Research Article Macrophage Migration Inhibitory...

Research ArticleMacrophage Migration Inhibitory Factor Interacting withTh17 Cells May Be Involved in the Pathogenesis of AutoimmuneDamage in Hashimotorsquos Thyroiditis

Haibo Xue1 Yuhua Yang2 Ying Zhang3 Shoujun Song1 Li Zhang1

Lei Ma4 Tingting Yang1 and Huan Liu1

1Department of Endocrinology and Metabolism Binzhou Medical University Hospital 661 Second Huanghe RoadBinzhou 256603 China2Department of Endocrinology Wudi County Peoplersquos Hospital Tengda Street Wudi 251900 China3Department of Clinical Nutrition Binzhou Medical University Hospital 661 Second Huanghe Road Binzhou 256603 China4Department of Dermatology Binzhou Medical University Hospital 661 Second Huanghe Road Binzhou 256603 China

Correspondence should be addressed to Haibo Xue xue haibohotmailcom and Lei Ma doctor maleihotmailcom

Received 16 January 2015 Accepted 2 March 2015

Academic Editor Amedeo Amedei

Copyright copy 2015 Haibo Xue et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Purpose To explore the possible role of MIF andTh17 cells in the thyroid-specific autoimmune damage of Hashimotorsquos thyroiditis(HT) Material and Methods We enrolled 40 HT patients and 30 healthy controls and divided HT patients into euthyroid subset(119899 = 22) and subclinical or overt hypothyroidism subset (119899 = 18)The percentages ofTh17 cells and expressions of MIF interleukin17A (IL-17A) mRNA in PBMCs as well as serum concentrations of MIF and IL-17A and thyroid functions and thyroid-specificautoantibodies (TPOAb TgAb) were detected by flow cytometry real-time RT-PCR ELISA and ECLIA in all subjectsResults MIFmRNA IL-17AmRNA expressions andTh17 cells percentages serumMIF and IL-17A protein levels were all significantly higher inHT patients even in euthyroid subgroup Additionally the differences becamemore obvious in dysfunction subgroup ImportantlybothMIF levels andTh17 cells percentage were positively correlated with serumTPOAb TgAb and thyrotropin (TSH) levels in HTpatients Conclusions These data suggest that MIF andTh17 cells increased dynamically and positively correlated with the markersof thyroid autoimmune damage which indicated that interaction between MIF andTh17 cells may participate in the pathogenesisand development of thyroid-specific autoimmunity in HT

1 Introduction

Hashimotorsquos thyroiditis (HT) is known as a typical autoim-mune thyroid disease (AITD) which affects up to 2 of gen-eral population and is 5 to 10 times more common in womenthan in men [1 2] However the prevalence of anti-thyroidantibodies without any clinical manifestation may be evenhigher [3 4] The features of HT mainly include lymphocyticinfiltration in the thyroid and increasing serum antibodiesto thyroid-specific antigens (thyroid peroxidase antibodyTPOAb thyroglobulin antibody TgAb) Most of HT patientsultimately evolve into hypothyroidism although patients canhave normal thyroid function or even hyperthyroidism atearly disease stageTherefore it has been considered themost

common cause which leads to hypothyroidism [5 6] Thecrucial factor in the development of HT is considered thebreakdown of immune tolerance Furthermore hereditarysusceptibility and environmental factors may increase theincidence of HT especially high iodine intake [7] So far theexact pathogenesis of HT has not been well elucidated

Macrophage migration inhibitory factor (MIF) is orig-inally discovered as a lymphokine relevant to delayed-typehypersensitivity which inhibits the random migration ofmacrophages and concentratesmacrophages at inflammatorylesions as well [8] As a multifunctional proinflammatorycytokine MIF is demonstrated to participate in innate andadaptive immune responses and is known to be implicatedin the pathogenesis of many autoimmune diseases such as

Hindawi Publishing CorporationMediators of InflammationVolume 2015 Article ID 621072 9 pageshttpdxdoiorg1011552015621072

2 Mediators of Inflammation

Table 1 Characteristics of patients with HT and healthy controls

HT HT-A HT-B Healthy control Normal rangeCases (119899) 40 22 18 30 mdashGender (FM) 355 193 162 264 mdashAge (years) 2893 plusmn 830 2891 plusmn 921 2894 plusmn 730 3097 plusmn 639 mdashTSH (mIUL) 413 (182ndash625)lowastlowast 194 (099ndash306) 695 (512ndash1618)lowastlowast 189 (115ndash259) 028ndash43FT3 (pmolL) 449 plusmn 132lowast 497 plusmn 117 389 plusmn 127lowastlowast 523 plusmn 098 28ndash71FT4 (pmolL) 1565 plusmn 304lowast 1658 plusmn 228 1452 plusmn 350lowastlowast 1729 plusmn 209 12ndash22TPOAb (IUmL) 20000 (14835ndash29988)lowastlowast 17100 (10953ndash21508)lowastlowast 29475 (19958ndash41085)lowastlowast 1425 (753ndash2400) 0ndash34TgAb (IUmL) 48975 (30070ndash77470)lowastlowast 33515 (25885ndash48548)lowastlowast 71755 (55153ndash105558)lowastlowast 5100 (1975ndash8425) 0ndash115MUI (120583gL) 16940 16845 17030 16335 mdashData are shown as mean plusmn SD or median (25thndash75th percentile) according to the distribution HT-A HT patient with normal thyroid function HT-B HTpatient with subclinical or overt hypothyroidism M male F female MUI median of urine iodine The P values represent different groups compared withhealthy control (HC) lowast119875 lt 005 lowastlowast119875 lt 001

inflammatory bowel disease (IBD) [9] rheumatoid arthritis[10] and vitiligo vulgaris [11] Additionally MIF gene poly-morphisms (rs755622 SNP) have been shown in associationwith the severity of goiter in patients with untreated Gravesrsquodisease (GD) which is another common AITD [12] Morerecently T helper 17 cells (Th17) a newly recognized subset ofCD4+ T helper cells have been demonstrated to play criticalroles in the pathogenesis of several autoimmune diseaseswhich is considered the main source of interleukin 17 (IL-17)[13ndash15] Given that subsequent investigations reported thatMIF can interact with other cytokines and lead to impairedimmune responses there is no in-depth study about the roleof MIF in HT Several studies have revealed that MIF isinvolved in promoting the differentiation and developmentof IL-17 in animal [16 17] Thus we speculate that MIFmay be implicated in inflammatory and autoimmune diseasethrough interacting withTh17 cells

Therefore in the present study we detected the expres-sions of MIF Th17 cells and IL-17A in HT patients andhealthy controls analyzed the relationships between MIFand Th17 cells and MIF Th17 cells and thyroid-specificautoantibodies and tried to explore the possible role of MIFand Th17 cells in the pathogenesis of thyroid autoimmunedamage in HT

2 Materials and Methods

21 Subjects and Clinical Assessments Forty patients withHTwere enrolled in this study and all the patients were newlydiagnosed and untreated previouslyThe diagnosis of HTwasbased on the classical criterion [18] HT patients were dividedinto two subgroups based on their thyroid functions HT-Asubset (euthyroidism 22 cases) and HT-B subset (subclinicalor overt hypothyroidism 18 cases) Meanwhile thirty healthyvolunteers withmatched age and sex features were selected asthe healthy controls (HC) who had no autoimmune diseasehistory The clinical features of all subjects were shown inTable 1 All study procedures were performed in accordancewith the guidelines of the Declaration of Helsinki withthe approval of the Ethics Committee of Binzhou MedicalUniversityHospital From all participants a written informedconsent was obtained

Electrochemiluminescence immunoassay analyzer(ECLIA Roche Cobas 6000 Germany) was used to measurethyroid function and thyroid-specific autoantibodies in allsubjects which include TSH free triiodothyronine (FT

3)

and free thyroxine (FT4) and TPOAb and TgAb (their

reference ranges were shown in Table 1) Color Dopplerultrasonography (LOGIQ9 GE USA) was performed on allsubjects by trained observers mainly observing thyroid sizeecho (especially hypoechogenicity) and blood flow Urinaryiodine excretion was detected in all participants to evaluatetheir iodine nutritional status [19]

22 Flow Cytometric Analysis of Th17 Cells Isolation ofperipheral bloodmononuclear cells (PBMCs) was performedvia Ficoll-Hypaque density gradient centrifugation (Sigma-Aldrich St Louis MO 1200 rpm 25min) Cells were washedtwice in phosphate buffered saline (PBS) incubated for5 hours with 25 ngmL phorbol myristate acetate (PMA)and 1 120583gmL ionomycin (Sigma USA) in the presence of2mmolmLmonensin at 37∘C under a 5 CO

2environment

and then transferred to each tube washed once with PBSand incubated with anti-CD4-FITC at 4∘C for 30 minutesin the dark Following being fixed and permeabilized cellswere stained with intracellular anti-IL-17A-PE Meanwhileisotype-matched controls were used to correct nonspecificbindingTh17 cell numbers were analyzed with a FACSCantoflow cytometer and data were collected and analyzed usingCellQuest software (BD Biosciences USA) The antibodiesmentioned above were obtained from eBioscience (USA)

23 Real-Time Quantitative RT-PCR Analysis of MIF and IL-17AmRNA Total RNAwas isolated fromPBMCswith Trizol(Invitrogen USA) Reverse transcriptionwas performedwithPrimeScript RT reagent kit (TaKaRa Japan) on an ABI 9700PCR meter (37∘C for 15min followed by 85∘C for 5 s) Real-time RT-PCR was performed on a Rotor-Gene 3000 (CorbettResearch Australia) using SYBR Premix Ex Taq II (TaKaRaJapan) and the given primers for MIF and IL-17A (Table 2)additionally 120573-actin was used as an internal control Thesame samplewas run in triplicate and datawere analyzedwiththe Rotor-Gene Real-Time Analysis Software 60

Mediators of Inflammation 3

Table 2 Primers for real-time quantitative RT-PCR

Primer Sequence (51015840 to 31015840)

MIF Forward 51015840-ACCAGCTCATGGCCTTCG-31015840

Reverse 51015840-CTTGCTGTAGGAGCGGTT-31015840

IL-17A Forward 51015840-TGTCCACCATGTGGCCTAAGAG-31015840

Reverse 51015840-GTCCGAAATGAGGCTGTCTTTGA-31015840

120573-actin Forward 51015840-AGTTGCGTTACACCCTTTCTTG-31015840

Reverse 51015840-TCACCTTCACCGTTCCAGTTT-31015840

24 Measurements of Serum MIF and IL-17A Using Enzyme-Linked Immunosorbent Assay (ELISA) The levels of serumMIF and IL-17A protein were measured in duplicate withhuman ELISA kits (RampD system USA) and the proto-cols were conducted in accordance with the manufacturerrsquosinstructions

3 Statistical Analysis

Data are shown as mean plusmn standard deviation or median(interquartile range) according to the distribution Thenormally distributed data were analyzed by independent-samples 119905-test and Pearson correlation Meanwhile the anal-ysis of abnormally distributed data was performed withKruskal-Wallis test followed by theMann-Whitney119880 test andSpearman test All analyses were performed using GraphPadPrism software version 60 (SanDiego CA)119875 values oflt 005were considered statistically significant

4 Results

41 Subject Characteristics Thyroid function test revealedthat TSH FT

3 and FT

4in HT and HT-B subgroup changed

obviously compared to HC (all 119875 lt 005) howeverthe differences between HT-A subgroup and HC were notsignificant (all 119875 gt 005) Moreover thyroid function inHT-B differed markedly from that in HT-A (119875 lt 001 or lt005) Additionally serum TPOAb and TgAb titers increasedsignificantly inHT andHT subgroups (all119875 lt 001) andHT-B patients had higher levels than HT-A (119875 lt 001) Moreoverthe results of median of urine iodine (MUI) indicated that allparticipants were in adequate iodine status and there wereno significant differences between different groups (all 119875 gt005) These data were shown in Table 1

42 MIF mRNA and IL-17 mRNA Levels in PBMCs TheMIF mRNA expressions of PBMCs in HT (298 plusmn 142) HT-A (250 plusmn 104) and HT-B (357 plusmn 163) subsets increasedsignificantly compared to HC (196 plusmn 078 119875 = 00010038 0001 resp) and HT-B patients had higher levelsthan HT-A (119875 = 0023) Figure 1(a) Additionally the IL-17A mRNA changes of PBMCs in HT (423 plusmn 113) HT-A (377 plusmn 118) and HT-B (479 plusmn 077) were also muchhigher than HC (202 plusmn 056 all 119875 lt 001) moreoverthe differences between HT-A and HT-B subgroups weresignificant (119875 = 0003) Figure 1(b) Furthermore Pearsoncorrelation analysis showed that MIF mRNA levels positively

correlated with IL-17A mRNA expressions (119903 = 0390119875 = 0013) Figure 1(c) These data suggested that both MIFand LI-17A mRNA increased markedly in HT patients andhad dynamic changes during different disease status MoreinterestinglyMIFmRNAexpressions had a close relationshipwith that of IL-17A

43 Percentage of Th17 Cells in PBMCs Flow cytometryanalysis showed that the proportions of circulatingTh17 cellsin HT (143plusmn043) HT-A (116plusmn032) and HT-B (176plusmn031)were all obviously higher than healthy controls (042plusmn014 all 119875 lt 001) In addition HT-B subgroup patientsalso had higher Th17 cell numbers than HT-A (119875 lt 001)Figure 2 These results revealed that peripheral Th17 cellsdynamically changed between the two HT patient subsetswhich suggested that there was a close relation betweenTh17cells and the course of HT

44 ELISA Results of MIF and IL-17A Concentrations inSerum Increased serum MIF protein levels were foundin both HT and HT subsets compared to HC (2538 plusmn1327 ngmL 1664 plusmn 893 ngmL and 3608 plusmn 926 ngmLversus 789 plusmn 189 ngmL all 119875 lt 001) meanwhilethere were remarkable differences between HT-A and HT-Bsubgroups (119875 lt 001) Figure 3(a) Additionally serum IL-17A concentrations also increased obviously in HT and HTsubsets patients compared toHC (3009plusmn518 pgmL 2780plusmn512 pgmL and 3288plusmn376 pgmL versus 1127plusmn223 pgmLall 119875 lt 001) and patients in HT-B subgroup had higherIL-17A levels than HT-A subgroup (119875 lt 001) Figure 3(b)Furthermore serum MIF protein levels positively correlatedwith serum IL-17A protein concentrations and Th17 cellspercentages of PBMCs in patients with HT (119903 = 04590442 119875 = 0003 0004 resp Figures 3(c) 3(d)) Similarly apositive correlation was found between serum IL-17A levelsand peripheral Th17 cells percentages in patients with HT(119903 = 0485 119875 = 0001)

45 Results of Correlation Analysis First we analyzed therelationships between serum MIF levels and serum TPOAband TgAb titers in HT patients The results revealed thatMIF had positive correlations with TPOAb (119903 = 037 119875 =0019) and TgAb (119903 = 0489 119875 = 0001) In addition Th17cells percentages also had positive correlations with TPOAb(119903 = 0459 119875 = 0003) and TgAb in HT patients (119903 =0401 119875 = 0010) More interestingly both MIF and Th17cells positively correlated with serum TSH levels in patientswith HT (119903 = 0444 0553 119875 = 0007 0001 resp) Thesedata suggested that there were close relationships betweenMIF and Th17 cells and the markers of thyroid-specificautoimmune damage as well as the sensitive index of thyroiddysfunction as shown in Figure 4

5 Discussion

Although immunological factors have been considered toplay crucial roles in the pathogenesis of AITD the precisemechanisms by which immunological factors contribute to


MIF

mRN

A ex

pres

sions

HC HT HT-A HT-B0

2

4

6

8lowastlowast

lowastlowast

lowast

lowast

(a)

HC HT HT-A HT-B0

2

4

6

8

IL-1

7A m

RNA

expr

essio

ns

lowastlowast

lowastlowast

lowastlowast

lowastlowast

(b)

MIF mRNA expressions

IL-1

7A m

RNA

expr

essio

ns

0 2 4 60

2

4

6

8r = 0390 P = 0013

(c)

Figure 1 (a) Expressions of MIF mRNA increased significantly in HT patients compared with HC (lowast119875 lt 005 lowastlowast119875 lt 001) In HT subsetsHT-B patients had higher MIF mRNA expressions than those in HT-A subgroup (lowast119875 lt 005) which indicated that there were dynamicchanges consistent with the severity of the disease (b) The same characteristics of IL-17A mRNA in HT and HT subgroups as MIF mRNAexpressions could be found (lowastlowast119875 lt 001) (c) MIF mRNA levels positively correlated with IL-17A mRNA levels in HT patients

the pathogenesis of HT have remained obscure In this workwe aimed to analyze the potential roles of MIF andTh17 cellsin the thyroid-specific autoimmune activity in patients withHT In accordance with what had been found in the patientswith other autoimmune diseases we also demonstratedincreasedMIFmRNA levels of PBMCs inHTpatientsMean-while serum MIF protein concentrations also increased aswell as positively correlating with MIF mRNA expressionsMoreover dynamic alterations in different disease statuswere found to be statistically significant even in euthyroidstage particularly more obvious in patients with subclinicaland overt hypothyroidism Further detections illustrated thatperipheral circulating Th17 cells and their main effectivecytokine IL-17A increased markedly in HT patients withsimilar features as MIF Meanwhile both IL-17A mRNA inPBMCs and serum IL-17A protein correlated positively withTh17 cells percentages Importantly we further found thatthere were positive correlations between MIF and Th17 cellspercentage IL-17A levels in HT patients Previous studies

demonstrated MIF could induce powerful proinflammatorybiological responses and has been shown to be an importanteffector molecule in infection [20] and also upregulate theexpression of Toll-like receptor 4 (TLR4) which mediateslipopolysaccharide binding and activation of macrophages[21] Therefore MIF has been recognized as a cytokinethat exhibits a broad range of immune and inflammatoryactivities including induction of inflammatory cytokinesand regulation of macrophage and lymphocyte proliferationMIF deficiency whether achieved through genetic dele-tion (119872119868119865minusminus) or anti-MIF antibodies (Abs) neutralizationresults in inhibiting inflammatory responses in a variety ofmurine models of human inflammatory and autoimmunediseases followed by significant reduction of inflammatorycell infiltration and cytokines expressions [22ndash24] whereasan increased number of skin-infiltrating eosinophils wereobserved in ovalbumin-sensitizedMIF transgenic mice com-pared with the wild-type [25] further suggesting MIF playsa dominant role in lymphocyte activation and cytokine


100

101

102

103

104

100

101

102

103

104

PE-A

IL-17

A

FITC-ACD4

HT patient Healthy control

CD4

220 045

100

101

102

103

104

FITC-A

100

101

102

103

104

PE-A

IL-17

A(a)

Th17

cells

()

0

1

2

3

HC HT HT-A HT-B

lowastlowast

lowastlowast

lowastlowast

lowastlowast

(b)

Figure 2 (a) The representative graphs of flow cytometric analysis CD4+IL-17A+ T cells were determined as Th17 cells (b) Comparisonsof Th17 cells percentages between subjects in HT and HC as well as between HT subsets and HC or between HT-A and HT-B subsets HTpatients hadmuch higherTh17 cells proportion than HC even in euthyroidism status (lowastlowast119875 lt 001) AdditionallyTh17 cells numbers in HT-Bsubgroup also increased significantly compared to HT-A (lowastlowast119875 lt 001) which provided the evidences of dynamic changes in different diseasestages

production A recent study reported that MIF-knockoutmice had severely impaired production of IL-17 IL-1120573 IL-6 IL-23 and TGF-120573 but treatment of lymph node cellswith recombinant MIF upregulated antigen-stimulated IL-17 expression and secretion [16] Because these cytokinesare essential for the differentiation and sustained generationof IL-17 from naıve T lymphocytes [26 27] thus MIF isconsidered to potently stimulate IL-17 production through acomplex cytokine network Th17 cells have been designatedas predominant producers of IL-17 and been demonstratedto play a pivotal role in many kinds of autoimmune diseases[28] including some commonAITD [29 30] Taken together

with our results MIF may be implicated in the pathogenesisand progression of HT through promoting the differentiationand development of Th17 cells

In addition to lymphocytes infiltration another impor-tant feature of HT is the increased levels of thyroid-specificautoantibodies mainly including TPOAb and TgAb whichindicate the severities of autoimmune damage in thyroid[31] In the present study we firstly found that MIF mRNAand protein expressions positively correlated with TPOAband TgAb titers in HT patients further supporting a closerelationship betweenMIF and thyroid autoimmune responseThe current opinion is thyroperoxidase (TPO) relating to


Seru

m M

IF le

vels

(ng

mL)

HC HT HT-A HT-B0

20

40

60 lowastlowast

lowastlowast

lowastlowast

lowastlowast

(a)

0

20

10

30

40

50

HC HT HT-A HT-B

lowastlowast

lowastlowast

lowastlowast

lowastlowast

Seru

m IL

-17A

leve

ls (p

gm

L)

(b)

Seru

m IL

-17A

leve

ls (p

gm

L)

r = 0459 P = 0003

Serum MIF levels (ngmL)200 40 60

20

10

30

40

50

(c)

Th17

cells

()

r = 0442 P = 0004

0

1

2

3


(d)

Serum IL-17A levels (pgmL)

Th17

cells

()

20 30 400

1

2

3r = 0485 P = 0001

(e)

Figure 3 (a) Differences of serum MIF protein levels between HT and HC HT subsets and HC and HT-B and HT-A subgroups were allsignificant (lowastlowast119875 lt 001) (b) Similar characteristics of serum IL-17A protein concentrations could be found like MIF (lowastlowast119875 lt 001) (c) and (d)Positive correlations between MIF protein levels and IL-17A protein concentrations Th17 cells percentages were found in patients with HT(e) Levels of serum IL-17A protein positively correlated with the proportion of Th17 cells

TPOAb is the main disease-causing antigen (Ag) in human[32 33] TPOAb is found in about 95 of HT patients but israre in healthy controls and correlates well with the numberof autoreactive lymphocytes infiltrating the thyroid [34]

Therefore it is now considered the best serological markerof thyroid dysfunction and its presence is predictive of thesubsequent occurrence of thyroid failure in AITD patients[6] Importantly another finding of our study is that MIF


Serum MIF levels (ngmL)

Seru

m T

POAb

tite

rs (I

Um

L)

0 20 40 600

200

400

600

800r = 0370 P = 0019

(a)

Seru

m T

gAb

titer

s (IU

mL)

Serum MIF levels (ngmL)0 20 40 60

0

1000

2000

3000

4000r = 0489 P = 0001

(b)


Seru

m T

SH le

vels

(mIU

L)

0 10 20 30 400

50

100

50

r = 0444 P = 0007

(c)

Seru

m T

POAb

tite

rs (I

Um

L)

0

200

400

600

800

Th17 cells ()05 10 15 20 25

r = 0459 P = 0003

(d)

Seru

m T

gAb

titer

s (IU

mL)

Th17 cells ()05 10 15 20 25

0

1000

2000

3000

4000r = 0401 P = 0010

(e)

Th17 cells ()

Seru

m T

SH le

vels

(mIU

L)

05 10 15 20 250

25

50

75

100r = 0553 P = 0001

(f)

Figure 4 (a)ndash(c)MIF protein levels positively correlated with serum TPOAb TgAb and TSH concentrations (d)ndash(f) Increasing percentagesof Th17 cells also had positive correlation with serum TPOAb TgAb and TSH levels These analyses all used Spearman test

also positively correlated with serum TSH levels which is themost sensitive index reflecting thyroid function In additionTh17 cells percentages in PBMCs had illustrated the samecharacteristics as MIF positively correlating with TPOAbTgAb and TSH It has been found that T cells activatedby specific Ag mitogens or anti-CD3 Abs show increased

expression of MIF mRNA and protein However anti-MIFAbs inhibit T cell proliferation and Abs production fromB cells [35] Thus MIF expression has been considered tobe critical to the generation of an antigen-specific immuneresponse Collectively we consider that the severities ofthyroid autoimmune damage and impaired thyroid function


status may be attributed to the interaction between MIF andTh17 cells

6 Conclusions

Taken together our data provide novel evidence thatincreasedMIF together with circulatingTh17 cells positivelycorrelated with thyroid-specific autoantibodies and differentthyroid dysfunction stages in HT patients in which wespeculate that MIF may also be involved in the patho-genesis and development of thyroid autoimmune responsesin patients with HT Given that current therapeutic inves-tigations revealed that MIF and IL-17 deficiency throughgenetic deletion or Abs neutralization results in protectionor release from several animal models of inflammatory andautoimmune disease [9 10 24ndash27 36] clinical applicationstill needs further in-depth research Anyhowbetter advancesofMIF andTh17 cells in the pathogenesis ofHTwill be helpfulfor the new target of treatment for patients with HT

Abbreviations

MIF Macrophage migration inhibitory factorHT Hashimotorsquos thyroiditisAITD Autoimmune thyroid diseaseTh17 T helper 17IL-17A Interleukin 17APBMC Peripheral blood mononuclear cellTPOAb Thyroid peroxidase antibodyTgAb Thyroglobulin antibodyTSH ThyrotropinFT3 Free triiodothyronine

FT4 Free thyroxine

MUI Median of urine iodineELISA Enzyme-linked immunosorbent assayECLIA Electrochemiluminescence immunoassay

analyzer

Conflict of Interests

The authors declare no conflict of interests

Acknowledgments

This study was financed by Shandong Province Science andTechnology Development Project of China (2011YD18063)ShandongProvinceHigher Educational Science andTechnol-ogy Program of China (J11LF66) Binzhou Medical Univer-sity Scientific Research Foundation (BY2010KYQD04) andShandong Province Natural Science Foundation of China(ZR2013HM049)

References

[1] D S A McLeod and D S Cooper ldquoThe incidence andprevalence of thyroid autoimmunityrdquo Endocrine vol 42 no 2pp 252ndash265 2012

[2] M P J Vanderpump ldquoThe epidemiology of thyroid diseaserdquoBritish Medical Bulletin vol 99 no 1 pp 39ndash51 2011

[3] S H Golden K A Robinson I Saldanha B Anton andP W Ladenson ldquoPrevalence and incidence of endocrine andmetabolic disorders in the united states a comprehensivereviewrdquo The Journal of Clinical Endocrinology and Metabolismvol 94 no 6 pp 1853ndash1878 2009

[4] B Delemer J-P Aubert P Nys F Landron and S BoueeldquoAn observational study of the initial management of hypothy-roidism in France The ORCHIDEE studyrdquo European Journal ofEndocrinology vol 167 no 6 pp 817ndash823 2012

[5] E N Pearce A P Farwell and L E Braverman ldquoThyroiditisrdquoTheNewEngland Journal ofMedicine vol 348 no 26 pp 2646ndash2655 2003

[6] A Antonelli S M Ferrari A Corrado et al ldquoAutoimmunethyroid disordersrdquo Autoimmunity Reviews vol 14 no 2 pp174ndash180 2015

[7] W Teng Z Shan X Teng et al ldquoEffect of iodine intakeon thyroid diseases in Chinardquo The New England Journal ofMedicine vol 354 no 26 pp 2783ndash2793 2006

[8] B R Bloom and B Bennett ldquoMechanism of a reaction in vitroassociated with delayed-type hypersensitivityrdquo Science vol 153no 3731 pp 80ndash82 1966

[9] J Nishihira ldquoMolecular function of macrophage migrationinhibitory factor and a novel therapy for inflammatory boweldiseaserdquo Annals of the New York Academy of Sciences vol 1271no 1 pp 53ndash57 2012

[10] J N Hoes M C van der goes J W G Jacobs F P G J LafeberJ W J Bijlsma and J A G van Roon ldquoChanges in macrophageinhibitory factor correlate with changes in bonemineral densityin glucocorticoid-treated patients with rheumatoid arthritisrdquoRheumatology vol 50 no 10 Article ID ker268 pp 1921ndash19242011

[11] L Ma H-B Xue X-H Guan et al ldquoRelationship ofmacrophage migration inhibitory factor levels in PBMCslesional skin and serum with disease severity and activity invitiligo vulgarisrdquo Brazilian Journal of Medical and BiologicalResearch vol 46 no 5 pp 460ndash464 2013

[12] Y H Liu C C Chen C M Yang Y J Chen and F J TsaildquoDual effect of a polymorphism in the macrophage migrationinhibitory factor gene is associated with new-onset gravesdisease in a Taiwanese Chinese populationrdquo PLoS ONE vol 9no 3 Article ID e92849 2014

[13] E Bettelli M Oukka and V K Kuchroo ldquoT119867-17 cells in the

circle of immunity and autoimmunityrdquoNature Immunology vol8 no 4 pp 345ndash350 2007

[14] W Ouyang J K Kolls and Y Zheng ldquoThe biological functionsof T helper 17 cell effector cytokines in inflammationrdquo Immu-nity vol 28 no 4 pp 454ndash467 2008

[15] T Korn E Bettelli M Oukka and V K Kuchroo ldquoIL-17 andTh17 cellsrdquo Annual Review of Immunology vol 27 pp 485ndash5172009

[16] I Stojanovic T Cvjeticanin S Lazaroski S Stosic-Grujicicand D Miljkovic ldquoMacrophage migration inhibitory factorstimulates interleukin-17 expression and production in lymphnode cellsrdquo Immunology vol 126 no 1 pp 74ndash83 2009

[17] J Li H-Y Mo G Xiong et al ldquoTumor microenviron-ment macrophage inhibitory factor directs the accumulationof interleukin-17-producing tumor-infiltrating lymphocytesand predicts favorable survival in nasopharyngeal carcinomapatientsrdquo The Journal of Biological Chemistry vol 287 no 42pp 35484ndash35495 2012


[18] P Caturegli A de Remigis and N R Rose ldquoHashimoto thy-roiditis clinical and diagnostic criteriardquoAutoimmunity Reviewsvol 13 no 4-5 pp 391ndash397 2014

[19] J T Dunn H E Crutchfield and R Gutekunst Methods forMeasuring Iodine in Urine International Council for the Con-trol of Deficiency Disorders Wageningen The Netherlands1993

[20] T Calandra ldquoMacrophage migration inhibitory factor and hostinnate immune responses tomicrobesrdquo Scandinavian Journal ofInfectious Diseases vol 35 no 9 pp 573ndash576 2003

[21] T Roger J David M P Glauser and T Calandra ldquoMIFregulates innate immune responses throughmodulation of Toll-like receptor 4rdquo Nature vol 414 no 6866 pp 920ndash924 2001

[22] G M Cox A P Kithcart D Pitt et al ldquoMacrophage migrationinhibitory factor potentiates autoimmune-mediated neuroin-flammationrdquo Journal of Immunology vol 191 no 3 pp 1043ndash1054 2013

[23] BWang X Huang P J Wolters et al ldquoCutting edge deficiencyof macrophage migration inhibitory factor impairs murineairway allergic responsesrdquo The Journal of Immunology vol 177no 9 pp 5779ndash5784 2006

[24] Y Nakamaru N Oridate J Nishihira D Takagi Y Furutaand S Fukuda ldquoMacrophage migration inhibitory factor (MIF)contributes to the development of allergic rhinitisrdquo Cytokinevol 31 no 2 pp 103ndash108 2005

[25] Y Yoshihisa T Makino K Matsunaga et al ldquoMacrophagemigration inhibitory factor is essential for eosinophil recruit-ment in allergen-induced skin inflammationrdquo Journal of Inves-tigative Dermatology vol 131 no 4 pp 925ndash931 2011

[26] E V Acosta-Rodriguez G Napolitani A Lanzavecchia and FSallusto ldquoInterleukins 1120573 and 6 but not transforming growthfactor-120573 are essential for the differentiation of interleukin 17-producing human T helper cellsrdquo Nature Immunology vol 8no 9 pp 942ndash949 2007

[27] N J Wilson K Boniface J R Chan et al ldquoDevelop-ment cytokine profile and function of human interleukin 17-producing helper T cellsrdquo Nature Immunology vol 8 no 9 pp950ndash957 2007

[28] R P Singh S Hasan S Sharma et al ldquoTh17 cells in inflamma-tion and autoimmunityrdquo Autoimmunity Reviews vol 13 no 12pp 1174ndash1181 2014

[29] N Figueroa-Vega M Alfonso-Perez I Benedicto F Sanchez-Madrid R Gonzalez-Amaro and M Marazuela ldquoIncreasedcirculating pro-inflammatory cytokines and Th17 lymphocytesin Hashimotorsquos thyroiditisrdquo Journal of Clinical Endocrinologyand Metabolism vol 95 no 2 pp 953ndash962 2010

[30] D Peng B Xu YWangH Guo andY Jiang ldquoA high frequencyof circulating Th22 and Th17 cells in patients with new onsetGravesrsquo diseaserdquo PLoS ONE vol 8 no 7 Article ID e684462013

[31] P Caturegli A de Remigis K Chuang M Dembele AIwama and S Iwama ldquoHashimotorsquos thyroiditis celebrating thecentennial through the lens of the Johns Hopkins hospitalsurgical pathology recordsrdquoThyroid vol 23 no 2 pp 142ndash1502013

[32] A Carle P Laurberg N Knudsen et al ldquoThyroid peroxidaseand thyroglobulin auto-antibodies in patients with newly diag-nosed overt hypothyroidismrdquo Autoimmunity vol 39 no 6 pp497ndash503 2006

[33] S M McLachlan and B Rapoport ldquoThyroid peroxidase as anautoantigenrdquoThyroid vol 17 no 10 pp 939ndash948 2007

[34] A A Pandit M V Warde and P S Menon ldquoCorrelation ofnumber of intrathyroid lymphocytes with antimicrosomal anti-body titer inHashimotorsquos thyroiditisrdquoDiagnostic Cytopathologyvol 28 no 2 pp 63ndash65 2003

[35] M Bacher C N Metz T Calandra et al ldquoAn essentialregulatory role formacrophagemigration inhibitory factor inT-cell activationrdquo Proceedings of the National Academy of Sciencesof the United States of America vol 93 no 15 pp 7849ndash78541996

[36] J Yang M S Sundrud J Skepner and T Yamagata ldquoTargetingTh17 cells in autoimmune diseasesrdquo Trends in PharmacologicalSciences vol 35 no 10 pp 493ndash500 2014

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


Table 1 Characteristics of patients with HT and healthy controls

HT HT-A HT-B Healthy control Normal rangeCases (119899) 40 22 18 30 mdashGender (FM) 355 193 162 264 mdashAge (years) 2893 plusmn 830 2891 plusmn 921 2894 plusmn 730 3097 plusmn 639 mdashTSH (mIUL) 413 (182ndash625)lowastlowast 194 (099ndash306) 695 (512ndash1618)lowastlowast 189 (115ndash259) 028ndash43FT3 (pmolL) 449 plusmn 132lowast 497 plusmn 117 389 plusmn 127lowastlowast 523 plusmn 098 28ndash71FT4 (pmolL) 1565 plusmn 304lowast 1658 plusmn 228 1452 plusmn 350lowastlowast 1729 plusmn 209 12ndash22TPOAb (IUmL) 20000 (14835ndash29988)lowastlowast 17100 (10953ndash21508)lowastlowast 29475 (19958ndash41085)lowastlowast 1425 (753ndash2400) 0ndash34TgAb (IUmL) 48975 (30070ndash77470)lowastlowast 33515 (25885ndash48548)lowastlowast 71755 (55153ndash105558)lowastlowast 5100 (1975ndash8425) 0ndash115MUI (120583gL) 16940 16845 17030 16335 mdashData are shown as mean plusmn SD or median (25thndash75th percentile) according to the distribution HT-A HT patient with normal thyroid function HT-B HTpatient with subclinical or overt hypothyroidism M male F female MUI median of urine iodine The P values represent different groups compared withhealthy control (HC) lowast119875 lt 005 lowastlowast119875 lt 001

inflammatory bowel disease (IBD) [9] rheumatoid arthritis[10] and vitiligo vulgaris [11] Additionally MIF gene poly-morphisms (rs755622 SNP) have been shown in associationwith the severity of goiter in patients with untreated Gravesrsquodisease (GD) which is another common AITD [12] Morerecently T helper 17 cells (Th17) a newly recognized subset ofCD4+ T helper cells have been demonstrated to play criticalroles in the pathogenesis of several autoimmune diseaseswhich is considered the main source of interleukin 17 (IL-17)[13ndash15] Given that subsequent investigations reported thatMIF can interact with other cytokines and lead to impairedimmune responses there is no in-depth study about the roleof MIF in HT Several studies have revealed that MIF isinvolved in promoting the differentiation and developmentof IL-17 in animal [16 17] Thus we speculate that MIFmay be implicated in inflammatory and autoimmune diseasethrough interacting withTh17 cells

Therefore in the present study we detected the expres-sions of MIF Th17 cells and IL-17A in HT patients andhealthy controls analyzed the relationships between MIFand Th17 cells and MIF Th17 cells and thyroid-specificautoantibodies and tried to explore the possible role of MIFand Th17 cells in the pathogenesis of thyroid autoimmunedamage in HT

2 Materials and Methods

21 Subjects and Clinical Assessments Forty patients withHTwere enrolled in this study and all the patients were newlydiagnosed and untreated previouslyThe diagnosis of HTwasbased on the classical criterion [18] HT patients were dividedinto two subgroups based on their thyroid functions HT-Asubset (euthyroidism 22 cases) and HT-B subset (subclinicalor overt hypothyroidism 18 cases) Meanwhile thirty healthyvolunteers withmatched age and sex features were selected asthe healthy controls (HC) who had no autoimmune diseasehistory The clinical features of all subjects were shown inTable 1 All study procedures were performed in accordancewith the guidelines of the Declaration of Helsinki withthe approval of the Ethics Committee of Binzhou MedicalUniversityHospital From all participants a written informedconsent was obtained

Electrochemiluminescence immunoassay analyzer(ECLIA Roche Cobas 6000 Germany) was used to measurethyroid function and thyroid-specific autoantibodies in allsubjects which include TSH free triiodothyronine (FT

3)

and free thyroxine (FT4) and TPOAb and TgAb (their

reference ranges were shown in Table 1) Color Dopplerultrasonography (LOGIQ9 GE USA) was performed on allsubjects by trained observers mainly observing thyroid sizeecho (especially hypoechogenicity) and blood flow Urinaryiodine excretion was detected in all participants to evaluatetheir iodine nutritional status [19]

22 Flow Cytometric Analysis of Th17 Cells Isolation ofperipheral bloodmononuclear cells (PBMCs) was performedvia Ficoll-Hypaque density gradient centrifugation (Sigma-Aldrich St Louis MO 1200 rpm 25min) Cells were washedtwice in phosphate buffered saline (PBS) incubated for5 hours with 25 ngmL phorbol myristate acetate (PMA)and 1 120583gmL ionomycin (Sigma USA) in the presence of2mmolmLmonensin at 37∘C under a 5 CO

2environment

and then transferred to each tube washed once with PBSand incubated with anti-CD4-FITC at 4∘C for 30 minutesin the dark Following being fixed and permeabilized cellswere stained with intracellular anti-IL-17A-PE Meanwhileisotype-matched controls were used to correct nonspecificbindingTh17 cell numbers were analyzed with a FACSCantoflow cytometer and data were collected and analyzed usingCellQuest software (BD Biosciences USA) The antibodiesmentioned above were obtained from eBioscience (USA)

23 Real-Time Quantitative RT-PCR Analysis of MIF and IL-17AmRNA Total RNAwas isolated fromPBMCswith Trizol(Invitrogen USA) Reverse transcriptionwas performedwithPrimeScript RT reagent kit (TaKaRa Japan) on an ABI 9700PCR meter (37∘C for 15min followed by 85∘C for 5 s) Real-time RT-PCR was performed on a Rotor-Gene 3000 (CorbettResearch Australia) using SYBR Premix Ex Taq II (TaKaRaJapan) and the given primers for MIF and IL-17A (Table 2)additionally 120573-actin was used as an internal control Thesame samplewas run in triplicate and datawere analyzedwiththe Rotor-Gene Real-Time Analysis Software 60













4 Results


3 and FT








5 Discussion



MIF

mRN

A ex

pres

sions

HC HT HT-A HT-B0

2

4

6

8lowastlowast

lowastlowast

lowast

lowast

(a)

HC HT HT-A HT-B0

2

4

6

8

IL-1

7A m

RNA

expr

essio

ns

lowastlowast

lowastlowast

lowastlowast

lowastlowast

(b)


IL-1

7A m

RNA

expr

essio

ns

0 2 4 60

2

4

6

8r = 0390 P = 0013

(c)





100

101

102

103

104

100

101

102

103

104

PE-A

IL-17

A

FITC-ACD4


CD4

220 045

100

101

102

103

104

FITC-A

100

101

102

103

104

PE-A

IL-17

A(a)

Th17

cells

()

0

1

2

3

HC HT HT-A HT-B

lowastlowast

lowastlowast

lowastlowast

lowastlowast

(b)






Seru

m M

IF le

vels

(ng

mL)

HC HT HT-A HT-B0

20

40

60 lowastlowast

lowastlowast

lowastlowast

lowastlowast

(a)

0

20

10

30

40

50

HC HT HT-A HT-B

lowastlowast

lowastlowast

lowastlowast

lowastlowast

Seru

m IL

-17A

leve

ls (p

gm

L)

(b)

Seru

m IL

-17A

leve

ls (p

gm

L)

r = 0459 P = 0003


20

10

30

40

50

(c)

Th17

cells

()

r = 0442 P = 0004

0

1

2

3


(d)


Th17

cells

()

20 30 400

1

2

3r = 0485 P = 0001

(e)






Seru

m T

POAb

tite

rs (I

Um

L)

0 20 40 600

200

400

600

800r = 0370 P = 0019

(a)

Seru

m T

gAb

titer

s (IU

mL)


0

1000

2000

3000

4000r = 0489 P = 0001

(b)


Seru

m T

SH le

vels

(mIU

L)

0 10 20 30 400

50

100

50

r = 0444 P = 0007

(c)

Seru

m T

POAb

tite

rs (I

Um

L)

0

200

400

600

800

Th17 cells ()05 10 15 20 25

r = 0459 P = 0003

(d)

Seru

m T

gAb

titer

s (IU

mL)

Th17 cells ()05 10 15 20 25

0

1000

2000

3000

4000r = 0401 P = 0010

(e)

Th17 cells ()

Seru

m T

SH le

vels

(mIU

L)

05 10 15 20 250

25

50

75

100r = 0553 P = 0001

(f)






6 Conclusions


Abbreviations


FT4 Free thyroxine


analyzer



Acknowledgments


References












































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




























4 Results


3 and FT








5 Discussion



MIF

mRN

A ex

pres

sions

HC HT HT-A HT-B0

2

4

6

8lowastlowast

lowastlowast

lowast

lowast

(a)

HC HT HT-A HT-B0

2

4

6

8

IL-1

7A m

RNA

expr

essio

ns

lowastlowast

lowastlowast

lowastlowast

lowastlowast

(b)


IL-1

7A m

RNA

expr

essio

ns

0 2 4 60

2

4

6

8r = 0390 P = 0013

(c)





100

101

102

103

104

100

101

102

103

104

PE-A

IL-17

A

FITC-ACD4


CD4

220 045

100

101

102

103

104

FITC-A

100

101

102

103

104

PE-A

IL-17

A(a)

Th17

cells

()

0

1

2

3

HC HT HT-A HT-B

lowastlowast

lowastlowast

lowastlowast

lowastlowast

(b)






Seru

m M

IF le

vels

(ng

mL)

HC HT HT-A HT-B0

20

40

60 lowastlowast

lowastlowast

lowastlowast

lowastlowast

(a)

0

20

10

30

40

50

HC HT HT-A HT-B

lowastlowast

lowastlowast

lowastlowast

lowastlowast

Seru

m IL

-17A

leve

ls (p

gm

L)

(b)

Seru

m IL

-17A

leve

ls (p

gm

L)

r = 0459 P = 0003


20

10

30

40

50

(c)

Th17

cells

()

r = 0442 P = 0004

0

1

2

3


(d)


Th17

cells

()

20 30 400

1

2

3r = 0485 P = 0001

(e)






Seru

m T

POAb

tite

rs (I

Um

L)

0 20 40 600

200

400

600

800r = 0370 P = 0019

(a)

Seru

m T

gAb

titer

s (IU

mL)


0

1000

2000

3000

4000r = 0489 P = 0001

(b)


Seru

m T

SH le

vels

(mIU

L)

0 10 20 30 400

50

100

50

r = 0444 P = 0007

(c)

Seru

m T

POAb

tite

rs (I

Um

L)

0

200

400

600

800

Th17 cells ()05 10 15 20 25

r = 0459 P = 0003

(d)

Seru

m T

gAb

titer

s (IU

mL)

Th17 cells ()05 10 15 20 25

0

1000

2000

3000

4000r = 0401 P = 0010

(e)

Th17 cells ()

Seru

m T

SH le

vels

(mIU

L)

05 10 15 20 250

25

50

75

100r = 0553 P = 0001

(f)






6 Conclusions


Abbreviations


FT4 Free thyroxine


analyzer



Acknowledgments


References












































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















MIF

mRN

A ex

pres

sions

HC HT HT-A HT-B0

2

4

6

8lowastlowast

lowastlowast

lowast

lowast

(a)

HC HT HT-A HT-B0

2

4

6

8

IL-1

7A m

RNA

expr

essio

ns

lowastlowast

lowastlowast

lowastlowast

lowastlowast

(b)


IL-1

7A m

RNA

expr

essio

ns

0 2 4 60

2

4

6

8r = 0390 P = 0013

(c)





100

101

102

103

104

100

101

102

103

104

PE-A

IL-17

A

FITC-ACD4


CD4

220 045

100

101

102

103

104

FITC-A

100

101

102

103

104

PE-A

IL-17

A(a)

Th17

cells

()

0

1

2

3

HC HT HT-A HT-B

lowastlowast

lowastlowast

lowastlowast

lowastlowast

(b)






Seru

m M

IF le

vels

(ng

mL)

HC HT HT-A HT-B0

20

40

60 lowastlowast

lowastlowast

lowastlowast

lowastlowast

(a)

0

20

10

30

40

50

HC HT HT-A HT-B

lowastlowast

lowastlowast

lowastlowast

lowastlowast

Seru

m IL

-17A

leve

ls (p

gm

L)

(b)

Seru

m IL

-17A

leve

ls (p

gm

L)

r = 0459 P = 0003


20

10

30

40

50

(c)

Th17

cells

()

r = 0442 P = 0004

0

1

2

3


(d)


Th17

cells

()

20 30 400

1

2

3r = 0485 P = 0001

(e)






Seru

m T

POAb

tite

rs (I

Um

L)

0 20 40 600

200

400

600

800r = 0370 P = 0019

(a)

Seru

m T

gAb

titer

s (IU

mL)


0

1000

2000

3000

4000r = 0489 P = 0001

(b)


Seru

m T

SH le

vels

(mIU

L)

0 10 20 30 400

50

100

50

r = 0444 P = 0007

(c)

Seru

m T

POAb

tite

rs (I

Um

L)

0

200

400

600

800

Th17 cells ()05 10 15 20 25

r = 0459 P = 0003

(d)

Seru

m T

gAb

titer

s (IU

mL)

Th17 cells ()05 10 15 20 25

0

1000

2000

3000

4000r = 0401 P = 0010

(e)

Th17 cells ()

Seru

m T

SH le

vels

(mIU

L)

05 10 15 20 250

25

50

75

100r = 0553 P = 0001

(f)






6 Conclusions


Abbreviations


FT4 Free thyroxine


analyzer



Acknowledgments


References












































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















100

101

102

103

104

100

101

102

103

104

PE-A

IL-17

A

FITC-ACD4


CD4

220 045

100

101

102

103

104

FITC-A

100

101

102

103

104

PE-A

IL-17

A(a)

Th17

cells

()

0

1

2

3

HC HT HT-A HT-B

lowastlowast

lowastlowast

lowastlowast

lowastlowast

(b)






Seru

m M

IF le

vels

(ng

mL)

HC HT HT-A HT-B0

20

40

60 lowastlowast

lowastlowast

lowastlowast

lowastlowast

(a)

0

20

10

30

40

50

HC HT HT-A HT-B

lowastlowast

lowastlowast

lowastlowast

lowastlowast

Seru

m IL

-17A

leve

ls (p

gm

L)

(b)

Seru

m IL

-17A

leve

ls (p

gm

L)

r = 0459 P = 0003


20

10

30

40

50

(c)

Th17

cells

()

r = 0442 P = 0004

0

1

2

3


(d)


Th17

cells

()

20 30 400

1

2

3r = 0485 P = 0001

(e)






Seru

m T

POAb

tite

rs (I

Um

L)

0 20 40 600

200

400

600

800r = 0370 P = 0019

(a)

Seru

m T

gAb

titer

s (IU

mL)


0

1000

2000

3000

4000r = 0489 P = 0001

(b)


Seru

m T

SH le

vels

(mIU

L)

0 10 20 30 400

50

100

50

r = 0444 P = 0007

(c)

Seru

m T

POAb

tite

rs (I

Um

L)

0

200

400

600

800

Th17 cells ()05 10 15 20 25

r = 0459 P = 0003

(d)

Seru

m T

gAb

titer

s (IU

mL)

Th17 cells ()05 10 15 20 25

0

1000

2000

3000

4000r = 0401 P = 0010

(e)

Th17 cells ()

Seru

m T

SH le

vels

(mIU

L)

05 10 15 20 250

25

50

75

100r = 0553 P = 0001

(f)






6 Conclusions


Abbreviations


FT4 Free thyroxine


analyzer



Acknowledgments


References












































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Seru

m M

IF le

vels

(ng

mL)

HC HT HT-A HT-B0

20

40

60 lowastlowast

lowastlowast

lowastlowast

lowastlowast

(a)

0

20

10

30

40

50

HC HT HT-A HT-B

lowastlowast

lowastlowast

lowastlowast

lowastlowast

Seru

m IL

-17A

leve

ls (p

gm

L)

(b)

Seru

m IL

-17A

leve

ls (p

gm

L)

r = 0459 P = 0003


20

10

30

40

50

(c)

Th17

cells

()

r = 0442 P = 0004

0

1

2

3


(d)


Th17

cells

()

20 30 400

1

2

3r = 0485 P = 0001

(e)






Seru

m T

POAb

tite

rs (I

Um

L)

0 20 40 600

200

400

600

800r = 0370 P = 0019

(a)

Seru

m T

gAb

titer

s (IU

mL)


0

1000

2000

3000

4000r = 0489 P = 0001

(b)


Seru

m T

SH le

vels

(mIU

L)

0 10 20 30 400

50

100

50

r = 0444 P = 0007

(c)

Seru

m T

POAb

tite

rs (I

Um

L)

0

200

400

600

800

Th17 cells ()05 10 15 20 25

r = 0459 P = 0003

(d)

Seru

m T

gAb

titer

s (IU

mL)

Th17 cells ()05 10 15 20 25

0

1000

2000

3000

4000r = 0401 P = 0010

(e)

Th17 cells ()

Seru

m T

SH le

vels

(mIU

L)

05 10 15 20 250

25

50

75

100r = 0553 P = 0001

(f)






6 Conclusions


Abbreviations


FT4 Free thyroxine


analyzer



Acknowledgments


References












































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















Seru

m T

POAb

tite

rs (I

Um

L)

0 20 40 600

200

400

600

800r = 0370 P = 0019

(a)

Seru

m T

gAb

titer

s (IU

mL)


0

1000

2000

3000

4000r = 0489 P = 0001

(b)


Seru

m T

SH le

vels

(mIU

L)

0 10 20 30 400

50

100

50

r = 0444 P = 0007

(c)

Seru

m T

POAb

tite

rs (I

Um

L)

0

200

400

600

800

Th17 cells ()05 10 15 20 25

r = 0459 P = 0003

(d)

Seru

m T

gAb

titer

s (IU

mL)

Th17 cells ()05 10 15 20 25

0

1000

2000

3000

4000r = 0401 P = 0010

(e)

Th17 cells ()

Seru

m T

SH le

vels

(mIU

L)

05 10 15 20 250

25

50

75

100r = 0553 P = 0001

(f)






6 Conclusions


Abbreviations


FT4 Free thyroxine


analyzer



Acknowledgments


References












































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















6 Conclusions


Abbreviations


FT4 Free thyroxine


analyzer



Acknowledgments


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 Macrophage Migration Inhibitory...

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Transcript of Research Article Macrophage Migration Inhibitory...