Neutrophil-derived chemokines on the road to immunity

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ContentslistsavailableatScienceDirect

Seminars

in

Immunology

jo u r n al h om ep a g e :w w w . e l s e v i e r . c o m / l o c a t e / y s m i m

Neutrophil-derived

chemokines

on

the

road

to

immunity

Cristina

Tecchio

a,∗

_,

_Marco

_A.

_Cassatella

b,∗,1

a_Department_of_Medicine,_Section_of_Hematology_and_Bone_Marrow_Transplant_Unit,_University_of_Verona,_Verona,_Italy b_Department_of_Medicine,_Section_of_General_Pathology,_University_of_Verona,_Verona,_Italy

a

r

t

i

c

l

e

i

n

f

o

Articlehistory: Received23March2016

Receivedinrevisedform4April2016 Accepted5April2016

Availableonline14April2016 Keywords: Neutrophils Chemokines Innateimmunity Adaptiveimmunity Infections Tumors Immune-mediateddiseases

a

b

s

t

r

a

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Duringrecentyears,ithasbecomeclearthatpolymorphonuclearneutrophilsareremarkablyversatile cells,whosefunctionsgofarbeyondphagocytosisandkilling.Infact,besidesbeinginvolvedin pri-marydefenseagainstinfections–mainlythroughphagocytosis,generationoftoxicmolecules,release oftoxicenzymesandformationofextracellulartraps–neutrophilshavebeenshowntoplayarolein ﬁnelyregulatingthedevelopmentandtheevolutionofinﬂammatoryandimmuneresponses.These lat-terneutrophil-mediatedfunctionsoccurbyavarietyofmechanisms,includingtheproductionofnewly manufacturedcytokines.

Herein,weprovideageneraloverviewofthechemotacticcytokines/chemokinesthatneutrophilscan potentiallyproduce,eitherunderinﬂammatory/immunereactionsorduringtheiractivationinmore

prolongedprocesses, suchasintumors.Wehighlightrecentobservationsgeneratedfromstudying

humanorrodentneutrophilsinvitroandinvivomodels.Wealsodiscussthebiologicalsigniﬁcanceof neutrophil-derivedchemokinesinthecontextofinfectious,neoplasticandimmune-mediateddiseases. Thepicturethatisemergingisthat,giventheircapacitytoproduceandreleasechemokines,neutrophils exertessentialfunctionsinrecruiting,activatingandmodulatingtheactivitiesofdifferentleukocyte populations.

Contents

1. Introduction...119

2. Neutrophil-derivedchemokinesinimmuneresponsesandinfections...120

2.1. Humanneutrophils...120

2.2. Mouse/ratneutrophils...122

3. Neutrophil-derivedchemokinesintumors...123

3.2. Mouseneutrophils...124

4. Neutrophil-derivedchemokinesinimmune-mediateddiseases...124

4.2. Mouseneutrophils...125

5. Conclusions...125

Conﬂictofintereststatement...125

Acknowledgments...125

References...125

∗ Correspondingauthorsat:DepartmentofMedicine,HematologyandBone Mar-rowTransplantUnit,PiazzaleL.A.Scuroand,SectionofGeneralPathology,Strada LeGrazie8,37134Verona,Italy.

E-mailaddresses:cristina.tecchio@univr.it(C.Tecchio),

marco.cassatella@univr.it(M.A.Cassatella).

1 _Department_of_Medicine,_Section_of_General_Pathology,_Strada_Le_Grazie_8,₃₇₁₃₄

Verona,Italy.

1. Introduction

Chemokines are 8- to12-kDa polypeptides, sharing 20–70%

homology in amino acid sequence, that are classiﬁedinto four

families(XC,CC,CXCandCX3Cfamilies)basedonthe

position-ingoftheirinitialcysteineresidues[1].CXCandCCchemokines

http://dx.doi.org/10.1016/j.smim.2016.04.003

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represent the two major and most studied groups, being the

CXC chemokinesfurtherdivided into two subfamilies,

depend-ing on the presence of the glutamate-leucine-arginine (ELR)

motif preceding the ﬁrst two cysteins [2]. CXC ELR-expressing

chemokinesaremostlychemotacticforneutrophilsandinclude,

among othermembers, CXCL8/IL-8, CXCL1/growth-related gene

product-␣(GRO-␣),CXCL2/macrophageinﬂammatoryprotein

2-alpha(MIP-2␣)/GRO␤,CXCL3/MIP-2␤/GRO␥andCXCL5/epithelial

cell-derived and neutrophil-activating 78-amino acid peptide

(ENA-78)[1,3,4].Bycontrast,CXCLmemberslackingtheELRmotif,

such as CXCL10/interferon (IFN)␥-inducible protein of 10kDa

(IP-10),CXCL9/monokineinducedbyIFN␥(MIG)andCXCL11/IFN

␥-inducibleT-cell␣chemoattractant(I-TAC),actinsteadonnatural

killer(NK)andactivatedTcells[1,5,6].CXCchemokines

contain-ingtheELRmotifalsodisplayapotentangiogenicactivity,while

CXCchemokineslackingtheELRmotifareangiostatic[2].TheCC

familyincludeschemokinessuchasCCL2/monocytechemotactic

protein/MCP-1, CCL3/macrophage inﬂammatory protein

(MIP)-1␣,CCL4/MIP-1␤,CCL5/Regulated onActivation,NormalTcells

ExpressedandActivated (RANTES),CCL7/MCP-3,CCL17/Thymus

and activation regulated chemokine (TARC), CCL18/Pulmonary

and activationregulatedchemokine (PARC), CCL19/MIP-3_␤ and

CCL20/MIP-3␣, that are mostly chemotactic and stimulatory

for monocytes, macrophages, dendritic cells (DCs), T cells, NK

cells,eosinophilsandbasophils[1,3,4,7].Chemokinesaremostly

secretedintotheextracellularspaceassolublefactorsorbound

totheextracellularmatrix,thusformingtransientorstable

con-centrationgradients,respectively[1].Theypromoteincreasedcell

motilityand directionalmigration upon binding totheir

corre-spondingcell-surface,seventransmembrane-spanningreceptors

(e.g.,CXCRsandCCRs),thatsignalthroughGprotein-mediated

cas-cades[8,9].Basedonthepatternofreceptorsexpressed,discrete

cellpopulationsarespeciﬁcallyrecruitedbydifferentchemokines

[1,10].Usuallyagivenleukocytepopulationhasreceptorsfor,and

respondsto,differentchemokines[11].Besidesregulating

leuko-cyte trafﬁcking,and therefore coordinating immune responses,

chemokines play an important role also in regulating T and B

cell-development[12],modulatingangiogenesis[13,14]and

inﬂu-encingtumorgrowth[15].Althoughvirtuallyallcelltypesmay

releasechemokines,innateandadaptiveimmunitycells,

includ-ingpolymorphonuclear neutrophils,representthemajorsource

ofthem[1,16],especiallyininﬂammatory(infectiousand/or

non-infectious)[17,18]ortumorsettings[17,19].

Neutrophilsarenotanymoreviewedassimple“suicide”killer

cellsatthebottomofthehierarchyofimmuneresponse[20,21].

The last two decades have, in fact, witnessed a new wave of

excitingstudies about thecapacity of neutrophils toexpress a

numberof geneswhose productslieat thecoreofcrucial

bio-logicalprocesses,includinginnateimmuneresponses[21,22].In

suchregard,neutrophilshavebeenshowntoexpressandproduce

a varietyof chemokines (Table 1)upon activation by

microen-vironmentalstimulisuchasmicrobial agents or theirproducts,

includingligandsforToll-likereceptors(TLRs)orother

pathogen-associated molecular patterns (PAMPs), even in a timely- and

speciﬁc stimulus-dependent manner [16,22,23]. In addition to

amplifytheirproductionofchemokinesbyautocrineloops[23–25],

neutrophilshavebeenalsoshowntopositively/negatively

modu-latetheeffectsofthechemokinespresentinthemicroenvironment

byreleasingenzymeswithproteolyticactivities,either in

asso-ciationwithextracellulartraps[26]ornot[27].Therefore,inthe

contextofaninﬂammatoryreactionthathastheultimategoalto

killandremovetheinvadingpathogens,neutrophilsmayrecruit,

viachemokinerelease,discreteinnateandadaptiveimmunitycells

tooptimallyorchestratethemostefﬁcaciousimmune response.

Neutrophils themselves have been shown to migrate into the

inﬂammatory site in tightly regulatedwaves, which are

medi-atedbychemoattractant/chemokinecascadesreleasedbyactivated

resident tissue cells and/or previously activated neutrophils or

macrophages[23,28,29].

Inthefollowingsections,wewillhighlightrecentliterature

con-cerningtheroleofchemokinesderivedbyhumanneutrophilsin

shapingtheinnateandadaptiveimmuneresponse,eithertowards

infections,orinthecontextofotherpathologicalconditionssuchas

cancerorimmune-mediateddiseases.Wewillalsodescribe

ﬁnd-ingsgeneratedininvivomousemodels,whicheitherextend,or

uncover,differences betweenspecies[10,22].For a morebroad

comprehensionoftheknowledgeexistingintheﬁeld,the

read-ersmayrefer topreviouslypublished, veryexhaustive,reviews

[16,28,30].

2. Neutrophil-derivedchemokinesinimmuneresponses andinfections

Moststudiesindicatethatneutrophilsupregulate

chemokine-encoding genes and/orrelease chemokineswhen appropriately

stimulated[30]. Hence,the pattern of chemokinesreleased by

neutrophilsisstrictlydependentonthetypeofstimulusand/or

associated to a speciﬁc inﬂammatory/immunological context,

in vitro and in vivo. A large but non-exhaustive list of the

stimuli able to induce the production of chemokines by

neu-trophilshasbeenpreviouslyreported[30],yetagoniststriggering

neutrophil-derived chemokines are continuously identiﬁed, for

instance:granulocytecolony-stimulatingfactor(G-CSF),shownto

induceCXCL5[31]andCXCL2/MIP-2␣[32];Wnt5a,aligandthat

activatesthenon-canonicalWntsignalingpathways(

␤-catenin-independentpathways),showntotriggerthereleaseofCXCL8and

CCL2[33];organicdust,showntotriggerthereleaseofCXCL8and

CCL3[25];andanincreasingnumberofmicrobial-derived

prod-ucts,asdescribedinthefollowingparagraphs.

2.1. Humanneutrophils

In vitro studies have demonstrated that, upon stimulation

with microbial agents or their derivatives, neutrophils release

chemokines potentially able to recruit neutrophils themselves,

monocytes,macrophages,DCsandNKcells,aswellasTcell

sub-sets(Fig.1), suggestingthat,bythis function,theymayamplify

boththeinnateandtheadaptiveimmuneresponse[16].For

exam-ple,neutrophilsculturedwitheitherMycobacteriumtuberculosisor

lipoarabinomann(itsmajorcellwallcomponent)havebeenshown

torelease CXCL1and CXCL8 [34], two chemokines involved in

neutrophilrecruitment.Similarly,neutrophilsreleaseCXCL8when

exposedinvitrotoCandidaalbicans[35],Helicobacterpyloriwater

soluble surface protein [36] and H. pylori neutrophil-activating

protein(HP-NAP)[37].Bycontrast,phagocytosedStaphylococcus

aureus hasbeen shown toreduce the production of CXCL8 by

neutrophils,concomitantlywithsuppressingphosphorylationof

nuclearfactor-␬Bandacceleratingcelldeath,inthismanner

favor-ingitsownsurvivalandpromotingdisease[38].

Furtherevidenceoftheroleofneutrophil-derivedchemokines

in amplifying localinnate responseshas beenprovided bythe

capacity of neutrophils to upregulate the expression of CXCL1,

CXCL2 and, mostly, CXCL3 when in vitro exposed to

Fusobac-terium nucleatum [39]. In other studies, neutrophils incubated

withLPS-ortumornecrosisfactor-␣(TNF␣)[40],aswellasin

the presence of Gram-positive or Gram-negative bacteria [41],

wereshowntosequentiallyexpressandreleasebiologicallyactive

CCL20 and CCL19 [40], as revealed by experiments in which

neutrophil-derivedsupernatantsinducedchemotaxisofimmature

andmatureDCs,respectively.Thesedatahavebeenfurther

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Table1

Chemokinesthathumanandmurineneutrophilscanpotentiallyexpressand/orproduce.

CHEMOKINE HUMAN REFERENCES MOUSE/RAT REFERENCES

CXCL1 + [34,39,105] + [79]a_,_[95,98,108] CXCL2/MIP−2␣b ₊ _[39,46] ₊ _[79]a_,_{[32,47,69,95,110]} CXCL3 + [39] ND CXCL4c ₊ _[22] ₊ _[22] CXCL5 + [31] ND CXCL6 + [22] ND CXCL8d ₊ _{[25,33–38,44,45,54,58,59,61,66,67,82–86,92,93,101,106]} _ND CXCL9 + [48,52,62,67,105] + [95,109] CXCL10 + [48,49,52–54,57,63] + [79]a_,_{[76,77,95,109]} CXCL11 + [48] + [79]a CXCL12 +c _[22] ₊ _[78] CXCL13c ₊ _[22] ₊ _[95] CXCL16 +c _unpublished_observation ₊c _[95] CCL2 + [33,43,44,54,57,64,82,89,90,92,93] + [79]a_,_{[74,75,90,95]} CCL3 + [25,37,44–46,59,92,103] + [47,69,70,72–74,95,108] CCL4 + [37,45,46,60,66–68,85,87,88] + [79]a_,_{[47,72,73,108]} CCL5 ND + [72] CCL7 ND +c _[79]a_,_[95] CCL9 ND +c _[79]a CCL12 ND +c _[79]a CCL17 + [90,91] + [90,91,95] CCL18 + [103] ND CCL19 + [40,41] +(?) [22] CCL20 + [40–42,54,57,67,104] + [72] CCL22 ND +c _[79]a

Expressionand/orproductionofthelistedchemokineshavebeendetectedinhumanandmouseneutrophilsbygeneexpressiontechniques,IHC,enzyme-linked immunoad-sorbentassays(ELISAs)orbiologicalassays.

(?):Itindicatescontroversialdata. ND:Notreportedintheliterature.

a_Detected_in_rat. b_Mouse_only.

c _It_refers_to_studies_performed_at_the_mRNA_level_only. d _Human_only.

Fig.1. Chemokinesproduced/expressedbyneutrophilsexperimentallyshowntochemoattracttheinnate(greenbackground)andadaptive(violet-purplebackground) immunitycellsdisplayedintheﬁgure.

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bacterial-derived chemoattractant known as

formyl-methionyl-leucyl-phenylalanine(fMLF)signiﬁcantlyincreasetheproduction

ofneutrophil-derivedCCL20inresponsetoLPS,throughentirely

unrelatedmolecularmechanisms[42].Inthecaseofneutrophils

incubatedwithIFN␥plusLPS,whichalsomaintainveryelevated

theproductionofCCL2[43],apotentantiapoptoticeffectexerted

byIFN␥likelysustainschemokineexpression[43].

Asmentioned,neutrophil-derivedchemokinesmayalso

orches-trate more sophisticated responses involvingadaptive immune

cells.Forexample,neutrophilsisolatedfrompulmonary

tuberculo-sispatientswereshowntodisplayaugmentedlevelsofCXCL8,CCL2

andCCL3,whichfurtherincreaseduponinfectionwith

mycobac-terialstrainsinvitro[44].Consistently,ithasbeendemonstrated

thatneutrophilsexposedtoMycobacteriumbovisBacillus

Calmette-Guerin(BCG)[45], orincubatedwithHP-NAP[37], producenot

onlyCXCL8,butalsoCCL3andCCL4,twochemokinesrecruiting

monocytes,DCsand Tcells tothesiteof infections. Insupport

of this notion, supernatants from BCG-conditioned neutrophils

were found to induce, in vitro, the chemotaxis of monocytes

and,indirectly(viamonocytes),ofTcells[45].Neutrophil-derived

chemokineshavebeenalsoinvolvedinregulatingthemigration

ofmonocytes/macrophages,Tcellsandneutrophils,whichentrap

Schistosomajaponicumeggsandultimatelyformthetypical

inﬂam-matorygranulomas[46].Accordingly,humanneutrophilsexposed

invitrotoS.japonicumeggswerefoundtoupregulatethetranscripts

encodingCCL3,CCL4andCXCL2[46],consistentwithaprevious

studyfromthesamegroupevidencingthepresenceofCCL3-,

CCL4-andCXCL2/MIP-2␣-positiveneutrophilswithintheneutrophil-rich

coreofS.japonicum-granulomasininfectedmice[47].

Humanneutrophilshavebeenfoundtosynergisticallyexpress

andreleasealsoCXCL9,CXCL10andexpressCXCL11after

incu-bationwithIFN␥in combinationwitheither LPSorTNF␣[48].

The molecular mechanisms underlying such synergistic effects

weresubsequentlyuncovered[49].Moreover,neutrophil-derived

CXCL9andCXCL10werefoundbiologicallyactiveinvitro,as

super-natantsharvestedfromIFN␥plusLPS-orTNF␣-treatedneutrophils

promotedthemigration, as wellas a rapid integrin-dependent

adhesion,ofCXCR3-expressingTh1cells, inCXCL9- or

CXCL10-dependentfashions[48].Theseobservationsfortheﬁrsttimehave

highlightedapotentialdirectcrosstalkbetweenneutrophilsand

Th1cells[48],subsequentlyconﬁrmedandexpanded[50],andare

importantbecauseTh1cellsarecrucialforcell-mediatedimmunity

andphagocyte-dependentprotectiveresponses[51].Thatthe

pro-ductionofCXCL9andCXCL10byneutrophilsmightberelevantfor

ﬁghtinginfectiousdiseaseswasfurtherevidencedbyother

stud-ies.Inoneofthem,Anaplasmaphagocytophilum-infectedhuman

neutrophilshavebeenfoundtoreleaseloweramountsofCXCL9

andCXCL10ascomparedtouninfectedneutrophils[52].Similarly,

Porphyromonasgengivaliswasshowntobeineffectivein

stimulat-ingthereleaseofCXCL10byneutrophils,thereforecontributing

tosuppressandevadingaTh1-immuneresponseinthesettingof

periodontaldisease[53].

In addition to Th1 cells [48], IFN␥ plus LPS-activated

neu-trophilshavebeenshowntoproduceandreleasealsoCCL2and

CCL20,and,inturn,chemoattractTh17cellsinvitro[54].Th17cells

arespecializedinorchestratingadaptiveimmunedefensetoward

extracellularpathogens,viatherecruitmentofneutrophilstothe

siteofinfection,bytriggeringtheproductionofCXCL8,CXCL1and

G-CSFfromtissuecells[55].However,Th17cellsarealsoinvolved

inthepathogenesisofchronicinﬂammatoryand/orautoimmune

diseases [56]. Also neutrophils incubated with the

neutrophil-activatingproteinA(NapA)fromBorreliaburgdorferiwerefound

torecruitbothTh1andTh17cellsviaCXCL10andCCL2/CCL20

pro-duction,respectively[57].Interestingly,becauseNapAfunctions

asoneofthemainbacterialproductsinvolvedinthepathogenesis

ofLymearthritis,whichischaracterizedbyajointinﬁltrationof

mainlyneutrophilsandTcells(Th1andTh17),thelatterdata[57]

wouldsuggestthattheinﬁltrationofThcellsmayrely,inpart,on

thechemokineslocallyproducedbyneutrophilsexposedtoNapA.

ThatneutrophilsandTh17mayundertakeacrosstalk,ultimately

leadingtotheampliﬁcationoflocalimmuneresponse,isvery

plau-sibleduetotheabilityofactivatedTh17cellstoproduceCXCL8and,

consequently,directlyattractneutrophils[54].

Muchlessisknownaboutneutrophilresponsivenesstoviruses

in terms of chemokine production. For instance, it has been

reportedthatneutrophilstreatedinvitrowiththehuman

immun-odeﬁciencyvirustransactivatorprotein(Tat)produceCXCL8[58],

whileneutrophilsincubatedwithRespiratorySyncytialVirus(RSV)

produce and release also CCL3 [59] and CCL4 [60], in addition

toCXCL8 [61], therefore disclosing their role as cells releasing

potentinﬂammatorymediatorsduringRSV-relatedbronchiolitis.

Similarly,neutrophilsexposed toHerpes SimplexVirus1

(HSV-1)-infectedcornealtissuewerefoundtoproduceelevatedCXCL9

levels[62],suggestingthattheycontributetotheattractionofCD4+

Tcells/Th1cells,whichareessentialforantiviralimmunity.In

pul-monarytuberculosis,neutrophilsfromthebronco-alveolarlavage

ﬂuids(BAL)ofpatientswithhumanImmunodeﬁciencyVirus

(HIV)-associatedsyndromehavebeenfoundtoexpressCXCL10[63],in

spiteofaverylownumberofIFN␥-producingCD4+_cells_in_BAL.

SinceCXCL10isatypicalIFN␥-responsivegene,datasuggestthat

HIVmightdirectlyorindirectlycauseadysregulatedCXCL10

pro-ductionbyhumanneutrophils[63].Inanotherstudy,neutrophils

pulse-treatedinvitrowithR5HIV(amacrophage-tropicHIVstrain)

havebeenfoundtoproduceCCL2andIL-10[64].Moreover,freshly

isolatedneutrophilseitherco-cultured,ortranswell-cultured,with

R5HIV-infectedsyngeneic monocyte-derivedmacrophageswere

showntoenhance,ina CCL2-andIL-10-dependentmanner,the

replicationofR5HIVinmacrophages,thussupportinga

neutrophil-mediatedroleinfavoring,atleastinvitro,R5HIVinfection[64].In

thiscontext,glycyrrhizin,anantiviralcompoundalreadyusedin

clinic,wasfoundabletoinhibit theinvitroproduction ofCCL2

andIL-10byneutrophils exposedtoR5HIV,thereforeproviding

anexampleofa potentialchemokine/cytokine-targetedtherapy

[64].Atthelightofrecentﬁndingsdemonstratingthat,inhuman

neutrophils,thechromatinattheIL-10locusappearsinan

inac-tiveconformation[65],theproductionofIL-10byR5HIV-treated

neutrophils should be conﬁrmed using highly pure neutrophil

populations.Inanothercontext, neutrophilsfromHumanTcell

lymphotropic virus type-1 (HTLV-1)-infected patients exposed

in vitro to LPS, or to Leishmania amazonensis, were found to

releaseamountsofCXCL8andCCL4similartoneutrophilsfrom

HTLV-1-seronegativecontrols[66].Finally,neutrophilstransfected

invitrowithpolyinosinic:polycytidylicacid[poly(I:C)],asynthetic

mimeticofviraldsRNAthatactsviaintracellularcytoplasmicRNA

helicases,havebeenshowntoexpresselevatedtranscriptlevels

encoding CXCL8,CXCL10, CCL4 and CCL20 [67]. Similarly,

neu-trophilsincubatedwithR848,animidazoquinolinemimickingthe

actionofsinglestrandviralRNAactingonTLR8,havebeenshown

toexpressCCL4[68],aswellasCCL3,CCL19,CXCL1,CXCL8and

CXCL16(ourunpublishedobservations),allchemokinespotentially

involvedintherecruitmentofbothinnateandadaptiveimmunity

cellsduringviralinfections.

2.2. Mouse/ratneutrophils

Evidencethatalsorodentneutrophilsproducechemokinesis

abundantlydocumented,asalreadyreviewed[30].Forinstance,

murineneutrophilsexposedinvitrotoM.tuberculosishavebeen

showntoproduceCXCL2/MIP-2␣andCCL3[69].Inanotherwork,

neutrophilshave beenfoundtoreleasebiologically activeCCL3

(e.g.,abletochemoattractimmatureDCs)uponinvitroexposure

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major-resistantmicewasshowntoabolishtherecruitmentofDCs

tothesiteofparasiteinoculation,aphenomenonadoptively

res-cuedbyinoculationofwild-typeneutrophils[70].Overall,these

latterdatapointtoaroleforneutrophil-derivedCCL3inthe

induc-tionofaprotectiveCD4+_Th1_immune_response_against_L._major

infectionviatherecruitmentofimmatureDCsandtheirsubsequent

differentiation[71].Inlinewiththeaforementionedmodelarealso

resultsobtainedusingmurineneutrophilsexposedinvitroto

Tox-oplasmagondii, whichwere foundtorelease manychemokines

abletorecruitimmatureDCs(e.g.,CCL3,CCL4,CCL5andCCL20),

asexperimentallyproved[72].Interestingly,undernon-infectious

setting,neutrophil-derivedCCL3andCCL4havebeenalsoshown

toinduceanearlymacrophageinﬂuxtothesitesofpolyacrylamide

gel-inducedcutaneousgranulomaformation[73].

In anothermodel, namelythe methicillin-resistant S.aureus

(MRSA)infection,neutrophilsfrommiceresistanttoMRSAhave

beenshowntoinduce,viaCCL3andIL-12release,thepolarization

ofmacrophagestowardsaphenotypecharacterizedbythe

expres-sionofcytokinesandchemokinespotentiallyassociatedwithaTh1

response(e.g.,IFN␥,IL-12,IL-18,CCL3,CCL5,CXCL9andCXCL10)

[74].Bycontrast,supernatantsofneutrophilsobtainedfrom

MRSA-susceptiblemice[74]werefoundtopolarizemacrophages,viathe

releaseofCCL2andIL-10,towardsaphenotypecharacterizedby

theexpressionofcytokinesandchemokinespotentiallyassociated

withaTh2response(e.g.,IL-1receptorantagonist,IL-10,CCL17,

CCL18,CCL22)[74].Similarresultswerealsoobservedinamodelof

compensatoryanti-inﬂammatoryresponsesyndrome[75].Finally,

inmiceinfectedwithPlasmodiumbergheiANKA,neutrophil-and

monocyte-derivedCXCL10havebeenshowntoinducethe

migra-tionofanti-Plasmodiumeffectorcellsoutoflymphoidsecondary

organs[76],thereforecausing animpairedcontrolof theblood

stageofmalariaandtheconsequententrapmentofparasitizedred

bloodcellsintothecerebral-microcirculation[76].

Asforhumanneutrophils,increasingevidencepointforaroleof

neutrophil-derivedchemokinesinorganizingimmuneresponses

towardvirusesalsoinrodents.Forinstance,neutrophilsisolated

from the lungs of inﬂuenza virus-infected wild-type mice

dis-played,intheacutephase,signiﬁcantlevelsofCXCL10mRNAand

werealsofoundtoexpressCXCL10byimmunohistochemical(IHC)

analysis[77].Bycontrast,micelackingCXCL10orCXCR3,besides

presenting reduced acuterespiratory distress syndrome(ARDS)

manifestationsandanincreasedsurvival,hadalowernumberof

inﬁltratinglungneutrophilsascomparedtothewild-typestrains

[77]. Data areconsistent withthenotionof neutrophil-derived

CXCL10asresponsiblefortherecruitmentofadditional

(CXCR3-positive) neutrophils and the consequent excessive pulmonary

inﬂammation/ARDS[77].Interestingly,similardatawereobtained

byinducingachemicalpneumoniausingthesamemodels[77].In

amousemodelofinﬂuenzaAvirusinfection,neutrophil-derived

CXCL12/stromal-cell derived factor 1 (SDF-1) hasbeen instead

demonstratedtobecrucialforCD8+_T_cell_recruitment,_and

there-forefortheinductionofaCD8+_T_{cell-mediated}_immune_response

towardsinﬂuenzavirusitself[78].Usingtwophotonmicroscopy

invivo, micehavingCXCL12 conditionally-depletedneutrophils

presentedfewerTcellswithintheinfectionsiteandhadaslower

clearanceofthevirus,similartothemicewithreducedneutrophil

counts [78]. Strikingly, the same study has also demonstrated

that,duringtheirmigration tothesiteof infection,neutrophils

depositlong-lasting,CXCL12-containingtrailsfromtheirelongated

uropods,inthismannerguidingTcellstowardsinfectedtissue[78].

Finally,ratneutrophilsincubatedinvitrowithconditionedmedium

fromcoronavirus(CoV)-infectedalveolarepithelialcellshavebeen

showntodisplayhighermRNAlevelsofCXCL1,CXCL2,CXCL10

andCXCL11,CCL2,CCL4,CCL7,CCL9/mMIP-1_␥,CCL12/MCP-5and

CCL22ascomparedtoneutrophilsincubatedinacontrolmedium

[79].Thepotentialvalidityoftheseinvitroobservationsinvivo

havebeenconﬁrmedinaratmodelofnon-fatallungCoVinfection,

inwhichneutrophil-depletedratsdisplayedfewermacrophages

andlymphocytesintheirrespiratorytract,ascomparedto

non-depletedrats,andlowerchemokinelevels[79].

3. Neutrophil-derivedchemokinesintumors

Increasing experimental evidence indicatesthat, in addition

tomacrophages,DCsandlymphocytes,alsoneutrophilsinﬁltrate

tumors[80],toplay aroleininﬂuencingtheneoplasticgrowth.

Thatoccursnotonlybecausetumor-associatedneutrophils(TANs)

maydirectlyinteractwiththeneoplasticcells,butalsobecause,

withinthetumormicroenvironment,TANsreleaseawidearrayof

molecules,includingchemokinesandcytokines[81].

Neutrophil-derived chemokines inﬂuence the tumor fate either indirectly,

becausetheyrecruitandactivateinnateandadaptiveimmunecells

[81],ordirectly,fortheircapacitytomodulateangiogenesisand

cellproliferation[13,14].Recentknowledgeontheinvolvementof

neutrophil-derivedchemokinesexertingpro-oranti-tumoractions

issummarizedinthefollowingsections.

Anumberofinvitrostudieshavedemonstratedthat

periph-eralneutrophilsmayexpressand/orproducechemokinesinthe

presenceof tumorcell-conditionedmediumand/ortumorcells.

For instance, neutrophils incubatedwith gastriccancer-derived

mesenchymalstemcell-conditionedmediumhavebeenshownto

upregulateCXCL8andCCL2 mRNA[82].CXCL8wasalsoshown

tobeproducedbyneutrophilseitherco-culturedwithmetastatic

melanoma [83] and glioma cell lines [84], or in the presence

ofsupernatantsfromaheadandnecksquamouscellcarcinoma

(HNSCC)cellline[85].Theseobservationsanticipatea potential

roleforTANstoregulateinvivo,viaCXCL8,notonlytherecruitment

of additional neutrophils, but also cancercell growth,survival,

motionandangiogenesis[86].Neutrophilsincubatedinvitrowith

HNSCC-derivedsupernatantswerealsofoundtorelease

consid-erableamountsof CCL4[85,87],achemokine thatmayelicitan

immuneresponsetowardsthetumorbyrecruitingNK,immature

DCsandTcells[87,88].Inanotherstudy,peripheralneutrophils

fromhepatocellular carcinoma (HCC) patientshavebeen found

to produce,in vitro, signiﬁcantly higher amounts of CCL2 than

neutrophilsfromhealthydonors[89].Moreover,theamountsof

neutrophil-derived CCL2 were found to beproportional to the

tumor sizes of patients [89]. Consistent with the observations

evidencingthatmouseneutrophilsexertingimmunosuppressive

functionsproduceCCL2andIL-10,whilethosewith

immunoacti-vatingpropertiesreleaseCCL3andIL-12[74],supernatantsfrom

HCCneutrophilswerefoundtoinhibit,invitro,theproductionof

IFN␥byperipheralbloodmononuclearcellsinaCCL2-dependent

fashion[89].Inamorerecentstudy,TANsisolatedfromHCC

sam-ples have beenshown torelease signiﬁcantlevelsof CCL2 and

CCL17invitro[90],consistentwiththecapacityofHCCcelllinesto

inducetheproductionofthesamechemokines(CCL2andCCL17)

whenco-culturedwithneutrophilsinvitro[90].Inagreementwith

thelatterdata,IHCanalysisofhumanHCCspecimenshas

demon-stratedthepresenceofCCL2-andCCL17-positiveTANswithinthe

tumorstroma[90].Notably,TAN-conditionedmediawerefound

toincrease,invitro,themigratoryactivityofmacrophagesandT

regulatorycells(Tregs)viaCCL2andCCL17,respectively[90],in

linewithaTAN-N2protumoralphenotypepreviouslydescribed

[80]. Aninverse correlationbetween thenumber of CCL2- and

CCL17-positiveTANsintumorsectionsandpatientsurvival

con-ﬁrmedtheprotumorroleforTANsinHCC[90].CCL17hasbeen

(6)

tumors[91],thussuggestingthat,alsointhistypeofcancer,TANs

mayrecruitTregs.

Bycontrast,TANsisolatedfromlungtumorsofpatientsatthe

earlystageofdiseasehavebeenshowntoproducesigniﬁcantlevels

ofCXCL8,CCL2andCCL3,otherthanIL-6andtheanti-inﬂammatory

IL-1Ra[92].Althoughtheprecisecontributionofeachchemokine

wasnotspeciﬁcallyaddressed,lungtumorTANswereshownto

induce,invitro,theproliferationandthereleaseofIFN␥byboth

CD4+_and_CD8+ _T_cells_[92]_,_pointing_for_their_anti-tumor_role._In

addition,invitroexperimentsuncoveredacrosstalkbetweenTANs

andactivatedTcellsleadingtoasubstantialupregulationof

costim-ulatorymoleculesinneutrophils,which,inturn,bolsteredafurther

Tcellproliferationin apositive-feedbackloop[92].Similarlyto

lungTANs[92],humanneutrophilsinfectedwithanoncolytic

vac-cinestrain of measlesvirus (MV) have been shown to secrete

CXCL8andCCL2,otherthanTNF␣andIFN␣[93].These

observa-tionssupportandextendpreviousﬁndings,madeinahumantumor

xenograftmousemodel,demonstratingthatneutrophilscontribute

totheantitumorefﬁcacyofMV,mostlyMVexpressing

granulocyte-macrophagecolony-stimulatingfactor(MVGM-CSF)[94].

3.2. Mouseneutrophils

Asa general notion, mouse TANshave been shown to

con-stitutively produce a variety of chemokines, including CXCL1,

CXCL2/MIP-2␣,CXCL9,CXCL10,CXCL13/Blymphocyte

chemoat-tractant/(BLC), CXCL16, CCL2, CCL3, CCL7 and CCL17 [95],

therefore suggesting that they can potentially recruit

mono-cytes/macrophages,DCs,NKcells,TandBcellsubsetsand/ormore

neutrophilstothetumor.Inturn,thetype ofimmuneresponse

elicited by TANs would depend on the pattern of chemokines

prevalentlyproducedineachsinglesituation.Forinstance,TANs

isolatedfromlungtumorswerefoundtoexpressandreleasehigher

CCL17levelsthanbone marrowneutrophilsfromthesame

ani-mals [91]. In this model, TAN-derived supernatants have been

showntopreferentiallyattractTregsinaCCL17-dependent

man-ner,inmigrationassaysbothinvitroandinvivo[91].Interestingly,

TANsisolated fromthesamemousemodel,but aftertreatment

withthetransforminggrowthfactor-␤(TGF-␤)-inhibitor SM16,

expressedandreleasedsigniﬁcantlyloweramountsofCCL17than

TANsfromuntreated mice[91], conﬁrminga role forTGF-␤in

conditioningneutrophilactivities towardstumors [96].In such

regard,thepresence/absenceofTGF-␤withinthetumor

microen-vironmenthasbeenshowntopolarizeTANsintotwophenotypes

displayingoppositeimmunoregulatoryfunctions,mainlybasedon

theircytokine/chemokine-producingrepertoire[97]:inthecaseof

chemokines,CCL3fortheantitumor(N1),CCL2plusCCL17forthe

protumor(N2),phenotype[95,96].InagreementwiththeN1/N2

hypothesis,tumorvolume,aswellasthenumberofpulmonary

metastases,wereshowntosigniﬁcantlyincreaseinamousetumor

modelobtainedbycoinjectinganimalswithHCCcelllinestogether

withCCL2andCCL17-producingTANs.Moreover,thesetumors

dis-playedanincreasednumber ofstromalmacrophages andTregs

[90].

Protumor,proinﬂammatoryactivitiesbyinﬁltratingneutrophils

have been also uncovered in another mouse model of

colitis-associated cancer, in which prostaglandin receptor (subtype

EP2)-positive colon neutrophils werefoundto express,byIHC,

CXCL1inadditiontoIL-6,TNF␣andCOX-2[98].Consistentwith

theseﬁndings,bonemarrow-derivedneutrophilsfromthesame

micewereshowntoupregulatetheexpressionofCXCL1,TNF␣,

IL-6 and COX-2 mRNA when incubated with prostaglandin E2

(PGE2) and TNF␣ in vitro [98]. This suggests that the PGE2

-prostaglandinreceptor-(EP2)pathwayformsanautocrineloopfor

neutrophilrecruitmenttothecolonbyinducingCXCL1and

conse-quentlybyamplifyingthepro-tumorinﬂammatoryresponse[98].

Notably,underthesameexperimentalsetting(e.g.,induced

colitis-associatedcancer)EP2-deﬁcientmicedisplayedareducednumber

of tumor lesions, therefore pointing for a role for neutrophils

inpromoting tumorigenesis[98].Notably,clinicalspecimens of

ulcerativecolitis-associatedcolorectalcancerdisplayEP2-positive

infiltratingneutrophils,confirmingthefindingsinmice[98].

4. Neutrophil-derivedchemokinesinimmune-mediated diseases

Immune-mediatedinﬂammatory diseasesarea groupof

dis-easeslackingadeﬁniteetiologyandcharacterizedbyprolonged

inﬂammatorysymptoms,oftentriggeredbyadysregulationofthe

normalimmuneresponse[99].Inadditiontootherleukocytes,

neu-trophilshavebeenoftenshowntobeinvolvedinthepathogenesis

ofthesediseases[100].

Evidence points for the involvement of neutrophil-derived

chemokinesinvarioustypesofarthritis.Forexample,peripheral

neutrophilshavebeenshowntoreleaseCXCL8uponphagocytosis

ofmonosodiumurate(MSU)crystalsinvitro[101].MSUcrystals

cause the typicalgout attacks when deposit in joints, tendons

andsurroundingtissues,towhichneutrophilsparticipateviathe

release ofproinﬂammatory mediators[102]. Therefore,because

of the capacity of CXCL8 to potently attract neutrophils, these

ﬁndingswoulduncoverapotentialmechanismthatmaysustain

neutrophil-mediatedinﬂammationingoutyarthritis.Neutrophils

isolatedfromthesynovialﬂuid(SF)ofpatientswithrheumatoid

arthritis(RA),asystemicautoimmuneinﬂammatorydisorder

pri-marilyinvolvingthejoints,havebeenshowntoproducehighlevels

ofCXCL8,CXCL10, CCL2and CCL20[54], inaddition toexpress

elevatedtranscriptlevelsofCCL18and CCL3[103].Data would

suggestthat, inRAarthritis, neutrophilsrecruitadditional

neu-trophils,aswellasTh1cellsandTh17cells,toundertakereciprocal

crosstalk[54].Consistent withthis hypothesis, neutrophils and

Th17cellshavebeenfoundtocloselycolocalizeinSFs fromRA

patients[54].Inaddition,invitroexperimentshavedemonstrated

thatsupernatantsfromIFN␥plusLPS-activatedneutrophilsinduce

chemotaxisofTh1andTh17cellsviaCCL2andCXCL10,andCCL2

andCCL20,respectively[54].Inthesamestudies,Th17,butnot

Th1,cellshave beenshown toreleaseCXCL8and chemoattract

neutrophilsinvitro,pointingfortheirdirect actioninrecruiting

neutrophils and therefore inamplifying thelocal inﬂammatory

response [54]. Interestingly, previous studies had also

demon-stratedthatneutrophilsfromSFsofRApatientsmayexpressCCL20

mRNA and that stimulation with SFs, containing or not TNF␣,

inducedCCL20mRNAinperipheralneutrophils[104].

Neutrophil-derived chemokines have been suspected to be

involvedinotherimmune-mediateddiseases,suchasulcerative

colitis (UC), aninﬂammatory bowel disease(IBD) characterized

byrelapsingmucosalinﬂammation.Accordingly,inbowel

speci-mensfromUCpatients,neutrophilshavebeenfoundtoexpress

CXCL1andCXCL9byIHC[105],consistentwitharoleof

neutrophil-derived CXCL1 and CXCL9 in recruiting neutrophils, NK and

T cells, which are known to be involved in the

immunologi-cal response causing lesions. Similarly, tissue specimens from

anotherIBD,namelyCrohn’sdisease(animmune-mediatedileitis),

displayedacolocalizationofneutrophilsand Th17cells by

con-focalmicroscopy[54].Theseﬁndingsare,onceagain,supporting

neutrophil-derivedCCL2andCCL20inthepathogenesisofCrohn’s

diseaseviaTh17cells recruitment.Finally, invitroexperiments

haveshownthat,uponstimulationwithanti-phospholipid

(7)

[106].Itisthereforeconceivabletohypothesizeaparticipationof

CXCL8inthepathogenesisoftheanti-phospholipidsyndrome,a

systemicautoimmunediseaselikelytriggeredbyTLRsactivation

andcharacterizedbytheoccurrenceofanti-phospholipid

antibod-ies,recurrentthrombosisandfetalloss[106].

4.2. Mouseneutrophils

Fulminanthepatitisdevelopsinabout1%ofpatientswithacute

hepatitisB,inwhomanexcessivehostdefensiveimmuneresponse

isdetected[107].In suchregard, amousemodel ofthehuman

disease has been developed, based on an adoptive transfer of

antigen-speciﬁccytotoxicTlymphocytes(CTLs) intohepatitis B

virus(HBV)-transgenicmice,which,inturn,triggersafulminant

hepatitis[108].Insuchamodel,injectionofantigen-speciﬁc

cyto-toxicTcellsinHBVtransgenicmiceisfollowedbytherecruitment

ofmononuclearcellsandneutrophilsintotheliver[108].Inthis

organ,CTLsreleaseIFN␥and TNF␣uponantigenrecognition,in

turninducingliverneutrophilstoreleaseelastaseandto

upregu-lateCCL3,CCL4andCXCL1mRNA[108].Importantly,micetreated

with anti-CCL3, −CCL4, and −CXCL1 antibodies were foundto

displayalower recruitmentof inﬂammatorycells intotheliver

and a reduced hepatic injury, indicating that these

neutrophil-derivedchemokinesmediatetheinﬂammatoryandimmunological

responseengagedbyCTLsagainsttheHBV-infectedhepatocytes

[108].Inamodelofdelayed-typehypersensibility(DTH)obtained

bysensitizingmicewithHerpesSimplexVirustypeI(HSV-1)

anti-genonthescariﬁedcornea,neutrophilshavebeendemonstratedto

recruitTcellsbyproducingCXCL9andCXCL10[109].Consistently,

neutrophildepletionwasaccompaniedbya markeddecreasein

thenumberofCD4+ _T_cells_to_the_site_of_DTH_and_a_drop_in_the

levelsofCXCL9andCXCL10inDTHtissuelysate[109].Moreover,

consistentwiththereleaseofCXCL9andCXCL10byneutrophils

stimulatedinvitrowithIFN␥,IFN␥-knockoutmicemanifesteda

depressedDTHuponHSV-1antigenchallenge,andonlythe

recon-stitutionofthesemicewithIFN␥re-inducedthesynthesisofboth

chemokines[109].Therefore, according tothemodel described

above,neutrophils activatedbyIFN␥releaseCXCL9andCXCL10

andrecruitCD4+_T_cells._The_latter_cells,_in_turn,_would_contribute

toa furtherproductionof IFN␥ andtherefore tothe

ampliﬁca-tionoftheinﬂammatorycascadeinsitesofDTH[109].Finally,in

amousemodelofcutaneoustypeIIIhypersensitivityobtainedby

injectingantibodiesintomouseearskin,andbysystemically

deliv-eringthecorrespondingantigens,immunecomplexes(ICs)-laden

neutrophilsisolatedfromtheearsdisplayedasigniﬁcantly

upreg-ulatedexpressionofCXCL2/MIP-2␣ascomparedtobone-marrow

neutrophils[110].Invitrostudiesconﬁrmedthatthedirect

stimula-tionofisolatedbonemarrow-derivedneutrophilswithICtriggers

asubstantialsecretionofCXCL2/MIP-2␣[110].Therefore,a role

ofCXCL2/MIP-2␣inrecruitingadditionalneutrophilsand

endoge-nouslyactivatingtheireffectorfunctions,includingreactiveoxygen

speciesproductionandphagocytosis,mightbeplausible[110].

5. Conclusions

Thereisnodoubtthatneutrophilsmayregulateleukocyte

traf-ﬁckingduringimmuneresponses.Asbrieﬂyoutlinedinthisshort

review,thisfunctionreliesontheneutrophilcapacitytoproducea

varietyofchemokines,butitshouldbenotforgottenthatalso

pre-formedfactorscontainedinneutrophilgranuleshavebeenshown

tobechemotactic formononuclear cells and neutrophils(for a

review,pleaseseereference[111]).Nonetheless, inspiteofthe

largebodyofdatadescribingtheexpressionpatternof

neutrophil-derivedchemokinesinvitro,weneedtoexpandourknowledgeon

whatistherealsigniﬁcanceofthisphenomenoninvivo,particularly

inhumans.Ofutmostimportanceistoimprovethetechniquesto

isolateneutrophilsfromtissuesandlymphoidorgans(e.g.,spleen

andlymph-nodes)atveryhighlevelsofpurity,toavoidfalse

pos-itivedatacausedbyeventualcontaminationwithothercelltypes.

Studiesaimedatgainingmoreinsightsonthemolecularregulation

ofchemokineexpressioninneutrophilsarealsoawaited.Infact,

similarlytootherleukocytes,chemokineexpressioninneutrophils

canbecontrolledatthetranscriptionaland/orpost-transcriptional

level[30],insomecasesbysophisticatedandcell-speciﬁc

regula-torymechanisms,includingtheinvolvementofmicroRNAs[69],

speciﬁctranscription factors[112]andchromatinmodiﬁcations

[65]. However, very little is still known on all these

phenom-ena.Furthermore,neutrophil-derivedchemokinescanbeinvolved

eitherinphysiologicalandpathological angiogenesis,afunction

thatisoftenunderestimated[13].Finally,it isknownthat

neu-trophilsmaybeengagedintocomplexbidirectionalinteractions

withotherleukocytesortissuecells[21].Asaresultofthiscrosstalk,

neutrophilsand targetcellsreciprocallymodulatetheirsurvival

andactivationstatus. Chemokinesmightcertainly contributeto

regulatesuchacrosstalk,buttheireffectiverolesremainsmostly

unsolved.Insuchregard,futurechallengesforscientistsintheﬁeld

willbetotranslateallthisknowledgeintoefﬁcacious

neutrophil-targetedtherapieswithoutcompromisingimmunity.

Conﬂictofintereststatement

Theauthorsdeclarenoﬁnancialorcommercialconﬂictof

inter-est.

Acknowledgments

M.A.C.issupportedbyagrantfromAssociazioneItalianaperla

RicercasulCancro(AIRC,IG-15454).CTissupportedbyagrantfrom

AlessandroMorettiFoundation,LionsClub(SanGiovanniLupatoto,

ZevioedestraAdige,Verona).

Weapologizefornothavingdiscussedallthepaperspublished

intheﬁeldduetorestrictedlengthlimitations.

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