise,SPcanbe foundinskinmastcells,makingitparticularlyrelevant
to allergicreactions.Earlyrecordsshowintradermal injectionof SPproducesflare,wheal anditching,andthat
antihistaminesinhibitsuchresponses (Hagermarket al., 1978). Thus,SP regulateshistaminergicitchanddoessovia
neurokininone (NK1) receptors (Andohetal.,1998). In1998, Andohetal. foundthatmast cell-deficientmice
exhibitscratchingbehaviour,implyingthatmastcellsmayin fact,be negligibleinSP-induceditching.Conversely
Tatemotoetal. (2006) suggestedthatMrgX2 receptorscontribute tomast cell degranulationbyactivatingG
proteins.Regardless,studieshave shownaprepitant,a selective NK1receptorantagonist,tobe effective inchronic
itch patients (Standeretal., 2010), as well asmurine modelsof atopicdermatitis (Ohmuraetal., 2004). Verylittle is
knownaboutmechanismsunderlyingSP-induceditch;although,nitricoxide (Andoh& Kuraishi,2003) and
leukotriene B4(Andohet al., 2001) have beenimplied.
Modulationof the ItchPathway
Peripheral Sensitisation
In conditionscausingpathological itch,the neural pathwaysare functionallyabnormal.Thisleadstosensitisationof
the peripheral and/orcentral neurocircuitries,thussubsequentexcitationof spontaneousorhypersensitive itch
response occurs (Schmelz,2014). Such exampleshave beenobservedinchronicprurituspatientswherebyelectrical
and pruriticstimuli induceanenhanceditchresponse incomparisontonormal skinof healthyindividuals (Ozawaet
al., 2009; can Laarhoven et al.,2010). Examplesof hyperinnervationare evidentinbothexperimental andclinical
studieswherebyepidermalnerve fibre densitiesare significantlyincreased (Kinkelinetal.,2000; Tominaga et al.,
2007). These resultsmaybe explainedbyremarkable increasesinnerve growthfactor(NGF) expressionseeninthe
epidermisandimmunecells (Kinkelinetal.,2000; Gronebergetal., 2005; Tominaga et al.,2007; Suga et al., 2013).

14. In line withthishypothesis,Rukwiedetal. (2013) demonstratedthatpre-treatmentof NGFcauses sensitisationof
cowhage-butnothistamine-induceditch.Thisalsosuggeststhathistaminergic- andnonhistaminergic-itch
sensitisationismediatedbyseparate mechanisms:cowhage-induceditchmaybe specifictoperipheral sensitisation
mechanismswhile histamine-induceditchmaybe modulatedbycentral sensitisationmechanisms.Ithasalsobeen
impliedthattumournecrosisfactor(TNF)-αactsas an upregulatorof epidermal NGFviaMAPKsignallingpathways
(Takaoka et al., 2009), and isultimatelyderivedfrommastcells (Kakurai etal., 2006). Additionally,class3
semaphorins(Sema3A),anerve growthinhibitor,were showntobe downregulatedinlesional skill of atopic
dermatitis (Tominagaeta., 2008). Interestingly,while Sema3A caninhibitNGF-inducednervesprouting,NGFcauses
collapsingof nerve growthcones (Tominaga& Takamori, 2014). Thisimpliesadelicate balance betweenSema3A
and NGF levelsisneededfornormal epidermal innervation.Understandingandtargetingappropriate regulationof
these growthfactorsmay be a promisingtargetforfuture therapeutics,especiallyconcerningchronicitch.
Central Sensitisation
The distinctionsbetweenpainanditchbecome all the more unclearasresearchdelvesintocentral sensitisation
mechanisms.Enhanceditchprocessingoccurssimilarlytohyperalgesia(anexaggeratedpainresponse tonoxious
stimuli due tohyperexcitabilityof nociceptors).PruriceptorslocalisedinCNSneuronsacquire alowerthresholdfor
actionpotential firing,thusitchtransmissionoccursinspite of non-pruriticstimulation (Schmelz,2014).This
phenomenonhasbeentermedalloknesis.Havingdevelopedareliable animal modelof alloknesis,Akiyamaetal.
(2012), foundthatupon injectionof histamineinthe rostral back,lightmechanical stimulationawayfromthe
applicationsite,elicitsscratchingbehaviour.ThisisinagreementwithIkomaetal.’s2004 studyinwhichnoxious
stimuli primarilyevokeditchinatopicdermatitispatients.Withregardtospecifictargetsparticipating incentral
sensitisation,recentdevelopmentisonlybeginningtoexpose suchcontributors.In2012, Liu etal.demonstrated
that spinal cordslicesof TLR3 knockoutmice revealedsignificantlyattenuatedpruritus.Thus,TLR3may be critically
involvedin the regulationof neuronalexcitability,itchtransmissionandcentral sensitisation.A later2013 paperby
Zhao etal. developedamouse model expressingconstitutivelyactive serine/threoninekinaseBRAFinsodium
channel Nav1.8-gatedneurons.Thesemice were foundtohave enhancedandpersistentexpressionof itch-sensing
genes(GRP& MrgprA3 inTRPV1-positivenociceptors) inthe spinal cord,whichwasreflectedinamplifieditch
response behaviours.Inhibitionof BRAFor GRP signallingrelievedsome of the itch,thus,itcan be deducedthat
BRAFand GRP are keyregulatorsincentral sensitisation (Zhangetal.,2013). Especiallyconcerningchronicpatients,
investigatingsensitisationof the itchpathwaymaybe extremelybeneficial.The difficultlywill be indifferentiating
betweenpainanditchprocessing,however,existingknowledge regardingpainsensitisationmayassistinthis
process.
Conclusion
Chronicitch isa seriousconditionthatcandetrimentallyaffectqualityof life.Despite the developmentof
antihistamines,the medical needsof suchpatientsare severelylacking.Currentunderstandingconcerningthe way
itch isperceivedhassetaside the intensitytheoryandtakenupa new,labelled-linetheory.Recentresearch

15. suggeststhatmajor mediatorsinvolvedinitchinclude HisRs,TRPchannels,TLRs,Mrgprs,inflammatorymolecules
and neuropeptides.Itchmaybe signalledbyeitherendogenousorexogenouspruriceptivestimuli andcanbe
categorisedintohistaminergicandnonhistaminergicitch.Whileevidenceimpliesthe activationof itchspecific
primaryafferents,contradictionarisesondeepercontemplation.Itseemsthatseveralstimuli andmediatorsare
sharedbetweenitchandpain,leadingtoexcitationof multipleafferentpathwaysincertaincases.Moreover,a
confusingantagonisticrelationshipexistsbetweenthe two.Takingintoaccountthe evidence forperipheral and
central sensitisation,populationcodingmayemerge asanall-encompassingitchtheory;nonetheless,further
researchis needed.Hopefully,combiningmethodsof geneticmanipulation,inculture research,brainimagingand
clinical observationwill improve researchimpactandsuccessfullyrevealthe mysteriesof itch.
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