Research Article IL-17 Genetic and Immunophenotypic Evaluation...

Research ArticleIL-17 Genetic and Immunophenotypic Evaluation inChronic Graft-versus-Host Disease

Renata Gonccedilalves Resende1 Jeane de Faacutetima Correia-Silva2 Tarciacutelia Aparecida Silva3

Ulisses Eliezer Salomatildeo4 Luciano Marques-Silva5 Eacuterica Leandro Marciano Vieira6

Walderez Ornelas Dutra7 and Ricardo Santiago Gomez3

1 Servico de Estomatologia Hospital Municipal Odilon Behrens Rua Formiga 50 31110-430 Belo Horizonte MG Brazil2 Centro Universitario Newton Paiva Faculdade de Odontologia Avenida Presidente Carlos Luz 22031230-010 Belo Horizonte MG Brazil

3 Departamento de Clınica Patologia e Cirurgia Odontologicas Faculdade de Odontologia Universidade Federal de Minas GeraisAvenida Antonio Carlos 6627 31270-901 Belo Horizonte MG Brazil

4Departamento de Odontologia Restauradora Faculdade de Odontologia Universidade Federal de Minas GeraisAvenida Antonio Carlos 6627 31270-901 Belo Horizonte MG Brazil

5 Ciodonto-Universidade FACSETE Rua Italia Pontelo 50 35700-170 Sete Lagoas MG Brazil6 Laboratorio Interdisciplinar de Pesquisa Medica Faculdade de Medicina Universidade Federal de Minas GeraisAvenida Alfredo Balena 190 30130-10 Belo Horizonte MG Brazil

7 Departamento de Morfologia Instituto de Ciencias Biologicas Universidade Federal de Minas GeraisAvenida Antonio Carlos 6627 31270-901 Belo Horizonte MG Brazil

Correspondence should be addressed to Renata Goncalves Resende renatagresendeyahoocombr

Received 17 April 2014 Revised 8 June 2014 Accepted 26 June 2014 Published 21 July 2014

Academic Editor Luca Cantarini

Copyright copy 2014 Renata Goncalves Resende et al This is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

Although interleukin-17 (IL-17) is a recently discovered cytokine associated with several autoimmune diseases its role in thepathogenesis of chronic graft-versus-host disease (cGVHD) was not established yet The objective of this study was to investigatethe association of IL17A and IL17F genes polymorphisms and IL-17A and IL-17F levels with cGVHD IL-17A expression was alsoinvestigated in CD4+ T cells of patients with systemic cGVHD For Part I of the study fifty-eight allo-HSCT recipients and donorswere prospectively studied Blood samples were obtained to determine IL17A and IL17F genes polymorphisms Cytokines levels inblood and saliva were assessed by ELISA at days +35 and +100 after HSCT In Part II for the immunophenotypic evaluation eightpatients with systemic cGVHD were selected and the expression of IL-17A was evaluated We found association between recipientAA genotype with systemic cGVHD No association was observed between IL-17A levels and cGVHD Lower IL-17A levels in theblood were associated with AA genotype In flow cytometry analysis decreased expression of IL-17A was observed in patients withcGVHD after stimulation In conclusion IL-17A may have an important role in the development of systemic cGVHD

1 Introduction

Hematopoietic stem cell transplant (HSCT) represents thedefinitive immunotherapy for malignancy and immunologicdiseases [1 2] However graft-versus-host disease (GVHD)is an important complication of HSCT that limits its successand can be fatal in approximately 15 of the HSCT patients[3] Acute GVHD (aGVHD) and chronic GVHD (cGVHD)

involve distinct pathological process where the first onehas strong inflammatory components and the second onedisplays more autoimmune and fibrotic features [4]

T lymphocytes play a critical role in the immune responseincluding allograft rejection graft failure and GVHD aftertransplant understanding the posttransplant immune regu-lation might help to generate new therapeutic strategies thatmight overcome these posttransplant problems [5]

Hindawi Publishing CorporationMediators of InflammationVolume 2014 Article ID 571231 7 pageshttpdxdoiorg1011552014571231

2 Mediators of Inflammation

Interleukin-17 (IL-17) is a recently discovered cytokinethat can be produced by many cells although its source fromCD4+ T cells is the most investigated [6] The Th17 cellsare a new lineage of CD4+ T helper cells [7] and generateproinflammatory cytokines such as IL-17A IL-17F IL-21 IL-22 tumor necrosis factor (TNF) granulocyte macrophage-colony stimulating (GM-CSF) and some chemokines [6] IL-17A the original member of this family was first identifiedin 1995 [8] and acts predominantly as a chemoattractantRecent dates have shown thatTh17 pathway is associated withcGVHD [6] AnotherTh17 cytokine is the IL-17F it shows thehighest overall amino acid sequence identity with IL-17A inthe IL-17 family The genes encoding IL-17A and IL-17F areboth located on 6p12 [9] IL-17A and IL-17F share biologicalfunctions and have many proinflammatory effects in a widevariety of cells including macrophage endothelial cells andfibroblasts [10]

In few recent studies IL-17A has been associated with thepathway of the GVHD and some studies have assessed theassociation between IL17A gene polymorphism and inflam-matory diseases including the cGVHD [6 11 12] Howeverthe role of IL-17F in the pathogenesis of cGVHD was notpreviously addressed

So the aim of the present study was to investigate theassociation of the IL17A and IL-17F gene polymorphisms andIL-17A and IL-17F levels with cGHVD as well as to determinethe relationship between IL-17A expression by CD4+ T cellsand the development of the disease

2 Methods

To achieve the proposed objectives the workwas divided intotwo parts which include two groups of individuals In Part 1we performed the genotypic and phenotypic analyses of thecytokines IL-17A and IL-17F and in Part 2 IL-17A immunemarker was assessed cGVHD was determinate according toNIHConsensus Development Project on Criteria for ClinicalTrials [13]

21 Part 1 Genotypic e Phenotypic Evaluation

211 Subjects Fifty-eight consecutive allo-HSCT recipientsand related donors from Hospital das Clınicas at Universi-dade Federal de Minas Gerais (HC-UFMG) were deemedeligible and included in this prospective study The patientswere followed between the days minus7 and +100 after it in a totalof sixteen weeks

All the patients were followed from pre-HSCT until thedevelopment of cGVHD so the number of samples includedin each essay varied because of patientrsquos death and lossof samples related to the process of collection Recipientswere conditioned for allo-HSCT according to the specificprotocols from the Stem Cell Transplant Unit at HC-UFMGand varied according to the type of the disease diseasestatus and the previous treatment at the time of transplan-tation Cyclosporine in combination with methotrexate ormycophenolate mofetil was used for GVHD prophylaxiswhereas 2mgkg ofmethylprednisolone in combinationwithcyclosporine was used for GVHD treatment

This study protocol was approved by the local Institu-tional Ethics Committee (ETIC 01240203000-11) Writteninformed consent was obtained from all patients

212 Subjects and Sample Collection Blood samples werecollected from recipients and donors one week before theHSTC and were submitted for DNA analysis Saliva sam-ples were collected using cotton swabs on the floor of themouth tongue and labial and buccal normal oral mucosaof the HSCT subjects Sites with localized injuries were notincluded The cotton swabs were removed with a sterilecytobrush placed immediately in sterile tubes containing500120583L of Krebs buffer (NaCl 20 KCl 2 CaCl

22H2O

2 MgSO4 KH2PO4 and C

6H12O6) and stored at minus20∘C

until processing Peripheral blood (4mL) was collected invacutainer tubes containing EDTA and stored at minus70∘C untilprocessing

213 DNA Isolation Total genomic DNA was extractedfrom saliva and blood samples using a QIAamp DNA bloodminikit (Quiagen Valencia CAUSA) in accordancewith themanufacturerrsquos instructions The final elution of saliva andblood DNA was performed in 50120583L of a specific AE bufferfrom the kit and stored at minus20∘C until use

214 IL17A and IL17F Gene Polymorphism Analysis Recip-ient and donor IL17 197 (AG) and IL17F 7488 (TC)polymorphism was assessed by means of polymerase chainreaction (PCR) amplification and digestion The sequencesof PCR primers for IL17A were reverse 51015840 AACAAGTAA-GAATGAAAAGAGGACATGGT 31015840 and ant-sense 51015840 CCC-CCAAATGAGGTCATAGAAGAGAATC 31015840 and for IL17Fwere reverse 51015840 GTTCCCATCCAGCAAGAGAC 31015840 and ant-sense 51015840 AGCTGGGAATGCAAACAAAC 31015840 as previouslydescribed [14 15] The PCR was carried out in a total volumeof 50120583L containing approximately 400 ng of DNA primers(20 pmolreaction) and 25 120583L of premix buffer (PhoneutriaBiotecnologia Belo Horizonte Brazil) According to themanufacturer the premix buffer contained 50mM KCl10mM Tris-HCl pH 84 01 triton X-100 15mM MgCl

2

deoxynucleoside triphosphates and 125 units of Taq DNApolymerase PCR products were digested overnight at 37∘Cwith XagI (Fermentas Life Sciences Vilnius Lithuania) forIL-17A and NlaIII (Fermentas Life Sciences Vilnius Lithua-nia) for IL-17F Visualization of the product was performedin a 65 polyacrylamide gel electrophoresis stained withsilver

215 Detection of IL-17A and IL-17F Levels To determine thecytokine levels saliva and blood samples were collected indays +35 and +100 after allo-HSCT The saliva sample wascollected in Salivette tubes (Sarstedt AG amp Co NumbrechtGermany) according to the manufacturerrsquos instructions Thesaliva samples were subsequently diluted (1 1) in a PBS solu-tion containing protease inhibitors (01mM PMSF 01Mmbenzethoniumchloride 10mMEDTA and 001mgmL apro-tinin A) and 005 Tween-20 and subsequently stored atminus20∘C until analysis The total protein content in the saliva

Mediators of Inflammation 3

0 200 400 600 800 1000FSC-A

0

200

400

600

800

1000

SSC-

A

30R1

(a)

100

100

101

101

102

102

103

103

104

104

FITC-A CD4 FITC-APE

-A I

L-17

PE-

A

334

0046

0011 0034

391609

R2R3

R4

(b)

100

101

102

103

104

PE-A IL-17 PE-A

0

50

100

15001

Num

ber o

f cel

ls

M1

(c)

Figure 1 Representative flow cytometry graphs of CD4+ lymphocytes expressing intracellular cytokine IL-17A Flow cytometry dot-plotsdemonstrate the lymphocyte region (R1) selected (a) and the data analyzed in total CD4+ T cells (R2) (b) A single-color histogram (M1) (c)expressing IL-17A in the CD4+ T cells was obtained from the R2 region (c) Double-positive CD4+IL-17A+ in total lymphocytes (R3) andtotal IL-17A production (R4) were also analyzed in a dot-plot graphic (b)

was determined using the Bradford reagent (Sigma SaintLouis MO USA) and the BSA standard (Fermentas LifeSciences Vilnius Lithuania) The total protein content wasused to correct the IL-17A values for each sample The serumsamples were obtained from venous blood samples and werecentrifuged within 2 hours after blood collection and storedat minus20∘C

22 Part 2 Immunophenotyping Evaluation

221 Subjects For immunophenotypic evaluation addi-tional patient selection was performed and 3 differentsubgroups were established HSCT patients with systemiccGVHD (119899 = 8) HSCT patients without systemic cGVHD(119899 = 4) and healthy individuals (119899 = 3)

222 Blood Collection and Cell Stimulation Nine milliliters(9mL) of blood was collected from each individual in avacuum tube containing sodium heparin by a qualifiedhealthcare professional complying with the rules for theuse of sharp instruments in an aseptic environment Wholeblood was subjected to 3 different conditions media stim-ulus with superantigen staphylococcal enterotoxin B (SEB)(120 ngmL) and stimulus with anti-CD3 plus anti-CD28(120572CD3120572CD28) monoclonal antibody (Abs) (10 120583gmL) for20 hours Brefeldin A (at 1 120583gmL) was added during thelast 4 hours of culture After culture red blood cell lysiswas performed using 1x RBC lysis buffer (eBioscience SanDiego CA USA) for flow cytometry as recommended by themanufacturer

223 Cell Surface and Intracellular Staining After lysisleukocytes from whole blood were stained with fluoresceinisothiocyanate- (FITC-) labeled anti-CD4 monoclonal anti-body (eBioscience BD Pharmingen San Diego CA USA)for 20 minutes at 4∘C The cells were then fixed using 2

formaldehyde (Sigma-Aldrich) The fixed cells were perme-abilized and stained using phycoerythrin- (PE-) labeled anti-cytokine monoclonal antibodies for IL-17A (eBioscience SanDiego CA USA) IFN-120574 (eBioscience San Diego CA USA)or Foxp3 (eBioscience San Diego CA USA) FITC- orPE-labeled isotype control antibodies and an unstimulatedcell control were included in all experiments Preparationswere acquired on a FACSCanto II (Becton amp DickinsonSan Jose CA USA) A minimum of 50000 gated events onthe lymphocyte population were acquired for analysis dueto the low frequency of positive events being analyzed Theacquisition was processed using the Diva software (Becton ampDickinson)

224 Flow Cytometry Data Analysis CD4+ T lympho-cytes were analyzed for their intracellular expression of IL-17A using the FlowJo program (Tree Star Ashland ORUSA) Analyses were performed using a lymphocyte gate(Figure 1(a)) We determined the total expression of IL-17 inthe lymphocyte gate (Figure 1(b) R4) we also determined thefrequency of Th1 and Th17 cells by analyzing the frequencyof CD4+IFN-gamma+ and CD4+IL-17+ cells respectively(Figure 1(b) R3) The expression of IL-17 within the CD4+T cells was assessed by further gating on the total CD4+population (Figure 1(b) R2) and requesting a histogramanalysis of such gating (Figure 1(c)) The same strategy wasused for IFN-120574 and Foxp3 Limits for the quadrant markerswere always set based on negative populations and isotypecontrols

23 Statistics The Shapiro-Wilk test was used to deter-mine if samples followed normal distribution Two inde-pendent groups were compared by Studentrsquos 119905-test andby the Wilcoxon test Univariate analyses were performedusing the Chi-square Mann-Whitney and Friedman testsand Studentrsquos 119905-test The SPSS software was used for the


Table 1 Clinical characteristics of allo-HSCT patients and donors(119899 = 58)

Parameters Total (119899 = 58)Recipient median age in years (range) 315 (5ndash56)Female gender 25 (432)Primary disease

(i) Malignant(ii) Chronic myeloid leukemia 9 (155)(iii) Acute myeloid leukemia 15 (26)(iv) Acute lymphoid leukemia 5 (86)(v) Non-Hodgkinrsquos lymphoma 4 (69)(vi) Hodgkinrsquos lymphoma 3 (51)(vii) Other malignancieslowast 4 (69)(viii) Bone marrow failure syndromelowastlowast 18 (31)

HLA match(i) HLA matched related 52 (896)(ii) HLA matched unrelated 4 (68)(iii) HLA mismatched related 2 (36)

Donor median age in years (range) 354 (6ndash69)Donor female gender 21 (363)Conditioning regimen

(i) BUCY 20 (345)(ii) CY +minus ATG or Alemtuzumab 14 (242)(iii) BU + FLU +minus Alemtuzumab 9 (155)(iv) CY + FLU +minus Alemtuzumab 8 (138)(v) MEL + FLU +minus Campath 5 (86)(vi) Otherslowastlowastlowast 2 (34)

Ethnic group Brazilian mixedpopulation

Source of stem(i) Bone marrow 32 (552)(ii) Peripheral blood stem cells 25 (431)(iii) Umbilical cord blood 1 (17)

lowastMyelodysplastic syndrome (119899 = 1) myelofibrosis (119899 = 1) and multiplemyeloma (119899 = 2)lowastlowastParoxysmal nocturnal hemoglobinuria (119899 = 2) severe aplastic anemia(119899 = 14) and Fanconi anemia (119899 = 2)lowastlowastlowastBUMEL (119899 = 1) CytarabineCampathFLUD (119899 = 1)BU busulfan CY cyclophosphamide FLUD fludarabine MEL melphalanATG antithymoglobulins

analyses (SPSS Inc version 190 Chicago IL) Differenceswere considered significant at 119875 lt 005

3 Results

31 Part I Genotypic e Phenotypic Evaluation

311 Clinical Outcomes Clinical data from patients anddonors is shown in Table 1

During the monitoring period the final ldquo119899rdquo value for theanalysis related to IL-17A was 34 and related to IL-17F was42 The initial ldquo119899rdquo selected was 58 but over time of patientsrsquo

followup losses occurred due to patient death or loss ofsamples for analysis related to the collection process

312 IL17A and IL17F Gene Polymorphisms in Recipientand Donor of Allo-HSCT and cGVHD Results of recipientand donor IL17A and IL17F gene polymorphism and theirassociation with systemic cGVHD are presented in Table 2We found association between recipient IL17A AA genotypeand the occurrence of systemic cGVHD (119875 = 003)

313 IL-17A and IL-17F Levels in Blood and Saliva and theIncidence of aGVHD No association was found between IL-17A or IL-17F levels in the blood or saliva samples and theoccurrence of systemic cGVHD (data not shown)

314 IL-17A Levels in Blood and Saliva and Recipient andDonors IL17A Genotypes Higher IL-17A levels in the bloodwere observed at day +35 in recipients with IL17 AG genotype(119875 = 0007) No association was found between recipientgenotypes and IL-17A levels in saliva or between donor IL17Agenotypes and IL-17A levels in blood or saliva (Table 3) Noassociation was also found between IL-17F blood or salivalevels and IL17F genotypes (data not shown)

32 Part II Immunophenotyping Evaluation

321 Clinical Outcomes Clinical data from patients andhealthy individuals is in Table 4 None of the patients fromthe group without cGVHD presented the systemic form ofthe disease

322 Expression of IL-17A in Total CD4 and in CD4+ T Cellsand cGVHD No statistical difference was found between theexpression of total IL-17A and IL-17A+CD4+ in total T cellsas well as percentage of IL-17A in CD4+ T cells and patientswith cGVHD (data not shown)

323 Expression of IL-17A in Total CD4 and in CD4+ T Cellsand cGVHD after Stimulus Decreased IL-17A expressionwas observed in total CD4 T cells of patients with cGVHDafter stimulation with SEB (119875 = 0008) and 120572CD3120572CD28(119875 = 0008) stimulus compared to media-stimulated controlcells (Figure 2(a)) In patients without cGVHD or in healthyindividuals the expression of IL-17A in total CD4T cells afterstimulation with SEB or 120572CD3120572CD2 was not statistically dif-ferent than that seen in media-stimulated control cells (datanot shown) Overlapping histograms for IL-17A fluorescenceare shown in Figure 2(b)

4 Discussion

Several studies have been devoted to analyze the role of IL-17 a new discovered cytokine in the pathogenesis of GVHD[12 16ndash18] However this is the first work that evaluatedboth cytokines IL-17A and IL17F involving the genetic andphenotypic aspects and investigated the immunophenotypicaspect of IL-17A regarding systemic cGVHD


Table 2 Association between recipient and donor IL17A (119899 = 34) and IL-17F (119899 = 42) genotypes and occurrence of systemic cGVHD

Gene IL17A (G197A)genotype 119899 () Systemic cGVHD

119875

lowast IL17F (7488TC)genotype 119899 () Systemic cGVHD

119875

lowast

A P A P

RecipientGG 12 (353) 9 3

003CC 10 (238) 7 3

NSAG 13 (382) 11 2 CT 18 (429) 9 9AA 9 (265) 3 6 TT 14 (333) 10 4

DonorGG 11 (324) 7 4

NSCC 9 (214) 7 2

NSAG 15 (441) 9 6 CT 19 (452) 12 7AA 8 (235) 7 1 TT 14 (334) 7 7

A absent P present NS not significant lowastChi-square test

Table 3 Association between saliva IL-17A levels and recipient IL17A genotypes (119899 = 34)

Days Genotype 119899

validampIL-17Amedian

Minimumlevels

Maximumlevels 119875

lowast

Blood levelsa+35

GG 14 2242 000 849800007AG 13 4925 000 48014

AA 11 000 000 3956

+100GG 8 359 000 17800

NSAG 12 000 000 11600AA 8 000 000 4402

Saliva levelsb

+35GG 10 000 000 578

NSAG 10 162 000 2174AA 9 000 000 1112

+100GG 9 000 000 2700

NSAG 8 076 000 5412AA 7 000 000 2123

NS not significant A absent P present lowastKruskal Wallis test apgmL bpgmg proteinampNumber of valid samples in each moment

Table 4 Clinical characteristics of recipients with cGVHD (119899 = 8) recipients without cGVHD (119899 = 4) and healthy individuals (119899 = 3)included in the immunophenotypic analysis

Groups 119899 Age median Individuals gender Donor gender Source of cells Media time after HSCT

With cGVHD 8 372 Female = 2 (25) Female = 1 (125) BMSC = 2 (25) 5 years and 8 monthsMale = 6 (75) Male = 7 (875) PBSC = 6 (75)

Without cGVHD 4 415 Female = 3 (75) Female = 2 (50) BMSC = 4 (100) 6 years and 1 monthMale = 1 (25) Male = 2 (50) PBSC = 0

Healthy individuals 3 266 Female = 2 (667) mdash mdash mdashMale = 1 (333)

BMSC bone marrow stem cells PBSC peripheral blood stem cells

In the current study we investigate the involvement ofthe IL17A and IL17F gene polymorphism in the cGVHDand our results showed association of AA recipient geno-types with the occurrence of systemic cGVHD Previousstudies showed association between G197A polymorphismand several chronic diseases [12] like ulcerative colitis [19]and rheumatoid arthritis [20] A study reported associationbetween allele A of IL17A polymorphism and high risk ofcGVHD [12] Until now no other work was reported in theliterature relating IL17F polymorphism and GVHD We didnot find association between IL17F genotypes and cGVHD

however other authors showed association between IL17Fpolymorphism (TC 7488) and Behcet disease [21] gastriccancer [14] and bone mineral density [22]

Although our results did not show association betweenIL-17A or IL-17F levels and the presence of cGVHD theimportance of both in the context of the disease cannot beexcluded because we do not understand all the temporaland dynamic factors involved in cGVHD development Thedetection of cytokines in a specific time of the diseaseprovides only a snapshot of a more complex process Th17pathway has been implicated in the development of acute


00

02

04

06

08

ConditionsMedia SEB 120572CD3120572CD28

0008lowast

0008lowast

IL-17

A in

CD4+

T ce

lls (

)

(a)

MediaSEB

120572CD3120572CD28

100

101

102

103

104

IL-17 PE

100

80

60

40

20

0

Max

()

(b)

Figure 2 IL-17A expression by CD4+ T cells from HSCT patients with cGVHD following culture with SEB and 120572CD3120572CD28 Whole bloodfrom HSCT patients with cGVHD was maintained in culture without stimulus (media) as well as with SEB and 120572CD3120572CD28 IL-17A (a)expression in CD4+ T cellsThe lowast indicates a 119875 value lt 005 between media and stimulus conditions usingWilcoxonrsquos matched pairs testTheoverlap histogram graphics represent IL-17 (b) expression in CD4+ T cells

and cGVHD [6] Previous preclinical and clinical studieshave shown the role of IL-17A in pathogenesis of cGVHDespecially in skin [16 23]

When we investigated the impact of the IL17A polymor-phism on the cytokine levels of HSCT patients we foundthat recipients with AA genotypes showed lower levels ofcytokine in the blood in moment +35 compared to the othergenotypes Several polymorphisms in cytokine genes withtranscriptional relevance in vitro have been identified [24]A modification in cytokine production is associated withgenetic variations in the promoter region [24ndash26] Just onework until now investigated the relationship between IL17Apolymorphism and IL-17A levels and the authors showed thatserum concentration of IL-17Awas significantly higher in thepresence of allele A (AA and AG genotypes) in a populationof Chinese patients with atopic dermatitis [27]

In our study we observed decreased percentage of IL-17A+CD4+ T cells after stimulus with SEB and 120572CD3120572CD28T-cell production of IL-17 induces epithelial endothelial andstromal cells to secrete proinflammatory cytokines (IFN-120574 IL-6 TNF-120572 IL-1120573 and G-CSF) and the levels of thesecytokines have been shown to be higher in patients withcGVHD [28] Therefore decreased expression of IL-17A inthe stimulated cells observed in this work may be caused bythe inhibitory effect of others cytokines like IFN-120574 [7] whichmay be relevant to the pathogenesis of cGVHD

5 Conclusion

In conclusion we demonstrated association between AAgenotype of recipients with systemic cGVHD and those withlower IL-17A levels and observed that stimulated lympho-cytes of patients with cGVHD present decreased expressionof IL-17A Our data indicate that this cytokine could be an

important mediator of the cGVHD andmay be an importantnew target for investigation and therapeutics of the disease

Conflict of Interests

The authors declare that they have no conflict of interests

Acknowledgment

This study was supported by CAPES (Coordenacao deAperfeicoamento de Pessoal de Nıvel Superior) Brazil

References

[1] B E Anderson J M McNiff D Jain B R Blazar W DShlomchik and M J Shlomchik ldquoDistinct roles for donor-and host-derived antigen-presenting cells and costimulatorymolecules inmurine chronic graft-versus-host disease require-ments depend on target organrdquo Blood vol 105 no 5 pp 2227ndash2234 2005

[2] M R van den Brink and S J Burakoff ldquoCytolytic pathwaysin haematopoietic stem-cell transplantationrdquo Nature ReviewsImmunology vol 2 no 4 pp 273ndash281 2002

[3] M C Pasquini L M Griffith D L Arnold et al ldquoHematopoi-etic stem cell transplantation for multiple sclerosis collabo-ration of the CIBMTR and EBMT to facilitate internationalclinical studiesrdquo Biology of Blood and Marrow Transplantationvol 16 no 8 pp 1076ndash1083 2010

[4] B R Blazar W J Murphy and M Abedi ldquoAdvances ingraft-versus-host disease biology and therapyrdquo Nature ReviewsImmunology vol 12 no 6 pp 443ndash458 2012

[5] K J Wood A Bushell and J Hester ldquoRegulatory immune cellsin transplantationrdquo Nature Reviews Immunology vol 12 no 6pp 417ndash430 2012


[6] J S Serody and G R Hill ldquoThe IL-17 differentiation pathwayand its role in transplant outcomerdquoBiology of Blood andMarrowTransplantation vol 18 no 1 pp S56ndashS61 2012

[7] L E Harrington R D Hatton P R Mangan et al ldquoInterleukin17-producing CD4+ effector T cells develop via a lineage distinctfrom the T helper type 1 and 2 lineagesrdquo Nature Immunologyvol 6 no 11 pp 1123ndash1132 2005

[8] Z Yao W C Fanslow M F Seldin et al ldquoHerpesvirus Saimiriencodes a new cytokine IL-17 which binds to a novel cytokinereceptorrdquo Immunity vol 3 no 6 pp 811ndash821 1995

[9] H Park Z Li X O Yang et al ldquoA distinct lineage of CD4 Tcells regulates tissue inammation by producing interleukin 17rdquoNature Immunology vol 6 no 11 pp 1133ndash1141 2005

[10] K Shibata H Yamada R Nakamura X Sun M Itsumi and YYoshikai ldquoIdentification of CD25+ 120574120575 T cells as fetal thymus-derived naturally occurring IL-17 producersrdquo The Journal ofImmunology vol 181 no 9 pp 5940ndash5947 2008

[11] J D CorreaM FMMadeira R G Resende et al ldquoAssociationbetween polymorphisms in interleukin-17A and -17F genes andchronic periodontal diseaserdquo Mediators of Inflammation vol2012 Article ID 846052 9 pages 2012

[12] J L Espinoza A TakamiMOnizuka et al ldquoA single nucleotidepolymorphism of IL-17 gene in the recipient is associatedwith acute GVHD after HLA-matched unrelated BMTrdquo BoneMarrow Transplantation vol 46 no 11 pp 1455ndash1463 2011

[13] A H Filipovich D Weisdorf S Pavletic et al ldquoNational Insti-tutes of Health consensus development project on criteria forclinical trials in chronic graft -versus-host disease I diagnosisand stagingworking group reportrdquoBiology of Blood andMarrowTransplantation vol 11 no 12 pp 945ndash956 2005

[14] X Wu Z Zeng B Chen et al ldquoAssociation between polymor-phisms in interleukin-17A and interleukin-17F genes and risksof gastric cancerrdquo International Journal of Cancer vol 127 no 1pp 86ndash92 2010

[15] M Kawaguchi D Takahashi N Hizawa et al ldquoIL-17F sequencevariant (His161Arg) is associatedwith protection against asthmaand antagonizes wild-type IL-17F activityrdquo Journal of Allergyand Clinical Immunology vol 117 no 4 pp 795ndash801 2006

[16] M J CarlsonM LWest JMCoghill A Panoskaltsis-MortariB R Blazar and J S Serody ldquoIn vitro differentiated TH17cells mediate lethal acute graft-versus-host disease with severecutaneous and pulmonary pathologic manifestationsrdquo Bloodvol 113 no 6 pp 1365ndash1374 2009

[17] C Iclozan Y Yu C Liu et al ldquoT helper17 cells are sufficient butnot necessary to induce acute graft-versus-host diseaserdquoBiologyof Blood andMarrow Transplantation vol 16 no 2 pp 170ndash1782010

[18] T Yi D Zhao C Lin et al ldquoAbsence of donor Thl7 leadsto augmented Thl differentiation and exacerbated acute graft-versus-host diseaserdquo Blood vol 112 no 5 pp 2101ndash2110 2008

[19] T Arisawa T Tahara T Shibata et al ldquoThe influence ofpolymorphisms of interleukin-17A and interleukin-17F geneson the susceptibility to ulcerative colitisrdquo Journal of ClinicalImmunology vol 28 no 1 pp 44ndash49 2008

[20] G B N Nordang M K Viken J E Hollis-moffatt et alldquoAssociation analysis of the interleukin 17A gene in Caucasianrheumatoid arthritis patients from Norway and New ZealandrdquoRheumatology vol 48 no 4 pp 367ndash370 2009

[21] W C Jang Y H Nam Y C Ahn et al ldquoInterleukin-17Fgene polymorphisms in Korean patients with Behcetrsquos diseaserdquoRheumatology International vol 29 no 2 pp 173ndash178 2008

[22] Y Oishi Y Watanabe S Shinoda et al ldquoThe IL6 gene poly-morphism -634CgtG and IL17F gene polymorphism 7488TgtCinfluence bone mineral density in young and elderly Japanesewomenrdquo Gene vol 504 no 1 pp 75ndash83 2012

[23] E Dander A Balduzzi G Zappa et al ldquoInterleukin-17-producing t-helper cells as newpotential playermediating graft-versus-host disease in patients undergoing allogeneic stem-celltransplantationrdquo Transplantation vol 88 no 11 pp 1261ndash12722009

[24] A Accardo Palumbo G I Forte D Pileri et al ldquoAnalysis ofIL-6 IL-10 and IL-17 genetic polymorphisms as risk factors forsepsis development in burned patientsrdquo Burns vol 38 no 2 pp208ndash213 2012

[25] E Crawley R Kay J Sillibiurne et al ldquoPolymorphic haplotypesof the interleukin-10 51015840 flanking region determine variableinterleukin-10 transcription and are associated with particularphenotypes of juvenile rheumatoid arthritisrdquo Arthritis andRheumatology vol 42 no 6 pp 1101ndash1108 1999

[26] O Schroder R A Laun H D Roher et al ldquoHeat shock protein70 genotypes HSPA1B and HSPA1L influence cytokine levelsand interfere with outcome after major injuryrdquo Critical CareMedicine vol 31 no 1 pp 73ndash77 2003

[27] L Ma H-B Xue X-H Guan et al ldquoPossible role of Th17 cellsand IL-17 in the pathogenesis of atopic dermatitis in NorthernChinardquo Journal of Dermatological Science vol 68 no 1 pp 66ndash68 2012

[28] X Chen R Das R Komorowski J van Snick C Uytten-hove and W R Drobyski ldquoInterleukin 17 is not requiredfor autoimmune-mediated pathologic damage during chronicgraft-versus-host diseaserdquo Biology of Blood and Marrow Trans-plantation vol 16 no 1 pp 123ndash128 2010

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


Interleukin-17 (IL-17) is a recently discovered cytokinethat can be produced by many cells although its source fromCD4+ T cells is the most investigated [6] The Th17 cellsare a new lineage of CD4+ T helper cells [7] and generateproinflammatory cytokines such as IL-17A IL-17F IL-21 IL-22 tumor necrosis factor (TNF) granulocyte macrophage-colony stimulating (GM-CSF) and some chemokines [6] IL-17A the original member of this family was first identifiedin 1995 [8] and acts predominantly as a chemoattractantRecent dates have shown thatTh17 pathway is associated withcGVHD [6] AnotherTh17 cytokine is the IL-17F it shows thehighest overall amino acid sequence identity with IL-17A inthe IL-17 family The genes encoding IL-17A and IL-17F areboth located on 6p12 [9] IL-17A and IL-17F share biologicalfunctions and have many proinflammatory effects in a widevariety of cells including macrophage endothelial cells andfibroblasts [10]

In few recent studies IL-17A has been associated with thepathway of the GVHD and some studies have assessed theassociation between IL17A gene polymorphism and inflam-matory diseases including the cGVHD [6 11 12] Howeverthe role of IL-17F in the pathogenesis of cGVHD was notpreviously addressed

So the aim of the present study was to investigate theassociation of the IL17A and IL-17F gene polymorphisms andIL-17A and IL-17F levels with cGHVD as well as to determinethe relationship between IL-17A expression by CD4+ T cellsand the development of the disease

2 Methods

To achieve the proposed objectives the workwas divided intotwo parts which include two groups of individuals In Part 1we performed the genotypic and phenotypic analyses of thecytokines IL-17A and IL-17F and in Part 2 IL-17A immunemarker was assessed cGVHD was determinate according toNIHConsensus Development Project on Criteria for ClinicalTrials [13]

21 Part 1 Genotypic e Phenotypic Evaluation

211 Subjects Fifty-eight consecutive allo-HSCT recipientsand related donors from Hospital das Clınicas at Universi-dade Federal de Minas Gerais (HC-UFMG) were deemedeligible and included in this prospective study The patientswere followed between the days minus7 and +100 after it in a totalof sixteen weeks

All the patients were followed from pre-HSCT until thedevelopment of cGVHD so the number of samples includedin each essay varied because of patientrsquos death and lossof samples related to the process of collection Recipientswere conditioned for allo-HSCT according to the specificprotocols from the Stem Cell Transplant Unit at HC-UFMGand varied according to the type of the disease diseasestatus and the previous treatment at the time of transplan-tation Cyclosporine in combination with methotrexate ormycophenolate mofetil was used for GVHD prophylaxiswhereas 2mgkg ofmethylprednisolone in combinationwithcyclosporine was used for GVHD treatment

This study protocol was approved by the local Institu-tional Ethics Committee (ETIC 01240203000-11) Writteninformed consent was obtained from all patients

212 Subjects and Sample Collection Blood samples werecollected from recipients and donors one week before theHSTC and were submitted for DNA analysis Saliva sam-ples were collected using cotton swabs on the floor of themouth tongue and labial and buccal normal oral mucosaof the HSCT subjects Sites with localized injuries were notincluded The cotton swabs were removed with a sterilecytobrush placed immediately in sterile tubes containing500120583L of Krebs buffer (NaCl 20 KCl 2 CaCl

22H2O

2 MgSO4 KH2PO4 and C

6H12O6) and stored at minus20∘C

until processing Peripheral blood (4mL) was collected invacutainer tubes containing EDTA and stored at minus70∘C untilprocessing

213 DNA Isolation Total genomic DNA was extractedfrom saliva and blood samples using a QIAamp DNA bloodminikit (Quiagen Valencia CAUSA) in accordancewith themanufacturerrsquos instructions The final elution of saliva andblood DNA was performed in 50120583L of a specific AE bufferfrom the kit and stored at minus20∘C until use

214 IL17A and IL17F Gene Polymorphism Analysis Recip-ient and donor IL17 197 (AG) and IL17F 7488 (TC)polymorphism was assessed by means of polymerase chainreaction (PCR) amplification and digestion The sequencesof PCR primers for IL17A were reverse 51015840 AACAAGTAA-GAATGAAAAGAGGACATGGT 31015840 and ant-sense 51015840 CCC-CCAAATGAGGTCATAGAAGAGAATC 31015840 and for IL17Fwere reverse 51015840 GTTCCCATCCAGCAAGAGAC 31015840 and ant-sense 51015840 AGCTGGGAATGCAAACAAAC 31015840 as previouslydescribed [14 15] The PCR was carried out in a total volumeof 50120583L containing approximately 400 ng of DNA primers(20 pmolreaction) and 25 120583L of premix buffer (PhoneutriaBiotecnologia Belo Horizonte Brazil) According to themanufacturer the premix buffer contained 50mM KCl10mM Tris-HCl pH 84 01 triton X-100 15mM MgCl

2

deoxynucleoside triphosphates and 125 units of Taq DNApolymerase PCR products were digested overnight at 37∘Cwith XagI (Fermentas Life Sciences Vilnius Lithuania) forIL-17A and NlaIII (Fermentas Life Sciences Vilnius Lithua-nia) for IL-17F Visualization of the product was performedin a 65 polyacrylamide gel electrophoresis stained withsilver

215 Detection of IL-17A and IL-17F Levels To determine thecytokine levels saliva and blood samples were collected indays +35 and +100 after allo-HSCT The saliva sample wascollected in Salivette tubes (Sarstedt AG amp Co NumbrechtGermany) according to the manufacturerrsquos instructions Thesaliva samples were subsequently diluted (1 1) in a PBS solu-tion containing protease inhibitors (01mM PMSF 01Mmbenzethoniumchloride 10mMEDTA and 001mgmL apro-tinin A) and 005 Tween-20 and subsequently stored atminus20∘C until analysis The total protein content in the saliva


0 200 400 600 800 1000FSC-A

0

200

400

600

800

1000

SSC-

A

30R1

(a)

100

100

101

101

102

102

103

103

104

104

FITC-A CD4 FITC-APE

-A I

L-17

PE-

A

334

0046

0011 0034

391609

R2R3

R4

(b)

100

101

102

103

104

PE-A IL-17 PE-A

0

50

100

15001

Num

ber o

f cel

ls

M1

(c)





















3 Results












4 Discussion





119875


119875

lowast

A P A P


003CC 10 (238) 7 3

NSAG 13 (382) 11 2 CT 18 (429) 9 9AA 9 (265) 3 6 TT 14 (333) 10 4

DonorGG 11 (324) 7 4

NSCC 9 (214) 7 2

NSAG 15 (441) 9 6 CT 19 (452) 12 7AA 8 (235) 7 1 TT 14 (334) 7 7





Minimumlevels


lowast

Blood levelsa+35

GG 14 2242 000 849800007AG 13 4925 000 48014

AA 11 000 000 3956

+100GG 8 359 000 17800

NSAG 12 000 000 11600AA 8 000 000 4402

Saliva levelsb

+35GG 10 000 000 578

NSAG 10 162 000 2174AA 9 000 000 1112

+100GG 9 000 000 2700

NSAG 8 076 000 5412AA 7 000 000 2123












00

02

04

06

08


0008lowast

0008lowast

IL-17

A in

CD4+

T ce

lls (

)

(a)

MediaSEB

120572CD3120572CD28

100

101

102

103

104

IL-17 PE

100

80

60

40

20

0

Max

()

(b)





5 Conclusion





Acknowledgment


References



































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0 200 400 600 800 1000FSC-A

0

200

400

600

800

1000

SSC-

A

30R1

(a)

100

100

101

101

102

102

103

103

104

104

FITC-A CD4 FITC-APE

-A I

L-17

PE-

A

334

0046

0011 0034

391609

R2R3

R4

(b)

100

101

102

103

104

PE-A IL-17 PE-A

0

50

100

15001

Num

ber o

f cel

ls

M1

(c)





















3 Results












4 Discussion





119875


119875

lowast

A P A P


003CC 10 (238) 7 3

NSAG 13 (382) 11 2 CT 18 (429) 9 9AA 9 (265) 3 6 TT 14 (333) 10 4

DonorGG 11 (324) 7 4

NSCC 9 (214) 7 2

NSAG 15 (441) 9 6 CT 19 (452) 12 7AA 8 (235) 7 1 TT 14 (334) 7 7





Minimumlevels


lowast

Blood levelsa+35

GG 14 2242 000 849800007AG 13 4925 000 48014

AA 11 000 000 3956

+100GG 8 359 000 17800

NSAG 12 000 000 11600AA 8 000 000 4402

Saliva levelsb

+35GG 10 000 000 578

NSAG 10 162 000 2174AA 9 000 000 1112

+100GG 9 000 000 2700

NSAG 8 076 000 5412AA 7 000 000 2123












00

02

04

06

08


0008lowast

0008lowast

IL-17

A in

CD4+

T ce

lls (

)

(a)

MediaSEB

120572CD3120572CD28

100

101

102

103

104

IL-17 PE

100

80

60

40

20

0

Max

()

(b)





5 Conclusion





Acknowledgment


References



































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



























3 Results












4 Discussion





119875


119875

lowast

A P A P


003CC 10 (238) 7 3

NSAG 13 (382) 11 2 CT 18 (429) 9 9AA 9 (265) 3 6 TT 14 (333) 10 4

DonorGG 11 (324) 7 4

NSCC 9 (214) 7 2

NSAG 15 (441) 9 6 CT 19 (452) 12 7AA 8 (235) 7 1 TT 14 (334) 7 7





Minimumlevels


lowast

Blood levelsa+35

GG 14 2242 000 849800007AG 13 4925 000 48014

AA 11 000 000 3956

+100GG 8 359 000 17800

NSAG 12 000 000 11600AA 8 000 000 4402

Saliva levelsb

+35GG 10 000 000 578

NSAG 10 162 000 2174AA 9 000 000 1112

+100GG 9 000 000 2700

NSAG 8 076 000 5412AA 7 000 000 2123












00

02

04

06

08


0008lowast

0008lowast

IL-17

A in

CD4+

T ce

lls (

)

(a)

MediaSEB

120572CD3120572CD28

100

101

102

103

104

IL-17 PE

100

80

60

40

20

0

Max

()

(b)





5 Conclusion





Acknowledgment


References



































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















119875


119875

lowast

A P A P


003CC 10 (238) 7 3

NSAG 13 (382) 11 2 CT 18 (429) 9 9AA 9 (265) 3 6 TT 14 (333) 10 4

DonorGG 11 (324) 7 4

NSCC 9 (214) 7 2

NSAG 15 (441) 9 6 CT 19 (452) 12 7AA 8 (235) 7 1 TT 14 (334) 7 7





Minimumlevels


lowast

Blood levelsa+35

GG 14 2242 000 849800007AG 13 4925 000 48014

AA 11 000 000 3956

+100GG 8 359 000 17800

NSAG 12 000 000 11600AA 8 000 000 4402

Saliva levelsb

+35GG 10 000 000 578

NSAG 10 162 000 2174AA 9 000 000 1112

+100GG 9 000 000 2700

NSAG 8 076 000 5412AA 7 000 000 2123












00

02

04

06

08


0008lowast

0008lowast

IL-17

A in

CD4+

T ce

lls (

)

(a)

MediaSEB

120572CD3120572CD28

100

101

102

103

104

IL-17 PE

100

80

60

40

20

0

Max

()

(b)





5 Conclusion





Acknowledgment


References



































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















00

02

04

06

08


0008lowast

0008lowast

IL-17

A in

CD4+

T ce

lls (

)

(a)

MediaSEB

120572CD3120572CD28

100

101

102

103

104

IL-17 PE

100

80

60

40

20

0

Max

()

(b)





5 Conclusion





Acknowledgment


References



































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of













































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Research Article IL-17 Genetic and Immunophenotypic Evaluation...

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Transcript of Research Article IL-17 Genetic and Immunophenotypic Evaluation...