Research Article All-Trans Retinoic Acid Improves the Effects of …downloads.hindawi.com/journals/sci/2015/484528.pdf · 2019-07-31 · Research Article All-Trans Retinoic Acid Improves

Research ArticleAll-Trans Retinoic Acid Improves the Effects of BoneMarrow-Derived Mesenchymal Stem Cells on the Treatment ofAnkylosing Spondylitis An In Vitro Study

Deng Li1 Peng Wang12 Yuxi Li1 Zhongyu Xie1 Le Wang1 Hongjun Su2

Wen Deng2 Yanfeng Wu2 and Huiyong Shen1

1Department of Orthopedics Sun Yat-sen Memorial Hospital Sun Yat-sen University Guangzhou 510120 China2Center for Biotherapy Sun Yat-sen Memorial Hospital Sun Yat-sen University Guangzhou 510120 China

Correspondence should be addressed to Yanfeng Wu wuyanfengcn126com and Huiyong Shen shenhuiyongaliyuncom

Received 8 January 2015 Revised 11 March 2015 Accepted 11 March 2015

Academic Editor Antonio Salgado

Copyright copy 2015 Deng Li et al This is an open access article distributed under the Creative Commons Attribution License whichpermits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Previous studies have demonstrated the immunosuppressive effects of both all-trans retinoic acid (ATRA) and mesenchymal stemcells (MSCs) The present study aimed to assess the immunoregulatory effects of ATRA on MSCs in the treatment of ankylosingspondylitis (AS) Bone marrow-derived MSCs from healthy donors were pretreated with ATRA and cocultured with CD328-activated peripheral blood mononuclear cells (PBMCs) derived from AS patients Frequencies of Th17 and regulatory T (Treg)cells were analyzed using flow cytometry The secretion and the mRNA level of key cytokines were measured with cytometric beadarray and quantitative real-time PCR respectively ATRA pretreatment increased interleukin-6 (IL-6) secretion of MSCsTh17 andTreg subset populations were increased and reduced by ATRA-pretreatedMSCs respectively ATRA-pretreatedMSCs significantlydecreased not only the vital pathogenic cytokine in AS tumor necrosis factor-120572 (TNF-120572) but also AS-boosting factors interleukin-17 (IL-17A) and interferon-120574 (IFN-120574) These results indicated that IL-6 may be a potential protective factor in AS and highlightedthe promising role of ATRA in improving the efficacy of MSC-based treatment of AS

1 IntroductionAnkylosing spondylitis (AS) is a chronic progressive inflam-matory disease affecting primarily the axial skeleton AS isconsidered as an autoimmune disease and interleukin-17A(IL-17A-) producing Th17 cells are involved in AS pathogen-esis [1 2]

For the first time we have reported that allogenic intrave-nous infusion of bone marrow-derived mesenchymal stemcells was an effective and safemethod for the treatment of ASespecially cases for which etanercept andNSAIDs are ineffec-tive [3] However the underlying mechanisms remain largelyunknown Mesenchymal stem cells (MSCs) are multipotentprogenitor cells which exhibited immunomodulatory capac-ity [4] and low immunogenicity [5] MSCs have been suc-cessfully used in the treatment of a number of autoimmuneinflammatory diseases such as Crohnrsquos disease [6] and sys-temic lupus erythematosus [7] on the base of their immuno-suppressive ability The mechanisms of immunomodulation

are quite complicated and still not completely clear Somestudies demonstrated that cell-cell contact is involved inthe immunosuppressive action such as inhibition of Th17differentiation and IL-17 secretion [8 9] MSCs can alsoattract effector T cells through secreting chemokines to facil-itate direct immunomodulation [10] or trigger FASFASL-induced apoptosis [11] Parallel to this MSC secretome is alsoreported contributing to cell-contact independent effectsThe most studied soluble molecules derived from MSCsinclude indoleamine 23-dioxygenase (IDO) prostaglandinE2 (PGE2) transforming growth factor-beta (TGF-120573) nitricoxide (NO) and hepatocyte growth factor (HGF) [10 12]

All-trans retinoic acid (ATRA) a representative metabo-lite of retinol is involved in a number of biological activities ofretinol ATRAplays a key role in cell proliferation differentia-tion andmaturation [13] In addition ATRA has been shownto regulate innate immunity through inducing regulatory Tcells (Tregs) and suppressingTh17 differentiation [14] Several

Hindawi Publishing CorporationStem Cells InternationalVolume 2015 Article ID 484528 10 pageshttpdxdoiorg1011552015484528

2 Stem Cells International

studies have documented the efficacy of ATRA in the treat-ment of autoimmune diseases [15ndash17] The retinol level in ASpatients was significantly lower than that in healthy controls[18] It has also been observed that ATRA administrationreduced the frequency of Th17 and inhibited the secretion ofTNF-120572 in peripheral blood of AS patients [19]

Taken together we hypothesize that ATRA is effective forthe treatment of AS through regulating the immunomodula-tory capacity of MSCs In the present study we coculturedMSCs and peripheral blood mononuclear cells (PBMCs)and examined the frequencies of Treg and Th17 subsets andthe secretion of key cytokines We defined the pathogenicTh17 cells with a combination of the surface markers ofCD4 CCR4 and CCR6 [20] To the best of our knowledgethis is the first study showing that ATRA improved theimmunomodulatory ability of MSCs for the treatment of AS

2 Materials and Methods

21 Isolation of MSCs and PBMCs This study was approvedby the Ethics Committee of Sun Yat-sen UniversityGuangzhou China Healthy volunteers (119899 = 20 15 males 5females age 18ndash28 years) were recruited as donors of bonemarrow Bone marrow was extracted from the posteriorsuperior iliac spine under a sterile condition and MSCs wereisolated according to standardized procedures [21] MSCs ofthe fourth passage were used in experiments PBMCs wereisolated from the peripheral blood of AS patients (119899 = 2418 males 6 females age 16ndash40 years) using Ficoll-Hypaquegradient centrifugation Patients were selected accordingto modified New York criteria [22] Informed consent wasobtained from all donors and patients

22 Trilineage Differentiation Potential Assays of MSCs

Osteogenic Differentiation MSCs were seeded into six-wellplates (1 times 105 cellswell) in a total volume of 3mL DMEMmedium supplemented with 10 fetal bovine serum (FBS)50mgL ascorbic acid (Sigma) 10mM 120573-glycerophosphate(Sigma) and 100 nM dexamethasone (Sigma) The mediumwas replaced every three days Total culture duration was 21days Alizarin red staining was used

Chondrogenic Differentiation MSCs (25 times 105 cells) werecentrifuged at 600 g for 5 minutes in a 15mL conical tubeCells were cultured with high-glucose DMEM supplement-ed with 1 ITS-Premix (Corning) 50mgL ascorbic acid(Sigma) 1mMsodiumpyruvate (Sigma) 100 nMdexametha-sone (Sigma) and 10 ngmL transforming growth factor-1205733(RampD) The medium was replaced every three days Totalculture duration was 21 days

Adipogenic Differentiation MSCs were seeded into six-wellplates (1 times 105 cellswell) DMEMmediumwas supplementedwith 10 FBS 1120583M dexamethasone (Sigma) 10 120583gmL insu-lin (Sigma) 05mM 3-isobutyl-1-methylxanthine (Sigma)and 02mM indomethacin (Sigma) After 3 daysrsquo inductionthe medium was replaced with DMEM containing 10120583gmLinsulin and 1 day later the medium was replaced with

the inducing medium mentioned above After three cyclesof such culture cells were coated with DMEM containing10 120583gmL insulin until 14th day Oil red O staining was usedafter paraformaldehyde fixation

23 ATRA Preparation and MSCs Pretreatment ATRA pow-der (Sigma) was dissolved in dimethyl sulfoxide (DMSO) andstocked according to the manufacturerrsquos instructions ATRAsolution was prepared with DMEM medium supplementedwith 10 FBS MSCs were seeded into six-well plates (1 times105 cellswell) in a total volume of 3mL DMEM mediumpretreated by 1 120583MATRA for 1 day 3 days and 5 days accord-ing to different experiments The MSCs cocultured withPBMCs were only pretreated for 1 day After pretreatmentthe medium containing ATRA was completely removed andMSCs were washed with phosphate buffer saline (PBS) forthree times Cells were then cultured in ATRA-free mediumMSCs of the control group were pretreated with DMSO ofequal volume to ATRA

24 Cell Culture MSCs were cocultured with PBMCs at aratio of 1 20 of MSC PBMC T cells were stimulated bypurified anti-CD3 (02 120583gmL BD Pharmingen) and anti-CD28 (1 120583gmL BD Pharmingen) antibodies Cells werecocultured in RPMI-1640 medium of an ultimate volume of3mL for 5 days The cell ratio and the coculture durationwere determined according to preliminary experiments ForCD4+ T cell proliferation analysis PBMCs were incubatedwith 5 120583M carboxyfluorescein diacetatesuccinimidyl ester(CFSE Life Technologies) for 15 minutes and washed withPBS containing 10 FBS three times before coculture Toevaluate the effects of proinflammatory cytokines on IL-6secretion of MSCs exogenous interferon-120574 (IFN-120574 Pepro-Tech) (10 ngmL) and TNF-120572 (10 ngmL RampD) were addedinto the culture medium of pretreated MSCs which werecultured for the following 5 days For cytokine measurementa noncontact coculture was also conducted using a six-welland 04 120583m pore Transwell plate system (Corning) in whichPBMCs were seeded in the upper chamber

25 Flow Cytometry MSCs were trypsinized for identifica-tion of a number of surface markers The antibodies forsurface markers were anti-CD29-PE anti-CD34-APC anti-CD44-FITC anti-CD45-FITC anti-CD90-PE anti-CD105-FITC and anti-HLA-DR-PE (all from BD Pharmingen)PBMCs were harvested through centrifugation at the endof coculture For proliferation analysis of CD4+ T cellsCFSE-incubated PBMCs were marked with anti-CD4-PE(BD Pharmingen) cells left-shifting into the square gatewere recognized as the proliferating cells and the cell countpercentage within this gate represented the proliferation rateFor Th17 analysis anti-CD4-FITC anti-CCR4-PerCP-Cy55and anti-CCR6-APC (all from BD Pharmingen) were usedThe human regulatory T cell staining kit (eBioscience) con-taining anti-CD4-FITCCD25-APC cocktail and anti-Foxp3-PE was used in Treg analysis according to the manufacturerrsquosinstruction Cells were measured in a flow cytometry system(BD FACSVerse)

Stem Cells International 3

26 Cytometric Bead Array (CBA) Culture supernatantwas collected for cytokine measurement using a CBA kit(Human Th1Th2Th17 Kit BD Pharmingen) according tothemanufacturerrsquos instruction Cytokine concentrationswerepresented as PE fluorescence intensities relative to a standardcurve

27 Quantitative Real-Time PCR (qPCR) Total RNA wasextracted using the TRIzol reagent (Life Technologies)Synthesis of cDNA was performed using a PrimeScript RTreagent kit (Takara) according tomanufacturerrsquos instructionsThe qPCR was performed on a LightCycler 480 Real-TimePCR System (Roche) using SYBR Premix Ex Taq (Takara)The PCR program was 30 s at 95∘C 40 cycles of 5 s at 95∘Cand 20 s at 60∘C Relative expression changes in mRNA levelsof genes were assessed using 2(minusΔΔct) method and normalizedto the housekeeping gene GAPDH The sequences of primersused in the qPCR assay were as follows IL-6 51015840-CCTGAA-CCTTCCAAAGATGGC-31015840 (forward) 51015840-TTCACCAGG-CAAGTCTCCTCA-31015840 (reverse) IL-17A 51015840-TCCCACGAA-ATCCAGGATGC-31015840 (forward) 51015840-GGATGTTCAGGTTGA-CCATCAC-31015840 (reverse) TNF-120572 51015840-CCTCTCTCTAATCAG-CCCTCTG-31015840 (forward) 51015840-GAGGACCTGGGAGTAGAT-GAG-31015840 (reverse) IFN-120574 51015840-TCGGTAACTGACTTGAAT-GTCCA-31015840 (forward) 51015840-TCGCTTCCCTGTTTTAGCTGC-31015840 (reverse)GAPDH 51015840-GGAGCGAGATCCCTCCAAAAT-31015840 (forward) 51015840-GGCTGTTGTCATACTTCTCATGG-31015840 (re-verse)

28 Statistical Analyses Data were presented as mean plusmn SDOne-way ANOVA or 119905-test was used according to the type ofdata Statistical analyses were conducted using SPSS software(SPSS Inc) A 119875 value less than 005 was considered to besignificantly different

3 Results31 Phenotypic Characterization and Trilineage Differentia-tion of MSCs Flow cytometric analysis was used to identifyphenotypic surface markers of MSCs All MSCs from differ-ent donors were positive of CD29 CD44 CD90 and CD105but negative of CD34 CD45 and HLA-DR confirming thetypical MSC phenotypes (Figure 1(a)) Osteogenic chon-drogenic and adipogenic differentiations were successfullyinduced (Figure 1(b))

32 MSCs Inhibited the Proliferation of CD4+ T Cells andATRA Reduced This Inhibitory Ability The proliferation ofCD4+ T cells was measured according to fluorescence inten-sities The 0 day PBMCs (noncocultured not activated byCD328) group was defined as the nonproliferation controlWhen stimulated byCD328 the proliferation rate of CD4+Tcells was extremely high (837plusmn67) suggesting an efficientproliferation When cocultured with MSCs the proliferationof CD328-stimulated CD4+ T cells was inhibited (119875 lt0001) but a slightly higher proliferation rate was observedin ATRA-pretreated group (588plusmn62) than that in DMSO-pretreated group (524 plusmn 55) (119875 lt 005) Thus theability inhibiting the proliferation of CD4+ T cells of ATRA-pretreated MSCs was lower than control (Figure 2)

33 ATRA-PretreatedMSCsReduced the Treg but Increased theTh17 Subpopulation The percentage of CD328-stimulatedTh17 subset (Th17lymphocyte) was increased from 49plusmn18to 82 plusmn 27 (119875 lt 005) by ATRA-pretreated MSCs ATRApretreatment led to an expansion of Th17 subset (Figures3(a) and 3(c)) but the control DMSO pretreatment did notcause a Th17 expansion compared with the noncoculturedgroup The percentage of CD328-stimulated Treg subset(Treglymphocyte) was decreased from 137 plusmn 21 to 25 plusmn09 (119875 lt 0001) and 36 plusmn 10 (119875 lt 0001) by ATRA-pretreated MSCs and DMSO-pretreated MSCs respectivelyThus MSCs caused a significant decrease in the Treg sub-population andATRApretreatment further reduced the Tregsubset (119875 lt 005) (Figures 3(b) and 3(d))

34 The Secretion and mRNA Level of Interleukin-6 (IL-6) Was Increased by ATRA Pretreatment We found MSCsconstitutively secreted only IL-6 but none of other cytokineswithin the spectrum of CBA kit and the secretion of IL-6 wasincreased by ATRA pretreatment in an approximately time-dependent manner which was not seen in DMSO-pretreatedgroups (Figure 4(a)) In the coculture of MSCs and PBMCsthe IL-6 level in the supernatantwas significantly increased byover 150-fold at average comparedwith the IL-6 level inMSCsor PBMCs cultured alone and was even higher in ATRA-pretreated cultures than relative DMSO-pretreated controlgroups (119875 lt 005) (Figure 4(b))These findings were found inboth contact and noncontact cocultures The mRNA level ofIL-6 was not significantly changed in PBMCs but it increaseddramatically in MSCs suggesting that IL-6 induced in thecoculture ofMSCs and PBMCs was mainly secreted byMSCsrather than PBMCs (Figure 4(c))

35 TNF-120572 and IFN-120574 Increased IL-6 Production by MSCsExogenous TNF-120572 and IFN-120574 added into the culturemediumof pretreatedMSCs significantly and synergistically enhancedthe IL-6 secretion of MSCs TNF-120572 was more efficient thanIFN-120574 in upregulating IL-6 production with an equal dose(119875 lt 0001) Just like the results presented earlier ATRA-pretreated MSCs produced higher level of IL-6 than MSCswithout ATRA stimulation (Figure 4(d))

36 Changes of Cytokines of IL-17A IFN-120574 and TNF-120572by ATRA Pretreatment MSCs increased IL-17A productionwhich was lower in the ATRA-pretreated group (1612 plusmn260 pgmL) compared with the DMSO-pretreated group(1910 plusmn 273 pgmL) (119875 lt 001) (Figure 5(a)) IFN-120574 wasincreased from 64202plusmn3740 pgmL to 98449plusmn15530 pgmL(119875 lt 0001) by DMSO-pretreated MSCs but decreased to49202 plusmn 9778 pgmL (119875 lt 005) by ATRA-pretreated MSCs(Figure 5(b))TheTNF-120572 level was significantly reduced from1185 plusmn 178 pgmL to 83 plusmn 18 pgmL (119875 lt 0001) and168 plusmn 41 pgmL (119875 lt 0001) by ATRA-pretreated MSCs andDMSO-pretreated MSCs respectively (Figure 5(c)) Thesefindings were found in both contact and noncontact cocul-tures

37 Gene Transcription of IL-17A IFN-120574 and TNF-120572 Thevariations of the mRNA levels of IL-17A IFN-120574 and


Y

Y

XX X X

SSC

0

0

FSC HLA-DR-PE0 102 103 104 105 0 102 103 104 105 0 102 103 104 105

0 102 103 104 1050 102 103 104 1050 102 103 104 1050 102 103 104 105

CD34-APC CD45-FITC

CD29-PE CD44-FITC CD90-PE CD105-FITC

250k

250k

200 k

200 k

150k

150k

100 k

100 k

50k

50k

(a)

Osteogenesis Chondrogenesis Adipogenesis

(b)

Figure 1 Identification ofMSCs (a) Phenotypic characterization based on a number of surfacemarkersMSCswere positive for CD29 CD44CD90 and CD105 but negative for HLA-DR CD34 and CD45 Red line background fluorescence of isotype controls Blue line fluorescenceintensity from selected markers119883-axis fluorescence intensity 119884-axis cell count (b) Trilineage differentiation

TNF-120572 from PBMCs were also measured MSCs significantlyincreased IL-17A gene transcription especially in ATRA pre-treatment group (119875 lt 0001) (Figure 5(d)) MSCs increasedthe mRNA level of IFN-120574 which was lower in the ATRA-pretreated group comparedwith theDMSO-pretreated group(119875 lt 001) (Figure 5(e)) MSCs unexpectedly increased thegene transcription of TNF-120572 even in ATRA pretreatmentgroup (119875 lt 0001) (Figure 5(f)) These findings were foundin both contact and noncontact cocultures

4 Discussion

CD4+ T cells are also known as helper T cells Our resultsshowed that MSCs inhibited CD4+ T cell proliferation and

the inhibitory ability of ATRA-pretreated MSCs was slightlyweaker than that of controlled DMSO-pretreated MSCsTherefore ATRA could probably affect the immune nature ofMSCs by regulating the proportions of T helper effector cells

We initially expected an increase of Treg subset anda decrease of Th17 subset populations after MSCs werecocultured with PBMCs However we observed oppositeresults Briefly MSCs caused a decline of Treg subset and aslight increase of Th17 subset populations Similar findingshave been reported by Guo et al [23] Interestingly ATRApretreatment further augmented these unexpected changesWe noted that ATRA pretreatment increased the secretionof IL-6 by MSCs and the level of IL-6 correlated with Th17subpopulation It has been confirmed that IL-6 inhibited


SSC

SSC

SSC

SSC

SSC

SSC

FSC

459

CD4-PE

317

CFSE CFSE

CFSE CFSE

PBMC

0271

PBMC + CD328

939

561 534

ATRA-MSC + PBMC + DMSO-MSC + PBMC +CD328CD328

(a)

ATRA-MSC + PBMC + CD328DMSO-MSC + PBMC + CD328PBMC + CD328

lowast

Prol

ifera

tion

rate

of C

D4+

T ce

lls (

)

100

80

60

40

20

0

(b)

Figure 2 The proliferation rate of CD4+ T lymphocytes incubated with CFSE (a) Nonactivated T cells were used as controls (b) Whenstimulated by CD328 the proliferation rate of CD4+ T cells was extremely high (837plusmn67)When cocultured withMSCs the proliferationof CD328-stimulatedCD4+T cells was inhibited but a slightly higher proliferation ratewas observed inATRA-pretreatedMSCs (588plusmn62)than that in DMSO-pretreated MSCs (524 plusmn 55) One-way ANOVA was used lowast119875 lt 005 and

119875 lt 0001

the generation of Tregs induced by TGF-120573 and caused T0

skews forTh17 [24]Therefore theTh17-skewing triggered bycoculture was probably regulated by increased production ofIL-6

IL-6 has long been recognized as a proinflammatorycytokine and a therapeutic target because IL-6 togetherwith TNF-120572 and IL-1 was upregulated in most inflammatoryconditions [25] Some studies suggested that blocking IL-6was an efficient way for the treatment of AS [26 27] How-ever the most recent randomized placebo-controlled trialsdemonstrated that blocking IL-6 with antibodies against IL-6receptor-120572 was not effective for the treatment of AS [28 29]Furthermore no axial improvements were observed in ASpatients treated with tocilizumab an antibody against IL-6receptor in a multiple center study [30] Currently no IL-6blocker is approved for the treatment of AS The level of IL-6 produced by MSCs was increased by over 150-fold whenMSCs were cocultured with activated PBMCs according toour results andwe demonstrated the upregulation of IL-6 wascell-contact independent Therefore we intended to figure

out the factors responsible We chose TNF-120572 and IFN-120574 forinvestigation Firstly both TNF-120572 and IFN-120574 were classicalproinflammatory cytokines and were elevated in variousinflammatory statuses [31] Secondly cell-free macrophageconditioned medium significantly increased the secretion ofIL-6 of MSCs [32] while TNF-120572 was the primary productionof macrophage Thirdly IFN-120574 was confirmed to be theboost signal forMSC licensing related to immunomodulationof MSCs [33] Our data showed that TNF-120572 and IFN-120574significantly and synergistically enhanced IL-6 secretion ofMSCs suggesting that the upregulation of IL-6 dependsprobably on environmental mediators and may be associatedwith the immunomodulation of MSCs These observationsraised a question on whether IL-6 is a proinflammatory or ananti-inflammatory cytokine in ASThe increased productionof IL-6 by MSCs would have aggravated the damage if IL-6was proinflammatory in AS But according to our previousstudy MSC infusion was safe and effective for the treatmentof AS [3] In addition ATRA pretreatment caused a subse-quent decline of TNF-120572 IL-17A and IFN-120574 the recognized


SSC

FSC

458SS

C

CD4-FITC

296

CCR6

-APC

CCR6

-APC

CCR6

-APC

CCR6

-APC

Isotype control

0117

PBMC + CD328

164

ATRA-MSC +PBMC + CD328

256

DMSO-MSC +PBMC + CD328

118

CCR4-PerCP-Cy55 CCR4-PerCP-Cy55


(a)

SSC

FSC

624

SSC

CD4-FITC

383

CD25

-APC

CD25

-APC

CD25

-APC

CD25

-APC

Isotype control

0337

PBMC + CD328

364


49


72

Foxp3-PE Foxp3-PE

Foxp3-PE Foxp3-PE

(b)


lowast

Th17

lym

phoc

yte (

)

15

10

5

0

(c)


lowast

Treg

lym

phoc

yte (

)

20

15

10

5

0

(d)

Figure 3 Effects of ATRA-pretreated MSCs on Th17 and Treg subpopulations (a and c) The percentage of CD328-stimulated Th17 subset(Th17lymphocyte) (b and d) The percentage of CD328-stimulated Treg subset (Treglymphocyte) One-way ANOVA was used lowast119875 lt 005and 119875 lt 0001


lowastlowast

lowastlowast

lowastlowast

lowastlowast

ATRA-pretreated MSCDMSO-pretreated MSC

Con

cent

ratio

n of

IL-6

(pg

mL)

2500

2000

1500

1000

500

0

1 1 1 3 3 3 5 5 5

1 3 5 1 3 5 1 3 5

Pretreatment(day)

Secretion (day)

(a)

ATRA-MSC + PBMC + CD328DMSO-MSC + PBMC + CD328

Con

cent

ratio

n of

IL-6

(pg

mL)

500000

450000

400000

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300000

250000

10000

8000

6000

4000

2000

0

lowast

lowastlowast lowastlowast

ATRA-pretreated MSCPBMC + CD328DMSO-pretreated MSC

PBMC alone

(b)

Relat

ive m

RNA

amou

nt o

f IL-6

Cell source of IL-6

25

20

15

10

5

0

MSC (+CD328)MSC (+PBMC+CD328)

PBMC (+CD328)PBMC (+MSC+CD328)

(c)

lowast

Con

cent

ratio

n of

IL-6

(pg

mL)

80000

60000

40000

20000

0

ATRA-pretreated MSC DMSO-pretreated MSC

MSC aloneMSC + IFN-120574

MSC + TNF-120572MSC + IFN-120574 + TNF-120572

(d)

Figure 4 Changes of IL-6 levels under different conditions (a) Concentration of IL-6 secreted by MSCs pretreated for different durations(1 day 3 days and 5 days) The secreting durations (1 day 3 days and 5 days) after pretreatment were different too Paired 119905-test was used(b) Concentration of IL-6 in coculture and noncoculture groups Paired 119905-test was used (c) Relative mRNA amount showed the principalcell source of IL-6 (d) Effects of IFN-120574 and TNF-120572 on production of IL-6 by MSCs One-way ANOVA was used lowast119875 lt 005 lowastlowast119875 lt 001 and119875 lt 0001

pathogenic cytokines in AS Taken together we speculate thatIL-6 secreted by MSCs is a reactive rather than an initiativecytokine responding to inflammatory environment and IL-6may be a protective agent rather than a pathogenic factor inthe pathogenesis of AS In fact some studies did indicate thatIL-6 displayed anti-inflammatory nature [34ndash36]

Higher frequency of peripheral Th17 and IL-17A hasbeen detected in AS patients compared to healthy controls[1 2] In the present study we found that IL-17A productionwas increased by MSCs Our results also revealed a positiveassociation between the mRNA level of IL-17A and the Th17subpopulation However we observed a reduction in IL-17Asecretion in the ATRA-pretreated group compared with theDMSO-pretreated control groupThis inconsistency suggeststhat ATRA pretreatment of MSCs caused the expansion of

Th17 subpopulation but inhibited the secretion activities ofTh17 cells A recentmultiple center clinical trial demonstratedthat anti-IL-17A monoclonal antibody secukinumab waseffective for the treatment of AS [37]Therefore we speculatethat the frequency of Th17 cells is not significantly related tothe severity of AS but the biological function of Th17 cellsplays a more important role Our results suggest that ATRApretreatment may improve the efficacy of MSC infusion forthe treatment of AS by inhibiting the secretion of IL-17A fromTh17 cells

Kezic et al [38] demonstrated that IFN-120574 worsenedboth peripheral joint and axial diseases in murine modelsAbe et al [39] found in a mouse model that upregulatedIFN-120574 was associated with ankylosing enthesitis Zhao etal [40] demonstrated that IFN-120574 significantly activated


lowast

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cent

ratio

n of

IL-17

A (p

gm

L)

250

200

150

100

50

0

(a)

lowast

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cent

ratio

n of

IFN

-120574(p

gm

L)

15000

10000

5000

0

(b)

lowast

Con

cent

ratio

n of

TN

F-120572

(pg

mL)

150

100

50

0

(c)

lowastlowast

Re

lativ

e mRN

A am

ount

of I

L-17

A

150

100

50

5

4

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1

0

(d)


lowastlowastlowastlowast

Relat

ive m

RNA

amou

nt o

f IFN

-120574

4

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2

1

0

(e)


lowast

Relat

ive m

RNA

amou

nt o

f TN

F-120572

3

2

1

0

(f)

Figure 5 Secretion and gene transcription of IL-17A IFN-120574 and TNF-120572 in the coculture ofMSCs and PBMCs (a) Results of IL-17A secretionfrom CBA (b) Results of IFN-120574 secretion from CBA (c) Results of TNF-120572 secretion from CBA (d) Results of IL-17A gene transcription fromqPCR (e) Results of IFN-120574 gene transcription from qPCR (f) Results of TNF-120572 gene transcription from qPCR One-way ANOVA was usedlowast119875 lt 005 lowastlowast119875 lt 001 and

119875 lt 0001


the promoter of the HLA-B27 gene IFN-120574 is also an activatoror a polarizing factor of classically activated macrophage(M1) the main source of TNF-120572 [41] Thus IFN-120574 is involvedin the pathogenesis of AS and associated with the severityof AS In the present study while IFN-120574 secreted by PBMCswas increased by DMSO-pretreated MSCs IFN-120574 secretionwas suppressed by ATRA-pretreated MSCs at both mRNAandprotein levelsThereforeATRApretreatmentmay be ableto improve the therapeutic effects of MSC infusion for thetreatment of AS through downregulating IFN-120574 production

TNF-120572 blockade is widely used for the treatment ofAS In the present study we observed that TNF-120572 secre-tion was significantly inhibited by MSCs although no suchchanges were found in gene transcriptions InterestinglyATRA pretreatment further enhanced the inhibition of TNF-120572 secretion These results suggested MSCs especially ATRA-pretreated MSCs were efficient TNF-120572 inhibitors The TNF-120572 inhibitor role of MSCs may be the major mechanismunderlying the therapeutic effects of MSCs for the treatmentof ASTherefore ATRA could be used to further improve theMSC infusion therapy for AS

In summary our data show that (1) ATRA pretreatmentaffects the immunomodulatory function of MSCs by influ-encing the proliferation of CD4+ T cells (2) ATRA pretreat-ment onMSCs results in a reduction ofAS-related pathogeniccytokines IL-17A IFN-120574 and TNF-120572 (3) a significant reduc-tion of TNF-120572 may be the main mechanism underlying thetherapeutic effects of MSC infusion in AS patients and ATRApretreatment may further improve the efficacy (4) IL-6 isupregulated by ATRA and environmental proinflammatorymediators and it may act as an anti-inflammatory cytokinein AS though it leads to an expansion ofTh17 subpopulationOur findings may improve the therapeutic effects of MSCinfusion for the treatment of AS But further in vivo studiesare needed to confirm these statements

Conflict of Interests

The authors declare no conflict of interests

Authorsrsquo Contribution

Deng Li and Peng Wang contributed equally to this work

Acknowledgments

The authors thank Dr Jing Wei for help with flow cytometricanalysis and Dr Xinjun Liang for insightful discussion Thiswork was supported by the National Natural Science Foun-dation of China (81271951 and 81401850) and EngineeringTechnology Research Center for Comprehensive Diagnosisand Treatment of Ankylosing Spondylitis of GuangdongHigher Education Institutes

References

[1] A Taylan I Sari D L Kozaci et al ldquoEvaluation of the T helper17 axis in ankylosing spondylitisrdquo Rheumatology Internationalvol 32 no 8 pp 2511ndash2515 2012

[2] H Shen J C Goodall and J S Hill Gaston ldquoFrequencyand phenotype of peripheral blood Th17 cells in ankylosingspondylitis and rheumatoid arthritisrdquo Arthritis amp Rheumatismvol 60 no 6 pp 1647ndash1656 2009

[3] P Wang Y Li L Huang et al ldquoEffects and safety of allogenicmesenchymal stem cell intravenous infusion in active ankylos-ing spondylitis patients who failed nsaids a 20-week clinicaltrial rdquo Cell Transplantation vol 23 no 10 pp 1293ndash1303 2014

[4] A Gebler O Zabel and B Seliger ldquoThe immunomodulatorycapacity of mesenchymal stem cellsrdquo Trends in MolecularMedicine vol 18 no 2 pp 128ndash134 2012

[5] K Le Blanc C Tammik K Rosendahl E Zetterberg andO Ringden ldquoHLA expression and immunologic properties ofdifferentiated and undifferentiated mesenchymal stem cellsrdquoExperimental Hematology vol 31 no 10 pp 890ndash896 2003

[6] G M Forbes M J Sturm R W Leong et al ldquoA phase 2 studyof allogeneic mesenchymal stromal cells for luminal Crohnrsquosdisease refractory to biologic therapyrdquoClinical Gastroenterologyand Hepatology vol 12 no 1 pp 64ndash71 2014

[7] J Liang H Zhang B Hua et al ldquoAllogenic mesenchymal stemcells transplantation in refractory systemic lupus erythemato-sus a pilot clinical studyrdquoAnnals of the Rheumatic Diseases vol69 no 8 pp 1423ndash1429 2010

[8] M M Duffy J Pindjakova S A Hanley et al ldquoMesenchymalstem cell inhibition of T-helper 17 cell- differentiation is trig-gered by cell-cell contact and mediated by prostaglandin E2 viathe EP4 receptorrdquo European Journal of Immunology vol 41 no10 pp 2840ndash2851 2011

[9] P Luz-Crawford D Noel X Fernandez et al ldquoMesenchymalstem cells repress Th17 molecular program through the PD-1pathwayrdquo PLoS ONE vol 7 no 9 Article ID e45272 2012

[10] G Ren L Zhang X Zhao et al ldquoMesenchymal stem cell-mediated immunosuppression occurs via concerted action ofchemokines and nitric oxiderdquo Cell Stem Cell vol 2 no 2 pp141ndash150 2008

[11] K Akiyama C Chen D Wang et al ldquoMesenchymal-stem-cell-induced immunoregulation involves FAS-ligand-FAS-mediated T cell apoptosisrdquoCell Stem Cell vol 10 no 5 pp 544ndash555 2012

[12] A Dorronsoro J Fernandez-Rueda K Fechter et al ldquoHumanmesenchymal stromal cell-mediated immunoregulation mech-anisms of action and clinical applicationsrdquo Bone MarrowResearch vol 2013 Article ID 203643 8 pages 2013

[13] B Cassani E J Villablanca J de Calisto S Wang and J RMora ldquoVitamin A and immune regulation role of retinoic acidin gut-associated dendritic cell education immune protectionand tolerancerdquoMolecular Aspects of Medicine vol 33 no 1 pp63ndash76 2012

[14] C Wang S G Kang H HogenEsch P E Love and CH Kim ldquoRetinoic acid determines the precise tissue tropismof inflammatory Th17 cells in the intestinerdquo The Journal ofImmunology vol 184 no 10 pp 5519ndash5526 2010

[15] Y-H Van W-H Lee S Ortiz M-H Lee H-J Qin andC-P Liu ldquoAll-trans retinoic acid inhibits type 1 diabetes byT regulatory (Treg)- dependent suppression of interferon-120574-producing T-cells without affectingTh17 cellsrdquoDiabetes vol 58no 1 pp 146ndash155 2009

[16] H Keino T Watanabe Y Sato and A A Okada ldquoAnti-inflammatory effect of retinoic acid on experimental autoim-mune uveoretinitisrdquo British Journal of Ophthalmology vol 94no 6 pp 802ndash807 2010


[17] S Xiao H Jin T Korn et al ldquoRetinoic acid increases Foxp3+regulatory T cells and inhibits development of Th17 cells byenhancing TGF-120573-driven Smad3 signaling and inhibiting IL-6and IL-23 receptor expressionrdquo Journal of Immunology vol 181no 4 pp 2277ndash2284 2008

[18] F D OrsquoShea FW L Tsui B Chiu HW Tsui M Yazdanpanahand R D Inman ldquoRetinol (vitamin A) and retinol-bindingprotein levels are decreased in ankylosing spondylitis clinicaland genetic analysisrdquo Journal of Rheumatology vol 34 no 12pp 2457ndash2459 2007

[19] K Bidad E Salehi A Jamshidi et al ldquoEffect of all-transretinoicacid on Th17 and T regulatory cell subsets in patients withankylosing spondylitisrdquo Journal of Rheumatology vol 40 no 4pp 476ndash483 2013

[20] E V Acosta-Rodriguez L Rivino J Geginat et al ldquoSurfacephenotype and antigenic specificity of human interleukin 17-producing T helper memory cellsrdquo Nature Immunology vol 8no 6 pp 639ndash646 2007

[21] B Yamout R Hourani H Salti et al ldquoBonemarrowmesenchy-mal stem cell transplantation in patients withmultiple sclerosisa pilot studyrdquo Journal of Neuroimmunology vol 227 no 1-2 pp185ndash189 2010

[22] S Van Der Linden H A Valkenburg and A Cats ldquoEvaluationof diagnostic criteria for ankylosing spondylitis A proposal formodification of the NewYork criteriardquoArthritis amp Rheumatismvol 27 no 4 pp 361ndash368 1984

[23] Z Guo C Zheng Z Chen et al ldquoFetal BM-derived mesenchy-mal stem cells promote the expansion of human Th17 cellsbut inhibit the production of Th1 cellsrdquo European Journal ofImmunology vol 39 no 10 pp 2840ndash2849 2009

[24] E Bettelli Y Carrier W Gao et al ldquoReciprocal developmentalpathways for the generation of pathogenic effector T

11986717 and

regulatory T cellsrdquoNature vol 441 no 7090 pp 235ndash238 2006[25] J Scheller A Chalaris D Schmidt-Arras and S Rose-John

ldquoThe pro- and anti-inflammatory properties of the cytokineinterleukin-6rdquo Biochimica et Biophysica Acta Molecular CellResearch vol 1813 no 5 pp 878ndash888 2011

[26] J D Cohen R Ferreira and C Jorgensen ldquoAnkylosingspondylitis refractory to tumor necrosis factor blockaderesponds to tocilizumabrdquo Journal of Rheumatology vol 38 no7 article 1527 2011

[27] J C Henes M Horger I Guenaydin L Kanz and I KoetterldquoMixed response to tocilizumab for ankylosing spondylitisrdquoAnnals of the Rheumatic Diseases vol 69 no 12 pp 2217ndash22182010

[28] J Sieper B Porter-Brown L Thompson O Harari and MDougados ldquoAssessment of short-term symptomatic efficacy oftocilizumab in ankylosing spondylitis results of randomisedplacebo-controlled trialsrdquoAnnals of the RheumaticDiseases vol73 no 1 pp 95ndash100 2014

[29] J Sieper J Braun J Kay et al ldquoSarilumab for the treatmentof ankylosing spondylitis results of a Phase II randomiseddouble-blind placebo-controlled study (ALIGN)rdquoAnnals of theRheumatic Diseases 2014

[30] F K Lekpa C Poulain D Wendling et al ldquoIs IL-6 anappropriate target to treat spondyloarthritis patients refractoryto anti-TNF therapy Amulticentre retrospective observationalstudyrdquo Arthritis Research amp Therapy vol 14 no 2 article R532012

[31] H Hemeda M Jakob A-K Ludwig B Giebel S Lang andS Brandau ldquoInterferon-120574 and tumor necrosis factor-120572 differ-entially affect cytokine expression and migration properties of

mesenchymal stem cellsrdquo Stem Cells and Development vol 19no 5 pp 693ndash706 2010

[32] K Anton D Banerjee and J Glod ldquoMacrophage-associatedmesenchymal stem cells assume an activated migratory pro-inflammatory phenotype with increased IL-6 and CXCL10secretionrdquo PLoS ONE vol 7 no 4 Article ID e35036 2012

[33] M Krampera ldquoMesenchymal stromal cell lsquolicensingrsquo a multi-step processrdquo Leukemia vol 25 no 9 pp 1408ndash1414 2011

[34] Z Xing J Gauldie G Cox et al ldquoIL-6 is an antiinflammatorycytokine required for controlling local or systemic acute inflam-matory responsesrdquoThe Journal of Clinical Investigation vol 101no 2 pp 311ndash320 1998

[35] R Starkie S R Ostrowski S Jauffred M Febbraio and BK Pedersen ldquoExercise and IL-6 infusion inhibit endotoxin-inducedTNF-alpha production in humansrdquoTheFASEB Journalvol 17 no 8 pp 884ndash886 2003

[36] S M Melief S B Geutskens W E Fibbe and H RoelofsldquoMultipotent stromal cells skew monocytes towards an anti-inflammatory interleukin-10-producing phenotype by produc-tion of interleukin-6rdquo Haematologica vol 98 no 6 pp 888ndash895 2013

[37] D Baeten X Baraliakos J Braun et al ldquoAnti-interleukin-17Amonoclonal antibody secukinumab in treatment of ankylosingspondylitis a randomised double-blind placebo-controlledtrialrdquoThe Lancet vol 382 no 9906 pp 1705ndash1713 2013

[38] J M Kezic M P Davey T T Glant J T Rosenbaum and HL Rosenzweig ldquoInterferon-120574 regulates discordant mechanismsof uveitis versus joint and axial disease in a murine modelresembling spondylarthritisrdquo Arthritis amp Rheumatism vol 64no 3 pp 762ndash771 2012

[39] Y Abe M Ohtsuji N Ohtsuji et al ldquoAnkylosing enthesitisassociated with up-regulated IFN-120574 and IL-17 production in(BXSB times NZB) F1 male mice a new mouse modelrdquo ModernRheumatology vol 19 no 3 pp 316ndash322 2009

[40] L Zhao Y Fong K Granfors J Gu and D Yu ldquoIdentificationof cytokines that might enhance the promoter activity of HLA-B27rdquo The Journal of Rheumatology vol 35 no 5 pp 862ndash8682008

[41] S Gordon and P R Taylor ldquoMonocyte and macrophageheterogeneityrdquo Nature Reviews Immunology vol 5 no 12 pp953ndash964 2005

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


studies have documented the efficacy of ATRA in the treat-ment of autoimmune diseases [15ndash17] The retinol level in ASpatients was significantly lower than that in healthy controls[18] It has also been observed that ATRA administrationreduced the frequency of Th17 and inhibited the secretion ofTNF-120572 in peripheral blood of AS patients [19]

Taken together we hypothesize that ATRA is effective forthe treatment of AS through regulating the immunomodula-tory capacity of MSCs In the present study we coculturedMSCs and peripheral blood mononuclear cells (PBMCs)and examined the frequencies of Treg and Th17 subsets andthe secretion of key cytokines We defined the pathogenicTh17 cells with a combination of the surface markers ofCD4 CCR4 and CCR6 [20] To the best of our knowledgethis is the first study showing that ATRA improved theimmunomodulatory ability of MSCs for the treatment of AS

2 Materials and Methods

21 Isolation of MSCs and PBMCs This study was approvedby the Ethics Committee of Sun Yat-sen UniversityGuangzhou China Healthy volunteers (119899 = 20 15 males 5females age 18ndash28 years) were recruited as donors of bonemarrow Bone marrow was extracted from the posteriorsuperior iliac spine under a sterile condition and MSCs wereisolated according to standardized procedures [21] MSCs ofthe fourth passage were used in experiments PBMCs wereisolated from the peripheral blood of AS patients (119899 = 2418 males 6 females age 16ndash40 years) using Ficoll-Hypaquegradient centrifugation Patients were selected accordingto modified New York criteria [22] Informed consent wasobtained from all donors and patients

22 Trilineage Differentiation Potential Assays of MSCs

Osteogenic Differentiation MSCs were seeded into six-wellplates (1 times 105 cellswell) in a total volume of 3mL DMEMmedium supplemented with 10 fetal bovine serum (FBS)50mgL ascorbic acid (Sigma) 10mM 120573-glycerophosphate(Sigma) and 100 nM dexamethasone (Sigma) The mediumwas replaced every three days Total culture duration was 21days Alizarin red staining was used

Chondrogenic Differentiation MSCs (25 times 105 cells) werecentrifuged at 600 g for 5 minutes in a 15mL conical tubeCells were cultured with high-glucose DMEM supplement-ed with 1 ITS-Premix (Corning) 50mgL ascorbic acid(Sigma) 1mMsodiumpyruvate (Sigma) 100 nMdexametha-sone (Sigma) and 10 ngmL transforming growth factor-1205733(RampD) The medium was replaced every three days Totalculture duration was 21 days

Adipogenic Differentiation MSCs were seeded into six-wellplates (1 times 105 cellswell) DMEMmediumwas supplementedwith 10 FBS 1120583M dexamethasone (Sigma) 10 120583gmL insu-lin (Sigma) 05mM 3-isobutyl-1-methylxanthine (Sigma)and 02mM indomethacin (Sigma) After 3 daysrsquo inductionthe medium was replaced with DMEM containing 10120583gmLinsulin and 1 day later the medium was replaced with

the inducing medium mentioned above After three cyclesof such culture cells were coated with DMEM containing10 120583gmL insulin until 14th day Oil red O staining was usedafter paraformaldehyde fixation

23 ATRA Preparation and MSCs Pretreatment ATRA pow-der (Sigma) was dissolved in dimethyl sulfoxide (DMSO) andstocked according to the manufacturerrsquos instructions ATRAsolution was prepared with DMEM medium supplementedwith 10 FBS MSCs were seeded into six-well plates (1 times105 cellswell) in a total volume of 3mL DMEM mediumpretreated by 1 120583MATRA for 1 day 3 days and 5 days accord-ing to different experiments The MSCs cocultured withPBMCs were only pretreated for 1 day After pretreatmentthe medium containing ATRA was completely removed andMSCs were washed with phosphate buffer saline (PBS) forthree times Cells were then cultured in ATRA-free mediumMSCs of the control group were pretreated with DMSO ofequal volume to ATRA

24 Cell Culture MSCs were cocultured with PBMCs at aratio of 1 20 of MSC PBMC T cells were stimulated bypurified anti-CD3 (02 120583gmL BD Pharmingen) and anti-CD28 (1 120583gmL BD Pharmingen) antibodies Cells werecocultured in RPMI-1640 medium of an ultimate volume of3mL for 5 days The cell ratio and the coculture durationwere determined according to preliminary experiments ForCD4+ T cell proliferation analysis PBMCs were incubatedwith 5 120583M carboxyfluorescein diacetatesuccinimidyl ester(CFSE Life Technologies) for 15 minutes and washed withPBS containing 10 FBS three times before coculture Toevaluate the effects of proinflammatory cytokines on IL-6secretion of MSCs exogenous interferon-120574 (IFN-120574 Pepro-Tech) (10 ngmL) and TNF-120572 (10 ngmL RampD) were addedinto the culture medium of pretreated MSCs which werecultured for the following 5 days For cytokine measurementa noncontact coculture was also conducted using a six-welland 04 120583m pore Transwell plate system (Corning) in whichPBMCs were seeded in the upper chamber

25 Flow Cytometry MSCs were trypsinized for identifica-tion of a number of surface markers The antibodies forsurface markers were anti-CD29-PE anti-CD34-APC anti-CD44-FITC anti-CD45-FITC anti-CD90-PE anti-CD105-FITC and anti-HLA-DR-PE (all from BD Pharmingen)PBMCs were harvested through centrifugation at the endof coculture For proliferation analysis of CD4+ T cellsCFSE-incubated PBMCs were marked with anti-CD4-PE(BD Pharmingen) cells left-shifting into the square gatewere recognized as the proliferating cells and the cell countpercentage within this gate represented the proliferation rateFor Th17 analysis anti-CD4-FITC anti-CCR4-PerCP-Cy55and anti-CCR6-APC (all from BD Pharmingen) were usedThe human regulatory T cell staining kit (eBioscience) con-taining anti-CD4-FITCCD25-APC cocktail and anti-Foxp3-PE was used in Treg analysis according to the manufacturerrsquosinstruction Cells were measured in a flow cytometry system(BD FACSVerse)













Y

Y

XX X X

SSC

0

0

FSC HLA-DR-PE0 102 103 104 105 0 102 103 104 105 0 102 103 104 105

0 102 103 104 1050 102 103 104 1050 102 103 104 1050 102 103 104 105

CD34-APC CD45-FITC


250k

250k

200 k

200 k

150k

150k

100 k

100 k

50k

50k

(a)


(b)



4 Discussion





SSC

SSC

SSC

SSC

SSC

SSC

FSC

459

CD4-PE

317

CFSE CFSE

CFSE CFSE

PBMC

0271

PBMC + CD328

939

561 534


(a)


lowast

Prol

ifera

tion

rate

of C

D4+

T ce

lls (

)

100

80

60

40

20

0

(b)


119875 lt 0001






SSC

FSC

458SS

C

CD4-FITC

296

CCR6

-APC

CCR6

-APC

CCR6

-APC

CCR6

-APC

Isotype control

0117

PBMC + CD328

164


256


118



(a)

SSC

FSC

624

SSC

CD4-FITC

383

CD25

-APC

CD25

-APC

CD25

-APC

CD25

-APC

Isotype control

0337

PBMC + CD328

364


49


72

Foxp3-PE Foxp3-PE

Foxp3-PE Foxp3-PE

(b)


lowast

Th17

lym

phoc

yte (

)

15

10

5

0

(c)


lowast

Treg

lym

phoc

yte (

)

20

15

10

5

0

(d)



lowastlowast

lowastlowast

lowastlowast

lowastlowast


Con

cent

ratio

n of

IL-6

(pg

mL)

2500

2000

1500

1000

500

0

1 1 1 3 3 3 5 5 5

1 3 5 1 3 5 1 3 5

Pretreatment(day)

Secretion (day)

(a)


Con

cent

ratio

n of

IL-6

(pg

mL)

500000

450000

400000

350000

300000

250000

10000

8000

6000

4000

2000

0

lowast



PBMC alone

(b)

Relat

ive m

RNA

amou

nt o

f IL-6

Cell source of IL-6

25

20

15

10

5

0



(c)

lowast

Con

cent

ratio

n of

IL-6

(pg

mL)

80000

60000

40000

20000

0




(d)







lowast

lowastlowast

Con

cent

ratio

n of

IL-17

A (p

gm

L)

250

200

150

100

50

0

(a)

lowast

Con

cent

ratio

n of

IFN

-120574(p

gm

L)

15000

10000

5000

0

(b)

lowast

Con

cent

ratio

n of

TN

F-120572

(pg

mL)

150

100

50

0

(c)

lowastlowast

Re

lativ

e mRN

A am

ount

of I

L-17

A

150

100

50

5

4

3

2

1

0

(d)



Relat

ive m

RNA

amou

nt o

f IFN

-120574

4

3

2

1

0

(e)


lowast

Relat

ive m

RNA

amou

nt o

f TN

F-120572

3

2

1

0

(f)


119875 lt 0001









Acknowledgments


References


























11986717 and



























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology













Y

Y

XX X X

SSC

0

0

FSC HLA-DR-PE0 102 103 104 105 0 102 103 104 105 0 102 103 104 105

0 102 103 104 1050 102 103 104 1050 102 103 104 1050 102 103 104 105

CD34-APC CD45-FITC


250k

250k

200 k

200 k

150k

150k

100 k

100 k

50k

50k

(a)


(b)



4 Discussion





SSC

SSC

SSC

SSC

SSC

SSC

FSC

459

CD4-PE

317

CFSE CFSE

CFSE CFSE

PBMC

0271

PBMC + CD328

939

561 534


(a)


lowast

Prol

ifera

tion

rate

of C

D4+

T ce

lls (

)

100

80

60

40

20

0

(b)


119875 lt 0001






SSC

FSC

458SS

C

CD4-FITC

296

CCR6

-APC

CCR6

-APC

CCR6

-APC

CCR6

-APC

Isotype control

0117

PBMC + CD328

164


256


118



(a)

SSC

FSC

624

SSC

CD4-FITC

383

CD25

-APC

CD25

-APC

CD25

-APC

CD25

-APC

Isotype control

0337

PBMC + CD328

364


49


72

Foxp3-PE Foxp3-PE

Foxp3-PE Foxp3-PE

(b)


lowast

Th17

lym

phoc

yte (

)

15

10

5

0

(c)


lowast

Treg

lym

phoc

yte (

)

20

15

10

5

0

(d)



lowastlowast

lowastlowast

lowastlowast

lowastlowast


Con

cent

ratio

n of

IL-6

(pg

mL)

2500

2000

1500

1000

500

0

1 1 1 3 3 3 5 5 5

1 3 5 1 3 5 1 3 5

Pretreatment(day)

Secretion (day)

(a)


Con

cent

ratio

n of

IL-6

(pg

mL)

500000

450000

400000

350000

300000

250000

10000

8000

6000

4000

2000

0

lowast



PBMC alone

(b)

Relat

ive m

RNA

amou

nt o

f IL-6

Cell source of IL-6

25

20

15

10

5

0



(c)

lowast

Con

cent

ratio

n of

IL-6

(pg

mL)

80000

60000

40000

20000

0




(d)







lowast

lowastlowast

Con

cent

ratio

n of

IL-17

A (p

gm

L)

250

200

150

100

50

0

(a)

lowast

Con

cent

ratio

n of

IFN

-120574(p

gm

L)

15000

10000

5000

0

(b)

lowast

Con

cent

ratio

n of

TN

F-120572

(pg

mL)

150

100

50

0

(c)

lowastlowast

Re

lativ

e mRN

A am

ount

of I

L-17

A

150

100

50

5

4

3

2

1

0

(d)



Relat

ive m

RNA

amou

nt o

f IFN

-120574

4

3

2

1

0

(e)


lowast

Relat

ive m

RNA

amou

nt o

f TN

F-120572

3

2

1

0

(f)


119875 lt 0001









Acknowledgments


References


























11986717 and



























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Y

Y

XX X X

SSC

0

0

FSC HLA-DR-PE0 102 103 104 105 0 102 103 104 105 0 102 103 104 105

0 102 103 104 1050 102 103 104 1050 102 103 104 1050 102 103 104 105

CD34-APC CD45-FITC


250k

250k

200 k

200 k

150k

150k

100 k

100 k

50k

50k

(a)


(b)



4 Discussion





SSC

SSC

SSC

SSC

SSC

SSC

FSC

459

CD4-PE

317

CFSE CFSE

CFSE CFSE

PBMC

0271

PBMC + CD328

939

561 534


(a)


lowast

Prol

ifera

tion

rate

of C

D4+

T ce

lls (

)

100

80

60

40

20

0

(b)


119875 lt 0001






SSC

FSC

458SS

C

CD4-FITC

296

CCR6

-APC

CCR6

-APC

CCR6

-APC

CCR6

-APC

Isotype control

0117

PBMC + CD328

164


256


118



(a)

SSC

FSC

624

SSC

CD4-FITC

383

CD25

-APC

CD25

-APC

CD25

-APC

CD25

-APC

Isotype control

0337

PBMC + CD328

364


49


72

Foxp3-PE Foxp3-PE

Foxp3-PE Foxp3-PE

(b)


lowast

Th17

lym

phoc

yte (

)

15

10

5

0

(c)


lowast

Treg

lym

phoc

yte (

)

20

15

10

5

0

(d)



lowastlowast

lowastlowast

lowastlowast

lowastlowast


Con

cent

ratio

n of

IL-6

(pg

mL)

2500

2000

1500

1000

500

0

1 1 1 3 3 3 5 5 5

1 3 5 1 3 5 1 3 5

Pretreatment(day)

Secretion (day)

(a)


Con

cent

ratio

n of

IL-6

(pg

mL)

500000

450000

400000

350000

300000

250000

10000

8000

6000

4000

2000

0

lowast



PBMC alone

(b)

Relat

ive m

RNA

amou

nt o

f IL-6

Cell source of IL-6

25

20

15

10

5

0



(c)

lowast

Con

cent

ratio

n of

IL-6

(pg

mL)

80000

60000

40000

20000

0




(d)







lowast

lowastlowast

Con

cent

ratio

n of

IL-17

A (p

gm

L)

250

200

150

100

50

0

(a)

lowast

Con

cent

ratio

n of

IFN

-120574(p

gm

L)

15000

10000

5000

0

(b)

lowast

Con

cent

ratio

n of

TN

F-120572

(pg

mL)

150

100

50

0

(c)

lowastlowast

Re

lativ

e mRN

A am

ount

of I

L-17

A

150

100

50

5

4

3

2

1

0

(d)



Relat

ive m

RNA

amou

nt o

f IFN

-120574

4

3

2

1

0

(e)


lowast

Relat

ive m

RNA

amou

nt o

f TN

F-120572

3

2

1

0

(f)


119875 lt 0001









Acknowledgments


References


























11986717 and



























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


SSC

SSC

SSC

SSC

SSC

SSC

FSC

459

CD4-PE

317

CFSE CFSE

CFSE CFSE

PBMC

0271

PBMC + CD328

939

561 534


(a)


lowast

Prol

ifera

tion

rate

of C

D4+

T ce

lls (

)

100

80

60

40

20

0

(b)


119875 lt 0001






SSC

FSC

458SS

C

CD4-FITC

296

CCR6

-APC

CCR6

-APC

CCR6

-APC

CCR6

-APC

Isotype control

0117

PBMC + CD328

164


256


118



(a)

SSC

FSC

624

SSC

CD4-FITC

383

CD25

-APC

CD25

-APC

CD25

-APC

CD25

-APC

Isotype control

0337

PBMC + CD328

364


49


72

Foxp3-PE Foxp3-PE

Foxp3-PE Foxp3-PE

(b)


lowast

Th17

lym

phoc

yte (

)

15

10

5

0

(c)


lowast

Treg

lym

phoc

yte (

)

20

15

10

5

0

(d)



lowastlowast

lowastlowast

lowastlowast

lowastlowast


Con

cent

ratio

n of

IL-6

(pg

mL)

2500

2000

1500

1000

500

0

1 1 1 3 3 3 5 5 5

1 3 5 1 3 5 1 3 5

Pretreatment(day)

Secretion (day)

(a)


Con

cent

ratio

n of

IL-6

(pg

mL)

500000

450000

400000

350000

300000

250000

10000

8000

6000

4000

2000

0

lowast



PBMC alone

(b)

Relat

ive m

RNA

amou

nt o

f IL-6

Cell source of IL-6

25

20

15

10

5

0



(c)

lowast

Con

cent

ratio

n of

IL-6

(pg

mL)

80000

60000

40000

20000

0




(d)







lowast

lowastlowast

Con

cent

ratio

n of

IL-17

A (p

gm

L)

250

200

150

100

50

0

(a)

lowast

Con

cent

ratio

n of

IFN

-120574(p

gm

L)

15000

10000

5000

0

(b)

lowast

Con

cent

ratio

n of

TN

F-120572

(pg

mL)

150

100

50

0

(c)

lowastlowast

Re

lativ

e mRN

A am

ount

of I

L-17

A

150

100

50

5

4

3

2

1

0

(d)



Relat

ive m

RNA

amou

nt o

f IFN

-120574

4

3

2

1

0

(e)


lowast

Relat

ive m

RNA

amou

nt o

f TN

F-120572

3

2

1

0

(f)


119875 lt 0001









Acknowledgments


References


























11986717 and



























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


SSC

FSC

458SS

C

CD4-FITC

296

CCR6

-APC

CCR6

-APC

CCR6

-APC

CCR6

-APC

Isotype control

0117

PBMC + CD328

164


256


118



(a)

SSC

FSC

624

SSC

CD4-FITC

383

CD25

-APC

CD25

-APC

CD25

-APC

CD25

-APC

Isotype control

0337

PBMC + CD328

364


49


72

Foxp3-PE Foxp3-PE

Foxp3-PE Foxp3-PE

(b)


lowast

Th17

lym

phoc

yte (

)

15

10

5

0

(c)


lowast

Treg

lym

phoc

yte (

)

20

15

10

5

0

(d)



lowastlowast

lowastlowast

lowastlowast

lowastlowast


Con

cent

ratio

n of

IL-6

(pg

mL)

2500

2000

1500

1000

500

0

1 1 1 3 3 3 5 5 5

1 3 5 1 3 5 1 3 5

Pretreatment(day)

Secretion (day)

(a)


Con

cent

ratio

n of

IL-6

(pg

mL)

500000

450000

400000

350000

300000

250000

10000

8000

6000

4000

2000

0

lowast



PBMC alone

(b)

Relat

ive m

RNA

amou

nt o

f IL-6

Cell source of IL-6

25

20

15

10

5

0



(c)

lowast

Con

cent

ratio

n of

IL-6

(pg

mL)

80000

60000

40000

20000

0




(d)







lowast

lowastlowast

Con

cent

ratio

n of

IL-17

A (p

gm

L)

250

200

150

100

50

0

(a)

lowast

Con

cent

ratio

n of

IFN

-120574(p

gm

L)

15000

10000

5000

0

(b)

lowast

Con

cent

ratio

n of

TN

F-120572

(pg

mL)

150

100

50

0

(c)

lowastlowast

Re

lativ

e mRN

A am

ount

of I

L-17

A

150

100

50

5

4

3

2

1

0

(d)



Relat

ive m

RNA

amou

nt o

f IFN

-120574

4

3

2

1

0

(e)


lowast

Relat

ive m

RNA

amou

nt o

f TN

F-120572

3

2

1

0

(f)


119875 lt 0001









Acknowledgments


References


























11986717 and



























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


lowastlowast

lowastlowast

lowastlowast

lowastlowast


Con

cent

ratio

n of

IL-6

(pg

mL)

2500

2000

1500

1000

500

0

1 1 1 3 3 3 5 5 5

1 3 5 1 3 5 1 3 5

Pretreatment(day)

Secretion (day)

(a)


Con

cent

ratio

n of

IL-6

(pg

mL)

500000

450000

400000

350000

300000

250000

10000

8000

6000

4000

2000

0

lowast



PBMC alone

(b)

Relat

ive m

RNA

amou

nt o

f IL-6

Cell source of IL-6

25

20

15

10

5

0



(c)

lowast

Con

cent

ratio

n of

IL-6

(pg

mL)

80000

60000

40000

20000

0




(d)







lowast

lowastlowast

Con

cent

ratio

n of

IL-17

A (p

gm

L)

250

200

150

100

50

0

(a)

lowast

Con

cent

ratio

n of

IFN

-120574(p

gm

L)

15000

10000

5000

0

(b)

lowast

Con

cent

ratio

n of

TN

F-120572

(pg

mL)

150

100

50

0

(c)

lowastlowast

Re

lativ

e mRN

A am

ount

of I

L-17

A

150

100

50

5

4

3

2

1

0

(d)



Relat

ive m

RNA

amou

nt o

f IFN

-120574

4

3

2

1

0

(e)


lowast

Relat

ive m

RNA

amou

nt o

f TN

F-120572

3

2

1

0

(f)


119875 lt 0001









Acknowledgments


References


























11986717 and



























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


lowast

lowastlowast

Con

cent

ratio

n of

IL-17

A (p

gm

L)

250

200

150

100

50

0

(a)

lowast

Con

cent

ratio

n of

IFN

-120574(p

gm

L)

15000

10000

5000

0

(b)

lowast

Con

cent

ratio

n of

TN

F-120572

(pg

mL)

150

100

50

0

(c)

lowastlowast

Re

lativ

e mRN

A am

ount

of I

L-17

A

150

100

50

5

4

3

2

1

0

(d)



Relat

ive m

RNA

amou

nt o

f IFN

-120574

4

3

2

1

0

(e)


lowast

Relat

ive m

RNA

amou

nt o

f TN

F-120572

3

2

1

0

(f)


119875 lt 0001









Acknowledgments


References


























11986717 and



























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology









Acknowledgments


References


























11986717 and



























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology










11986717 and



























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Documents

Research Article All-Trans Retinoic Acid Improves the Effects of …downloads.hindawi.com/journals/sci/2015/484528.pdf · 2019-07-31 · Research Article All-Trans Retinoic Acid Improves