The Effect of Macrophage Addition to Interleukin 10 (IL-10) in

Tuberculosis Granuloma Model In Vitro

Dini Puspodewi

1,2,3

, Agung Dwi Wahyu W

and Jusak Nugraha

Post Graduate Student of Master of Immunology, Faculty of Pasca Sarjana, Universitas Airlangga,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: Mycobacterium tuberculosis, Granuloma, IL-10

Abstract: Tuberculosis (TB) caused by the bacterium Mycobacterium tuberculosis has caused more deaths than other

infectious diseases over the past 200 years. Granulomas are characteristic of mycobacterial infection.

Doubling of Mycobacterium tuberculosis bacilli in any place will cause specific inflammation to form

characteristic granulomas. Granulomas contains of different types of cells, but the main cellular component

of the structure is macrophages. Macrophages are cells for the formation of granulomas and major cell types

in most granulomas. One function of macrophages in granulomas is the production of anti-inflammatory

cytokines IL-10. IL-10 can reduce inflammation and maintain homeostasis. This study aims to determine

the effect of the addition of macrophages to levels of interleukin-10 (IL-10) in granuloma model in vitro.

This type of research is true experimental with the object of research in the form of PBMC which comes

from 1 healthy volunteer. Analysis using twoway ANNOVA with P <0.05, and tukey comparing for

comparison. The results showed that without macrophage group with 1x10

, 2x10

, and 3x10

macrophage

groups, the value of p = 0.3197 was greater than 0.05 (p> 0.05), so there was no significant difference.

While the variation day in the treatment group obtained p = 0.2407 which showed no effect of macrophage

addition to interleukin-10 (IL-10) level on granuloma model in vitro, with IL-10 concentration on the 1

day

until the 5

day are low. There is no effect of adding macrophages to IL-10 levels in tuberculosis granuloma

models in vitro.

1 INTRODUCTION

Tuberculosis (TB) has caused more deaths compared

to other infectious diseases over the past 200 years

(Heemskerk et al., 2015). This disease is caused by

Mycobacterium tuberculosis mostly attacks people

living in low-income and middle-income countries

where it reaches 9.4 million people a year

worldwide. Most individuals who are exposed do

not develop the disease because it can produce an

immune response to resist or eliminate bacteria (Fox

and Menzies, 2013).

Tuberculosis granuloma is a hallmark of

mycobacterial infection (Heemskerk et al., 2015).

Characteristic granuloma formation originates from

the presence of specific inflammation caused by

multiplication of Mycobacterium tuberculosis bacilli

in any place (Elorriaga et al., 2015). Granulomas

have a dynamic process in which the structure of the

size will increase as more cells move in (Orme and

Basaraba, 2014).

Mycobacterium tuberculosis infection causes

granuloma formation where bacteria enter in an

inactive state and can live for decades before

reactivation to develop active disease when the

host's immune system is weakened (Kapoor et al.,

2013). Bacteria will become latent when trapped in

granulomas and are under hypoxic conditions, lack

of nutrition, and pressure from adaptive immunity

(Orme and Basaraba, 2014). A person infected with

latent TB can store a small amount of inactive

Mycobacterium tuberculosis bacilli contained in

microgranuloma. This organism can continue to live

but is not active (Kapoor et al., 2013).

IL-10 is called an anti-inflammatory cytokine, an

inhibiting cytokine for a balance between

inflammatory and immunopathological responses

(Cyktor et al., 2013). IL-10 plays an important role

386

Puspodewi, D., W, A. and Nugraha, J.

The Effect of Macrophage Addition to Interleukin 10 (IL-10) in Tuberculosis Granuloma Model In Vitro.

DOI: 10.5220/0007543503860391

In Proceedings of the 2nd International Conference Postgraduate School (ICPS 2018), pages 386-391

ISBN: 978-989-758-348-3

in Mtb infection, where cytokines have been shown

to reduce immunity (Cavalcanti et al., 2012; Cyktor

et al., 2013; Adrian et al., 2015).Immunosuppressive

cytokines IL-10 have been associated with

susceptibility to TB in both humans and animals

(Cyktor et al., 2013). IL-10 plays an early inhibitory

role in preventing the development of protective

immunity associated with the containment of Mtb

infection (Cyktor et al., 2013).

This study aims to detect whether there is an

effect of the addition of macrophages on IL-10

levels in tuberculosis granuloma models in vitro.

2 MATERIALS AND METHODES

2.1 RPMI

The growth media used in this study was the

Roswell Park Memorial Institute (RPMI) 1640

which was obtained already in the form of ready-to-

use solution. RPMI 1640 media is a medium used

for cell and tissue culture, usually used for the

growth of human lymphoid cells. This medium

contains a large amount of phosphate and is

formulated for use in air with a 5% CO

atmosphere.

RPMI 1640 uses the bicarbonate buffer system so

that it enables the growth of several types of cells,

especially T lymphocytes, hybridomas. There are

several series of RPMI most often used is RPMI

1640.

2.2 PBMCs

Peripheral Blood Mononuclear Cells = PBMCs are

cells made from human blood which are then

processed for the PBMC cell capture. Sample

criteria are adult blood, the blood used should be

new blood taken, it can not be blood that has been

stored for too long, blood comes from healthy

people and does not suffer from tuberculosis

infection, there is no specific provision for sex either

male or female. Suggestions from the blood

researcher used should come from one person only,

because the immune response of each individual is

different so if coming from more than one person in

worry affects the outcome. The number of PBMC

used in this study was 10

in each well.

2.3 Mycobacterium tuberculosis

This study used bacterial isolates Mycobacterium

tuberculosis H37Rv obtained from the Laboratory of

Microbiology, Institute of Tropical Disease,

Airlangga University Surabaya with concentration

in each well.

2.4 Macrophage

Taken buffy-coat about 60 ml. Then prepare 50 ml

conical tubes with histopags each 15 ml. Prepared

one tube every 10 ml of buffy-coat. Histopags are

used at room temperature. Plate at 10 cm culture

dishes (10 ml / dish) incubated at 1-2 hours at 37ºC

5% CO

. Observe macrophage cells under a

microscope and then make doses 1,2 and 3x10

2.5 Procedure

There are four groups on this procedure. Group I

was given PBMC and Mycobacterium tuberculosis

bacteria on RPMI media as control. Group II was

given PBMC, Mycobacterium tuberculosis and

1x10

macrophages on RPMI media. Group III was

given PBMC, Mycobacterium tuberculosis and

2x10

macrophages on RPMI media. Group IV was

given PBMC, Mycobacterium tuberculosis and

3x10

macrophages on RPMI media. Prepared well

plates that already contain RPMI media. Enter

1x10

PBMC cells into all wells. Inoculated with

1x10

M. tuberculosis strain H37Rv bacterial

isolates into all wells. Added macrophages as much

as 1x10

, 2x10

and 3x10

cells into each well of

group II, III, and IV. Plate was incubated at 37°C

with 5% CO

condition. Observed on days 1,2, 3, 4

and 5. Every day 100 uL of supernatant was taken

to test IL-10 levels using the ELISA test at 450 nm

wavelength.

3 RESULTS

3.1

Direct Granuloma Observation

Results from observations under an inverted

microscope with 400x magnification of PBMC

samples from 1 healthy volunteer were added with

1x10

, 2x10

, 3x10

and Mtb macrophages showing

the difference from day one to fifth day. On the first

day there has not been a good aggregate on culture

without macrophages with a culture plus

macrophages (Figure 1). The second day has already

begun to form aggregate on both treatments i.e.,

without the addition of macrophages and the added

macrophages (Figure 2). On the third day (Figure 3)

and the fourth day (Figure 4) is still aggregate, while

on the fifth day the aggregate has begun to decrease

(Figure 5).

The Effect of Macrophage Addition to Interleukin 10 (IL-10) in Tuberculosis Granuloma Model In Vitro

387

Figure 1. First Day Culture. A. Without Macrophages, B.

Addition of macrophages 1x10

C. Addition of macrophages 2x10

, D. Addition of

macrophages 3x10

Figure 2. Culture Second day. A. Without Macrophages,

B. Addition of macrophages 1x10

C. Addition of macrophages 2x10

, D. Addition of

macrophages 3x10

Figure 3. Third Day Culture. A. Without Macrophages, B.

Addition of macrophages 1x10

C. Addition of macrophages 2x10

, D. Addition of

macrophages 3x10

Figure 4. Fourth Day Culture. A. Without Macrophages,

B. Addition of macrophages 1x10

C. Addition of macrophages 2x10

, D. Addition of

macrophages 3x10

ICPS 2018 - 2nd International Conference Postgraduate School

388

Figure 5. 5th Day Culture. A. Without Macrophages, B.

Addition of macrophages 1x10

C. Addition of macrophages 2x10

, D. Addition of

macrophages 3x10

3.2 Examination the levels of IL-10

Result of examination of level of IL-10 with elisa

method from 4 group that is without the addition of

macrophages, with the addition of 1x10

, 2x10

3x10

macrophages were analyzed by day variation.

From the research, it was found that the levels of

IL-10 without addition the highest value of

macrophages occurred on the 2nd day with an

average of 3,733 and the lowest average value

occurred on the 3rd day of 1,595 shown on figure 6.

Figure 6. Levels of IL-10 without the addition of

macrophages

The level of IL-10 with the addition of 1x10

macrophage the lowest value occurred on the 1st day

with an average of 1.435 and the highest average

value occurred on the 3rd day ie 3,506 shown in

Figure 7.

Figure 7. Levels of IL-10 with the addition of 1x10

macrophages

The level of IL-10 with the addition of 2x10

macrophage the highest value occurred on the 2nd

day with an average of 3.135 and the lowest average

value occurred on the 5th day that is 0.804 shown in

figure 8.

Figure 8. Levels of IL-10 with the addition of 2x10

macrophages

It was found that the level of IL-10 with the

addition of 3x105 macrophage of the lowest value

occurred on the 5th day with an average of 1,191

and the highest average value occurred on the

second day that is 3,521 shown in Figure 9.

Figure 9. Levels of IL-10 with the addition of 3x10

macrophages

The results of IL-10 examination with elisa

method of 4 groups, i.e., without the addition of

macrophages with the addition of macrophages

1x10

, 2x10

, 3x10

showed low levels of IL-10.

All groups experienced increased concentrations

on the second day. On the third day, the fourth and

fifth groups of 1x105 macrophages were different

from the other groups, i.e., the third and fifth day

The Effect of Macrophage Addition to Interleukin 10 (IL-10) in Tuberculosis Granuloma Model In Vitro

389

increases and the decrease on the fourth day, while

the group without macrophages, the 2x105 and

3x105 macrophages decreased the concentration on

the third and fifth days and increased the fourth day

(Figure 10).

Figure 10. IL-10 levels on day variations

The data obtained are then analyzed by two-way

ANOVA which aims to determine the significance

of the price of the proportion (p). In groups without

macrophages with a group of 1x105, 2x105, and

3x105 macrophages, the value p = 0.3197 was

greater than 0.05 (p> 0.05). Likewise with day

variations of groups without macrophages with

groups of macrophages added 1x105, 2x105, and

3x105 obtained value p = 0.2407 value is greater

than 0.05 (p> 0.05) so it shows no difference.

3.3 Discussion

Tuberculosis granuloma is a hallmark of

mycobacterial infection (Heemskerk et al., 2015).

Characteristic granuloma formation originates from

the presence of inflammation Specifically caused by

doubling the Mycobacterium tuberculosis bacilli in

any place (Elorriaga et al., 2015). Granuloma is a

pathological sign host response to Mycobacterium

tuberculosis infection. Development Granuloma is a

defense tool designed as a wall and contains

pathogens (Orme and Basaraba, 2014). Granuloma

formation facilitates host in accommodating

Mycobacterium tuberculosis bacilli and prevent

spread of bacteria, but can also be used by bacteria

for breed (Heemskerk et al., 2015). Granulomas

have a dynamic process because the more cells

move in, the size structure will increase (Orme and

Basaraba, 2014).

M. tuberculosis IL-10 is an immunoregulatory

cytokine with activity immunosuppressive is potent

against APC and Th1 cells. IL-10 production level

by Macrophages and dendritic cells are infected,

although promoted by Th1 ability, IL-10 is secreted

more by dendritic cells than macrophages. IFN-γ

decreases IL-10 production from macrophages

infected with M. tuberculosis. In this

microenvironment, IFN-γ secreted by activated T

cells can synergize with M. tuberculosis to reduce

IL-10 production (Hickman et al, 2002).

IL-10 is not always produced by T cells that

make other pro-inflammatory cytokines, although

checking granulomas in a small subset, there is a

small population of T cells (1.2%) that make IL-10

and IL-17. T cells with this phenotype have been

associated with control of several bacterial

infections rather than with autoimmune diseases

(Gideon et al., 2015).

4 CONCLUSIONS

There w no effect of adding macrophages to IL-10

levels in tuberculosis granuloma models in vitro.

ACKNOWLEDGEMENTS

The authors would like to thank the technicians of

the Stem cell Research Centre and Tuberculosis and

Leprosi Laboratory of Tropical Diseases (ITD) of

Airlangga University and all those who have assisted

in the completion of this research.

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