Effect of Macrophage Addition and Incubation Time to Interferon

Gamma (IFN-) Levels on Tuberculosis Granulomas In Vitro Models

Yusuf Eko Nugroho

1,2

, Agung Dwi Wahyu Widodo

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: granuloma, macrophage, IFN-.

Abstract: Tuberculosis (TB) is an infectious disease caused by stem bacteria Mycobacterium tuberculosis (Mtb)

which can cause latent infection. Mtb enters through aerosolization which then infects and activates

macrophages and dendritic cells present in the lungs. The active dendritic cells of coumadin present an

antigen which has been processed in peptide form into the lymphocyte cell which later becomes granuloma.

Granulomas are compounds of tissue composed of infected macrophages and multinucleated giant cells,

known by aggregates of new monocytes or macrophages, and neutrophils and lymphocytes. Macrophages

have a duty to kill Mtb germs. Different types of macrophage phenotype in granulomas with various

functions, including anti-mycobacterial effector action, produce cytokines ie IFN-γ. In humans, IFN-,

released by activated Th1 cells, is the major lymphokine to activate the search process and antimicrobial

activity that eliminates mycobacteria. This study aims to determine the relationship between IFN- and In-

vitro method using PBMC infected with Mtb germ with the addition of Macrophage with concentration

1,2,3x105 with incubation time 1,2,3,4 and 5 days to determine the level of IFN - using ELISA test. With

use with day variables of macrophages 1x105, 2x105, and 3x105 analized with One-Way ANOVA obtained

values p = P = 0.7201 The value is greater than 0.05 (p> 0.05). There is no meaningful difference.

1 INTRODUCTION

Tuberculosis (TB) is an infectious disease caused by

stem bacteria Mycobacterium tuberculosis (Mtb).

TB is a disease with the highest rates of morbidity

and mortality especially in developing countries and

is also a problem of chronic infection in the world

(Santoso et al., 2017). In addition to causing active

disease, Mtb can cause latent infection. Latent

infection results in one third of the world's

population carrying asymptomatic infections that

can produce 8 million new TB cases and 2 million

deaths annually (WHO, 2011; Birkness et al., 2007).

Mtb enters through aerosolization which then

infects and activates macrophages and dendritic cells

present in the lungs. The active dendritic cells of

kemuadin present treated antigens in peptide form to

CD4 T cells. The activated lymphocytes and

infected macrophages, in the inflammatory response

of cytokines and chemokines migrate to the infection

section and form an arrangement called granuloma,

where Mtb is in the dorman phase (Kapoor et al.,

2013).

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 have an important role

of granuloma. Macrophages serve as early cells of

granuloma formers. Macrophages have a duty to kill

Mtb germs. Different types of macrophage

phenotype in granulomas with various functions,

including anti-mycobacterial effector mechanisms,

produce proinflammatory and anti-inflammatory

cytokines, chemokine secretions and proteins

associated with tissue remodeling. These cells play a

major role in infection control in granulomas (Flynn

et al, 2011).

IFN-γ is a major cytokine involved in the

immune response to mycobacteria, and its primary

function is the activation of macrophages, enabling

them to use their microbicide role function (Khan et

392

Nugroho, Y., Widodo, A. and Nugraha, J.

Effect of Macrophage Addition and Incubation Time to Interferon Gamma (IFN-g) Levels on Tuberculosis Granulomas Invitro Models.

DOI: 10.5220/0007543603920397

In Proceedings of the 2nd International Conference Postgraduate School (ICPS 2018), pages 392-397

ISBN: 978-989-758-348-3

al., 2016). IFN-γ along with IL-12, IL-6, and TNF-α

stimulate the production of oxidative explosions,

thus mediating the function of tuberculostatic

macrophages, as well as stimulating immune cell

migration to the site of infection, contributing to

granuloma formation, which controls disease

progression (Khan et al., 2016).

Based on this background, researchers wanted to

know the role of adding macrophages to IFN-y

levels in granuloma models using ELISA.

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 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 10

in each well. Comparison of

PBMC and bacteria concentrations used in this study

was MOI 1: 0.1.

2.4 Macrophage Issolation

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% CO2. Observe macrophage cells under a

microscope and then make doses 1,2 and 3x10

2.5 Procedures

Divided into 4 groups. 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 1x105

M. tuberculosis strain H37Rv bacterial isolates into

all wells. Added macrophages as much as 1x10

2x10

and 3x10

cells into group II, III and IV.

Plate was incubated at 37°C with 5% CO2

condition. Observed on days 1,2, 3, 4 and 5. Every

day 100uL of supernatant was taken to test IL-10

levels using the ELISA test at 450nm wavelength.

3 RESULTS

3.1 Direct Granuloma Observation

The method used for direct observation is performed

directly under an inverted microscope using

specimens of living cell cultures in the plate / well

with the above lighting system followed by the lens

system on the microscope base. The direct

observational images present in this journal against

the control group (no macrophage), macrophage

addition of 1,2 and 3x10

and then incubate for 1

until 5 days in 37

Effect of Macrophage Addition and Incubation Time to Interferon Gamma (IFN-g) Levels on Tuberculosis Granulomas Invitro Models

393

Figure 1: PBMC infected M.Tb without macrophage

Figure 2: Direct observation of the group with the addition

of 2x10

macrophage (400x magnification

Figure 3: Direct observation of the group with the addition

of 2x10

macrophage (400x magnification)

Figure 4: Direct observation of the group with the addition

of 3x10

macrophage (400x magnification)

Day three (each dose) is the culmination of

granuloma formation. It is clearly visible from the

solid structure, with the many number of cells. Cell

aggregation appears larger than the first and second

days. Immune cell cells on the third day begin to

respond to Mtb for further elimination.

ICPS 2018 - 2nd International Conference Postgraduate School

394

3.2 Examination the levels of IFN-

Samples in the form of supernatant were then examined by

IFN-g using ELISA method and got the average result

from each treatment either control or sample.

Figure 5: IFN- secretion control without the addition of

macrophages by day variation (pg / mL)

Figure 6: IFN-y secretion with the addition of 1x10

macrophages based on day variation (pg / mL)

Figure 7: IFN-y secretion with the addition of 2x10

macrophages based on day variation (pg / mL)

Figure 8: IFN-y secretion with the addition of 3x10

macrophages based on day variation (pg / mL)

IFN-y examination results with ELISA method of 4

groups ie without the addition of macrophages with the

addition of macrophages 1x10

, 2x10

, 3x10

showed high

levels. The highest level occurred on day 2 at

concentrations of 3x105. On the 3rd day showed an

increase in levels which then tend to fall on days 4 and 5.

Effect of Macrophage Addition and Incubation Time to Interferon Gamma (IFN-g) Levels on Tuberculosis Granulomas Invitro Models

395

Figure 9: average IFN-y levels

The test results using One-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, p

= 0.451 was obtained. the value is greater than 0.05

(p> 0.05) thus indicating no significant difference.

3.3 Discussion

The formation of granulomas is a dynamic process

that begins immediately after infection and

continues to develop over time. Typically,

granulomas can be divided into three distinct phases:

(1) "congenital granuloma," a loose aggregate

consisting of macrophages and recruited neutrophils;

(2) "immune granuloma" is formed after the

emergence of antigen-specific T cells; and (3)

"chronic granulomas," resulting from different

morphological changes in granuloma structures

(Shaler et al., 2013).

After innate activation, APC cells are recruited to

the lungs and transport mycobacteria to mediastinal

lymph nodes. APC activates antigen-specific T cells.

Because of the nature of M.tb infection, the majority

of bacilli and antigen are in the endosome, and most

efficiently loaded into the major histocompatibility

complex (MHC) class II. Class II MHC loading

facilitates priming of the interferon gamma TH1

(IFN-γ) which is T cell discretion, which rapidly

returns the lung. While the dominant subset of T

cells is CD4 +, the cross presentation also allows

strong induction of CD8 + T cells, collectively

resulting in a polarized type 1 adaptive immune

response

Macrophages are important effector cells in

immunity against intracellular bacteria. In infection,

macrophages (MO) recognize mycobacteria with

Toll Like Receptor (TLR) involvement (mainly

TLR1 / 2 and TLR2 / 6) followed by phagocytosis

and mycobacterial growth control. In addition,

macrophages and dendritic cells also secrete

cytokines such as IL-12 and IL23 to induce IFN-

produksi production by T and NK cells, which, in

turn, increase phagocytosis, fagolososomes fusion,

oxidative bursts (Khan et al., 2016).

The addition of macrophages with different

doses does not affect the levels of IFN-y. this is

because IFN-y levels tend to be produced by T cells,

especially Th1 to stimulate macrophages more

actively in phagocytosis mtb. In this case the T cell

in generating IFN-y is independent.

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..

REFERENCES

Birkness A K, Jeannette G, Suraj B S, Ralph A T,

Kathryn L K, Jeanine B, Frederick D Q.

2007. An In Vitro Model Of The Leukocyte

Interactions Associated With Granuloma

Formation In Mycobacterium Tuberculosis

Infection. Immunology and Cell Biology vol.

85, 160–168.

Cowley Siobhán C. and Karen L. Elkins (2003).

CD4 T Cells Mediate IFN Independent

Control of Mycobacterium tuberculosis

Infection Both In Vitro and In Vivo .The

Journal of Immunology. 171:4689-4699.

Flynn JR, Chan J, Lin PL. 2011. Macrophages And

Control Of Granulomatous Inflammation In

Tuberculosis. Mucosal Immunol. Vol. : 4(3) ;

271-278.

Kapoor N, Santosh P, Tatiana D S, Chirajyoti D,

William L W, Pappachan E K. 2013. Human

Granuloma In Vitro Model, for TB Dormancy

And Resuscitation. PLOS ONE vol. 8 : issue

Parasa V R, Muhammad J R, Anh T N H, Mattias S,

Susanna B, Maria L. 2014. Modeling

Mycobacterium tuberculosis Early

Granuloma Formation In Experimental

Human Lung Tissue. Disease Models &

Mechanisms vol. 7 : 281-286.

Santoso G A, Hidayat S, Dwi YNH. 2017. Pengaruh

Infeksi Mycobacterium Tuberculosis Strain

ICPS 2018 - 2nd International Conference Postgraduate School

396

H37rv Terhadap Ekspresi Tnf-Α Pada

Jaringan Otak. MNJ , Vol.03, No.01.

WHO.(2011). GlobalTuberculosisCon- trol.

Geneva:WHO Press,World Health

Organization.

Effect of Macrophage Addition and Incubation Time to Interferon Gamma (IFN-g) Levels on Tuberculosis Granulomas Invitro Models

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