The Effect of Macrophage Dose to Secretion Interleukin 6 (IL-6) on

Model of Tuberculosis Granuloma In Vitro

Imam Agus Faizal

1ab

, Agung Dwi Wahyu Widodo

Jusak Nugraha

1 a

Post Graduate Master of Immunology, Sekolah Pascasarjana, Universitas Airlangga, Surabaya, Indonesia

Department of Stem Cell Institute of Tropical Disease, Universitas Airlangga, Indonesia

Department of Microbiology Clinic, Faculty of Medicine, Dr. Soetomo Hospital, Indonesia

Department of Patology Clinic, Faculty of Medicine, Dr. Soetomo Hospital, Indonesia

Keywords: Granuloma, Macrophage, Interleukin 6.

Abstract: Granuloma is a pathological sign of host response as a system of defense against infection of

Mycobacterium tuberculosis (Mtb) causing Tuberculosis (TB) disease in humans estimated to be 8.7 million

new cases, 1.4 million deaths, and about 2 billion latent infections. Macrophages are responsible for

activating protective immune responses in innate and adaptive immune to control or eliminate infection. The

inner body protects against Mtb infection producing various secretions of cytokines interleukin 6 (IL-6) that

play the role of activating multinucleated giant cells, differentiation of macrophage T cells and thus

stimulating CD4

and CD8

T-cells to strengthen macrophage antimicrobial capacity as early response

reactions (early phase). The aim of this research is to see the effect of dose of macrophage on secretion and

IL-6 expression on a TB granuloma model in vitro. Human blood was made by Peripheral Blood

Mononuclear Cell (PBMC) and treated with the addition of a dose of macrophages of 1x10

cells/well,

2x10

cells/well and 3x10

cells/well and control (without macrophages). Then the bacterium

Mycobacterium tuberculosis was added and then observing the secretion The enzyme-linked

immunosorbent assay (ELISA) method of IL-6 cytokine during the 1st, 2nd, 3rd, 4th, and 5th days. The

results of IL-6 examination on ELISA obtained p value of 0.7520 (p> 0.05). The conclusion of the study

was that there was no effect of adding macrophage dose to cytokine secretion of IL-6 levels on granuloma

TB model in vitro.

1 INTRODUCTION

Bacteria Mycobacterium tuberculosis (Mtb) is the

cause of Tuberculosis (TB) disease in humans

causing death and one of the infectious agents in

humans worldwide. There are an estimated 8.7

million new cases, 1.4 million deaths and about 2

billion latent infections caused by Mtb (Fitzgerald et

al., 2014). Based on data from the World Health

Organization (WHO) in 2014, the cases of TB in

Indonesia reach 1 million and the number of deaths

due to TB is estimated at 110,000 each year

(Kesehatan and Indonesia, 2017).

Granulomas are a pathological sign of the host

response to Mycobacterium tuberculosis infection

that has innate immunity, inflammation, but has

developed into a complex and dynamic structure

capable of being mediated by T cell response (Orme

and Basaraba 2014). Granulomas are tissue

compounds consisting of infected macrophages and

multinucleated giant cells, surrounded by

aggregations of new monocytes or macrophages, and

neutrophils and lymphocytes (Parasa, 2014).

Macrophages are responsible for the activation of

protective immune responses, innate and adaptive

immune, thus playing an important role in ongoing

cross-cell communication that is needed to control or

eliminate host cell infection in the early phase

(Murugesan V.S., Rajaram et al., 2015).

Pathophysiology begins with internalization of Mtb

by host cell macrophages so that activated

macrophages secrete various cytokine markers such

as IL-8, IFN-γ, and TNF-α (Kapoor et al., 2013).

Mycobacterial infected macrophages also release

large amounts of IL-6 that play the role of active

macrophage differentiation into multinucleated giant

cells (Fitzgerald et al., 2014). IL-6 secreted by Mtb

infected macrophages suppresses an uninfected

306

Agus Faizal, I., Dwi Wahyu Widodo, A. and Nugraha, J.

The Effect of Macrophage Dose to Secretion Interleukin 6 (IL-6) on Model of Tuberculosis Granuloma In Vitro.

DOI: 10.5220/0007541803060311

In Proceedings of the 2nd International Conference Postgraduate School (ICPS 2018), pages 306-311

ISBN: 978-989-758-348-3

macrophage response to IFN-γ. Increased levels of

IL-6 correlate with elevated levels of IL-1β and IL-

11 so that IL-6 can play many roles (pleotropic

cytokines) contributing positively or negatively to

host control cells against Mtb infection (Romero-

Adrian., 2015).

Based on the above phenomenon, this research

was conducted to analyze the role of the addition of

dose of macrophage to the formation of granuloma

TB and the role of cytokine levels of IL-6 in the

formation of granuloma TB in vitro so that a

granuloma model can be used in latent and active

stage with kedapannya done to develop effective

diagnosis and develop a vaccine.

2 MATERIALS AND METHODS

Research data is quantitative. The independent

variables were the addition of the dose of

macrophages as 1x10

(dose 1), 2x10

(dose 2), and

3x10

(dose 3) cells/well and duration of incubation

days 1, 2, 3, 4 and 5 on granuloma model in vitro.

While the dependent variable is the amount of IL-6

secretion level.

2.1 PBMC

PBMC is made from adult blood, a healthy criterion.

After the blood was taken it was immediately

isolated. Blood sentrifuge blood plasma was taken

1.5 ml conical tube and added 3 ml PBS and 5 ml

histopague and then centrifuge 30 minutes/ 1700

rpm taken buffycoat centrifuge plus PBS centrifuge

5 minutes/1300 rpm then transferred to tube 1.5 ml

save temperature -30°C.

2.2 Macrophages

Buffycoat is taken 60 ml. 50 ml conical tubes were

prepared with a histopague of 15 ml each. Prepared

one tube every 10 ml of buffycoat. Histopagues are

used at room temperature. Then sterile scissors and

tubes for buffycoat with ethanol 70%, cut the ends

of the tube and buffycoat poured into tissue culture

flask 75 cm

. Buffycoat was diluted with 2% FBS

and PBS volume and then stirred slowly then

centrifuge was taken ring cell on the surface

between histopag and plasma cell as much as 50 ml.

Buffycoat is taken 60 ml. 50 ml conical tubes

were prepared with a histopague of 15 ml each.

Prepared one tube every 10 ml of buffycoat.

Histopague are used at room temperature. Then

sterile scissors and tubes for buffycoat with ethanol

70%, cut the ends of the tube and buffycoat poured

into tissue culture flask 75 cm

. Buffycoat was

diluted with 2% FBS and PBS volume and then

stirred slowly then centrifuge was taken ring cell on

the surface between histopague and plasma cell as

much as 50 ml. Discarded supernatant and

centrifuge. Plate at 10 cm culture dishes (10 ml /

dish) incubated for 1-2 hours at 37ºC 5% CO

Observe macrophage cells under a microscope then

discard the supernatant and then add PBS and

homogenize. Let the cell differentiate for 7 days

observed macrophage cells daily.

2.3 Model Granuloma In Vitro

PBMC added media Roswell Park Memorial

Institute (RPMI). Plate 96 well (control, treatment

day 1, 2, 3, 4, 5 per/well). Bacterial strain

Mycobacterium tuberculosis strain H37Rv (obtained

from ITD department Tbc) inoculated as much as

1x10

CFU into all wells. Then macrophages were

added at doses 1, 2, 3. Plate was incubated at 37ºC

5% to 5 days per day granuloma harvested and

50 μl for ELISA examination.

2.4 ELISA

Examination of ELISA content using reagent kit

Elabscience Biotechnology Inc. All Rights

Reserved, 2017 (Interleukin 6). Preparation of

reagent consists of wash buffer, standard working

solution, Biotinylated Detection Ab working

solution, Concentrated HRP Conjugate working

solution. Advanced examination can be seen in the

guide book and determine the optical density (OD

value) of each well with 450 nm.

3 RESULTS

Granuloma was taken by BTA dye to confirm Mtb.

Then haematoxylin-eosin (HE) staining looked at

the morphology of the granuloma structure. Before

PBMC made media observed macrophage cells

under a microscope.

The Effect of Macrophage Dose to Secretion Interleukin 6 (IL-6) on Model of Tuberculosis Granuloma In Vitro

307

Figure 1: A: bacteria Mtb (red arrows) B: granuloma

morphology of clusters (black arrows) C:macrophage cells

(yellow arrows).

3.1 Direct Granuloma Observation

Granulomas are observed directly in an inverted

microscope per day. Seen granuloma morphology,

aggregation and cells form granuloma.

Figure 2: PBMC infected M.Tb without macrophage

(control). Figure 1, 2, 3, 4 and 5 have not formed

granuloma aggregation.

Figure 3: Direct observation of the group with the

addition of 1x10

macrophage (400x magnification). The

brown arrow indicates 2, 3 have formed granuloma

aggregations and 4 granulomas are more clustered.

Figure 4: Direct observation of the group with the addition

of 2x10

macrophage (400x magnification). The brown

arrow indicates 2, 4 have formed granuloma aggregation

and 3 perfect granulomas are more clustered and dense.

ICPS 2018 - 2nd International Conference Postgraduate School

308

Figure 5: Direct observation of the group with the addition

of 3x10

macrophage (400x magnification).

The brown arrow 2, 3, 4 indicates the formation of

granuloma aggregation and formation. It is clearly visible

from the solid structure, with the high number of cells.

The granuloma on the second and third days,

while on the first day the granuloma was still in the

aggregation formation stage consisting of

macrophages containing lipids (foamy

macrophages), epitheloid cells and Langhans cells.

However, the fifth day of granuloma aggregation

observation began to rupture.

3.2 The Examination the levels of IL-6

Supernatant samples were then examined for levels

of IL-6 secretion using ELISA method and obtained

the average result from each treatment, either control

or sample.

Figure 6: Examination the levels of IL-6

Results of IL-6 examination by ELISA method

from 4 groups were control with the addition of dose

of macrophage 1x10

, 2x10

, 3x10

showed high

level. The highest levels of IL-6 secretion occurred

on day 5 with a dose of 3x10

. While the lowest

levels of IL-6 secretion occurred on day 2 with a

dose of 2x10

The Effect of Macrophage Dose to Secretion Interleukin 6 (IL-6) on Model of Tuberculosis Granuloma In Vitro

309

Figure 7: Test results using One-Way ANOVA

The test results using One-Way ANOVA which

aims to determine the significance of the price of the

proportion (p). In the control group with the addition

of dose of macrophage 1x10

, 2x10

, and 3x10

, p

value = 0.4624 was higher than 0.05 (p> 0,05) so it

showed no significant difference.

4 DISCUSSION

Granulomas create a balance between both

preventing the spread of infection in the host and the

protection of Mycobacterium tuberculosis from

immune reactions (Majeed, Mir, and Sharma, 2015).

The advantage of this study is to know the effect of

dose of macrophage on the level of IL-6 secretion so

as to explain the early phase macrophage mechanism

on host cells infected by Mtb. It was also observed

that granulomas from the early stages of formation,

clustered from granuloma aggregation to rupture,

were seen based on the amount of secretion of IL-6

cytokine. The addition of the expected macrophage

dose suggests adding to the availability of

macrophages as a therapeutic strategy for

tuberculosis (Randolph 2015). The ability of Mtb

through the early secreted antigenic target of 6 kDa

(ESAT-6) is capable of replicating active Mtb to

modulate the number of cells secreted through the

mycobacterial type VII secretion system (ESX-1)

(V. R. Parasa et al., 2014).

The ability of Mtb to secrete ESAT-6 plays an

important role in inducing aggregate aggregation of

monocyte and macrophage cells from early stage

granuloma formation. This is in line with

observations made in the in vivo zebrafish embryo

that transparency permits a picture of the life of

neutrophil cells against granuloma formation (Je et

al., 2016). IL-6 is involved in the differentiation of

macrophage and cytotoxic T cells. IL-12 induces

IFN-γ to differentiate CD4 + T cells on Th1

effectors. Cytokines can also direct neutrophils,

monocytes, lymphocytes to CD4 + and CD8 + T-

cell-inducing infections to strengthen macrophage

antimicrobial capacity. ESAT-6 and culture filtrate

protein 10 (CFP-10) in Mycobacterium tuberculosis

are among the candidates of the Tb vaccine because

they induce immune-strong T cells in animal models

so that some previous studies raised extraordinary

problems related to ESAT-6/ CFP-10 candidates as

an effective vaccine against Tb (Abebe et al., 2017).

Using a model for studying human-like human

papillomavirus granulomas is important because: (1)

it is difficult to directly study human lung biopsy

specimens due to access limitations; (2) biopsy

specimens only show static images and should

foresee various possibilities for understanding

dynamic process in granuloma and (3) M.

tuberculosis has no natural host other than humans

so it takes an in vitro granuloma model to study the

initial steps of granuloma formation and treatment,

and has the potential to address more of the

translational aspects of human M. tuberculosis

infection. These in vitro models are less similar in

structures such as the lung and micro-tissue

environment (Guirado and Schlesinger, 2013). This

granuloma model has the potential to provide insight

into host-pathogen interactions at different stages of

human granuloma Tb formation (Kapoor et al., 2013

granuloma Tb manusia (Kapoor et al., 2013).

ACKNOWLEDGEMENTS

The authors would like to thank the technicians of

the Stem Cell Research Centre and Tuberculosis and

Leprosy Laboratory of Tropical Diseases (ITD) of

Airlangga University and all those who have assisted

in the completion of this research.

REFERENCES

Abebe, F., M. Belay, M. Legesse, A. Mihret, and K. S.

Franken. 2017. “Association of ESAT-6/CFP-10-

Induced IFN-Γ, TNF-α and IL-10 with Clinical

Tuberculosis: Evidence from Cohorts of Pulmonary

Tuberculosis Patients, Household Contacts and

Community Controls in an Endemic Setting.” Clinical

and Experimental Immunology 189 (2): 241–49.

doi:10.1111/cei.12972.

Fitzgerald, Liam E, Naiara Abendaño, Ramon A Juste, and

Marta Alonso-hearn. 2014. “Three-Dimensional In

Vitro Models of Granuloma to and Resuscitation of

Dormant Mycobacteria” 2014.

Guirado, Evelyn, and Larry S. Schlesinger. 2013.

“Modeling the Mycobacterium Tuberculosis

ICPS 2018 - 2nd International Conference Postgraduate School

310

Granuloma - the Critical Battlefield in Host Immunity

and Disease.” Frontiers in Immunology 4 (APR): 1–7.

doi:10.3389/fimmu.2013.00098.

Je, Sungmo, Hailian Quan, Yirang Na, Sang-nae Cho,

Bum-joon Kim, and Seung Hyeok Seok. 2016. “An in

Vitro Model of Granuloma-like Cell Aggregates

Substantiates Early Host Immune Responses against

Mycobacterium Massiliense Infection,” 1118–27.

doi:10.1242/bio.019315.

Kapoor, Nidhi, Santosh Pawar, Tatiana D Sirakova,

Chirajyoti Deb, William L Warren, and Pappachan E

Kolattukudy. 2013. “Human Granuloma In Vitro

Model , for TB Dormancy and Resuscitation” 8 (1).

doi:10.1371/journal.pone.0053657.

Kesehatan, Kementerian, and Republik Indonesia. 2017.

“Penanggulangan Tb Kini Lebih Baik,” no. 1990: 7–8.

Majeed, Shahnawaz, Shabir Ahmad Mir, and Sadhna

Sharma. 2015. “Dual Role of Inflammation in

Prognosis and Prevention of Tuberculosis” 6 (1): 1–9.

doi:10.4172/2155-9899.1000298.

Murugesan V.S. Rajaram, Bin Ni, Claire E. Dodd, and

Larry S. Schlesinger. 2015. “Macrophage

Immunoregulatory Pathways In Tuberculosis” 26 (6):

471–85.

doi:10.1016/j.smim.2014.09.010.Macrophage.

Orme, Ian M, and Randall J Basaraba. 2014. “Seminars in

Immunology The Formation of the Granuloma in

Tuberculosis Infection.” Seminars in Immunology 26

(6). Elsevier Ltd: 601–9.

doi:10.1016/j.smim.2014.09.009.

Parasa, V. R., M. J. Rahman, A. T. Ngyuen Hoang, M.

Svensson, S. Brighenti, and M. Lerm. 2014.

“Modeling Mycobacterium Tuberculosis Early

Granuloma Formation in Experimental Human Lung

Tissue.” Disease Models & Mechanisms 7 (2): 281–

88. doi:10.1242/dmm.013854.

Parasa, Venkata Ramanarao, Muhammad Jubayer

Rahman, Anh Thu, Ngyuen Hoang, Susanna

Brighenti, Maria Lerm, Venkata Ramanarao Parasa, et

al. 2014. “Modeling Mycobacterium Tuberculosis

Early Granuloma Formation in Experimental Human

Lung Tissue Modeling Mycobacterium Tuberculosis

Early Granuloma Formation in Experimental Human

Lung Tissue,” no. 7: 281–88.

doi:10.1242/dmm.013854.

Randolph, Gwendalyn J. 2015. “Previews Macrophage

Supply and Demand at the Core of the Necrotic

Granuloma.” Cell Host and Microbe 18 (1). Elsevier

Inc.: 3–4. doi:10.1016/j.chom.2015.06.014.

Romero-Adrian, Tania Beatriz. 2015. “Role of Cytokines

and Other Factors Involved in the Mycobacterium

Tuberculosis Infection.” World Journal of

Immunology 5 (1): 16. doi:10.5411/wji.v5.i1.16.

The Effect of Macrophage Dose to Secretion Interleukin 6 (IL-6) on Model of Tuberculosis Granuloma In Vitro

311