Fibrocyte
accumulation
in
the
lungs
of
cystic
fibrosis
patients
Rajesh K. Kasam
a ,b, Prathibha R. Gajjala
a ,b, Anil G. Jegga
a ,c, Jennifer A. Courtney
d,
Scott H. Randell
e, Elizabeth L. Kramer
a ,b, John P. Clancy
f, Satish K. Madala
a ,b ,∗ a DepartmentofPediatrics,UniversityofCincinnatiCollegeofMedicine,Cincinnati,OHbDivisionsofPulmonaryMedicine
cBioinformatics,CincinnatiChildren’sHospitalMedicalCenter,Cincinnati,Ohio,45229USA
d MolecularandDevelopmentalBiologyGraduateProgram,CincinnatiChildren’sHospitalMedicalCenter,Cincinnati,Ohio,45229 e DepartmentofCellBiology&Physiology,TheUniversityofNorthCarolinaatChapelHill,NorthCarolina,27599USA f CysticFibrosisFoundataion,Bethesda,Maryland,20814USA
a
r
t
i
c
l
e
i
n
f
o
Articlehistory:
Received11October2019 Revised1June2020 Accepted6June2020 Availableonlinexxx
Keywords:
Cysticfibrosis Pulmonaryfibrosis Fibrocytes Lung CFTR
a
b
s
t
r
a
c
t
Background: Cysticfibrosis(CF)patientsdevelopseverelungdiseaseincludingchronicairwayinfections, neutrophilicinflammation,andprogressivefibroticremodelinginairways.However,cellularand molec-ularprocessesthatregulateexcessivecollagendeposition inairwaysinthesepatients remainunclear. Fibrocytesarebonemarrow(BM)-derivedmesenchymalcellsthatexpressthehematopoieticcellmarker CD45,andmesenchymalcellmarkersandimplicatedincollagendepositioninseveralfibroticdiseases. ItisunknownwhetherfibrocytesaccumulateinthelungsofCFpatients,sothecurrentstudyevaluates thepresenceoffibrocytesinthefibroticlesionsofairways inexplantedCFlungscomparedtonon-CF unuseddonorlungs(control).
Methods: Weused immunofluorescencestainingtodetermineiffibrocytesaccumulateinexplantedCF lungscomparedtohealthydonorlungs.Simultaneously,weevaluatedcellscollectedbybronchoalveolar lavage(BAL)inCFpatientsusingmulti-colorflowcytometry.Finally,weanalyzedtranscriptsdifferentially expressedinfibrocytesisolatedfrom theexplanted CFlungscomparedto control toassess fibrocyte-specificpro-fibroticgenenetworks.
Results: Ourfindingsdemonstratefibrocyteaccumulation inCFlungs comparedto non-CFlungs. Ad-ditionally,fibrocytes weredetectedintheBALofall CFchildren. Transcriptomic analysisoffibrocytes identifieddysregulatedgenesassociatedwithfibroticremodelinginCFlungs.
Conclusions: Withsignificantlyincreasedfibrocytesthatshowincreasedexpressionofpro-fibroticgene transcriptscomparedtocontrol,ourfindingssuggestaninterventionforfibroticremodelingasapotential therapeutictargetinCF.
1. Introduction
Cystic fibrosis (CF) is an inheritable multi-system disease due todeficientchlorideiontransportatthecellmembraneof epithe-lial cells,especially inthelower airways, that leadsto significant lungdisease.CFiscausedbymutationsinthegenecodingforthe cystic fibrosis transmembrane conductance regulator (CFTR) pro-tein [1 ,2] .CF-associatedmutationsresultindefectiveCFTRprotein synthesis,processingorfunction,andoftenleadtofibrosisin mul-tipleorgansincludingtheliver,pancreasandlung [1 ,2] .Inaddition todefectiveCFTR-dependentchloridetransport,bicarbonate trans-port may be impaired and sodium absorption may be enhanced
∗ Correspondingauthorat:DivisionsofPulmonaryMedicineandBioinformatics,
CincinnatiChildren’sHospitalMedicalCenter,Cincinnati,OH45229USA
E-mailaddress:[email protected](S.K.Madala).
in the airways. Thick mucus glandsecretions, airway mucus ob-structionandsusceptibilityto chronicinfectionthen ensue [2–4] . ThepathophysiologicmechanismsunderlyingCFlungdiseasethat leadtobronchiectasis,myofibroblastaccumulationandprogressive collagendeposition, alsoreferred to asbronchial wall thickening, remainundefined [3 ,5] .PreviousfetalandanimalworkinCF sug-gestsCFpro-fibroticabnormalitiesmanifestduringtheearlystages ofCF lung diseases beforethe establishment ofchronic infection ofthelowerairways [6 ,7] .However,strongevidenceindicatesthat theCFhostinflammatoryresponseisexcessiveandchronic inflam-mation and fibrosis directly cause bronchiectasis and respiratory failureasthediseaseprogresses [6] .
Themechanismsgoverningfibroticlung remodelingduetothe CFTR mutations in CF are not clearly defined. Currently, there is uncertainty whether early infection and inflammation initiate this pro-fibrotic phenotype. Also, the lack of an animal model
that mimics clinical features of CF lung disease has reducedthe progressonidentifyingmechanisms underlyingfibrosisandother clinical abnormalities in CF. However, recent development of CF pigs that lack CFTR or express mutant CFTR (F508del) has cre-ated a phenotype similar to human disease including intestinal, pancreaticandairwaydisease.Moreover,newbornpigletswithCF havedeveloped airflow obstruction and air trapping in part due to tracheal structuralabnormalities, in the absence of inflamma-tionand mucusobstruction [8] .Several studies provide evidence thatpro-fibroticgrowthfactors,suchasTGF
β
,functionas modi-fiergenesinCFandmaycontributetotheseverityoflungdisease byactivatingpro-fibroticpathways [3 ,5] .Morphometricanalysisof TGFβ
signaling,myofibroblastdifferentiation, andcollagen depo-sitioninthelungsofCF,IPFandnon-CFpatientsdemonstratethat pulmonaryfibrosisisprominentinCFandiscomparabletoIPF [3] . Additionally,unresolvedneutrophilicinflammation,andaltered IL-17A-andTh2cytokine-drivensignalingmayalsocontribute to fi-brosisandseverelungdisease [3 ,5 ,9] .Otherstudieshavesuggested thatEGFRligands,suchasTGFα
andcytokinesincludingTNFα
, IL-13,andIL-17,areabundantinCFcomparedtonon-CFlungs [5 ,10] . Experimental models of chemical-inducedlung injury or overex-pressionofTGFα
havedemonstratedthatmesenchymalcells accu-mulateandexcessiveproduction ofECM leadstofibrotic remod-eling [11 ,12] . Furtherunderstandingof thecellularandmolecular mechanismsunderlyingfibrosis in CFshould be a priorityasthe developmentoftherapiesthatcaninhibitordelaytheonsetof fi-brosisandsubsequentorganfailureinCFcouldbeessentialin ex-tendingthelifeand/ordelayingtheneedforlungtransplantation.Severalfibroticlungdiseases arecharacterized bythe progres-siveaccumulation oflung mesenchymalcells, thesubsequent ex-pansionoffibroticlesions and theultimate loss oflung function [13] .InCF,thecellularsourcesofthelungfibroblastsinfibrotic le-sionsremainunknown. Fibrocytesarebone marrow(BM)-derived stromalcellsthatexpressavarietyofcell-surfacemarkersrelated toleukocytes, hematopoieticprogenitor cells,andfibroblasts [14] . In tissues, fibrocytes have been shown to become alpha-smooth muscle actin (
α
SMA)-positive myofibroblasts, but this occurs in limitednumbers [15] .Inpatientswithinterstitiallungdisease,the numberofcirculatingfibrocyteshasbeenshowntocorrelatewith lungremodeling [16] .Modulatingthemechanismsthatdrive fibro-cyterecruitmenttothelung,andtargetingchemokinesinvolvedin fibrocytemigration,havebeenshowntoreducefibroticburdenin mice [17] .Studies fromourlaboratory andothershaveidentified aspecific,endogenousmechanismofpronounced andprogressive pulmonaryfibrosisdrivenbyfibrocytesinthelung [15 ,18] . Specif-ically, we have found that fibrocytes are tissue invasive and suf-ficienttoaugmentthe proliferativeexpansion oflung-resident fi-broblastsofthefibroticlesionsinthelung [15] .Currently, it isnot clearwhetherfibrocytes accumulate inthe pathogenesis of fibrotic lung disease in humans with defective CFTR.Inthisstudy,wetestedwhetherfibrocytesaccumulatein ex-plantedCFlungswiththeF508delmutationfrompatientswho un-derwentlungtransplantation.Weperformedsystematicvalidation offibrocytepresenceinthelungsandidentifiedfibrocyte-specific genenetworksthatmayinvolveinfibroticlungremodeling.
2. Materials and methods
2.1. Human tissue samples
CellsfromBALfluidwerecollectedaccordingtotheCincinnati Children’s Hospital Medical Center (CCHMC) Institutional Review Board(IRB)approvedprotocol(IRB#2014–3309).Inaddition, de-identifiedexplantedlung tissuespecimenscollectedfromthe dis-talareasoflung wereacquiredusingresearch protocolsapproved bytheUniversityofNorthCarolinaandCCHMC(IRB#2017–6850).
CFwasdiagnosedaccordingtoclinicalstandardswithan elevated sweat chloride test andtwo copies ofa CFTR-deficient mutation. LungtissuesampleswereobtainedfromCFpatientswithF508del CFTR homozygousmutation who had undergone lung transplan-tation. Inaddition,lungtissue wasacquiredfromdonorswithno historyofpriorchroniclungdisease(controlgroup)(agerange16– 39years,attemptingtomatchtheCFcohort).
2.2. Lung mesenchymal cell culture
Human lung stromal cell culture wasperformed asdescribed previously [19] . We performeda detailed macroscopicevaluation of the explanted lung that was cut sagittally resulting in two gross sectionsto identifynormal or remodeled areas in the dis-tal lung. From the normal donor lungs, we selected distal lung tissue with a soft consistency to finger palpation, appeared red incolor dueto normaltissue perfusion andhadno mucus plug-gingorcystformation.We usedthe CFlung tissueswith moder-ate to severe damage that had a firm consistency to finger pal-pation, appeared pale in color (due to scar) and that had air-waysfilledwithmucus.Thus,thedistallungparenchymaselected with smallairways wascut into 2X2 cmpieces. Eachpiece was finely minced and digested in DMEM media containing collage-nase(2mg/ml)byincubatingat37°Cfor1hr.Digestedtissue was passedthrougha100μmfilter,washedtwice bycentrifugationat 100 Xg for5min.Afterthe digestion,ten millioncellswere cul-tured onto100-mm tissue-cultureplates in 10mlDMEM supple-mentedwith10%bovinecalfserum,2mMl-glutamineanda
cock-tail of antibiotics including Ceftazidime (100μg/ml), Tobramycin (80μg/ml), Vancomycin(100μg/ml), Ciprofloxacin(20μg/ml), Nys-tatin(100U/ml)andDoxycycline(4μg/ml).Mesenchymalcells ad-hered to platesand migrateaway from thelarger remaining tis-suepieces.Thelungmesenchymalcellsweremaintainedinculture untiltheyreached80%confluency,andnon-adherentfloatingcells anddeadcellswerewashedawaywithregularmediachanges. Fi-brocytes(CD45+ Col1+) fromcontrol and CF lung cultures were isolatedonday9usinganti-CD45magneticbeadsandthecell pu-ritywasgreaterthan94%,asassessedbyflowcytometry [15] .
2.3. RNA-sequencing, bioinformatics analysis and RT-PCR
Fig.1. Presenceoffibrocytesinthelungsofcysticfibrosispatients.(A)Frozenlungsectionsfromnon-CF(n=4)andCF(n=6)individualswerecoimmunostainedusing CD45andvimentin.Imagesarerepresentativeofeachgroup.ArrowsindicatingCD45&vimentindouble-positivecells.(60X,Scalebar:50μm)(B)Percentof vimentin-positivecellswerequantifiedfromeachgroupusingmetamorphimagingsoftware.(C)Percentfibrocytes(CD45+ andVimentin+ )werecalculatedusingmetamorphimaging software.DAPIstainedcellswerequantifiedfortotalcellnumber.∗P<0.05,∗∗P<0.005.Unpairedt-test.
Fig.2. IdentificationoffibrocytesinBALcellsofcysticfibrosispatients.Representativeflowcytometryplotsshowingthegatingstrategyusedtoidentifyfibrocytesthatare double-positiveforCD45andcollagen1observedusingFACSanalysisoftotalBALcellsisolatedfromcysticfibrosispatients.ForFACSanalysis,thegatesweresetbasedon isotypeantibodystainedcontrols.
enriched biologicalprocessesregulatedby differentiallyexpressed genes in CF fibrocytes compared to normal fibrocytes. Real-time PCR was performedusing the CFX384 Touch Real Time PCR de-tectionsystemandSYBRselectmastermix(Bio-Rad,Hercules,CA) asdescribedpreviously [19] . Wehaveincluded humanbetaactin geneasaninvariantendogenouscontroltocorrectsampleto sam-plevariationsandhaveavoidederrorsinthenormalizationby us-ing good qualityRNA inequalamounts andalso performedmelt
curve analysisto exclude primersets producing non-specific PCR products.Real-TimePCRprimerdetailsareprovidedintableS1.
2.4. Flow cytometry
cy-Fig.3. DifferentialgeneexpressionanalysisofCFFibrocytesshowsfibrosis-associatedtranscriptionalchangesinCFlungs.Heatmapoffibrocyte-specifictranscriptsisolated fromfibroticCFlungscomparedtonormallungswasanalyzedusingRNA-sequencing(N=3).Geneontologyclassificationofhighlyregulatedprocessesandselectedexample genesarelistedontheright.
tometer(BD Biosciences). BAL cellsstained with isotypeIgG and single stain control antibodies were used for compensation and gating.Data were analyzed usingFlowJo software(TreeStar, Ash-land,OR).
2.5. Immunofluorescence and confocal imaging
Human lung tissue sections were embedded in OCT medium and frozen lung sections were prepared as described previ-ously [20] .Lungsectionwere co-immunostainedusingantibodies againstCD45(ab10558,Abcam)andvimentin(sc-7557,SantaCruz Biotechnology)followedby secondaryantibodiesconjugated with AlexaFluor488 andAlexa Fluor568.Lungsectionswere stained with DAPI for nucleus visualization and confocal Z-stack images were obtained using a Nikon AIR-A1 laser scanning microscope. 3DrenderingfromconfocalZ-stackswasperformedusingthe “an-imation” toolin Imaris (version 9.2, Bitplane) software. To quan-tifythevimentinorCD45andvimentin-positivecells,sixrandom highmagnification imageswere obtainedfrom each lung section andquantifiedusingMetaMorphimagingsoftware(Molecular De-vices,CA,USA)asdescribed [19] .Also,immunostainingswere per-formed on serial lung sections with antibodies against vimentin (anti-humanvimentin,ab137321;Abcam), andCD45(rabbit anti-CD45;Ventana,Tucson,AZ,USA)asdescribedpreviously [15] . Im-ages were obtained using a Leica DM2700 M bright-field micro-scope(LeicaMicrosystems,BuffaloGrove,IL,USA).
2.6. Statistics
All data were analyzed using Prism (Version5; GraphPad, La Jolla,CA,USA).Student’s t -test wasusedto comparetwo
experi-mentalgroups. Data wereconsidered statisticallysignificant for P valueslessthan0.05.
3. Results
3.1. Fibrocytes accumulate in fibrotic lung lesions of CF patients
Inorder toidentify fibrocytes,we co-immunostainedthelung sectionsfromF508delCFTRhomozygous(n =6)andnon-CF(n =4) donors withantibodiesagainst CD45and vimentin.We observed an increasednumberofmesenchymal cellsthat aredual positive forvimentin andCD45 inCF lungs comparedto non-CF controls (Fig. 1 A).Also,weobservedasignificantincreaseinthetotal num-berofvimentin-positivemesenchymalcellsinCFlungscompared tonon-CFcontrols(Fig. 1 B).Further,wequantified thenumberof fibrocytesandobserved asignificantly greatertotalnumber of fi-brocytesinCFlungscomparedtonon-CFcontrols(Fig. 1 C).3D ren-deringof confocal images revealsthe presence of fibrocytes that arepositiveforbothCD45andvimentininCF lungs (Supplemen-taryVideo1).Inordertofurtheridentifyanincreasein mesenchy-malcells, we performedimmunostaining on serialsectionsof CF andnon-CFlungsusingantibodiesagainstvimentin andCD45. In the serial sections, we observed an increase in cells positive for bothvimentin andCD45inCF lungscomparedtonon-CF disease controls(FigureS1).
3.2. Fibrocytes identified in BAL specimens of pediatric CF patients
Fig.4. CFfibrocytesshowdysregulatedtranscriptome.(A)QuantificationofgenesCFTR,KCNN4andSPINT1infibrocytesisolatedfromnon-CFandCFpatientlungsusing RT-PCR.(B)QuantificationofgenesCDH3andCTSDinfibrocytesisolatedfromnon-CFandCFpatientlungsusingRT-PCR.(C)QuantificationofgenesSERPINA1andELNin fibrocytesisolatedfromnon-CFandCFpatientlungsusingRT-PCR.(D)QuantificationofgenesSELP,SERPINB2andFGF1infibrocytesisolatedfromnon-CFandCFpatient lungsusingRT-PCR.Therelativeexpressionlevelisexpressedasfoldinductionrelativetothatincorrespondingnon-CFfibrocytesfollowingnormalizationtotheexpression ofhumanβ-actin.Alldataarepresentedasmean±SEM(n=6).∗P<0.05,∗∗P<0.005,∗∗∗P<0.0005,and∗∗∗∗P<0.00005.Unpairedt-test.
all patientsbetween2–16yearsofage.Theclinicalcharacteristics of the CF patients are summarized in Table 1 . Toidentify fibro-cytes intheBALfluidofCF patients,cellswere stainedforCD45, and Collagen1 and analyzed using flow cytometry. FACS analy-sis identified that about10.3% ofthe population inthe totalBAL cellswere positiveforfibrocyte markers,CD45, andCol1 (Fig. 2 & Table 1 ).
3.3. Differential expression of fibrosis-associated transcripts by fibrocytes in CF patients
Toidentifyfibrocyte-specifictranscriptsinvolvedinCFlung dis-ease,weisolatedfibrocytesfromlungstromalcellculturesof
non-Table1
Clinicalparametersofcysticfibrosispatients.
Clinicalparameters CF(N=11)
CFandF508delCFTRhomozygousdonorlungtissueandcompared transcripts that were differentially expressed more than 1.5-fold (TableS2). As shownin Fig. 3 , therewasa largergroup ofgenes subjectto changeinthefibrocytes ofCFlungs comparedto non-CF lungs.The F508delCFTR mutation resulted inup- and down-regulationof 879 and 1939 transcripts, respectively. Gene ontol-ogyanalysisrevealedtheupregulationofgenesinvolvedinpositive regulationofcellmigration,negativeregulationofwoundhealing, muscle tissue contraction and development, and downregulation ofgenes involvedinCFTR dependentionchannels,F508del inter-actome,Nrf2interactome, andepithelial-stromal cellcrosstalk.In addition, we performed the validation of differentially expressed genes using RT-PCR. Transcripts of genes involved in CFTR de-pendent regulation of ion channels such as CFTR, KCNN4, and SPINT1aredownregulated inCF lung derivedfibrocytes (Fig. 4 A). Notably, gene transcripts associated with the F508del interac-tome (CDH3 and CTSD), and Nrf2 interactome (SERPINA1 and ELN)weresignificantly downregulatedinCF lung fibrocytes com-paredto control fibrocytes(Fig. 4 Band 4 C). Conversely, the tran-scripts of profibrotic genes such as SELP, SERPINB2, and FGF1 are upregulated in CF fibrocytes compared to normal fibrocytes. Thesegenes havebeenimplicatedinincreasedmigration,delayed wound healing, and muscle tissue development and contraction (Fig. 4 D).
4. Discussion
Fibrocytesareasubsetofmacrophagesthatoriginatefrombone marrowandmigratetoinjuredareastoperformpro-fibrotic func-tions. Fibrocytes have been shown to increase in circulation and progressivelyaccumulateininjuredlungsinmultiplechroniclung diseases including asthma, acute respiratory distress syndrome (ARDS),systemic sclerosis, and IPF [28–31] . We identified that a significantnumberoffibrocytesaccumulateinexplantedCF lungs thatareextensivelyremodeled.
Unremitting inflammation andfibrosis ofthe lowerairways is amajor cause ofmorbidity andmortalityin CF patients. For the first time, we identified that fibrocytes marked withCD45+ and Col1 accumulate in the distal areasof the CF lungs.Importantly, we alsoidentified that fibrocytes are presentin the BAL fluid of youngerCFpatients.Althoughwecannotdefinethatfibrocytesare associatedwithdiseaseduetoourstudydesign,ourfindings sug-gestthat their presence could be contributingtoCF lung disease astheir levels are typically undetectable in BAL fluid of healthy controls [32] . Therefore, our findings support the need for addi-tionalstudieswithalargercohortofCF patientstofurther inves-tigateiffibrocytesinBALfluidorcirculationofCFaccumulateand can contribute to lung disease. Multiple studies using chemical-inducedlunginjurymodelsdemonstratethatcirculatingfibrocytes migrateto the lung in response to the local release of cytokine and chemoattractants such as the C–C motif chemokine ligand CCL2,CCL5andCXCL12/CXCR4 axis [17 ,33 ,34] .Notably,disruption ofthesepathwaysthat drive recruitmentofcirculatingfibrocytes tothelung,andtargetingchemokinesinvolvedinfibrocyte migra-tion,reducefibroticburdeninmousemodelsofpulmonaryfibrosis [15 ,17 ,35] .Studiesfromourlaboratoryandothersshow that mul-tiple factors secreted by fibrocytes act in a paracrine manner to activateresidentfibroblasts,stimulatingtheirproliferationand dif-ferentiationduringthepathogenesisofpulmonaryfibrosis [20 ,36] . Inparticular, the adoptive transfer of fibrocytes wassufficient to augmentthefibroticburdenininjuredlungsbutnotinuninjured lungsduringbleomycin-induced pulmonaryfibrosis [20 ,37] . How-ever,it is plausible that lung resident macrophages orfibrocytes expandedlocally to contribute to increasedfibrocyte numbers in CFlungsorother fibroticlung diseases. Thelack ofCFTR
expres-sion has been shown to cause impaired resolution of inflamma-torycells andincreased damage in CF airway epithelia andlung fibrosiswithLPSchallengeornaphthaleneinjuryinCFmice com-paredto wildtype mice [38 ,39] .Therefore, it isimportant to un-derstandthemechanisms underlyingfibrocyterecruitment or ac-tivation, and their contribution to the fibrotic burden in the CF lung.
Here, starting from the identification of fibrocytes in CF air-ways, we identified gene networks that were selectively dysreg-ulatedinCFfibrocytescomparedtocontrol fibrocytesusing next-generationRNA-seqanalysis.ThelossofCFTR expressioninCF fi-brocytessuggeststhatCFTR exertsitsregulatoryrolein fibrocyte-drivenfibroticlungdisease.Specifically,geneontologyanalysis re-vealedthedownregulationofCFTR-dependentionchannels, delta-508interactome,Nrf2interactome,andgenesinvolvedin stromal-epithelial crosstalk. The expression of CFTR by fibrocytes is con-sistentwithpreviousreportsonCFTRexpressionbynon-epithelial cellsincludingmacrophages,smoothmusclecells,andendothelial cells [40 ,41] .Zhang etal.showedthatmacrophages derivedfrom peripheralbloodmonocytesofCFpatientshavelowlevelsofCFTR expressioncomparedto non-CFmacrophages [42] .Moreover, pre-viousstudiesshowthatduringoxidativestressdecreasedCFTR ex-pression is due to the transcriptional repression but not due to decreasedstability [43 ,44] .However,futurestudiesare warranted tounderstandthetranscriptionalregulationandfunctionsofCFTR in fibrocytes. Detailed transcriptome analysis of CF fibrocytes al-lowedustohypothesizethatthelossofCFTRwasassociatedwith theactivationofapro-fibroticgeneprograminCFfibrocytes.Also, theobserved decreaseinthe expressionofgenes associatedwith delta-508 interactome(CDH3,CTSD),nrf2 interactome(SERPINA1, ELN),andstromalepithelialcross-talk(MMP9,LCN2)suggest that CFTR in fibrocytes might play a role in fibroticlung remodeling. Therefore, future studies are warranted to identify the potential roleofCFTRinfibrocyteactivityandfibroticremodelingintheCF lung.Additionally,variousgenes involvedinpositive regulationof cell migration(SELP, SMOC2,andPTGER3), negativeregulationof woundhealing(SERPINB2,AJAP1,GP5),andmuscletissue develop-mentandcontraction(FGF1,MYH13,GATA4) wereupregulatedin CFfibrocytes.CFfibrocytesdisplayanincreaseinthetranscriptsof FGF1, apro-fibroticgrowthfactor implicatedin muscletissue de-velopment andregeneration. Published studies have shownFGF1 to cause growth and migration of both endothelial and smooth musclecells.Moreover,vascularabnormalitiesandsmoothmuscle remodelingaresignificantfactorsthatcontributetolungpathology inpatientswithCF [45–47] .
CF lung disease in humans shares several features of fibrotic remodelingseen inIPF,includingmyofibroblastaccumulationand excessive collagen deposition [48] . Fibrocytes have been impli-catedinthepathogenesisofseveralchronicinflammatorydiseases such as necrotizing enterocolitis (NEC), asthma, Graves’ disease, andrheumatoidarthritis [49–52] .Consistent withprevious work, we detected a significant number of fibrocytes in the BAL fluid ofCFpatients. Inparticular,fibrocyteinductioninGrave’sdisease andNEChasbeenshowntoaggravateaninflammatory response. Nonetheless,definingthepresenceandroleoffibrocytesinchronic fibroticlung regions andBAL fluid warrants futureinvestigations intothe contributionoffibrocytesin CFlung fibrosis and inflam-mation.
Declaration of Competing Interests
Theauthorsdeclarenofinancialandpersonalrelationshipswith otherpeopleororganizationsthatcouldinfluenceourwork.
CRediT authorship contribution statement
Rajesh K. Kasam: Conceptualization, Methodology, Formal analysis, Writing - original draft, Writing - review & editing. Prathibha R. Gajjala: Resources, Methodology, Investigation, For-malanalysis,Writing -review &editing. Anil G. Jegga: Resources, Methodology, Investigation, Formal analysis, Writing - review & editing. Jennifer A. Courtney: Resources, Methodology, Investiga-tion, Formal analysis, Writing - review & editing. Scott H. Ran- dell: Resources,Methodology,Investigation,Formal analysis, Writ-ing - review & editing. Elizabeth L. Kramer: Resources, Method-ology, Investigation, Formal analysis, Writing - review & edit-ing. John P. Clancy: Resources,Methodology, Investigation,Formal analysis, Writing - review & editing. Satish K. Madala: Concep-tualization,Methodology, Formalanalysis,Writing-original draft, Writing-review&editing.
Acknowledgments
This research was supported by funds from Cystic Fibrosis Foundation (CFF) CLANCY15R0 (JPC) , CFF BOUCHE15R0 (SHR),DoD W81XWH-17 –1 –0 6 6 6 (SKM), NIH 1R01 HL134801 (SKM), NIH AI137309 (SKM) and NIH P30DK065988 (SHR). We wouldlike to thankFlowCytometryCoreatCincinnatiChildren’sHospital Med-icalCenterforassistancewithflowcytometryanalysis.
Supplementary materials
Supplementary material associated with this article can be found,intheonlineversion,atdoi:10.1016/j.jcf.2020.06.011 .
References
[1] ZhangW,FujiiN,NarenAP.Recentadvancesandnewperspectivesintargeting CFTRfortherapyofcysticfibrosisandenterotoxin-inducedsecretorydiarrheas. FutureMedChem2012;4(3):329–45.
[2] RoweSM,ClancyJP.Advancesincysticfibrosistherapies.CurrOpinPediatr 2006;18(6):604–13.
[3] HarrisWT,KellyDR,ZhouY,WangD,MacEwenM,HagoodJS,etal. Myofi-broblastdifferentiationandenhancedTGF-Bsignalingincysticfibrosislung disease.PLoSONE2013;8(8):e70196.
[4] LivraghiA,Randell SH.Cysticfibrosisand otherrespiratory diseasesof im-pairedmucusclearance.ToxicolPathol2007;35(1):116–29.
[5] Hardie WD, Bejarano PA, Miller MA, Yankaskas JR, Ritter JH, Whitsett JA, etal.Immunolocalizationoftransforminggrowthfactoralphaandepidermal growthfactorreceptorinlungsofpatientswithcysticfibrosis.PediatricDev Pathol1999;2(5):415–23.
[6] HubeauC, Puchelle E, GaillardD. Distinctpattern ofimmune cell popula-tioninthelungofhumanfetuseswithcysticfibrosis.JAllergyClinImmunol 2001;108(4):524–9.
[7] AwadallaM,MiyawakiS,AbouAlaiwaMH,AdamRJ,BouzekDC,MichalskiAS, etal.Earlyairwaystructuralchangesincysticfibrosispigsasadeterminantof particledistributionanddeposition.AnnBiomedEng2014;42(4):915–27. [8] AdamRJ,Michalski AS, Bauer C,Abou AlaiwaMH, Gross TJ, AwadallaMS,
etal.Airtrappingandairflowobstructioninnewborncysticfibrosispiglets. AmJRespirCritCareMed2013;188(12):1434–41.
[9] TanHL,RegameyN,BrownS,BushA,LloydCM,DaviesJC.TheTh17pathway incysticfibrosislungdisease.AmJRespirCritCareMed2011;184(2):252–8. [10] TiringerK,TreisA,FucikP,GonaM,GruberS,RennerS,etal.ATh17-and
Th2-skewedcytokine profile incystic fibrosis lungs represents a potential riskfactorforPseudomonasaeruginosainfection.AmJRespirCritCareMed 2013;187(6):621–9.
[11]SinghB,KasamRK,SontakeV,WynnTA,MadalaSK.Repetitiveintradermal bleomycininjectionsevokeT-helpercell2cytokine-drivenpulmonaryfibrosis. AmJPhysiolLungCellMolPhysiol2017;313(5):L796–806.
[12] Hardie WD, Le Cras TD, Jiang K, Tichelaar JW, Azhar M, Korfhagen TR. Conditionalexpression oftransforminggrowthfactor-alphain adultmouse lung causes pulmonary fibrosis. Am J Physiol Lung Cell Mol Physiol 2004;286(4):L741–9.
[13]SelmanM, KingTE, PardoA.Idiopathic pulmonaryfibrosis: prevailingand evolvinghypothesesaboutitspathogenesisandimplicationsfortherapy.Ann InternMed2001;134(2):136–51.
[14]PillingD,FanT,HuangD,KaulB,GomerRH.Identificationofmarkersthat distinguishmonocyte-derivedfibrocytesfrommonocytes,macrophages,and fi-broblasts.PLoSONE2009;4(10):e7475.
[15]MadalaSK,EdukullaR,SchmidtS,DavidsonC,IkegamiM,HardieWD.Bone marrow-derived stromalcells areinvasive and hyperproliferative and alter transforminggrowthfactor-alpha-inducedpulmonaryfibrosis.AmJRespirCell MolBiol2014;50(4):777–86.
[16]MoellerA,GilpinSE,AskK,CoxG,CookD,GauldieJ,etal.Circulating fibro-cytesareanindicatorofpoorprognosisinidiopathicpulmonaryfibrosis.Am JRespirCritCareMed2009;179(7):588–94.
[17]Moore BB, Kolodsick JE, Thannickal VJ, Cooke K, Moore TA, Hogaboam C, etal.CCR2-mediatedrecruitmentoffibrocytestothealveolarspaceafter fi-broticinjury.AmJPathol2005;166(3):675–84.
[18]Keeley EC, Mehrad B, Strieter RM. Fibrocytes: bringing new insights into mechanisms of inflammation and fibrosis. Int J Biochem Cell Biol 2010;42(4):535–42.
[19]KasamRK,ReddyGB,JeggaAG,MadalaSK.Dysregulationofmesenchymalcell survivalpathwaysinseverefibroticlungdisease:theeffectofnintedanib ther-apy.FrontPharmacol2019;10:532.
[20]Sontake V, Shanmukhappa SK, DiPasquale BA, Reddy GB, Medvedovic M, HardieWD,etal.FibrocytesregulateWilmstumor1-positivecell accumula-tioninseverefibroticlungdisease.JImmunol2015;195(8):3978–91. [21]TrapnellC,PachterL, SalzbergSL.TopHat:discoveringsplice junctionswith
RNA-Seq.Bioinformatics2009;25(9):1105–11.
[22]LiH,HandsakerB,WysokerA,FennellT,RuanJ,HomerN,etal.Thesequence alignment/mapformatandSAMtools.Bioinformatics2009;25(16):2078–9. [23]MorganM,AndersS,LawrenceM,AboyounP,PagesH,GentlemanR.
Short-Read:abioconductorpackageforinput,qualityassessmentandexplorationof high-throughputsequencedata.Bioinformatics2009;25(19):2607–8. [24]IhakaRaGR.R:alanguagefordataanalysisandgraphics.JComputGraphStat
1996;5:299–344.
[25]Anders S, McCarthy DJ, Chen Y, Okoniewski M, Smyth GK, Huber W, etal.Count-baseddifferentialexpressionanalysisofRNAsequencingdata us-ingRandBioconductor.NatProtoc2013;8(9):1765–86.
[26]FreudenbergJM,SivaganesanS,WagnerM,MedvedovicM.A semi-paramet-ricBayesianmodelforunsuperviseddifferentialco-expressionanalysis.BMC Bioinformatics2010;11:234.
[27]Chen J, Bardes EE, Aronow BJ, Jegga AG. ToppGene Suite for gene list enrichment analysis and candidate gene prioritization. Nucleic Acids Res 2009;37(WebServerissue):W305–11.
[28]MooreBB,KolbM.Fibrocytesandprogressionoffibroticlungdisease.Ready forshowtime?AmJRespirCritCareMed2014;190(12):1338–9.
[29]LaParDJ,BurdickMD,EmaminiaA, HarrisDA, StrieterBA,Liu L,etal. Cir-culatingfibrocytescorrelatewithbronchiolitisobliteranssyndrome develop-mentafterlungtransplantation:anovelclinicalbiomarker.AnnThoracSurg 2011;92(2):470–7discussion7.
[30]MathaiSK,GulatiM,PengX,RussellTR,ShawAC,RubinowitzAN,etal. Circu-latingmonocytesfromsystemicsclerosispatientswithinterstitiallungdisease showanenhancedprofibroticphenotype.LabInvest2010;90(6):812–23. [31] MehradB,BurdickMD,ZismanDA,KeaneMP,BelperioJA,StrieterRM.
Cir-culatingperipheralbloodfibrocytesinhumanfibroticinterstitiallungdisease. BiochemBiophysResCommun2007;353(1):104–8.
[32]BorieR,QuesnelC,PhinS,DebrayMP,Marchal-Somme J,Tiev K,etal. Detec-tionofalveolarfibrocytesinidiopathicpulmonaryfibrosisandsystemic scle-rosis.PLoSONE2013;8(1):e53736.
[33]SunL,LouieMC,VannellaKM,WilkeCA,LeVineAM,MooreBB,etal.New concepts of IL-10-induced lung fibrosis: fibrocyte recruitment and M2 activation in a CCL2/CCR2 axis. Am J Physiol Lung Cell Mol Physiol 2011;300(3):L341–53.
[34]PhillipsRJ,BurdickMD,HongK,LutzMA,MurrayLA,XueYY,etal.Circulating fibrocytestraffictothelungsinresponsetoCXCL12andmediatefibrosis.JClin Invest2004;114(3):438–46.
[35]Inomata M,Kamio K,AzumaA, MatsudaK, KokuhoN,MiuraY, etal. Pir-fenidoneinhibitsfibrocyteaccumulationinthe lungsinbleomycin-induced murinepulmonaryfibrosis.RespirRes2014;15:16.
[36]Sontake V, Gajjala PR, Kasam RK, Madala SK. New therapeutics based on emerging concepts in pulmonary fibrosis. Expert Opin Ther Targets 2019;23(1):69–81.
[37]KleavelandKR,Velikoff M,YangJ,AgarwalM,RippeRA,MooreBB,etal. Fi-brocytesarenotanessentialsourceoftypeIcollagenduringlungfibrosis.J Immunol2014;193(10):5229–39.
[38]BrusciaEM, ZhangPX, BaroneC,ScholteBJ,HomerR,KrauseDS,etal. In-creased susceptibilityofCftr-/-mice toLPS-inducedlungremodeling.AmJ PhysiolLungCellMolPhysiol2016;310(8):L711–19.
[39]Carvalho-Oliveira IM, Charro N, Aarbiou J, Buijs-Offerman RM, Wilke M, Schettgen T, etal. Proteomic analysis of naphthalene-induced airway ep-ithelialinjuryand repairinacystic fibrosismouse model.JProteome Res 2009;8(7):3606–16.
[41] VandebrouckC,MelinP,NorezC,RobertR,GuibertC,MetteyY,etal.Evidence thatCFTRisexpressedinrattrachealsmoothmusclecellsandcontributesto bronchodilation.RespirRes2006;7:113.
[42]Zhang S, Shrestha CL, Kopp BT. Cystic fibrosis transmembrane conduc-tanceregulator(CFTR)modulatorshavedifferentialeffectsoncysticfibrosis macrophagefunction.SciRep2018;8(1):17066.
[43]RabA,BartoszewskiR,JurkuvenaiteA,WakefieldJ,CollawnJF,BebokZ. Endo-plasmicreticulumstressandtheunfoldedproteinresponseregulategenomic cysticfibrosistransmembraneconductanceregulatorexpression.AmJPhysiol CellPhysiol2007;292(2):C756–66.
[44] BartoszewskiR, Rab A, TwittyG, StevensonL, FortenberryJ, PiotrowskiA, etal.Themechanismofcysticfibrosistransmembraneconductanceregulator transcriptionalrepressionduringtheunfoldedproteinresponse.JBiolChem 2008;283(18):12154–65.
[45]HannonK,KudlaAJ,McAvoyMJ,ClaseKL,OlwinBB.Differentiallyexpressed fibroblastgrowthfactorsregulateskeletalmuscledevelopmentthrough au-tocrineandparacrinemechanisms.JCellBiol1996;132(6):1151–9.
[46]CookDP,Rector MV,BouzekDC,MichalskiAS,GansemerND,ReznikovLR, etal. Cysticfibrosis transmembrane conductanceregulator insarcoplasmic reticulumofairwaysmoothmuscle.implicationsforairwaycontractility.AmJ RespirCritCareMed.2016;193(4):417–26.
[47]HaysSR,FerrandoRE,CarterR,WongHH,Woodruff PG.Structuralchangesto airwaysmoothmuscleincysticfibrosis.Thorax2005;60(3):226–8.
[48]HarrisWT,BoydJT,McPhailGL,BrodyAS,SzczesniakRD,KorbeeLL,etal. Con-strictiveBronchiolitisinCysticFibrosisAdolescentswithRefractoryPulmonary Decline.AnnAmThoracSoc2016;13(12):2174–83.
[49]LiuY,QingjuanS,GaoZ,DengC,WangY,GuoC.Circulatingfibrocytesare in-volvedininflammationandleukocytetraffickinginneonateswithnecrotizing enterocolitis.Medicine(Baltimore)2017;96(26):e7400.
[50]DouglasRS,AfifiyanNF,HwangCJ,ChongK,HaiderU,RichardsP,etal. In-creasedgenerationoffibrocytesinthyroid-associatedophthalmopathy.JClin EndocrinolMetab2010;95(1):430–8.
[51]Shipe R,BurdickMD,StrieterBA,Liu L,ShimYM,SungSS, etal. Number, activation,anddifferentiationofcirculatingfibrocytescorrelatewith asthma severity.JAllergyClinImmunol2016;137(3)750–7e3.
