IL-17 and TB

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IMMUNOLOGICAL ASPECTS

Regulatory T cell frequency and modulation of IFN-gamma and IL-17 in active andlatent tuberculosis

Nancy D. Marin a,c, Sara C. Pars a, Viviana M. Vlez a,d, Carlos A. Rojas b, Mauricio Rojas a, Luis F. Garca a,*

a Grupo de Inmunologa Celular e Inmunogentica, Centro de Investigaciones Mdicas, Universidad de Antioquia, Medelln, Colombiab Grupo de Epidemiologa, Facultad de Salud Pblica, Universidad de Antioquia, Medelln, Colombiac NDM is recipient of a predoctoral scholarship from Colciencias, Bogot, Colombiad VMV is recipient of a Joven Investigador award from Vicerrectora de Investigaciones, Universidad de Antioquia, Medelln, Colombia

a r t i c l e i n f o

Article history:

Received 24 February 2010

Received in revised form

5 May 2010

Accepted 20 May 2010

Keywords:

Tuberculosis

Latent infection

Regulatory T cells

Interferon gamma

IL-17

s u m m a r y

Regulatory T cells (Tregs) play an essential role in immune homeostasis. In infectious diseases Tregs may

inhibit protective responses facilitating pathogen multiplication and dissemination, but they may also

limit the inflammatory response diminishing tissue damage. Although there is experimental and clinical

evidence that Tregs are induced during Mycobacterium tuberculosis infection, their role in the immu-

nopathogenesis of tuberculosis is still not completely understood. In this study, the phenotype, frequency

and activity of circulating Tregs in active and latent tuberculosis were evaluated. Phenotypic analysis

showed that Tregs were CD4CD25highFOXP3CD45ROCD127-. High levels of circulating Tregs were

found in patients with active pulmonary tuberculosis, compared to individuals with latent infection. Treg

activity was evaluated by ELISPOT by determining the effect of CD25 cell depletion on the frequency of

IFN-g and IL-17 producing cells after in vitro stimulation with ESAT-6, CFP-10 and PPD. Treg depletion

increased the frequency of IFN-g producing cells, without affecting the frequency of IL-17 producing cells,

in both active and latent tuberculosis, irrespective of the antigen used. Neutralization of IL-10 did not

have any effect on the frequency of IFN-g and IL-17 producing cells. Altogether, these results suggest that

during active tuberculosis Tregs inhibit protective Th1 responses, but not the proinfl

ammatory Th17responses, facilitating mycobacterial replication and tissue damage.

2010 Published by Elsevier Ltd.

1. Introduction

According to the World Health Organization, one third of the

word population is infected with Mycobacterium tuberculosis (Mtb),

but only 10% of the infected individuals would develop active TB

during their lifetime.1 It is well known that bacterial, host and

environmental factors influence the development of active TB.2e4

In most of cases, the host immune response controls the Mtb

replication and a latent infection (LTBi) is established, but when the

host immune response fails to control the tubercle bacilli replica-tion, active TB (ATB) is developed.5 Latency is maintained by a fine

balance between the pathogen persistence and the immune

response, therefore perpetuating the risk of reactivation. Thus, the

immune response against Mtb infection is associated with

the establishment of latency, but the phenomena responsible for

the development or reactivation of ATB in absence of an immu-

nosuppressive event are not well understood.

Tuberculosis has many clinical manifestations, but the most

common form is pulmonary tuberculosis. Individuals with

pulmonary tuberculosis spread bacilli in aerosol, transmitting the

infection to other persons. The extent and the strength of the

exposure affect the transmission rate, thus a higher exposure to M.

tuberculosis results in a higher risk of infection andactive disease.6,7

Household contacts (HHC) in close contact to patients with ATB are

exposed to high bacterial loads and therefore, they have a higherprobability to be infected and develop active disease.6

T cell responses are critical components of the protective

immunity against M. tuberculosis. IFN-g producing Th1 cells are

essential to control the mycobacterial replication by inducing

macrophages antimycobacterial mechanisms and activating CD8

cytotoxic cells,8e10 butTh1 cells alone do not explainthe resistance/

susceptibility to infection and disease.11,12 Th1 cells are important

for protection, but they are also involved in the inflammation and

tissue damage that occurs during active TB.13 The more recently

described Th17 cells have also been associated with Mtb infec-

tion.12,14 IL-17 is produced early during immune response against

* Corresponding author. LFG. Grupo de Inmunologa Celular e Inmunogentica,

Sede de Investigacin Universitaria, Cra 53 N 61-30, Lab. 410, Medelln, Colombia. .

Tel.: 57 4 219 6446; fax: 57 4 219 6450

E-mail address: [email protected] (L.F. Garca).

Contents lists available at ScienceDirect

Tuberculosis

j o u r n a l h o m e p a g e : h t t p : / / i n t l . e l s e v i e r h e a l t h . c o m / j o u r n a l s / t u b e

1472-9792/$ e see front matter 2010 Published by Elsevier Ltd.

doi:10.1016/j.tube.2010.05.003

Tuberculosis 90 (2010) 252e261
mailto:[email protected]://www.sciencedirect.com/science/journal/14729792http://intl.elsevierhealth.com/journals/tubehttp://dx.doi.org/10.1016/j.tube.2010.05.003http://dx.doi.org/10.1016/j.tube.2010.05.003http://dx.doi.org/10.1016/j.tube.2010.05.003http://dx.doi.org/10.1016/j.tube.2010.05.003http://dx.doi.org/10.1016/j.tube.2010.05.003http://dx.doi.org/10.1016/j.tube.2010.05.003http://intl.elsevierhealth.com/journals/tubehttp://www.sciencedirect.com/science/journal/14729792mailto:[email protected]

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Mtb and it has been proposed to be associated with reactivation in

latent TB infected individuals.15,16 Importantly, the kinetics of IFN-g

and IL-17 production and the phenotypic and functional charac-

teristics of Th1 and Th17 cells are different,15,17,18 as well the

susceptibility to Treg suppression, which is diminished in Th17

cells.19 Knowing the role of IFN-g in the defense against Mtb and its

ability to inhibit IL-17 production,20 in addition to the proposedrole

for IL-17 in tuberculosis, it is important to understand their regu-

lation and function during latent and active TB. There is evidence

that many ATB patients present suppression of Mtb specific T cell

responses, including decreased production of IL-2 and IFN-g,[21e23]

suggesting that T cell responses during infection are subject to

regulatory mechanisms; however, little is known about IL-17

producing cells during Mtb infection and the development of active

disease.

The suppressive mechanisms described in the immune response

against Mtb include increased activity of regulatory T cells.24e34

Tregs are recruited to infected organs down-regulating the

immune response against Mtb infection and preventing the clear-

ance of M. tuberculosis by suppressing antigen specific CD4 cells

and interfering with antigen presenting cells.28,30,33 Thus Tregs

have the capacity to control the tissue damage while dampening

the adequate control of mycobacterial replication,29 allowing thepersistence and the establishment of a chronic infection, but they

may also be involved in the reactivation and dissemination of Mtb.

Regulatory T cells with the CD4CD25FOXP3 phenotype

(Tregs) represent 5e10% of circulating CD4 cells,25,27,35 but in

humans only thesubset expressing higher levels of CD25 (a chain of

IL-2R) exhibit a strong suppressive capacity.36 Tregs are a key

component of peripheral tolerance suppressing auto-reactive T

cells and preventing autoimmune diseases. However, there is

strong evidence that Tregs are involved in the immune response

against Mtb and have been detected in a higher frequency in TB

patients peripheral blood mononuclear cells (PBMC) associated

with decreased effectors responses.25,27,30e32

The immunological and physiological events triggered after the

establishment of active TB have been extensively studied, but thoseevents responsible for maintaining the latency and causing reac-

tivation in immunocompetent individuals are not yet well defined.

Therefore, in this study the frequency of Tregs and their effect on

IFN-g and IL-17 production in response to mycobacterial antigens

were studied in individuals with ATB, LTBi individuals with a high

level of exposure (HHC) and LTBi individuals with a low level of

exposure (no HHC) to Mtb. Results show an increased frequency of

circulating CD4CD25high and CD4CD25highFOXP3 cells in ATB

patients compared to LTBi individuals. The functional evaluation of

these cells showed a higher capacity of CD4CD25high cells to

inhibit the IFN-g production and a lesser capacity to inhibit IL-17

producing cells in both ATB and LTBi individuals. These results

suggest an important role of Tregs in the reactivation of the latent

infection and the development of active tuberculosis by decreasingIFN-g responses, while IL-17 may continue facilitating the accu-

mulation of cells in the inflamed tissues.

2. Materials and methods

2.1. Study population

Thirty-one newly diagnosed, active TB (ATB) patients were

recruited at the Tuberculosis Control Program in Medelln

(Colombia) and its metropolitan area. ATB patients had acid fast

smear or culture positive for Mtb. ATB patients were studied before

or within the first 2 weeks of anti-TB treatment. Thirty-eight

subjects with latent TB infection (LTBi) were selected according to

an IFN-g positive response to CFP-10, as evaluated by ELISA in

seven-days whole blood culture supernatants, as previously

reported by our Group.5 LTBi individuals included 26 HHC of

pulmonary TB patients who were followed for 3 years between

2005 and 2008 in our cohort study, remaining healthy without

clinical evidence of active TB.6 A household contact was considered

to be someone who had spent time regularly (weekly) in the same

household as the index case (active TB) for at least one month prior

to the time when the index cases diagnosis was confirmed. Twelve

LTBi individuals who were not household contacts (no HHC) and

who did not have a recent documented exposure to active TB were

selected among laboratory personal according a positive response

to CFP-10 as described by del Corral H et al. 6 All subjects studied

were HIV negative, as tested by DoubleCheckGold HIV 1&2 kit

(Orgenics, Courbevole, France), following the manufacturer

instructions. The study was approved by the Ethical Committee of

the Instituto de Investigaciones Mdicas of the Universidad de

Antioquia and a written informed consent was obtained from all

participants. Individuals infected with HIV, using immunosup-

pressive drugs, with diabetes, or younger than 15 years old were

excluded.

2.2. Sample preparation

Ten to 20 mL of blood were obtained using heparin as antico-

agulant, and the PBMC were obtained by Ficoll-Hypaque density

gradient centrifugation (Biowittaker, Walkersville, MD). PBMC

were washedtwice in PBS (Invitrogen,Carlsbad, CA) andcountedin

a hemocytometer. Viability, as tested by trypan blue staining, was

always !95%.

2.3. Mycobacterial antigens

Recombinant ESAT-6 and CFP-10 were provided by the

Department of Microbiology and Immunology at Colorado State

University, Fort Collins, CO through the Tuberculosis Vaccine

Testing and Research Material Contract No. HHSN26266400091C

NIH, NIAID (N01-AI-40091). PPD (RT50) was obtained from StatensSerum Institute (Copenhagen, Denmark).

2.4. Phenotypic analyses

The expression of CD4, CD25 and FOXP3 were determined in

freshly isolated PBMC. One million PBMCs were incubated at

room temperature for 30 min with anti-CD4-FITC (clone RPA-T4)

plus anti-CD25-PeCy5 (clone M-A251) (BD Biosciences, San Diego,

CA). Mouse IgG1k-PeCy5 (clone MOPC-21) was used as isotype

control. Thereafter, cells were washed with PBS and non-per-

meabilized cells were fixed with 2% paraformaldehyde (J.T.Baker.

Phillipsburg, NJ). For FOXP3 detection, cells stained for CD4 and

CD25 were fixed and permeabilized using anti-human FOXP3

staining buffer (eBioscience). Thereafter, anti-FOXP3-PE (clonePCH101) or rat IgG2a-PE isotype control (Clone eBR2a) was added

for 30 min 4 C. One hundred cells were acquired and the analysis

included identification of CD25FOXP3 cells and

CD25highFOXP3 cells within the CD4 gate. The CD25 pop-

ulation was defined by isotype control and the CD25h population

was defined as the population expressing higher CD25 MFI on

a dotplot. To further evaluated the memory phenotype of Tregs as

previously reported,37 CD45RO expression on CD4CD25/high-

FOXP3

cells was evaluated in some representative samples, using

anti-CD45RO-APC (clone UCHL-1) plus anti-CD4-FITC (clone RPA-

T4) and anti-CD25-PeCy5 (clone M-A251) (BD Biosciences. San

Diego, CA), followed by intracellular staining with anti-FOXP3-PE.

Mouse IgG1k-PeCy5 (clone MOPC-21), mouse IgG2ak-APC (Clone

eBM2a) and rat IgG2a-PE (Clone eBR2a) were used as isotype

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controls. One hundred thousand cells were acquired in FACS

Canto II Flow Cytometer (San Jose, CA) and analyzed using

Cytomation Summit (Fort Collins, CO) and BD FACSDiva Software

v6.1.2. (San Jose, CA). Furthermore, for a better characterization of

Tregs, the CD127 expression that had been associated with cells

with effector phenotype, and lacking in Tregs,26,38 was evaluated

in 1 106 PBMC cells using anti-CD127-FITC (clone eBioRDR5)

(eBioscience) plus anti-CD4-ECD (clone SFCl12T4D11) (T4)

(Beckman Coulter. San Diego, CA) and anti-CD25-PeCy5 (clone M-

A251) (BD Biosciences), followed by intracellular staining with

anti-FOXP3-PE. Mouse IgG1k-FITC (clone MOPC-21) and mouse

IgG1k-PeCy5 (clone MOPC-21) were used as isotype controls. To

determine CD127 expression, 1 105 cells were acquired in

a Coulter EPICS XL Flow Cytometer (Hialeah, FL).

2.5. ELISPOT

The frequency of IFN-g and IL-17 producing cells was evaluated

by ELISPOT using human IFN-g and IL-17 ELISPOT kit (eBioscience),

according to the manufacturers instructions. Briefly, each well in

MultiScreenHTS 96-well filter plates (Millipore. Billerica, MA) was

covered overnight with either anti-IFN-g or anti-IL-17 capture

antibody at room temperature, followed by blockade with RPMI-1640 (Invitrogen) supplemented with 10% Fetal Bovine Serum

(Invitrogen) plus Penicillin/streptomycin (Biowittaker) (complete

medium) at room temperature for 1e2 h. Then, 1.5 105 PBMC per

well were cultured in duplicate wells with complete medium in the

presence of ESAT-6 (1 mg/ml), CFP-10 (5 mg/ml), and PPD (10mg/ml)

for 48 h at 37 C, 5% CO2. Non-stimulated wells were used as

controls. After incubation, supernatants were discarded and the

plates washed, followed by incubation with anti-IFN-g or anti-IL-17

detection antibodies for 2 h at room temperature. The wells were

washed again and HRP-streptavidin was added for 45 min, light

protected, and after washing, the AEC substrate (BD Pharmingen)

was added. The reaction was stopped with distilled water. When

theplates were dried,the spot forming unitswere determined in an

ImmnunoSpot Reader

(CTL, Shaker Heights, OH). Readingsobtained in the nil control were subtracted from samples stimu-

lated with antigens. The spot forming units (SFU) are reported as

SFU 106 cells.

2.6. Evaluation of the suppressive function of Tregs

To evaluate Treg activity two strategies were used. First, the

frequency of IFN-g and IL-17 producing cells in non-depleted and

CD25high-depleted PBMC cultures stimulated with ESAT-6, CFP-10

and PPD was compared. Second, CD4CD25high cells, obtained by

sorting, were added back into CD25high-depleted PBMC cultures,

and the IFN-g and IL-17 production in response to CFP-10 and PPD

was compared with CD25high-depleted, non-reconstituted PBMC

cultures, and non-depleted PBMC cultures.

2.6.1. Depletion of regulatory T cells

The depletion of Tregs was performed using the Human

Regulatory T Cell Isolation kit (R&D systems, Minneapolis, USA)

following manufacturer instructions. Briefly, 6e7106 PBMC were

washed in MagCellet buffer 1 and incubated for 15 min at 4 C

with suboptimal amounts of anti-CD25 ferrous beads (8 ml

compared to 15 ml recommended by manufacturers), ensuring the

depletion of the CD25high population. Thereafter, 1 ml of Mag-

Cellet buffer 1 was added and the mix incubated for 6 min on

the MagCellet magnet, allowing the CD25 cells to attach to the

magnet. CD25-depleted PBMC were collected and washed with

complete medium. Cells were counted and the efficiency of

CD4

CD25

high

depletion was confi

rmed byfl

ow cytometry using

anti-CD4 plus anti-CD25 followed by intracellular staining with

anti-FOXP3 antibodies, as described above.

To determine the effect of CD25high depletion, 1.5 105 non-

depleted PBMC or Treg-depleted PBMC were cultured in ELISPOT

plates in duplicate wells in absence or presence of ESAT-6 (5mg/ml),

CFP-10 (5 mg/ml) and PPD (10 mg/ml) for 48 h at 37 C, 5% CO2. Non-

stimulated plates were used as controls. After incubation, the spot

forming units were determined as described above.

2.6.2. CD4CD25high Treg reconstitution assay

Ten million PBMCs were stained with anti-CD4-FITC and

anti-CD25-PECy5 antibodies and sorted in a MoFlo XDP Cell

Sorter (Beckman Coulter. Brea, CA). CD4 cells were gated on

a dotplot allowing the selection of CD4CD25high positive cells

to be sorted. Sorted cells were collected in RPMI-1640 plus

penicillin/streptomycin, and added to Treg-depleted PBMC

cultures at the same proportion of Tregs that were present

before depletion of Tregs with magnetic beads. The effect of

CD4CD25high reconstitution on IFN-g and IL-17 producing cells

was compared with the response of non-depleted PBMC

cultures and CD4CD25high-depleted PBMC in response to CFP-

10 and PPD and evaluated by ELISPOT as described above. The

purity of CD4CD25high cells sorted was !80% with a FOXP3expression !85%.

2.7. Neutralization of IL-10

For IL-10 neutralization, 1.5 105 PBMCs were preincubated

in duplicate wells in ELISPOT plates in absence or presence of

0.125 mg/mle2 mg/ml of neutralizing anti-human IL-10 antibody

(R&D systems), as suggested by the manufacturer, or 0.25 mg/

mle2 mg/ml of goat IgG Isotype control (R&D systems) for

30 min at 37 C. Thereafter, CFP-10 (5 mg/ml) and PPD (10 mg/ml)

were added for 48 h at 37 C, 5% CO2. The ELISPOT was per-

formed as described above and the SFU are reported as

SFU 106 cells.

2.8. Statistical analysis

The frequency of CD4CD25/highFOXP3 Tregs in LTBi no

HHC, LTBi HHC and ATB individuals were compared by Krus-

kalleWallis and Dunns post test. Wilcoxon test was used for

evaluate differences between non-depleted and Treg-depleted

PBMCs. Statistical differences and significance are shown in each

graph. Statistical significance was considered when p 0.05. All

analyses were carried out using the Prism 5 software (GraphPad,

San Diego, CA).

3. Results

3.1. Clinical characteristics of studied groups

Twelve individuals with LTBi no HHC, 26 individuals with LTBi

HHC and 31 smear or culture positive ATB patients were studied.

Their median (range) ages were: 31(27e61) years for LTBi no HHC,

38(15e68) years for LTBi HHC and 45 (16e70) years for ATB

patients. The male/female ratio for each group was: 4/8 for LTBi no

HHC, 9/17 for LTBi HHC and 22/9 for ATB (Table 1). Twenty-eight

ATB patients had pulmonary tuberculosis, 2 patients had military

tuberculosis and another one had laryngeal tuberculosis. Most

pulmonary TB individuals had a high bacterial load as detected by

acid fast staining of sputum smear.

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3.2. Regulatory T cells CD4CD25FOXP3 are CD127 and

CD45RO

For the characterization of regulatory T cells, different surface

and intracellular markers associated to Tregs were evaluated.

Although CD25 is a marker of regulatory T cells, it is also expressed

by effector cells after activation, albeit at lower expression.39

Additionally, the transcription factor FOXP3 is considered the best

marker for regulatory T cells40 and therefore the phenotypic anal-

ysis of Tregs was done within the CD4 population according to the

total and high CD25 expression, in addition to FOXP3 (Figure 1A).

Previous reports have shown the differential expression of CD127

(a chain of IL-7R) between effector and regulatory T cells. Regula-

tory T cells are CD127 negative whereas effector cells are positive

for this marker.26,38 Thus, CD127 expression on

CD4CD25/highFOXP3 Tregs was evaluated and !97% of them

were negative for CD127 (Figure 1B). Additionally, Tregs have been

reported to exhibit a memory phenotype,37 thus the expression of

CD45RO was also evaluated and more than 95% of

CD4CD25/highFOXP3 cells were found CD45RO (Figure 1C).

Therefore, the complete phenotype of Tregs studied under ourexperimental conditions was CD4CD25highFOXP3 CD127

CD45RO.

3.3. Active TB patients have a higher frequency of Tregs and HHC

have lower levels

To determine whether the frequency of circulating CD4 cells

expressing low or high CD25 is different in LTBi and ATB, the

percentage of CD4CD25 and CD4CD25high in LTBi no HHC, LTBi

HHC and ATB individuals were compared. There were not differ-

ences in the frequency of CD4CD25 cells among the groups (data

not shown). ATB patients had a higher frequency of CD4CD25high

cells (5.35% [Interquartile range, IQR 2.6e

6.3]), compared to LTBiHHC individuals who had the lowest frequency of CD4CD25high

cells (median 1.7 [IQR 1.2e2.8]) (p < 0.001) (Figure 2A). The

frequency of CD4CD25FOXP3 cells was similar among LTBi no

HHC, LTBi HHC and ATB individuals (Figure 2B). Active TB patients

had a higher frequency of CD4CD25highFOXP3 (median 2.0 [IQR

1.3e2.7]) compared to LTBi HHC (median 0.95 [IQR 0.6e1.8])

(p < 0.001). No differences were observed between LTBi no HHC

compared to LTBi HHC and ATB patients (Figure 2C). When ATB

patients were compared with the two groups of LTBi individuals

considered together, the percentages of CD4CD25high and

CD4CD25highFOXP3 (data not shown) were still increased in the

ATB group (p 0.0025 and p 0.004, respectively). Thus in the

remaining sections of results, the 2 groups of HHCs will be shown

together. These results indicate that during active TB there is

a higher frequency of circulating CD4CD25highFOXP3 Tregs

compared with latent TB infection.

3.4. Active TB patients have more IFN-g producing cells in response

to mycobacterial antigens than LTBi individuals

It has been reported that during ATB there is a reduced

production of IFN-g in response to different stimuli.21,41,42 There-

fore, the frequency of IFN-g and IL-17 producing cells in response to

ESAT-6, CFP-10 and PPD was evaluated by ELISPOT in 48 h cultures.

ATB patients, compared to LTBi individuals, showed higher

frequency of IFN-g producing cells in response to CFP-10

(p 0.0071) and PPD (p 0.0009). No differences were found in

response to ESAT-6 between the studied groups (Figure 3A), nor in

the frequency of IL-17 producing cells in response to ESAT-6, CFP-10

and PPD. In addition the IFN-g/IL-17 ratio in response to ESAT-6,

CFP-10 and PPD wasevaluated andthe IFN-g/IL-17 ratioin response

to CFP-10 was 14.7 [IQR 9.9e32.9] for ATB patients and 8.7 [IQR

2.9e14.6] for LTBi individuals (p 0.0097). No differences were

found in the IFN-g/IL-17 ratio in cultures stimulated with ESAT-6 or

PPD (Figure 3B).

3.5. Tregs suppress IFN-g producing cells but not IL-17 producingcells

There is evidence that Tregs can suppress both Th1 and Th17

responses,34,43 but IL-17 producing cells seem to be less susceptible

to suppression by Tregs.19 To investigate the effectof Tregs on IFN-g

and IL-17 production in response to mycobacterial antigens in LTBi

individuals and ATB patients, non-depleted PBMC and Treg-

depleted-PBMC cultures were stimulated with ESAT-6, CFP-10 and

PPD and the frequency of the IFN-g and IL-17 producing cells was

evaluated by ELISPOT. Depletion reduced the number of

CD4CD25high cells by 94 5.6% in LTBi individuals and 91 11.8%

in ATB patients (Figure 4 and data not shown). The depletion of

CD25high cells resulted in a significant increase in the frequency of

IFN-g producing cells responding to ESAT-6, CFP-10 and PPD in LTBiand ATB subjects (Figure 5A). On the contrary, depletion of

CD4CD25high cells did not affect the frequency of IL-17 producing

cells, with the exception of the cultures of LTBi individuals stimu-

lated with PPD that showed a lower frequency of IL-17 in CD25high-

depleted cultures (p 0.034) (Figure 5B). These findings suggest

that Tregs have a less suppressive capacity on IL-17 production, and

a higher susceptibility of IFN-g producing cells to the suppression

by regulatory T cells.

Tofurther confirm that Tregs areresponsiblefor the suppression

observed, reconstitution of Tregs in Treg-depleted PBMC cultures

was performed. CD4CD25high Tregs were purified by sorting and

added back into Treg-depleted PBMC cultures maintaining the

initial proportion of Tregs observed before depletion. The results

were not conclusive (data not shown) because there was a highvariability among LTBi and ATB individuals studied.

3.6. Tregs suppress IFN-g and IL-17 production is not IL-10-

dependent

Regulatory T cells suppress effector responses by different

mechanisms.44,45 One of these mechanisms is controlled by IL-10.

To evaluate whether the effect of CD4CD25highFOXP3 cells on

IFN-g producing cells observed under our experimental conditions

is IL-10-dependent, different concentrations of neutralizing anti-IL-

10 or isotype control antibody (0.125 mg/ml to 2 mg/ml) were added

to PBMC cultures stimulated with CFP-10 and PPD. However, the

addition of IL-10 did not affect the response to CFP-10 and PPD in

either LTBi or ATB individuals (Figure 6). These results suggest that

Table 1

Demographic and clinical characteristics of the populations studied.

Latent TB no

HHC

Latent TB

HHC

Active TB

Median age (range) 31 (27e63) 36 (15e68) 42 (16e70)

Gender Male 4 9 22

Female 8 17 9

AFB sputum 3

6 15

Without

data

4

Type of clinical

disease

28

pulmonary

1 laryngeal

2 miliary


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IL-10 is not involved in the suppression of IFN-g and IL-17responses to mycobacterial antigens under our experimental

conditions.

4. Discussion

The events responsible for reactivation of tuberculosis in indi-

viduals latently infected with M. tuberculosis are poorly understood.

Patients with active tuberculosis frequently have decreased levels

of IFN-g and IL-2, and high levels of immunomodulatory cytokines

IL-10 and TGF-b in response to mycobacterial antigens.21,25,46,47

Tregs, which are increased during active TB, have been associated

with the regulation of immune functions such as self-tolerance,

autoimmunity and anti-tumor response,48,49 but they also have

been associated with the regulation of the immune response in

infectious disease.50,51

In some conditions Tregs may regulateeffector cells during long-persistent diseases protecting them from

the tissue damage caused by effector cells,52,53 but during a chronic

infection, like M. tuberculosis infection, they may be deleterious

because they may down regulate antigen specific T cells, damp-

ening the effective macrophage activation and therefore the Mtb

replication control.29,30,33,54 However, the role of Tregs in TB is not

well understood; nor it is clear whether their expansion is a cause

or a consequence of the disease. Probably they are expanded as an

adaptive host response to limit the inflammatory reaction and

tissue damage induced during the immune reaction against the

mycobacteria. But it is also possible that they are expanded in

response to M. tuberculosis infection by recognition of particular

bacterial products, such as ManLAM that promotes Treg expansion

in a PGE2-dependent manner

33

or through the induction of IL-10

Figure 1. Phenotypic characterization of Tregs according to CD4, CD25, FOXP3, CD127 and CD45RO expression. Cells were stained with anti-CD4, anti-CD25, anti-CD27 and anti-

CD45RO followed by intracellular staining with anti-FOXP3. One hundred thousand cells were analyzed for CD25/FOXP3 expression and the CD127, CD45RO expression were

evaluated among CD4 cells. (A) Top CD4CD25FOXP3 T cells and bottom CD4CD25highFOXP3 T cells. (B) More than 97% of CD4CD25hFOXP3 cells were CD127 negative and

(c) more than 95% were CD45RO positive. A representative experiment is shown.


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and TGF-b produced during the infection, as supported by previous

reports showing increased levels of IL-10 and TGF-b in patients

with active TB.46,47,55,56

In this study, we compared the frequency of Tregs and their

suppressive capacity on IFN-g and IL-17 production in individuals

with active TB and two groups of latently infected individuals.

Latent TB infection in our study was confirmed by a positive IFN-g

response to CFP-10 as previously defined.6 They were classified as

HHC or not-HHC according to whether or not they had a recent and

prolonged exposure to a TB index case. HHCs were exposed to high

bacterial loads and probably this exposure had conditioned their

specific immune response and the ability of establish an effective

response against M. tuberculosis infection.

The phenotypic characterization of human Tregs is difficult

because they lack specific markers. In this study we used the most

accurate available markers: CD25high and FOXP3 expression,57 and

additionally, the expression of CD45RO and the lack of CD127

expression confirmed their phenotype as regulatory T cells. The

expression of CD45RO and the lack of CD127 suggest an activated

memory phenotype of these cells and it is in concordance with the

high expression of CD25.26,37,38 As previously reported by other

authors,we found a higher frequency of CD4CD25highFOXP3 cells

in patients with active TB, compared to individuals latently infectedwith M. tuberculosis,25,30,32 but no differences were observed

between no HHC and HHC LTBi individuals. This finding might be

explained by the time elapsed (about 2 years) between the initia-

tion of anti-TB treatment of the index cases and the recruitment of

their HHCs for this study. The low levels of CD4CD25highFOXP3

cells in HHC LTBi support their ability to control the mycobacterial

replication, despite their high exposure to the mycobacteria, pre-

venting reactivation of latent TB and the development of active

disease.

In agreement with previous reports using the same procedure,58

we found a higher frequency of IFN-g producing cells in ATB

patients in response to CFP-10 and PPD, compared to LTBi indi-

viduals. However, these results are not in agreement with other

reports that show decreased IFN-g production during ATB.23,41,42The explanation for such discrepancy could be the culture time

and the type of T cell involved. Whereas in short-term cultures

(24e48 h), as used herein, IFN-g is produced mainly by effector T

cells that do not require proliferation to initiate cytokine produc-

tion, in long-term cultures (120e144 h) IFN-g is produced mainly

by central memory T cells that require IL-2-dependent prolifera-

tion.59,60 It is also known that TB patients have decreased IL-2

production in response to different mycobacterial antigens.21,41

The hallmark of the regulatory T cells is their capacity of control

effector T cell responses, like cytokine production and cell prolif-

eration. Thus to assess Treg activity in ATB patients and LTBi indi-

viduals, the frequency of IFN-g and IL-17 producing cells in

non-depleted and Treg-depleted PBMC cultures stimulated with

ESAT-6, CFP-10 andPPD was evaluated by ELISPOT. The depletionofTregs in both ATB patients and LTBi individuals PBMC cultures led

to an increased frequency of IFN-g producing cells, indicating that

Tregs were actively functioning. However, the depletion of Tregs in

PBMC cultures did not affect the frequency of IL-17 producing cells

in response to the antigens used, except in response to PPD in

latently infected individuals, indicating a differential susceptibility

of Th1 and Th17 cells to the suppression exerted by Tregs. The

reason why IL-17 producing cells are less susceptible to the

Figure 2. Frequency of circulating (A) CD4CD25high, (B) CD4CD25FOXP3, and (C)

CD4CD25highFOXP3 cells in individuals with latent and active TB. PBMC were stained

with anti-CD4-FITC plus anti-CD25-PeCy5, followed by intracellular staining with anti-

FOXP3-PE. One hundred thousand cells were analyzed and the total and high CD25

expression was evaluated among CD4 cells. (A) ATB patients had higher frequency of

CD4CD25high T cells, whereas LTBi HHC had the lowest frequency. No differences were

observed in LTBi no HHC compared to LTBi HHC and ATB individuals. (B) The

proportion of CD4CD25FOXP3 Tregs was not different among studied groups. (C)

Active TB group had a higher frequency of CD4CD25highFOXP3 compared to LTBi

HHC individuals. KruskalleWallis test and Dunns multiple comparison post test

***p < 0.001.


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Figure 3. Comparison of the frequency of IFN-g and IL-17 producing cells in ATB patients and LTBi individuals in response to ESAT-6, CFP-10 and PPD. (A) PBMC from ATB patients

and LTBi individuals were stimulated with ESAT-6, CFP-10 and PPD and the frequency of IFN-g and IL-17 producing cells was evaluated by ELISPOT as described in Materials and

Methods. ATB patients compared to LTBi individuals had a higher frequency of IFN-g producing cells in response to CFP-10 and PPD. (B) The IFN-g/IL-17 ratio was compared between

LTBi individuals and ATB patients in response to ESAT-6, CFP-10 and PPD. ATB patients had a higher IFN- g/IL-17 ratio, compared to LTBi individuals, in response to CFP-10

(p 0.0097). Mann Whitney test was used and p values are shown in the graphs.

Figure 4. Effectiveness of CD4CD25high depletion. Seven million PBMC were depleted of CD25high using anti-CD25 ferrous beads as described in Materials and Methods. The

effectiveness of depletion was evaluated in the cell fraction that was not attached to the magnet. (A). Thefi

gure shows a representative example of LTBi ( n

22) and ATB (n

15) .


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suppressive effect of Tregs is not yet clear; however, it is possible

that Th17 cells are dependent on TGF-b, produced by Tregs for their

expansion or differentiation.61 In fact, in LTBi individuals, PBMC

stimulation with PPD down regulated IL-17 producing cells in Treg-

depleted PBMC, compared to non-depleted PBMC. This finding is

not in agreement with a recent report34 showing in TST individ-

uals that both IFN-g and IL-17 are susceptible to the suppressive

effect of Tregs. One possible explanation may be the differences in

the methodology used to evaluate this phenomenon. Also, it must

be noted that our ATB patients were studied before or within the

first 2 weeks of anti-TB treatment. Although it is unlikely that such

a short time under treatment would decrease the number or the

activity of Tregs, we cannot rule out this possibility, since in the

guinea pig model of TB it has been demonstrated that the standard

anti-TB treatment eliminates Tregs.62 Unfortunately, our experi-

ments of Treg reconstitution did not provide consistent results. It is

possible that manipulation of the Tregs during the sorting proce-

dure affected their suppressive capacity, but more probably that the

Figure 5. Effect of Treg depletion on the frequency of IFN-g and IL-17 producing cells in response to mycobacterial antigens. One hundred and fifty thousand non-depleted and

Tregs-depleted PBMC, as described in Materials and Methods, were stimulated with ESAT-6, CFP-10 and PPD for 48 h at 37 C, thereafter, the SFU were determined by ELISPOT. (A)

Frequency of IFN-g producing cells in response to ESAT-6, CFP-10 and PPD. The depletion of CD25high cells increased the frequency of IFN-g producing cells in LTBi individuals and

ATB patients in response to ESAT-6, CFP-10 and PPD. (B) Frequency of IL-17 producing cells in response to ESAT-6, CFP-10 and PPD. The frequency of IL-17 producing cells was lower

in Treg-depleted cultures from LTBi individuals in response to PPD, but no differences were observed in response to CFP-10 and PPD in LTBi and ATB individuals. Wilcoxon test was

used and p values are shown for each graph.


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amount of Treg cells added back to the depleted cells, which in our

experiments was equivalent to the pre-sorting percentage, was not

enough to suppress the effector cell response, since other authorshave used a higher proportion of Treg/effector cells.32,63

Despite the high levels of Tregs in inflamed tissues during

tuberculosis disease,25 the persistence of the inflammatory process

may indicate a selective suppressive function of different compo-

nents of the adaptive immune response. It is possible to suggest

that the high suppressive capacity of Tregs on IFN-g producing cells

and the low suppressive capacity over IL-17 producing cells

observed in our experiments, allow the persistence of IL-17

producing cells in inflamed tissues and thus the perpetuation or

recrudescence of the inflammatory reaction.15,64

IL-17 is considered a proinflammatory cytokine and it has been

proposed to participate early in the defense against M. tuberculosis,

due to its capacity to induce cytokine and chemokine production,

favoring the recruitment of other cells, including neutrophils, to thesites infected with M. tuberculosis.65 The role of neutrophils in

tuberculosis is controversial.66e68 Neutrophils are important

components of the innate immune system and are considered the

first line of defense against many invading microorganisms.69 On

the other hand, previous studies considered the neutrophils to be

deleterious in the defense against M. tuberculosis. High levels of

these cells have been observed in patients with active disease70 and

TB susceptible animals were found to accumulate neutrophils in TB

lesions compared to resistant animals.67,71

Regulatory T cells have an arsenal of suppressor mechanisms

including IL-10 production,44 high level of this cytokine have been

detected in patients with active TB.72 In tuberculosis, IL-10 is

produced as a result of the chronic stimulation by mycobacterial

antigens and produced by Tregs and Tr1 regulatory cells.73,74

IL-10production in patients with active disease is associated with anergy

to the stimulation with mycobacterial antigens,47 dampening

proliferation and cytokine production by effector cells. However, in

agreement with previous reports,33 the neutralization of IL-10 in

cultures stimulated with CFP-10 and PPD showed no association of

IL-10 with the inhibition of IFN-g and IL-17 production, suggesting

that Tregs use another suppressive mechanism, different from IL-10

production, to inhibit effector T cell responses. Future studies

should focus on the elucidation of the suppressive mechanisms

used by Tregs to inhibit proliferation and cytokine production in

response to mycobacterial antigens by TB patients, since the

increased frequency of circulating Tregs and their suppressive

activity on IFN-g production during active disease and their low

suppressive capacity on IL-17 producing cells support their

involvement in the development of tuberculosis disease. Currently

the role of Tregs in the latency phenomenon is not clear and more

studies are necessary to clarify it, and to possibly used them as

predictive biomarkers for the development of active TB in people

with a high risk of infection or disease by M. tuberculosis.

Acknowledgements

The authors thank the patients and the healthy volunteers for

their acceptance to participate in this study. We also thank the

Tuberculosis Control Programs of the Servicio Seccional de Salud de

Antioquia andthe Secretaria de Salud de Medelln forallowing us to

have access to the clinical records of patients. This work was sup-

ported by Colciencias, (Bogot, Colombia) grant 1115-408-20488

Funding: None

Competing interests: None declared.

Ethical approval: Not required.

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IL-17 and TB