Research Article Increased Frequency of CD4+ CD25+ FoxP3 ...downloads.hindawi.com/journals/jir/2015/985302.pdf · Increased Frequency of CD4+ CD25+ FoxP3+ T Regulatory Cells in Pulmonary

Research ArticleIncreased Frequency of CD4+ CD25+ FoxP3+ T RegulatoryCells in Pulmonary Tuberculosis PatientsUndergoing Specific Treatment and Its Relationship withTheir Immune-Endocrine Profile

Ariana Diacuteaz1 Natalia Santucci1 Bettina Bongiovanni1

Luciano DrsquoAttilio1 Claudia Massoni2 Susana Lioi2 Stella Radcliffe2

Griselda Diacutedoli1 Oscar Bottasso1 and Mariacutea Luisa Bay1

1 Institute of Immunology School of Medical Sciences National University of Rosario Rosario Santa Fe Argentina2Central Laboratory Centenary Provincial Hospital Rosario Santa Fe Argentina

Correspondence should be addressed to Ariana Dıaz arianadiazdgmailcom

Received 24 October 2014 Revised 22 January 2015 Accepted 23 January 2015

Academic Editor Marco Antonio Velasco-Velazquez

Copyright copy 2015 Ariana Dıaz 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

Tuberculosis (TB) is a major health problem requiring an appropriate cell immune response (IR) to be controlled Since regulatoryT cells (Tregs) are relevant in IR regulation we analyzed Tregs variations throughout the course of TB treatment and its relationshipwith changes in immune-endocrine mediators dealing with disease immunopathology The cohort was composed of 41 adultpatients 20 of them completing treatment and follow-up Patients were bled at diagnosis (T0) and at 2 (T2) 4 (T4) 6 (T6) and 9months following treatment initiation Twenty-four age- and sex-matched healthy controls (HCo) were also included Tregs (flowcytometry) from TB patients were increased at T0 (versus HCo 119875 lt 005) showing even higher values at T2 (versus T0 119875 lt 001)and T4 (versus T0 119875 lt 0001) While IL-6 IFN-120574 TGF-120573 (ELISA) and Cortisol (electrochemiluminescence EQ) were augmentedDHEA-S (EQ) levels were diminished at T0 with respect to HCo with cytokines and Cortisol returning to normal values at T9Tregs correlated positively with IFN-120574 (119877 = 0868 119875 lt 005) at T2 and negatively at T4 (119877 = minus0795 119875 lt 005) Lowered levels ofproinflammatory cytokines together with an increased frequency of Tregs of patients undergoing specific treatment might reflecta downmodulatory effect of these cells on the accompanying inflammation

1 Introduction

According the World Health Organization one third of theworld population is infected withMycobacterium tuberculosis(Mtb) and the majority of affected people are found indeveloping countries The situation becomes even moreproblematical because of the increased susceptibility of HIVinfected persons to develop the disease and the emergence ofstrains resistant to the antimicrobial therapy [1]

Most people infected with Mtb have a clinically latentinfection and 10 of them further progress to active TB dur-ing their lifetime Bacterial host and environmental factorsinfluence the development of active TB [1 2] Commonlythe host immune response (IR) controls Mtb replication

collaborating with the establishment of latent infectionwhich ultimately depends on a fine balance between thepathogen persistence and the specific IR [3] T cell-mediatedimmunity is the main response against TB mainly throughinterferon-gamma (IFN-120574) production by antigen specific Tcells However an exacerbated effector IR and the ensuingexcessive secretion of inflammatory mediators turn out to bedetrimental damaging host tissues through immunopatho-logical processes It follows that a properly balanced IR isessential to successfully cope with this pathogen [4]

Regulatory T cells which tend to modulate the anti-infectious host immunity are currently viewed as one of thesuitable mechanisms for Th-1-dependent immune responsesuppression Among these T cell subpopulations those

Hindawi Publishing CorporationJournal of Immunology ResearchVolume 2015 Article ID 985302 8 pageshttpdxdoiorg1011552015985302

2 Journal of Immunology Research

expressing intracellular transcription factor FoxP3 seem todisplay the most functional activity in terms of immuno-suppression FoxP3-positive regulatory T cells (Tregs) can beeither natural in the course of antigen-dependent thymicdifferentiation or induced in the periphery during adaptiveimmune responses [5] Tregs represent 5ndash10 of circulatingCD4+ cells but in humans only the subset expressing higherlevels of CD25 (120572 chain of IL-2R) exhibits a strong suppressivecapacity [6] The main mechanisms of suppression by Tregcells include inhibition of IFN-120574 production by T cellsthrough the production of IL-10 and transforming growthfactor beta (TGF-120573) as well as by cell to cell contact [7]Whileevidence indicates that Tregs are involved in the immuneresponse against Mtb there is no consensus regarding theprecise role of Treg in TB

Some studies reported Treg cell expansion in the bloodlung or other tissues of patients with active TB [8 9]suggesting that these cells may suppress T cell immunity andhence contribute to disease development [6 10] In contrastother studies reported no increase of Treg in TB patients [1112] A recent work carried out in nonhuman primates raisedthe view of increased Treg as representing a counterbalancinganti-inflammatory response instead of an immunosuppressoreffect [7] Collectively it may be assumed that Tregs arelikely to have a differential role on infection outcomesdepending on the proper balance between protective immunemechanisms and those involved with tissue damage

In parallel other extrinsic mechanisms may also con-tribute to reduce inflammation during immunologicallybased diseases partly because of the neuroimmunoendocrinecommunication in particular the hypothalamus-pituitary-adrenal (HPA) axis that attempts to maintain immunehomeostasis As key molecules of the HPA axis endoge-nous adrenal steroids (Cortisol Dehydroepiandrosterone(DHEA)) contribute to modulate the immune response toinfectious agents and other insults In turn the HPA axisis modulated by cytokines like IFN-120574 and IL-6 which areproduced during the immune response Within this settingwe have observed that patients with pulmonary TB haveimbalanced immune-endocrine responses with increasedplasma concentrations of proinflammatory cytokines andCortisol in presence of decreased DHEA levels associatedwith disease severity [13 14]

In expanding the knowledge about the participation ofTreg in TB the analysis of this T cell population throughoutthe course of TB treatment and its eventual relationship withchanges in immune-endocrine mediators involved in diseaseimmunopathology is informative According to this we haveproceeded to characterize this T cell population at differenttime points following treatment initiation along with theassessment of cytokines and hormones critically to containan infectious threat

2 Materials and Methods

21 Subjects Between January 2010 and January 2014 adultswho were diagnosed with lung TB based on clinical andradiological findings and identification of TB bacilli in

sputum were enrolled and followed for up to nine months ina prospective cohort study of Mtb infection and treatmentThis observational cohort included forty-one patients withneither HIV coinfection nor multidrug resistant TB Patientshad mild (119899 = 7) moderate (119899 = 16) or advanced(119899 = 18) disease according to the radiological findings aspreviously described [13] Antituberculosis therapy consistedof six months of rifampicin and isoniazid initially supple-mented by two months of pyrazinamide and ethambutolOf the 41 recruited patients blood samples at all timepoints were available in 20 of them (mild = 1 moderate =12 and severe = 7) Twenty-four matched healthy subjects(HCo) living in the same area and without antecedents ofcontactwithTBpatients were included as controls Exclusioncriteria for all participants included pathologies affecting thehypothalamus-pituitary-thyroid- or gonadal-axis or directcompromise of the adrenal gland pregnancy contraceptivedrugs age under 18 or systemic or localized pathologiesrequiring treatment with corticosteroids or immunosuppres-sants All subjects were BCG vaccinated and providedwrittenconsent to participate in the studyThe studywas approved bythe Ethical Committee of the Facultad de Ciencias MedicasUniversidad Nacional de Rosario and Centenario Hospital ofRosario

22 Sample Collection Blood samples were obtained fromTB patients at the time of diagnosis (before initiation of thetreatment T0) and 2 4 and 6 months (T2 T4 and T6) afterstarting the specific antituberculosis treatment An additionalsample was obtained three months after the end of treatmentcompletion (T9) All samples were obtained between 800and 900 am with and without EDTA and then centrifugedAprotinin (100UmL Aprotinin from bovine lung Sigma-Aldrich St LouisMOUSA)was added to the plasma shortlyafter collection and the samples were preserved at minus20∘CSerum samples were immediately processed for hormonequantificationOne blood samplewas obtained from age- andsex-matched HCo and processed in the same way

23 Sample Preparation Twenty milliliters of blood wasobtained using EDTA as anticoagulant and the PBMC wereobtained by Ficoll-Hypaque density gradient centrifugation(Biowittaker Walkersville MD) PBMC were washed twicein PBS and counted by trypan blue staining in a Neubauerchamber Viability was always of 95

24 Flow Cytometry For surface marker analyses a sampleof 50 120583L of well-mixed EDTA anticoagulated whole bloodwas incubated for 30min at room temperature with 20120583Lof BD Tritest CD4 FITCCD8 PECD3 PerCP Reagent (BDBiosciences San Jose CA USA) according to manufacturerinstructions Following incubation red blood cells were lysedwithNaHCO

3ClNH

4solution Cells were washed twice with

PBS and preserved for cytometric analysisAnother sample of 1 times 106 PBMC was incubated for

30min with anti-CD4-FITC and anti-CD25-PEcy55 (BDBiosciences San Jose CA USA) and for intracellular stainingof FoxP3 the cells in the tubes were resuspended in FixPerm

Journal of Immunology Research 3

buffer (eBioscience San Diego CA USA) and left at 4∘Cfor 30min Subsequently these cells were washed with coldPBS and with permeabilization buffer (eBioscience SanDiego CA USA) and then 20 120583L anti-human FoxP3-PE (BDBiosciences San Jose CAUSA) was added and the cells wereincubated at 4∘C for 30min Stained cells were analyzed witha FACSAria II flow cytometer (BD Biosciences San JoseCA USA) The percentage of positive cells and the meanfluorescence intensity (arbitrary units) for a specific markerwere calculated using FACSDiva software (BD BiosciencesSan Jose CA USA) For purpose analysis 30000 events wererecorded

25 Evaluation of Immunological Mediators and HormonesLevels of TGF-120573 IFN-120574 and IL-6 were measured in plasmausing commercially available high-sensitivity ELISA kitsaccording to the instructions of the manufacturer Detec-tion limits were 007 pgmL for IL-6 (RampD Systems IncMinneapolis USA) 125 pgmL for TGF-120573 and 47 pgmLfor IFN-120574 (both from BD Bioscience San Jose CA USA)Cortisol and DHEA-sulphated (DHEA-S) serum concen-trations were assessed by electrochemiluminescence (Cobase411 Roche Germany)

26 Statistical Analysis Comparisons between groups (TBversus HCo) were performed by nonparametric methods(Kruskall-Wallis analysis of variance and Mann-Whitney 119880test) Correlations between variables were assessed by Spear-manrsquos rank test Data were considered statistically significantwhen119875 lt 005The analyseswere performedusingGraphPadPrism 4 Software

3 Results

31 CD4+CD25+FoxP3+ T Cells Are Increased in the Blood ofPatients with TB Table 1 shows some general characteristicsof study groups All patients showed a decreased BodyMass Index (BMI) (119875 lt 0001) There were no differenceswith respect to age sex distribution BCG-vaccination andperipheral CD8+ T cells frequency However we found thatTB patients presented a significant diminution inCD4+T celllevels (119875 lt 005)Thenwe investigatedwhether active TBwasassociated with a CD4+CD25+FoxP3+ Treg expansion byassessing their frequency in PBMC of TB patients at the timeof diagnosis by flow cytometry (Figure 1(a)) Results depictedin Figure 1(b) indicate that the frequency of Treg cells withinthe total CD4+ population was significantly increased in TBpatients compared with HCo (119875 lt 005) We next analyzedthe percentage of Treg cells in TB patients according to theirseverity As seen in Figure 1(c) moderate TB patients differedfrom HCo

32 Treg Frequency during Antituberculosis Therapy In ear-lier studies we have evaluated immune and endocrineparameters during TB treatment showing that certaincytokines were augmented at the time of diagnosis (IL-1120573 IL-6 and IFN-120574) and even 2 months after treatmentinitiation (IFN-120574) [15] In relation to peripheral T cells we

Table 1 Characteristics of study groups

HCo (119899 = 24) TB (119899 = 41) 119875 valueAge (years)lowast 505 (258ndash573) 450 (215ndash548) nsSex (FM) 123 437 nsBCG () 95 80 nsBMI (Kgm2)lowast 274 (251ndash308) 199 (183ndash238) 119875 lt 0001CD4+lowast 514 (430ndash551) 394 (326ndash526) 119875 lt 005CD8+lowast 330 (246ndash358) 252 (218ndash334) nslowastResults are shown as median values (25ndash75 percentiles) HCo healthycontrols

found a decrease in the percentages of CD3+ CD4+ cellsat the time of diagnosis that attained normal values uponstarting specific treatment (data not shown) When assessingthe frequency of Tregs throughout specific treatment thesecell populations remained significantly increased during thatperiod compared to values in HCo reaching their highestrelative levels at T4 (HCo versus T4 119875 lt 001 Figure 2)

33 Correlations between Treg Frequency and Cytokine andHormone Levels TGF-120573 IFN-120574 and IL-6 plasma levelswere assessed at the beginning and at different time pointsduring anti-TB treatment Table 2 shows median values andinterquartile range for each cytokine at the study time pointsThe three cytokines were increased at the time of diagnosiswith respect HCo returning to normal values once patientsinitiated specific treatment Pairwise correlations betweencytokines and Treg frequency revealed that IFN-120574 levelswere positively associated with Treg at month two undertreatment (119877 = 0868 119875 lt 005 119899 = 8) Notably thiscorrelation became negative when patients underwent fourmonths of specific treatment (119877 = minus0798 119875 lt 005119899 = 9 see Supplementary Material available online athttpdxdoiorg1011552015985302) In a further step TBpatients were categorized according to their IFN-120574 levels atT4 In this way patients with values below the median valuehad significantly higher levels of Treg cells when comparedwith cases whose plasma IFN-120574 is situated above the medianconcentration (119875 = 0027)

With regard to adrenal hormones serum Cortisol wasincreased at the time of diagnosis (119875 lt 001) and at T2(119875 lt 001) reaching values comparable toHCo at T4DHEA-S levels which were found decreased at T0 (versus HCo 119875 lt005) remained so during the whole treatment period butwere augmented at T9 reaching values similar to those seen inHCo Unlike this DHEA-S levels which were found stronglydecreased at diagnosis started to increase earlier followingtreatment initiation (data not shown) Both adrenal steroidsshowed a negative correlation with the frequency of Tregcells DHEA-S at the time of diagnosis (119877 = minus051 119875 lt 004119899 = 13) and Cortisol at T4 (119877 = minus0504 119875 lt 005 119899 = 12 seeSupplementary Material)

4 Discussion

The involvement of Treg cells in the control of immuneresponses to self-antigens and in immune homeostasis is well


250

200

150

100

50

SSC-

A

250

200

150

100

50

25020015010050

SSC-

A

FSC-A

990o-CD4CD25FoxP3 990o-CD4CD25FoxP3

102

103

104

105

102

103

104

105

102

103

104

105

102

103

104

105

102

103

104

105

102

103

104

105

102

103

104

105

CD4 FITC-A

990o-CD4CD25FoxP3

CD4

FITC

-A

CD4

FITC

-A

FoxP3 PE-A

FoxP3 PE-A

990o-isotype control

IgG PE-A

990o-CD4CD25FoxP3

CD25

PE-C

y5-A

(I) (II)

(III) (IV)

(V)

times103

times103

times103

(a)

Figure 1 Continued


HCo versus TB P lt 005

lowastTr

eg (

)

5

4

3

2

1

0

HCo TB

(b)

lowast

Treg

() 4

6

2

0

HCo versus moderate P lt 005

HCo Mild Moderate Severe

(c)

Figure 1 CD4+CD25+FoxP3+ T cells (Tregs) frequency in TB patients (a) Plot of cytometric analysis from a representative sample (I) FSCand SSC identifying mononuclear cells (II) CD4+ cells within the lymphocyte gate (III) assessment of FoxP3+ cells gated on the CD4+ cellsas shown in panel II (IV) isotype control from the same sample and (V) an additional control showing that the CD4+FoxP3+ population isalso CD25 high (b) Treg comparisons between HCo and TB (c) Comparisons of disease severity of TB patients Box plots show medianvalues 25ndash75 percentiles of data in each group with maximum and minimum values HCo healthy controls

HCo T0 T2 T4 T6 T90

2

4

6

8

lowast

998400998400

Treg

()

Figure 2 Frequency of CD4+CD25+FoxP3+ T cells (Tregs) withinthe total CD4+population of TBpatients throughout treatment Boxplots show median values 25ndash75 percentiles of data in each groupwith maximum and minimum values HCo healthy controls T0time of diagnosis T2 T4 T6 2 4 and 6 months after starting thespecific anti-TB treatment T9 3 months after the end treatmentcompletion lowastHCo versus T0 and T6 119875 lt 005 HCo versus T2 andT4 119875 lt 001 T0 versus T2 119875 lt 005 T0 versus T4 119875 lt 001 10158401015840T4versus T9 119875 lt 005

established Also there is increasing evidence for a role of Tregin the regulation of immunity to infection [16] It is believedthat Treg downmodulate the IR after pathogen eradicationto avoid exacerbated pathology Although this mechanismbenefits the host during acute infections it may be worryingin the chronic setting notably when pathogen persistenceis sustained in spite of an active IR [10] During chronicinfections a complex interplay is established between the IRto the infectious agent and the ability of the microorganismto evade this protective IR In TB a predominantTh1 cellularresponse is necessary to effectively eradicate the mycobac-terium However if this response exacerbates certain regu-latory mechanisms would be activated to downmodulate it

with the aimof safeguarding the affected organ at the expenseof some interference with pathogen clearance One of theseregulatory mechanisms involves Treg [5] CD4+CD25 high Tcells have been reported to be present at elevated levels in TBand to be able to depress T-cell-mediated IFN-120574 productionin these patients [4 8 9 17] Here we show that TB patientshave an increased frequency of CD4+CD25+FoxP3 Treg incomparison with the group of healthy donors Furthermorethe frequency remained augmented at two and four monthsof treatment commencement reaching values comparable tothose seen HCo once the treatment was completed Tregsare known to display a diverse range of Treg-mediatedimmunological effects in terms of antimicrobial and tissue-damage protection In this case the increased presence ofTreg cells seems to be more compatible with an attemptto downmodulate immunological damage considering ourformer [14 18] and present demonstrationswherein advancedTB coexisted with increased levels of circulating proinflam-matory cytokines

The role of Tregs in TB is not well understood and itis unclear whether their expansion is a cause or a diseaseconsequence As stated they are probably expanded as anadaptive host response to limit the inflammatory reaction andtissue damage induced Tregs expansion duringMtb infectionupon recognition of particular bacterial antigens seems tobe achieved by the presence of IL-10 and TGF-120573 which areknown to be highly produced in patients with active TB[14 17 19 20] Some studies reported a higher suppressivecapacity of Tregs on IFN-120574 producing cells with respect tothose producing IL-17 which allowed the persistence of thelatter ones in inflamed tissues and thus the perpetuationor recrudescence of inflammation [6 21 22] Our resultsabout a significant positive correlation between Tregs andIFN-120574 plasma levels at two months of treatment suggest thatincreased Tregs are not downmodulating inflammation atthis stage However at T4 when the clinical improvement wasquite evident this correlation turned out to be negative It


Table2Cy

tokine

andho

rmon

elevelsinTB

patie

ntsd

uringspecifictreatment

Cytokinesa

ndadrenal

steroid

horm

ones

HCogrou

pTB

grou

pT0

T2T4

T6T9

IL-6

(pgmL)

105a

(04ndash22)

1211

asect(57ndash

239)

47a

(18ndash110

)29(17ndash

36)

31(16

ndash36)

31(10

ndash47)

IFN-120574

(pgmL)

47b

(47ndash82)

136

b(107ndash243)

69b

(52ndash125)

61b(47ndash

107)

47(47ndash

69)

47(47ndash

72)

TGF-120573

5721c(5313ndash5924)

6885c120585(5916ndash8040)

6290

(5733ndash7481)

5975

(5190ndash7283)

5970

(5524ndash6

924)

5932(5296ndash6

836)

Cortisol(120583gdL

)146

d(123ndash163)

223

dΥ(158ndash281)

196

d(156ndash

213)

166(114

ndash219

)181(126ndash

206)

164(131ndash209)

DHEA

-S(120583gdL

)2000e

(1334ndash2339)

1295

edagger(605ndash1835)

1128e

(584ndash

1807)

125e

(577ndash1650)

1202e

(8015ndash1970)

204(1303ndash3345)

Com

paris

onsb

etweengrou

ps

a HCoversus

T0119875lt001versusT

2119875lt005bHCoversus


2119875lt005versusT

4119875lt005cHCoversus

T0119875lt005dHCoversus


2119875lt005eHCoversus

T0

119875lt001versusT

2119875lt005versusT

4119875lt005versusT

6119875lt005

Com

paris

onsw

ithin

TBpatie

nts

sect T0versus

T2T

4T6

T9119875lt005T0

versus

T2T

4T6

T9119875lt005120585T0

versus

T2T

4T6

T9119875lt005Υ

T0versus

T9119875lt005daggerT0

versus

T9119875lt005

Results

aresho

wnas

medianvalues(25ndash75

percentiles)HCohealthyc

ontro

lsT0

tim

eofd

iagn

osis

T2T

4T6

24and

6mon

thsafte

rstartingthe

specifica

nti-T

Btre

atmentT9

3mon

thsafte

rthe

endtre

atment

completionCom

paris

onsfor

independ

entgroup

swerem

adeb

ythe

Kruskall-Wallis

analysisofvaria

ncefollowed

bypo

stho

ctestw

henapplicable

Com

paris

onsfor

relatedsampleswerep

erform

edby

theF

riedm

ananalysisforv

ariance


follows that month 4 might represent the best time whena proper balance between Treg functioning and clinicalresponse is established

The negative correlations between adrenal steroids andthe frequency of Treg cells are also worth discussing Glu-cocorticoids are potent anti-inflammatory and immunosup-pressive agents playing a role in the feedback inhibition ofimmuneinflammatory responses to maintain homeostasisXiang andMarshall Jr demonstrated that after 24 h of cultureof PBMC with dexamethasone (10minus8M) FoxP3 mRNA wassignificantly decreased suggesting a rapid sensitivity of Tregto corticosteroids [23] In the same sense DHEA was alsofound to inhibit FoxP3 expression in HIV-coinfected TBpatients [24] In line with these in vitro findings in ourpatients the frequency of Treg cells was inversely correlatedwith the amount of adrenal steroids implicating an extra loopof influence on this T cell population which is beyond theintrinsic immunological components

Ribeiro-Rodrigues et al showed that frequencies of Tregwere increased in PBMC of patients with active tuberculosisand did not decline at the end of 6 months of curativeantibiotic therapy At this time point Treg still preservedtheir capacity to suppress IFN-120574 production [25] Chen etal evaluated Treg frequencies in pulmonary tuberculosispatients after 2 years of chemotherapy and found a directcorrelation between the decreased levels of Treg towardnormality in patients successfully recovered as seen in ourcase This group also found that Tregs from tuberculosispatients not only suppress IFN-120574 production but also inhibitIL-10 production by CD4+ T cells [26]

To conclude the persistent increase of Tregs until 4months of specific treatment in presence of diminishedamounts of proinflammatory cytokines alongwith the inverseassociation between both immune parameters lend somesupport to the view of a damage-protective function of Tregsin TB Further functional studies are now being planned toassess the validity of the assumption of Tregs as counterbal-ancing the excessive inflammatory response encompassingthis disease

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contribution

Ariana Dıaz and Natalia Santucci made equal contributionsto the present study

Acknowledgments

This work was supported by grants from FONCYT (PID160 PAE 37245 and PICT 2012-1523) and the Fellowship ofNational Council for Science and Technology (CONICET)The authors thank Mara Ojeda for her technical assistance inflow cytometry

References

[1] WorldHealthOrganization ldquoGlobal tuberculosis reportrdquoWHOreport 2013 httpwwwwhointtbdata

[2] T H M Ottenhoff F A W Verreck M A Hoeve and E vande Vosse ldquoControl of human host immunity to mycobacteriardquoTuberculosis vol 85 no 1-2 pp 53ndash64 2005

[3] J D Ernst ldquoThe immunological life cycle of tuberculosisrdquoNature Reviews Immunology vol 12 no 8 pp 581ndash591 2012

[4] J-M Hougardy S Place M Hildebrand et al ldquoRegulatory Tcells depress immune responses to protective antigens in activetuberculosisrdquo The American Journal of Respiratory and CriticalCare Medicine vol 176 no 4 pp 409ndash416 2007

[5] E G Churina O I Urazova and V V Novitskiy ldquoTherole of foxp3-expressing regulatory T cells and T helpers inimmunopathogenesis of multidrug resistant pulmonary tuber-culosisrdquo Tuberculosis Research and Treatment vol 2012 ArticleID 931291 9 pages 2012

[6] N D Marin S C Parıs V M Velez C A Rojas M Rojasand L F Garcıa ldquoRegulatory T cell frequency and modulationof IFN-gamma and IL-17 in active and latent tuberculosisrdquoTuberculosis vol 90 no 4 pp 252ndash261 2010

[7] H J Lim J S Park Y-J Cho et al ldquoCD4+FoxP3+ T regulatorycells in drug-susceptible and multidrug-resistant tuberculosisrdquoTuberculosis vol 93 no 5 pp 523ndash528 2013

[8] V Guyot-Revol J A Innes S Hackforth T Hinks and ALalvani ldquoRegulatory T cells are expanded in blood and diseasesites in patients with tuberculosisrdquo The American Journal ofRespiratory and Critical Care Medicine vol 173 no 7 pp 803ndash810 2006

[9] X Y He L Xiao H B Chen et al ldquoT regulatory cellsand Th1Th2 cytokines in peripheral blood from tuberculosispatientsrdquo European Journal of Clinical Microbiology and Infec-tious Diseases vol 29 no 6 pp 643ndash650 2010

[10] M Kursar M Koch H-W Mittrucker et al ldquoCutting edgeregulatory T cells prevent efficient clearance of Mycobacteriumtuberculosisrdquo Journal of Immunology vol 178 no 5 pp 2661ndash2665 2007

[11] J Nemeth H-M Winkler L Boeck et al ldquoSpecific cytokinepatterns of pulmonary tuberculosis in Central Africardquo ClinicalImmunology vol 138 no 1 pp 50ndash59 2011

[12] T Chiacchio R Casetti O Butera et al ldquoCharacterizationof regulatory T cells identified as CD4+CD25highCD39+ inpatients with active tuberculosisrdquo Clinical and ExperimentalImmunology vol 156 no 3 pp 463ndash470 2009

[13] C Mahuad V Bozza S M Pezzotto et al ldquoImpaired immuneresponses in tuberculosis patients are related to weight lossthat coexists with an immunoendocrine imbalancerdquo NeuroIm-munoModulation vol 14 no 3-4 pp 193ndash199 2007

[14] A D Rey C V Mahuad V V Bozza et al ldquoEndocrine andcytokine responses in humans with pulmonary tuberculosisrdquoBrain Behavior and Immunity vol 21 no 2 pp 171ndash179 2007

[15] B Bongiovanni A Dıaz L DrsquoAttilio et al ldquoChanges in theimmune and endocrine responses of patients with pulmonarytuberculosis undergoing specific treatmentrdquo Annals of the NewYork Academy of Sciences vol 1262 no 1 pp 10ndash15 2012

[16] H W Mittrucker and S H Kaufmann ldquoRegulatory T cells andinfection suppression revisitedrdquo European Journal of Immunol-ogy vol 34 no 2 pp 306ndash312 2004

[17] D Dlugovitzky M L Bay L Rateni et al ldquoIn vitro synthesis ofinterferon-gamma interleukin-4 transforming growth factor-beta and interleukin-1 beta by peripheral blood mononuclear


cells from tuberculosis patients relationship with the severity ofpulmonary involvementrdquo Scandinavian Journal of Immunologyvol 49 no 2 pp 210ndash217 1999

[18] N Santucci L DrsquoAttilio L Kovalevski et al ldquoA multifacetedanalysis of immune-endocrine-metabolic alterations in patientswith pulmonary tuberculosisrdquo PLoS ONE vol 6 no 10 ArticleID e26363 2011

[19] C Y Chen D Huang S Yao et al ldquoIL-2 simultaneouslyexpands Foxp3+ T regulatory and T effector cells and confersresistance to severe tuberculosis (TB) implicative treg-T effec-tor cooperation in immunity to TBrdquo Journal of Immunology vol188 no 9 pp 4278ndash4288 2012

[20] V A Boussiotis E Y Tsai E J Yunis et al ldquoIL-10-producingT cells suppress immune responses in anergic tuberculosispatientsrdquoThe Journal of Clinical Investigation vol 105 no 9 pp1317ndash1325 2000

[21] S A Khader G K Bell J E Pearl et al ldquoIL-23 and IL-17 in the establishment of protective pulmonary CD4+ Tcell responses after vaccination and during Mycobacteriumtuberculosis challengerdquo Nature Immunology vol 8 no 4 pp369ndash377 2007

[22] M Umemura A Yahagi S Hamada et al ldquoIL-17-mediatedregulation of innate and acquired immune response against pul-monary Mycobacterium bovis bacille Calmette-Guerin infec-tionrdquo Journal of Immunology vol 178 no 6 pp 3786ndash37962007

[23] L Xiang and G D Marshall Jr ldquoImmunomodulatory effectsof in vitro stress hormones on FoxP3 Th1Th2 cytokine andcostimulatorymoleculemRNAexpression in humanperipheralbloodmononuclear cellsrdquoNeuroimmunomodulation vol 18 no1 pp 1ndash10 2011

[24] M F Quiroga M T Angerami N Santucci et al ldquoDynamicsof adrenal steroids are related to variations in Th1 and tregpopulations during mycobacterium tuberculosis infection inHIV positive personsrdquo PLoS ONE vol 7 no 3 Article IDe33061 2012

[25] R Ribeiro-Rodrigues T Resende Co R Rojas et al ldquoArole for CD4+ CD25+ T cells in regulation of the immuneresponse during human tuberculosisrdquo Clinical and Experimen-tal Immunology vol 144 no 1 pp 25ndash34 2006

[26] X Chen B Zhou M Li et al ldquoCD4+CD25+FoxP3+ regulatoryT cells suppress Mycobacterium tuberculosis immunity inpatients with active diseaserdquo Clinical Immunology vol 123 no1 pp 50ndash59 2007

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Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


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of


Behavioural Neurology

EndocrinologyInternational Journal of



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OncologyJournal of



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The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

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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


expressing intracellular transcription factor FoxP3 seem todisplay the most functional activity in terms of immuno-suppression FoxP3-positive regulatory T cells (Tregs) can beeither natural in the course of antigen-dependent thymicdifferentiation or induced in the periphery during adaptiveimmune responses [5] Tregs represent 5ndash10 of circulatingCD4+ cells but in humans only the subset expressing higherlevels of CD25 (120572 chain of IL-2R) exhibits a strong suppressivecapacity [6] The main mechanisms of suppression by Tregcells include inhibition of IFN-120574 production by T cellsthrough the production of IL-10 and transforming growthfactor beta (TGF-120573) as well as by cell to cell contact [7]Whileevidence indicates that Tregs are involved in the immuneresponse against Mtb there is no consensus regarding theprecise role of Treg in TB

Some studies reported Treg cell expansion in the bloodlung or other tissues of patients with active TB [8 9]suggesting that these cells may suppress T cell immunity andhence contribute to disease development [6 10] In contrastother studies reported no increase of Treg in TB patients [1112] A recent work carried out in nonhuman primates raisedthe view of increased Treg as representing a counterbalancinganti-inflammatory response instead of an immunosuppressoreffect [7] Collectively it may be assumed that Tregs arelikely to have a differential role on infection outcomesdepending on the proper balance between protective immunemechanisms and those involved with tissue damage

In parallel other extrinsic mechanisms may also con-tribute to reduce inflammation during immunologicallybased diseases partly because of the neuroimmunoendocrinecommunication in particular the hypothalamus-pituitary-adrenal (HPA) axis that attempts to maintain immunehomeostasis As key molecules of the HPA axis endoge-nous adrenal steroids (Cortisol Dehydroepiandrosterone(DHEA)) contribute to modulate the immune response toinfectious agents and other insults In turn the HPA axisis modulated by cytokines like IFN-120574 and IL-6 which areproduced during the immune response Within this settingwe have observed that patients with pulmonary TB haveimbalanced immune-endocrine responses with increasedplasma concentrations of proinflammatory cytokines andCortisol in presence of decreased DHEA levels associatedwith disease severity [13 14]

In expanding the knowledge about the participation ofTreg in TB the analysis of this T cell population throughoutthe course of TB treatment and its eventual relationship withchanges in immune-endocrine mediators involved in diseaseimmunopathology is informative According to this we haveproceeded to characterize this T cell population at differenttime points following treatment initiation along with theassessment of cytokines and hormones critically to containan infectious threat

2 Materials and Methods

21 Subjects Between January 2010 and January 2014 adultswho were diagnosed with lung TB based on clinical andradiological findings and identification of TB bacilli in

sputum were enrolled and followed for up to nine months ina prospective cohort study of Mtb infection and treatmentThis observational cohort included forty-one patients withneither HIV coinfection nor multidrug resistant TB Patientshad mild (119899 = 7) moderate (119899 = 16) or advanced(119899 = 18) disease according to the radiological findings aspreviously described [13] Antituberculosis therapy consistedof six months of rifampicin and isoniazid initially supple-mented by two months of pyrazinamide and ethambutolOf the 41 recruited patients blood samples at all timepoints were available in 20 of them (mild = 1 moderate =12 and severe = 7) Twenty-four matched healthy subjects(HCo) living in the same area and without antecedents ofcontactwithTBpatients were included as controls Exclusioncriteria for all participants included pathologies affecting thehypothalamus-pituitary-thyroid- or gonadal-axis or directcompromise of the adrenal gland pregnancy contraceptivedrugs age under 18 or systemic or localized pathologiesrequiring treatment with corticosteroids or immunosuppres-sants All subjects were BCG vaccinated and providedwrittenconsent to participate in the studyThe studywas approved bythe Ethical Committee of the Facultad de Ciencias MedicasUniversidad Nacional de Rosario and Centenario Hospital ofRosario

22 Sample Collection Blood samples were obtained fromTB patients at the time of diagnosis (before initiation of thetreatment T0) and 2 4 and 6 months (T2 T4 and T6) afterstarting the specific antituberculosis treatment An additionalsample was obtained three months after the end of treatmentcompletion (T9) All samples were obtained between 800and 900 am with and without EDTA and then centrifugedAprotinin (100UmL Aprotinin from bovine lung Sigma-Aldrich St LouisMOUSA)was added to the plasma shortlyafter collection and the samples were preserved at minus20∘CSerum samples were immediately processed for hormonequantificationOne blood samplewas obtained from age- andsex-matched HCo and processed in the same way

23 Sample Preparation Twenty milliliters of blood wasobtained using EDTA as anticoagulant and the PBMC wereobtained by Ficoll-Hypaque density gradient centrifugation(Biowittaker Walkersville MD) PBMC were washed twicein PBS and counted by trypan blue staining in a Neubauerchamber Viability was always of 95

24 Flow Cytometry For surface marker analyses a sampleof 50 120583L of well-mixed EDTA anticoagulated whole bloodwas incubated for 30min at room temperature with 20120583Lof BD Tritest CD4 FITCCD8 PECD3 PerCP Reagent (BDBiosciences San Jose CA USA) according to manufacturerinstructions Following incubation red blood cells were lysedwithNaHCO

3ClNH

4solution Cells were washed twice with

PBS and preserved for cytometric analysisAnother sample of 1 times 106 PBMC was incubated for

30min with anti-CD4-FITC and anti-CD25-PEcy55 (BDBiosciences San Jose CA USA) and for intracellular stainingof FoxP3 the cells in the tubes were resuspended in FixPerm





3 Results








4 Discussion



250

200

150

100

50

SSC-

A

250

200

150

100

50

25020015010050

SSC-

A

FSC-A


102

103

104

105

102

103

104

105

102

103

104

105

102

103

104

105

102

103

104

105

102

103

104

105

102

103

104

105

CD4 FITC-A

990o-CD4CD25FoxP3

CD4

FITC

-A

CD4

FITC

-A

FoxP3 PE-A

FoxP3 PE-A


IgG PE-A

990o-CD4CD25FoxP3

CD25

PE-C

y5-A

(I) (II)

(III) (IV)

(V)

times103

times103

times103

(a)

Figure 1 Continued



lowastTr

eg (

)

5

4

3

2

1

0

HCo TB

(b)

lowast

Treg

() 4

6

2

0



(c)


HCo T0 T2 T4 T6 T90

2

4

6

8

lowast

998400998400

Treg

()






Table2Cy

tokine

andho

rmon

elevelsinTB

patie

ntsd


Cytokinesa

ndadrenal

steroid

horm

ones

HCogrou

pTB

grou

pT0

T2T4

T6T9

IL-6

(pgmL)

105a

(04ndash22)

1211

asect(57ndash

239)

47a

(18ndash110

)29(17ndash

36)

31(16

ndash36)

31(10

ndash47)

IFN-120574

(pgmL)

47b

(47ndash82)

136

b(107ndash243)

69b

(52ndash125)

61b(47ndash

107)

47(47ndash

69)

47(47ndash

72)

TGF-120573

5721c(5313ndash5924)

6885c120585(5916ndash8040)

6290

(5733ndash7481)

5975

(5190ndash7283)

5970

(5524ndash6

924)

5932(5296ndash6

836)

Cortisol(120583gdL

)146

d(123ndash163)

223

dΥ(158ndash281)

196

d(156ndash

213)

166(114

ndash219

)181(126ndash

206)

164(131ndash209)

DHEA

-S(120583gdL

)2000e

(1334ndash2339)

1295


1128e

(584ndash

1807)

125e

(577ndash1650)

1202e

(8015ndash1970)

204(1303ndash3345)

Com

paris

onsb

etweengrou

ps

a HCoversus




2119875lt005versusT





T0

119875lt001versusT

2119875lt005versusT

4119875lt005versusT

6119875lt005

Com

paris

onsw

ithin

TBpatie

nts

sect T0versus

T2T

4T6

T9119875lt005T0

versus

T2T

4T6

T9119875lt005120585T0

versus

T2T

4T6

T9119875lt005Υ

T0versus


versus

T9119875lt005

Results

aresho

wnas



ontro

lsT0

tim

eofd

iagn

osis

T2T

4T6

24and

6mon

thsafte

rstartingthe

specifica

nti-T

Btre

atmentT9

3mon

thsafte

rthe

endtre

atment

completionCom

paris

onsfor

independ

entgroup

swerem

adeb

ythe

Kruskall-Wallis

analysisofvaria

ncefollowed

bypo

stho

ctestw

henapplicable

Com

paris

onsfor

relatedsampleswerep

erform

edby

theF

riedm

ananalysisforv

ariance










Acknowledgments


References


































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




















3 Results








4 Discussion



250

200

150

100

50

SSC-

A

250

200

150

100

50

25020015010050

SSC-

A

FSC-A


102

103

104

105

102

103

104

105

102

103

104

105

102

103

104

105

102

103

104

105

102

103

104

105

102

103

104

105

CD4 FITC-A

990o-CD4CD25FoxP3

CD4

FITC

-A

CD4

FITC

-A

FoxP3 PE-A

FoxP3 PE-A


IgG PE-A

990o-CD4CD25FoxP3

CD25

PE-C

y5-A

(I) (II)

(III) (IV)

(V)

times103

times103

times103

(a)

Figure 1 Continued



lowastTr

eg (

)

5

4

3

2

1

0

HCo TB

(b)

lowast

Treg

() 4

6

2

0



(c)


HCo T0 T2 T4 T6 T90

2

4

6

8

lowast

998400998400

Treg

()






Table2Cy

tokine

andho

rmon

elevelsinTB

patie

ntsd


Cytokinesa

ndadrenal

steroid

horm

ones

HCogrou

pTB

grou

pT0

T2T4

T6T9

IL-6

(pgmL)

105a

(04ndash22)

1211

asect(57ndash

239)

47a

(18ndash110

)29(17ndash

36)

31(16

ndash36)

31(10

ndash47)

IFN-120574

(pgmL)

47b

(47ndash82)

136

b(107ndash243)

69b

(52ndash125)

61b(47ndash

107)

47(47ndash

69)

47(47ndash

72)

TGF-120573

5721c(5313ndash5924)

6885c120585(5916ndash8040)

6290

(5733ndash7481)

5975

(5190ndash7283)

5970

(5524ndash6

924)

5932(5296ndash6

836)

Cortisol(120583gdL

)146

d(123ndash163)

223

dΥ(158ndash281)

196

d(156ndash

213)

166(114

ndash219

)181(126ndash

206)

164(131ndash209)

DHEA

-S(120583gdL

)2000e

(1334ndash2339)

1295


1128e

(584ndash

1807)

125e

(577ndash1650)

1202e

(8015ndash1970)

204(1303ndash3345)

Com

paris

onsb

etweengrou

ps

a HCoversus




2119875lt005versusT





T0

119875lt001versusT

2119875lt005versusT

4119875lt005versusT

6119875lt005

Com

paris

onsw

ithin

TBpatie

nts

sect T0versus

T2T

4T6

T9119875lt005T0

versus

T2T

4T6

T9119875lt005120585T0

versus

T2T

4T6

T9119875lt005Υ

T0versus


versus

T9119875lt005

Results

aresho

wnas



ontro

lsT0

tim

eofd

iagn

osis

T2T

4T6

24and

6mon

thsafte

rstartingthe

specifica

nti-T

Btre

atmentT9

3mon

thsafte

rthe

endtre

atment

completionCom

paris

onsfor

independ

entgroup

swerem

adeb

ythe

Kruskall-Wallis

analysisofvaria

ncefollowed

bypo

stho

ctestw

henapplicable

Com

paris

onsfor

relatedsampleswerep

erform

edby

theF

riedm

ananalysisforv

ariance










Acknowledgments


References


































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















250

200

150

100

50

SSC-

A

250

200

150

100

50

25020015010050

SSC-

A

FSC-A


102

103

104

105

102

103

104

105

102

103

104

105

102

103

104

105

102

103

104

105

102

103

104

105

102

103

104

105

CD4 FITC-A

990o-CD4CD25FoxP3

CD4

FITC

-A

CD4

FITC

-A

FoxP3 PE-A

FoxP3 PE-A


IgG PE-A

990o-CD4CD25FoxP3

CD25

PE-C

y5-A

(I) (II)

(III) (IV)

(V)

times103

times103

times103

(a)

Figure 1 Continued



lowastTr

eg (

)

5

4

3

2

1

0

HCo TB

(b)

lowast

Treg

() 4

6

2

0



(c)


HCo T0 T2 T4 T6 T90

2

4

6

8

lowast

998400998400

Treg

()






Table2Cy

tokine

andho

rmon

elevelsinTB

patie

ntsd


Cytokinesa

ndadrenal

steroid

horm

ones

HCogrou

pTB

grou

pT0

T2T4

T6T9

IL-6

(pgmL)

105a

(04ndash22)

1211

asect(57ndash

239)

47a

(18ndash110

)29(17ndash

36)

31(16

ndash36)

31(10

ndash47)

IFN-120574

(pgmL)

47b

(47ndash82)

136

b(107ndash243)

69b

(52ndash125)

61b(47ndash

107)

47(47ndash

69)

47(47ndash

72)

TGF-120573

5721c(5313ndash5924)

6885c120585(5916ndash8040)

6290

(5733ndash7481)

5975

(5190ndash7283)

5970

(5524ndash6

924)

5932(5296ndash6

836)

Cortisol(120583gdL

)146

d(123ndash163)

223

dΥ(158ndash281)

196

d(156ndash

213)

166(114

ndash219

)181(126ndash

206)

164(131ndash209)

DHEA

-S(120583gdL

)2000e

(1334ndash2339)

1295


1128e

(584ndash

1807)

125e

(577ndash1650)

1202e

(8015ndash1970)

204(1303ndash3345)

Com

paris

onsb

etweengrou

ps

a HCoversus




2119875lt005versusT





T0

119875lt001versusT

2119875lt005versusT

4119875lt005versusT

6119875lt005

Com

paris

onsw

ithin

TBpatie

nts

sect T0versus

T2T

4T6

T9119875lt005T0

versus

T2T

4T6

T9119875lt005120585T0

versus

T2T

4T6

T9119875lt005Υ

T0versus


versus

T9119875lt005

Results

aresho

wnas



ontro

lsT0

tim

eofd

iagn

osis

T2T

4T6

24and

6mon

thsafte

rstartingthe

specifica

nti-T

Btre

atmentT9

3mon

thsafte

rthe

endtre

atment

completionCom

paris

onsfor

independ

entgroup

swerem

adeb

ythe

Kruskall-Wallis

analysisofvaria

ncefollowed

bypo

stho

ctestw

henapplicable

Com

paris

onsfor

relatedsampleswerep

erform

edby

theF

riedm

ananalysisforv

ariance










Acknowledgments


References


































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















lowastTr

eg (

)

5

4

3

2

1

0

HCo TB

(b)

lowast

Treg

() 4

6

2

0



(c)


HCo T0 T2 T4 T6 T90

2

4

6

8

lowast

998400998400

Treg

()






Table2Cy

tokine

andho

rmon

elevelsinTB

patie

ntsd


Cytokinesa

ndadrenal

steroid

horm

ones

HCogrou

pTB

grou

pT0

T2T4

T6T9

IL-6

(pgmL)

105a

(04ndash22)

1211

asect(57ndash

239)

47a

(18ndash110

)29(17ndash

36)

31(16

ndash36)

31(10

ndash47)

IFN-120574

(pgmL)

47b

(47ndash82)

136

b(107ndash243)

69b

(52ndash125)

61b(47ndash

107)

47(47ndash

69)

47(47ndash

72)

TGF-120573

5721c(5313ndash5924)

6885c120585(5916ndash8040)

6290

(5733ndash7481)

5975

(5190ndash7283)

5970

(5524ndash6

924)

5932(5296ndash6

836)

Cortisol(120583gdL

)146

d(123ndash163)

223

dΥ(158ndash281)

196

d(156ndash

213)

166(114

ndash219

)181(126ndash

206)

164(131ndash209)

DHEA

-S(120583gdL

)2000e

(1334ndash2339)

1295


1128e

(584ndash

1807)

125e

(577ndash1650)

1202e

(8015ndash1970)

204(1303ndash3345)

Com

paris

onsb

etweengrou

ps

a HCoversus




2119875lt005versusT





T0

119875lt001versusT

2119875lt005versusT

4119875lt005versusT

6119875lt005

Com

paris

onsw

ithin

TBpatie

nts

sect T0versus

T2T

4T6

T9119875lt005T0

versus

T2T

4T6

T9119875lt005120585T0

versus

T2T

4T6

T9119875lt005Υ

T0versus


versus

T9119875lt005

Results

aresho

wnas



ontro

lsT0

tim

eofd

iagn

osis

T2T

4T6

24and

6mon

thsafte

rstartingthe

specifica

nti-T

Btre

atmentT9

3mon

thsafte

rthe

endtre

atment

completionCom

paris

onsfor

independ

entgroup

swerem

adeb

ythe

Kruskall-Wallis

analysisofvaria

ncefollowed

bypo

stho

ctestw

henapplicable

Com

paris

onsfor

relatedsampleswerep

erform

edby

theF

riedm

ananalysisforv

ariance










Acknowledgments


References


































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Table2Cy

tokine

andho

rmon

elevelsinTB

patie

ntsd


Cytokinesa

ndadrenal

steroid

horm

ones

HCogrou

pTB

grou

pT0

T2T4

T6T9

IL-6

(pgmL)

105a

(04ndash22)

1211

asect(57ndash

239)

47a

(18ndash110

)29(17ndash

36)

31(16

ndash36)

31(10

ndash47)

IFN-120574

(pgmL)

47b

(47ndash82)

136

b(107ndash243)

69b

(52ndash125)

61b(47ndash

107)

47(47ndash

69)

47(47ndash

72)

TGF-120573

5721c(5313ndash5924)

6885c120585(5916ndash8040)

6290

(5733ndash7481)

5975

(5190ndash7283)

5970

(5524ndash6

924)

5932(5296ndash6

836)

Cortisol(120583gdL

)146

d(123ndash163)

223

dΥ(158ndash281)

196

d(156ndash

213)

166(114

ndash219

)181(126ndash

206)

164(131ndash209)

DHEA

-S(120583gdL

)2000e

(1334ndash2339)

1295


1128e

(584ndash

1807)

125e

(577ndash1650)

1202e

(8015ndash1970)

204(1303ndash3345)

Com

paris

onsb

etweengrou

ps

a HCoversus




2119875lt005versusT





T0

119875lt001versusT

2119875lt005versusT

4119875lt005versusT

6119875lt005

Com

paris

onsw

ithin

TBpatie

nts

sect T0versus

T2T

4T6

T9119875lt005T0

versus

T2T

4T6

T9119875lt005120585T0

versus

T2T

4T6

T9119875lt005Υ

T0versus


versus

T9119875lt005

Results

aresho

wnas



ontro

lsT0

tim

eofd

iagn

osis

T2T

4T6

24and

6mon

thsafte

rstartingthe

specifica

nti-T

Btre

atmentT9

3mon

thsafte

rthe

endtre

atment

completionCom

paris

onsfor

independ

entgroup

swerem

adeb

ythe

Kruskall-Wallis

analysisofvaria

ncefollowed

bypo

stho

ctestw

henapplicable

Com

paris

onsfor

relatedsampleswerep

erform

edby

theF

riedm

ananalysisforv

ariance










Acknowledgments


References


































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

























Acknowledgments


References


































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Documents

Research Article Increased Frequency of CD4+ CD25+ FoxP3 ...downloads.hindawi.com/journals/jir/2015/985302.pdf · Increased Frequency of CD4+ CD25+ FoxP3+ T Regulatory Cells in Pulmonary