Secretion of IFN-γ and IL-17 after Stimulation of ESAT-6-CFP10

(EC610) Fusion Antigen from PBMC in Groups Active TB and

Latent TB

Nika Andriani

, Nova Kurniati

, Muhammad Irsan Saleh

, Eddy Mart Salim

Zen Hafy

, Jusak Nugraha

5 f,

, Kemas Ya’kub Rahadiyanto

FranciscaSrioetamiTanoerahardjo

Master Program in Biomedical Sciences, Medical Faculty, Sriwijaya University, Palembang, Indonesia

Department of Internal Medicine, Medical Faculty, Sriwijaya University, Palembang, Indonesia

Department of Pharmacology, Medical Faculty, Sriwijaya University, Palembang, Indonesia

Department of Histology, Medical Faculty,Sriwijaya University, Palembang, Indonesia

Department of Clinical Pathology, Medical Faculty, Airlangga University, Surabaya, Indonesia

Department of Clinical Pathology, Medical Faculty, Sriwijaya University, Palembang, Indonesia

Consultant of Molecular and Microbiology Laboratory in TB Research, Center for Biomedical and Basic Health

Keywords: Tuberculosis, IL-17,PBMC, EC610 Antigen

Abstract: Tuberculosis is an infectious disease that is transmitted by the bacteria Mycobacterium tuberculosis. The

immune system has an important role in the pathogenesis of TB. The protective response to TB involves the

secretion of proinflammatory cytokines, namely Th1 cells that produce IFN-γ and Th 17 which produce IL-

17, which plays a very important role in the body's defense system, especially in dealing with intracellular

bacterial infections. The EC610 fusion antigen is a specific M.TB antigen which has antigenicity to T cells so

that T cells secrete cytokines. The study aimed to determine the immune response of proinflammatory

cytokines against Mycobacterium tuberculosis infection by looking at the secretion of IFN-γ and IL-17 levels

after stimulation of the ESAT 6-CFP 10 (EC610) fusion antigen in active and latent TB patients. This type of

research was a quasi experimental in vitro. The research was conducted at the Palembang Lung Special

Hospital. The research subjects were 21 samples of active TB and 28 samples of latent TB. PBMC blood

samples were isolated using Ficoll-Paque, induced with ESAT-6 - CFP-10 Fusion Antigen (EC610) for 24 -

72 hours at 37 ° C. IFN-γ and IL-17 were measured by ELISA Reader. Analysis used the Mann Whitney test

ρ <0.05. IFN-γ levels and IL-17 levels in active TB were higher than latent TB, but statistically there was no

significant difference between IFN-levels (ρ = 0.769) and IL-17 levels with a value of ρ = 0.000, meaning

that there was a significant difference between both groups. The cut-off points were IFN-γ (6850 pg / mL)

and IL-17 (85 pg / mL) using Receiver Operating Curve (ROC) curve analysis. As a conclusions, IFN levels

were not different and IL-17 levels were different. This shows that IL-17 levels play a role in the protective

immune response against Mycobacterium tuberculosis during the progression of TB disease.

https://orcid.org/0000-0002-2588-0556

https://orcid.org/0000-0002-1520-7421

https://orcid.org/0000-0003-4788-8409

https://orcid.org/0000-0002-5654-0757

https://orcid.org/0000-0001-9682-2591

https://orcid.org/0000-0001-6700-9921

https://orcid.org/0000-0001-9557-467X

https://orcid.org/0000-0001-6948-0645

Andriani, N., Kurniati, N., Saleh, M., Salim, E., Hafy, Z., Nugraha, J., Rahadiyanto, K. and Tanoerahardjo, F.

Secretion of IFN- and IL-17 after Stimulation of ESAT-6-CFP10 (EC610) Fusion Antigen from PBMC in Groups Active TB and Latent TB.

DOI: 10.5220/0010491602890297

In Proceedings of the 1st Jenderal Soedirman International Medical Conference in conjunction with the 5th Annual Scientiﬁc Meeting (Temilnas) Consortium of Biomedical Science Indonesia

(JIMC 2020), pages 289-297

ISBN: 978-989-758-499-2

289

1 INTRODUCTION

Tuberculosis is an infectious disease that is

transmitted by the bacteria Mycobacterium

Tuberculosis which attacks various organs, especially

the lungs. Tuberculosis transmission occurs through

droplets of patients infected with the bacteria

Mycobacterium tuberculosis (Ministry of Health of

the Republic of Indonesia,2014).

Tuberculosis is a health problem that is of global

concern today. Indonesia is a country with the second

highest number of new cases in the world after India.

The number of new cases of TB BTA + in Indonesia

was 156,723 with the distribution of cases in several

provinces, especially in South Sumatra, the number

of new cases of TB BTA + was 5674 cases (Ministry

of Health of the Republic of Indonesia, 2017).

According to the Palembang Health Office in

2015, the number of tuberculosis cases in Palembang

was 1305 from the total population. Based on gender,

the number of cases in males is higher than in

females. Based on the age group, most tuberculosis

cases were found at the age of 25-34 years at 18.07%,

age 45-54 years at 17.25% and age 35-44 years at

16.81% (Ministry of Health of the Republic of

Indonesia, 2016).

At this time, experts suspect that there is an

immune system disorder in tuberculosis sufferers.

Helper-1 (Th1) cells play a very important role in the

body's defense system, especially in dealing with

intracellular bacterial infections. One of the cytokines

produced by Th1 cells is IFN-γ which plays an

important role in eliminating Mycobacterium

tuberculosis. IFN-γ serves to strengthen the potential

of phagocytes from macrophages infected with

Mycobacterium tuberculosis by stimulating the

formation of phagolysosomes. IFN-γ also stimulates

the formation of free radicals to destroy bacterial

components Mycobacterium tuberculosis, namely

DNA and bacterial cell walls(Widjaja J.T et al, 2010)

Recent studies have shown that IL-17 plays an

important role in the initial immune response against

Mycobacterium tuberculosis infection by forming

granulomas. Interleukin 17 (IL-17) is a pro-

inflammatory cytokine produced by Th 17 which has

an important role in the pathogenesis of TB. IL-17 is

important for modulator of inflammation and recall

memory response. The role of IL-17 as a

proinflammatory cytokine can recruit neutrophils and

induce an optimal Th1 response to stimulate IFN-γ

production and stimulate chemokines. However,

IFN-γ has the effect to suppress IL-17(Saraiva and

O’Garra, 2010; Javan et al, 2016).

Research over the last decade has resulted in the

development of Interferon-gamma realease assays

(IGRA) to detect Mycobacterium tuberculosis

infection. Based on the principle that individual T

cells that have TB infection can respond to re-

stimulation with the specific antigen Mycobacterium

tuberculosis. This test measures the production of

cytokines secreted by T lymphocytes that have been

sensitized by the specific antigen Mycobacterium

tuberculosis(CDC, 2011). The Food and Drug

Administration (FDA) has approved two IGRA

testing techniques, QuantiFERON-TB and T-

SPOT.TB to detect Mtb infection (Pai M et al., 2014).

QuantiFERON (QFT) is a measurement of IFN-γ

secreted from T cells previously exposed to

Mycobacterium tuberculosis when stimulated in vitro

with specific antigen Mycobacterium tuberculosis

ESAT-6, CFP-10 and TB 7.7 using the enzyme-

linked immunosorbent assay (ELISA) method. This

test is used to detect the amount of IFN-γ against a

specific antigen produced from the subject's T cells

exposed to M. tuberculosis with using a peptide

cocktail that simulates the proteins ESAT-6, CFP-10

and TB7.7. Antigen exposure generates an immune

response to aid screening for Latent TB (Pratomo and

Setyanto, 2013)

The T-SPOT.TB test is an in vitro diagnostic test

based on the enzyme-linked immunospot (ELISPOT)

method. This test is used to count the number of

effector T cells that respond to stimuli with a

combination of peptides that stimulate ESAT-6 and

CFP10 antigens. The immune response to

Mycobacterium tuberculosis infection is mainly

mediated through T cell activation. Activation of T

cells will fight Mycobacterium tuberculosis both CD4

+ and CD8 + which produce several cytokines

including IFN-γ and IL-17 after stimulated by antigen

ESAT-6 and CFP10. Peripheral blood mononuclear

cells (PBMC) were separated from whole blood,

washed and counted before adding to assay. Isolated

PBMCs (white blood cells) are placed into microtiter

orifices where they are exposed to

phytohemagglutinin (PHA) control (a mitogenic

stimulator that demonstrates cell function), nil

control, and tuberculosis-specific antigen. PBMCs

are incubated with antigens to allow stimulation of

sensitized T cells to produce cytokines(Oxford

Imunotec,2017).

A study conducted by Eunkyoung et al, 2016.On

patients with active TB and latent TB infection by

detecting whole blood levels of IFN-γ with IL-17 in

study subjects with TB before receiving treatment

using the QuantiFERON method found that IFN-γ

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and IL-17 were lower in active TB patients rather than

latent TB.

Another study using T-SPOT (ELISPOT) in vitro

conducted by Cowan J et al with research subjects TB

patients with new cases before receiving OAT

treatment found that there was a significant increase

in IFN-γ and IL-17 levels in active TB patients and

latent in PBMC. Research conducted by Marin ND

using TB patients who were given OAT in the first 2

weeks stated that the levels of IFN-γ and IL 17 in

active TB patients were higher than latent TB by

ELISPOT (enzyme-linked immunospot) method in

PBMC.

Based on previous research regarding the

measurement of cytokine secretion, the researchers

were interested in measuring the secretion of

proinflammatory cytokines in vitro, namely IFN-γ

and IL-17 after stimulation of the ESAT 6 - CFP 10

(EC610) fusion antigen in PBMC groups of active TB

and latent TB. Measurement of cytokines using the T-

SPOT and QuantiFERON methods in this study was

used for screening latent TB. Research using PBMC

is still limited, especially in Indonesia, so this

research is expected to have a novelty in evaluating

the pathogenesis of tuberculosis, especially to see the

immune response to Mycobacterium tuberculosis

infection.

2 MATERIAL AND METHODS

This type of research is a quasi experimental study or

quasi experimental study in vitro with a non-

equivalent post test only design. The research was

conducted at the Palembang Lung Special Hospital in

the period August 2018 - January 2019 This research

has received a certificate of ethical approval from

MoehammadHoesin Hospital and Sriwijaya

University Medical Faculty.

The number of research samples was divided into

two groups, namely the first group of latent TB came

from nurses in Palembang Paru Hospital without

clinical symptoms of TB who served more than 6

months in the outpatient and inpatient unit of

Palembang City Lung Hospital who had direct

contact with pulmonary TB patients, TST

examination. positive with induration> 10 mm and

negative smear examination and or radiological

examination did not show lung abnormalities

(normal). The second group of active TB was

diagnosed by pulmonary specialist doctors at the

Palembang City Lung Hospital as new TB cases,

there were BTA examination results, radiological

examinations / chest X-rays showing a picture of

active TB and anti-tuberculosis drug therapy (OAT)

for less than 1 month. The research subjects were

selected by purposive sampling. Pulmonary TB

patients receiving corticosteroid therapy or

immunosuppressant drugs with complaints of

respiratory infections such as bronchitis and allergies,

suffering from liver disorders, kidney disorders,

diabetes mellitus, hepatitis B and HIV infection were

not included in the study (exclusion criteria).

The study sample was a supernatant after

stimulation of the ESAT-6-CFP 10 antigen (EC610)

with the following research stages as a 16 ml venous

blood sample and put into five heparin anticoagulant

tubes. Then one tube of the sample was treated with

stimulation of the ESAT-6-CFP 10 antigen contained

in TB 1 and TB 2 tubes which were incubated for 16-

24 hours using the QuantiFERON method. Four

sample tubes were isolated by Peripheral Blood

Mononuclear Cells (PBMC) using Ficoll-Paque and

induced with ESAT-6 - CFP-10 Fusion Antigen for

24 - 72 hours using the T-SPOT method. Then the

levels of IFN-γ and IL-17 were checked with an

ELISA reader. The difference in mean levels of IFN-

γ and IL-17 was analyzed statistically by the Mann-

Whitney test with a significance level (ρ <0.05). To

get the cut-off-point, the Receiver Operating Curve

(ROC) curve analysis will be used.

3 RESULTS

The research subjects were 49 people who met the

inclusion criteria of the researcher. The research

subjects were divided into 2 patients, namely active

TB and latent TB. Active TB was 21 new TB patients

collected since January 2018, while 28 people with

latent TB had contact history of TB patients

consisting of 23 nurses and 5 family of patients. who

has TB.

The characteristics of the research subjects

included gender, age, education, BCG status, BMI,

chest X-ray, QFT results, and laboratory experiment

results were depicted inTable 1 and Table 2.

The latent TB group, as many as 28 people were

tested for TST and IGRA which were used to screen

for the LatentTB group. Obtained TST induration

varied. The results of TST induration in the Latent TB

group can be seen in the Table 3 and IGRA using

QuanTIFERON in the Latent TB Group can be seen

in the Table 4.

The value of ρ <0.05 was 0.000, which means that

there was a significant difference between IL-17

levels between the two groups after stimulation with

EC610 Fusion Antigen.

Secretion of IFN- and IL-17 after Stimulation of ESAT-6-CFP10 (EC610) Fusion Antigen from PBMC in Groups Active TB and Latent TB

291

Table 1. Demographic Data of Research Subjects

Characteristics Active TB Latent TB

n (%) n (%)

Number of

Sub

ects

21 (100) 28 (100)

Gender

Women

13 (61,90) 21 (75,0)

Male

8 (38,10) 7 (25,0)

39,10±10,4 36,32±8,773

Junior High

School

3 (14,3) 0 (0,0)

Senior High

School

12 (57,1) 7 (25,0)

Diploma 2 (9,5) 12 (42,9)

S1 4 (19,0) 9 (32,1)

BCG status

Yes 14 (66,7) 20 (71,4)

No 1 (4,8) 0 (0,0)

Unknown 6 (28,6) 8 (28,6)

IMT

Normal 11 (52,4) 19 (67,9)

Fat 1 (4,8) 4 (14,3)

Obesity 0 (0,0) 3 (10,7)

Table 3. Results of TST Induration in the Latent TB Group

Diameter of

Induration TST

(

)

n (%)

10 9

(

32.1

)

11 1(3.6)

12 3(10.7)

15 3(10.7)

16 3

(

10.7

)

18 2

(

7.1

)

20 3

(

10.7

)

21 1(3.6)

22 2(7.1)

27 1(3.6)

Total 28

(

100.0

)

Table2. Laboratory Data

Characteristics

TB Active TB Laten

n (%) n (%)

Thoracic Photo

Normal

0 (0,0) 28 (100)

Minimal lesions

15 (71,4) 0 (0,0)

Moderate lesions

6 (28,6) 0 (0,0)

BTA examination

ative

6 (28,6) 28 (100)

Positive

15 (71,4) 0

BTA +1

13 (61,9)

BTA +2

1 (4,8)

BTA +3

1 (4,8)

Qualitative Examination Results

IFN-γ results

T-SPOT

ative

4 (19.0) 22 (78.6)

Positive

17 (81.0) 6 (21.4)

IL-17 results

T-SPOT

ative

3 (14.3) 24 (85.7)

Positive

18 (85.7) 4 (14.3)

Table 4. IGRA (QuantiFERON) examination results in the

Latent TB Group

Result of QFT

n (%)

Negative

15 (53,6)

Positive

13 (46,4)

Table 5. IFN-γ and IL-17 levels in patients with Active TB

and Latent TB

IFN-γ ClinicalStatus N Median

(

Min-Max

)

EC610

Fusion

Antigen

Active TB 21 6700

(2000-9100)

Latent TB 28 6000

(2500-9000)

IL-17

EC610

Fusion

Antigen

Active TB 21 160 (80-210)

Latent TB 28 60 (40-90)

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Figure 1. Analysis of the ROC curveCurve IFN-γ (A)and CurveIL-17 (B) using cut-off-point IFN-γ 6850 pg / mL and cut-

off-point IL-17 85 pg / mL.

The sensitivity value of IFN-γ levels was 80.95%

with a specificity of 78.57%. Postive Predictive Value

(PPV) of 98.63%. Negative Predictive Value (NPV)

of 17.84%. Meanwhile, the sensitivity value of IL-17

levels was 85.71% with a specificity of 85.71%.

Postive Predictive Value (PPV) of 85.71%. Negative

Predictive Value (NPV) of 85.71%.

Table 5 informs that IFN-γ levels were higher in

active TB, namely 6700 (2000-9100) pg / mL while

latent TB was 6000 (2500-9000) pg / mL but

statistically there was no significant difference

between the two groups after stimulation of Fusion

Antigen EC610 with a value of ρ> 0.05, namely

0.769.IL-17 levels were higher in active TB, namely

160 (80-210) pg / mL and latent TB 60 (40-90) pg /

mL.

The area under the ROC curve of IFNγ levels to

predict active TB and latent TB is 0.475 (95% CI =

0.311 to 0.639) while the IL-17 level to predict active

TB and latent TB is 0.994 (95% CI = 0.980 s / d

1,000) (Figure 1). The ROC curve also shows that the

IFNγ level has a very low diagnostic value while the

IL-17 level is categorized as <85pg / mL and> 85 pg

/ mL has a good diagnostic value because the curve

moves away from the 50% line and approaches the

100% line. This suggests that IL-17 levels can be used

as a predictor or diagnosis of active TB and latent TB.

4 DISCUSSION

The proportion of subjects in this study based on

gender was found to be more women than men in

active TB and latent TB. As for several factors,

namely social and economic factors (financial

conditions) that cause women of childbearing age to

suffer from tuberculosis (TB) are more common,

these are found in Afghanistan, Pakistan and

Iran(Dotulong J.F.J et al, 2015)

The active TB and latent TB groups in this study

were found in the age range between 25-36 years.

This result is in accordance with the provisions of the

Ministry of Health of the Republic of Indonesia,

which is mostly found in the 25-34 year age group.

productive age is very dangerous to the level of

transmission because patients easily interact with

other people, high mobility allows it to be transmitted

to other people and the environment. In this study,

there were in the range of 25-36 years of age who

were classified as having a regular job every day

outside the home, especially the latent TB group

because they were nurses / hospital staff. This makes

it easy for patients to interact with other people,

thereby increasing the risk of contracting TB

(Dotulong J.F.J et al, 2015)

Based on the level of education in this study, the

most active TB was obtained with a high school

background while latent TB with a recent diploma

education. Higher education does not always behave

Secretion of IFN- and IL-17 after Stimulation of ESAT-6-CFP10 (EC610) Fusion Antigen from PBMC in Groups Active TB and Latent TB

293

well. Therefore education is not an indicator of

healthy life behavior (Marieta K.S, 2014).

Most of the patients with Active TB and Latent

TB have received BCG immunization when they

were children. However, BCG immunization does not

fully protect children from tuberculosis attacks. The

factors that cause someone to be infected with TB

include household contact with tuberculosis patients,

low nutritional status so that the body's immune

system is not optimal, high humidity that makes

tuberculosis bacteria thrive and an unclean

environment(Rachim R, 2014).

Body Mass Index (BMI) is a way of directly

assessing nutritional status using height and weight.

In this study most were found in BMI which was

classified as normal. The increase in BMI in TB

patients is a good marker of decreasing the likelihood

of relapse (relapse) from TB infection and a sign that

the TB infection process is reduced (Priyantomoet al

, 2014).

In this study all latent TB with TST induration ≥

10 mm (positive). The tuberculin test is done to find

out whether a person has immunity to TB bacilli or

not so it is very good for detecting TB infection. If the

tuberculin test result is positive or abnormal, it means

that the person is infected with TB bacilli and there

are antibodies to the TB bacilli that can become

active. Positive tuberculin test results should be

confirmed by chest X-ray and sputum examination. If

the chest X-ray is normal, then latent TB therapy can

be done, but if the chest X-ray is abnormal and shows

TB, it can be included in active Mycobacterium

tuberculosis (Kenyorini et al,. 2012).

Chest X-ray laboratory data shows abnormal

results of active pulmonary TB which is common

with minimal lesions and normal latent TB. This is

related to one's immunity and the virulence of

Mycobacterium tuberculosis. The lower the

immunity and virulence of a person against

Mycobacterium tuberculosis, the more damage there

is to the radiological image (chest X-ray) (Afif E et al

, 2013).

In this research, BTA examination was mostly

found in BTA +1. There are several factors that affect

the results of the sputum BTA examination including

too few germs due to sputum extraction (not

according to operational procedures (SOP), methods

and methods of examination that are not in

accordance with the SOP, and the effect of anti-

tuberculosis drug treatment. Coughing is effective in

removing sputum can help find BTA germs on

sputum examination in the pulmonary TB group (Ors

et al.,2007)

Two commercial kits that can be used to test for

M. Tuberculosis are QuantiFERON and T-SPOT.

The basic principle of this examination is that the

cells produced by TCD4 lymphocytes if these cells

are incubated with M. Tuberculosis antigen when the

examination uses QuantiFERON, whereas when

using T-SPOT, the number of spots formed in the

membrane is calculated which indicates the presence

of IFN-producing TCD4 lymphocytes. states that this

examination cannot distinguish between active and

latent TB infections so that in order to be able to

diagnose TB in addition to the results of the IGRA

examination, it still takes into account the clinical

situation, laboratory results and other examinations

(CDC, 2010).

Where in this study the latent TB group was

declared if the TST or IGRA results were positive,

both (TST and IGRA) were positive. QuantiFERON

in this study was used for screening latent TB. In

measuring the levels of IFN and IL-17 in this study

using T-SPOT. This QFT and T-SPOT examination

must be accompanied by other supporting

examinations such as Latent TB, TST examination

and active TB must be BTA examination and chest

X-ray to detect TB. QFT and T-SPOT are indirect

markers of M. tuberculosis exposure and indicate a

cellular immune response to M. tuberculosis.

4.1 Secretion of IFN-γ after EC610

Stimulation in Active TB and

Latent TB

IFN-γ is a pro-inflammatory cytokine produced by

activated T cells due to an immune response to a

specific antigen stimulus. These cytokines play an

important role in the activation of macrophages to

eliminate Mycobacterium tuberculosis by recruiting

phagocytic cells to eliminate Mycobacterium

tuberculosis. IFN-γ strengthens the phagocyte

potency of macrophages by stimulating

phagolisosome fusion which can destroy M.

tuberculosis bacteria(Widjaja JT et al, 2010;

Wahyuniati N, 2017)

In this study, IFN-γ levels after stimulation with

EC-610 antigen were higher in patients with active

TB than latent TB but statistically there was no

significant difference between the two groups.. These

results are in line with Setiawan and Nugraha that

active TB levels of IFN-γ were higher than latent TB

but there was no statistically significant difference.

IFN-γ levels are higher because there is a protective

immune response against infection with TB germs.

And the results of IFN-γ levels showed no significant

difference between active TB and latent TB. This is

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294

due to the nutritional status of active TB sufferers

who tend to be malnourished so that the immune

response does not function optimally. Patients who

have been diagnosed with TB but did not immediately

seek anti-TB treatment. This condition results in a

decrease in immune response, so that the IFN-γ level

is not too high (Setiawan H and Nugraha J, 2016

). The

development of recent research is that IFN-can induce

the autophagy mechanism in cells infected with

mycobacteria. The induction of autophagy will

deliver mycobacteria into the lysosome and there will

be a phagolysosome fusion which functions as an

anti-microbial so that the bacteria will be killed

(Wahyuniati N, 2017 )

The results of this study differ from previous

studies possibly because the sample in this study were

new TB patients who had received OAT <1 month

where the inflammatory response was still increasing

in the early phase of TB infection. When the initial

TB infection, the immune system will respond by

carrying out an inflammatory reaction that occurs

within 2-10 weeks after exposure to bacteria by

forming a body defense system called granuloma,

thusrecruiting immune cells to eliminate bacteria by

increasing the production of proinflammatory

cytokines. TB patients who consume OAT <1 month,

the possibility of developing M. tuberculosis bacteria

will decrease because the patient's immune system

increases so that macrophage activation occurs which

is marked by increased production of cytokines. The

increase in proinflammatory cytokines, namely the

levels of IFN-γ and IL-17 can also occur due to the

influence of memory T cells that have been

previously described (in vivo) and then stimulated

with a more specific EC610 antigen so that the

expression of IFN-IL and IL-17 levels increases. The

effect of the strength of antigen presentation is

thought to determine the antigenity that affects

stimulation of T cell proliferation and cytokine

production(Wibowo R.Y et al.,2017).

Low IFN-γ levels in latent TB are due to the

reduced number of effector memory T-cells secreting

IFN-in individuals with latent TB infection, due to the

absence of M. tuberculosis replication and antigen

load. This suggests that IFN-γ secreting T-cells

predominatse during active TB disease (Biselli R et

al.,2010)

4.2 Secretion of IL-17 after EC610

Stimulation in Active TB and

Latent TB

IL-17 is a pro-inflammatory cytokine that plays a role

in the pathogenesis of TB. The role of IL-17, among

others, is to induce an optimal Th1 response and form

granulomas, which are protective immunity against

MTB infection. IL-17 also plays a role in attracting

and activating neutrophils (Torado E and Cooper

M.A, 2010).

IL-17 levels in this study in patients with active

TB and latent TB showed a significant difference in

the two groups where the IL-17 levels in active TB

were higher than latent TB. This study is in line with

Luo et al. Stated that IL-17 levels were increased in

active TB compared to latent TB. In the early phase

of active TB, Th17 will produce IL-17 which will

recruit neutrophils to the infected site. In addition, as

TB disease progresses, there is an increase in the

production of IL-17 in peripheral blood for

aprotective immune response against M.Tuberculosis

(Luo et al., 2017)

Based on the results of previous studies, an

increase in IL-17 levels in TB patients stimulated

with an antigen increased IL-17 production, in

response to M. tuberculosis. IL-17 plays a significant

role in the protection induced by the ESAT-6 antigen.

However IL-17 production in the lungs is generally

immunosuppressive to IFN-γ. Thus, T cells in the

presence of ESAT-6 reduce the proliferation and

production of Th1 cytokines, but increase IL-17

production. ESAT-6 plays a role in changing the

function of T cells to suppress protective immunity

and eliciting a potential immunopathological

response. During tuberculosis, IL-17 is a strong

inflammatory cytokine capable of inducing

chemokine expression that promotes cell recruitment

and granuloma formation during infection. A balance

between Th1 and Th17 responses is needed to control

bacterial growth and limit immunopathology (Wang

X et al, 2013). In this study, there was a shift in the

response towards the excessive production of IL-17

which could lead to the recruitment of large numbers

of neutrophils and tissue damage. Thus, regulation of

Th1 and Th17 responses during tuberculosis is

essential for enhancing anti-mycobacterial immunity

and preventing widespread immunopathology. In this

study, T cells inhibited and suppressed the

proliferation of IFN-γ production. It can be seen from

the mean increase in IFN-levels which were not too

high and statistically insignificant. IL-17 as an initial

response to M. tuberculosis plays a role in granuloma

formation and controlling bacterial growth.

Therefore, the role of IL-17-secreting cells during

active TB patient disease represents the highest

proportion of T lymphocytes to produce IL-17 to the

site of infection ( Jurado et al.,2012).

By using the ROC curve, it can be seen that IFN-

γ levels have very low values to predict or diagnose

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active TB and latent TB. Another study by Khan et al

(2013) based on the ROC analysis of IFN-γ levels can

be used for tuberculosis biomarkers

The level of IL-17 in this study has a very good

value for predicting or diagnosing active TB and

latent TB because the curve moves away from the

50% line and approaches 100%. This suggests that

IL-17 levels can be used as a predictor or diagnosis of

active TB and latent TB. This is in accordance with

Seyedhosseini et al., (2019) from the area under curve

(AUC) value, it is known that IL-17 is more specific

in differentiating TB infection (Seyedhosseiniet

al.,2019)

Based on the analysis of the ROC curve and the

results of the study, IL-17 levels can be used to

diagnose active TB and latent TB, but must be

investigated for TB because the cytokine IL-17 is not

only in tuberculosis but can occur in other

inflammatory reactions.

5 CONCLUSION

IFN-γ levels and IL-17 levels after stimulation of

ESAT-6-CFP-10Fusion Antigen (EC610) were

higher in active TB than in latent TB. But statistically,

there was no difference in the meaning of IFN-γ

levels in the two groups and IL-17 levels were

significantly different in the two groups.

This shows that IL-17 levels play a role in the

protective immune response against Mycobacterium

tuberculosis during the progression of TB disease.

The author's suggestion for further research can

see the effect of the duration of use of OAT treatment

with the secretion pattern of IFN-γ and IL-17

stimulated by EC610 and compare active TB and

latent TB with healthy groups.

ACKNOWLEDGEMENTS

Thanks to Dr.dr. Francisca SrioetamiTanoehardjo,

SpPK., M.Si at the National Health Research and

Development Center, Kemenkes RI, Jakarta for her

support and assistance in procuring the EC610 fusion

antigen

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