Background and Objective:

Materials and Methods:

Results:

Conclusion:CD14+ cells purified from bone marrow and blood of RA and OA patients undergoinghip replacement surgery were profiled with Affymetrix HG-U133 Plus 2.0 arrays. TheBioRetis database was used for array analyses. For functional interpretation of arraydata Ingenuity Pathway Analysis and Gene Ontology were applied. 68 differentreference transcriptomes of healthy bone marrow progenitors and various stages ofactivated and differentiated monocytes were used for more detailed functionalinterpretation. Flow cytometry was applied for profiling of monocyte subsets:CD14+CD16-, CD14+CD16+ and CD14dimCD16+ in bone marrow, blood and synovialfluid samples. immunoClust algorithm was applied for automated analyses of FACSdata.

Alteration of RA monocytes was evident already in bone marrow and wascharacterized by increased monocytopoiesis and/or premature release intocirculation. Comprehensive analyses of Mo profiles by reference transcriptomesprovided a very detailed insight into gene patterns related to maturation,differentiation and activation of Mo. Flow cytometry analyses of Mo subsets in RAbone marrow, blood and synovial fluid depicted increased expression of CD16 on Moduring their maturation and differentiation from bone marrow into blood and duringtheir migration and activation from blood into synovium. The most obvious activationof Mo occurs in the joint and is depicted by a specific Mo subset that expresses thehighest level of CD14, CD16 & CD163.

Most biologics for rheumatoid arthritis (RA) target processes involved in monocyte activation. To determine when, where and how monocytes become involved in pathogenesisof RA, we analysed monocytes from bone marrow, blood and synovial fluid by gene-expression profiling and cytometry, and compared their activation patterns withosteoarthritis (OA).

ArthroMarkgrantno01EC1009A

Geneexpressionprofilingandcytometryanalysisfrombonemarrowmonocytes,blood

andsynovialfluidfromrheumatoidandosteoarthritispatients

BiljanaSmiljanovic1,TillSorensen1,MarcBonin-Andresen1,BrunoStuhlmuller1,GerdR.Burmester1,AndreasGrutzkau2,ThomasHaupl1

1DepartmentofRheumatologyandClinicalImmunology,CharitéUniversityHospital,Berlin,Germany,2GermanArthritisResearchCenter,Berlin,Germany

MarcBoninDepartmentofRheumatologyandClinicalImmunologyCharitéUniversityHospitalCharitéplatz1D-10117BerlinGermany

Tel:+49(0)30450513296Fax:+49(0)30450513968E-Mail: marc.bonin@charite.deWeb:www.charite-bioinformatik.de

Contacts:

www.charite-bioinformatik.de

1.TranscriptomesofRAbonemarrowandbloodmonocytesshowedaminoroverlapandmoreprominentalterationsinblood

2.FunctionalanalysisofRA-BM&RA-PBtranscriptomesemphasisedgenesinvolvedininflammationandhemopoiesis

3.FunctionalanalysesofRA-BM&RA-PBprofilesby68referencetranscriptomesshowedtheshiftstowardimmatureprofiles(shifttotheleft)

4.MonocytematurationduringmigrationfrombonemarrowviabloodtosynovialfluidinRApatientsanalyzedbyFACS

RAbonemarrowMoprofileanalysedbyMolecularnetwork- Ingenuity(IPA)&Geneontology(GO)

RAbloodMoprofileanalysedbyMolecularnetwork- Ingenuity(IPA)&Geneontology(GO)

(1)RAtranscriptomeofbonemarrow(BM)Mo (2)RAtranscriptomeofblood(PB)Mo (3)Principalcomponentanalysis(PCA)ofBM&PBprofilesfromRA&OAMo

221differentiallyexpressedprobe-setsbetweenRA&OAbonemarrowMo

379differentiallyexpressedprobe-setsbetweenRA&OAbloodMo

571differentiallyexpressedprobe-setsinBM&PBMofromRA&OApatients

OA-BMOA-PBRA_BMRA_PB

RA

OA

RA

OA

Biologicalprocesses Genesanti-apoptosis Up-reg:CLU,FAS,IL10,SOCS3,YWHAZ

Down-reg:ANXA1,NOTCH2NL,VNN1hemopoiesis Up-reg:FLT3,IL10,PICALM,ZBTB16

Down-reg:NOTCH2NLInflammatory Up-reg:FPR2,IL10,IL8,TNFAIP6,TPST1

Down-reg:ANXA1,C3AR1,VNN1responsecelladhesion Up-reg:ALCAM,ARF6,FPR2,ITGA4,ITGA6,ITGB1

Down-reg:CD36

Biologicalprocesses Genesinflammatoryresponse Up-reg:CCR2,CD163,FPR2,LTB4R,NLRC4,PXK

Down-reg:CAMK1D,CCL2,CSF1R,ITGAL,MIF,TNFanti-apoptosis Up-reg:BAG4,CLEC5A,NAIP,SERPINB2,THBS1,VNN1

Down-reg:CCL2,FOXO1,RIPK2,TCF7L2,TNFcellcyclearrest Up-reg:CDKN2B,GADD45A,MAP2K6,MYC,THBS1

Down-reg:CDKN1C,GAS2L1,TCF7L2hemopoiesis Up-reg:PICALM,RUNX1

Down-reg:BCL11A,CSF1R,IKZF1,ROGDI

• RA-BMprofileshowedamoreimmatureprofilethanOA-BMandischaracterisedbyearlymyelopoieticgenesandgenesup-regulatedbyG-CSFthatindicatedfasteregressofMofromBM

• RA-PBprofileexhibitedalsoamoreimmatureprofilethanOA-PB,characterizedbylatemyelopoieticgenesfromBM,pronouncedG-CSFeffectsandabsenceofgenesspecificforCD16+Mo

- Mofrombonemarrow,bloodandsynovialfluidwereanalysedbyFACS

- Mosubsets:CD14+CD16- &CD14~CD16+- Frequency,CD14,CD16&CD163expressionofMowereanalysed

- immunoClustsoftwareforautomatedanalysisofFACSdata- CD14+CD16- MoarethedominantsubsetinBMfromRA&OApatients.

- FrequencyofCD14~CD16+Mo,whichisthemoredifferentiatedMosubset,increasedinPBandexpressionofCD14decreasedwhileexpressionofCD16increasedonthissubset.

- CD14++CD16+subsetappearsonlyinSFandrepresentsthemostdifferentiated(thehighestCD16expression)andthemostactivated(thehighestlevelofCD14&CD163)Mosubset.

AnalysisofRA-BMprofilebyreferencetranscriptomes68Referencetranscritpomesofbonemarrow&bloodsamples AnalysisofRA-PBprofilebyreferencetranscriptomes

Bonemarrow(RA&OA) Blood(RA&OA) SF&Blood(RA)Mosubsets

CD14+CD16- &CD14~CD16+BM&PBfromRA&OA

MosubsetsCD14+CD16+&CD14~CD16+

&CD14++CD16+fromPB&SFofRA

RA

OA

RA

OA

RA-BMprofileUp-reginRA-BM

Down-reginRA-BM

Frequency CD14expression CD16expression CD163expression

1.Latemyelopoiesis

2.WeakTNF/LPSeffect

1.Earlymyelopoiesis

2.WeakTNF/LPSeffect

3. G-CSF effect

1.Myelopoiesis

2.TNF/LPSeffect

1. Early&Latemyelopoiesis

2.TNF/LPSeffect

3. G-CSF effect

RA-PBprofile

Up-reginRA-PB

Down-reginRA-PB

3.CD16+pattern