The Effect Collagen to Granuloma Structure and Immune Response

on Granuloma Tuberculosis In Vitro Models

Ira Pangesti

ab1

, 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: Structure Granuloma, TNF-α, Mycobacterium tuberculosis.

Abstract: Mycobacterium tuberculosis is a bacterium causes pulmonary tuberculosis, acid-resistant bacteria,

intracellular life and cause of death than other infectious diseases. The principle of immune response by the

formation of granulomas that prevent bacteria spread to other cells. TNF-α plays an important role in the

body's defense against intracellular bacterial infections and the invitro model maintaining granulomas and

the role of collagen in maintaining granulomas that prevent infection from becoming active. The purpose of

this study is to analyze the number of lymphocyte, monocytes/macrophages and levels of TNF-α between

collagen and without collagen. This study used healthy PBMC cells in mycobacterium tuberculosis bacterial

infection with MOI 1: 0.1 then divided into 2 groups the addition collagen and without collagen observation

in day 1, day 3, day 5, and day 7. HE stained was performed to calculate lymphocyte, monocyte/

macrophage and supernatant cells used to check TNF-α levels by ELISA method. The results showed no

effect of the addition of collagen to the number of lymphocytes, monocytes/macrophages and TNF-α levels.

Levels of TNF-α highest on day 3 and decresed on day 7.

1 INTRODUCTION

Tuberculosis (TB) is an infectious disease caused by

the bacterium Mycobacterium tuberculosis has

caused more deaths during the last 200 years

compared to other infectious diseases (Paulson,

2013). World Health Organization (WHO) data by

2015, an estimated 10.4 million new TB cases

worldwide. Six countries accounted for 60% of new

TB cases, including India, Indonesia, China, Nigeria,

Pakistan and South Africa (WHO, 2016).

Tuberculosis disease in addition to causing active

infection can also cause latent infections that are

asymptomatic conditions making it difficult for

treatment because it does not cause symptoms. In the

host body infected with Mycobacterium tuberculosis

mostly develops into a latent infection in comparison

with active infection at the host's primary defense

against Mycobacterium tuberculosis infection with

granuloma formation, ie the formation of organized

cell aggregates. (Fitzgerald et al., 2014).

Mycobacteria belong to the host by means of

aerosol will then be in the alveolar macrophages by

internalization by way of Mycobacterium

tuberculosis bacteria secrete virulence factors

namely ESAT-6 (Early Secreted Antigenic Target of

6 kDa), which in the secretion of through the system

the secretion of Mycobacterial type VII (ESX-1).

ESX-1 mediated translocation m.tuberculosis from

the macrophage cytoplasm into a phagolysosome is

the virulence of pathogenic Mycobacteria (Parasa et

al., 2014).

Mycobacterium tuberculosis bacteria are

internalized by macrophages releasing Interleukin 8

(IL-8) which is a strong chemoatractant for T

lymphocytes because it produces a bacterial antigen

so as to recruit T cells. T lymphocytes then become

active and produce gamma interferon (IFN-ɣ) which

serves to activate other macrophages , increasing the

production of Tumor Necrosis Factor Alpha (TNF-

α) and activating the metabolism of oxidative

macrophages and antimicrobial activity. TNF-α

plays a very important role in the development of

granulomas for cellular organization and

maintenance of granulomas, TNF-α mediates acute

phase responses that give rise to systemic symptoms,

356

Pengesti, I., Widodo, A. and Nugraha, J.

The Effect Collagen to Granuloma Structure annd Immune Response on Granuloma Tuberculosis Invitro Models.

DOI: 10.5220/0007542803560360

In Proceedings of the 2nd International Conference Postgraduate School (ICPS 2018), pages 356-360

ISBN: 978-989-758-348-3

and is necessary for the development and the

strength of granulomas that play an important role in

the accumulation and differentiation of macrophages

into epithelioid cells on mature granulomas

(Birkness et al., 2007 and Fitzgerald et al., 2014).

Granulomas are not always awake, bacteria

Mycobacterium tuberculosis can be reactivated.

During reactivation, granulomas become catalase

and then necrotic, and infected macrophages secrete

MMP-1, excessive secretion leads to collagen

degradation and tissue damage, causing M.

tuberculosis to enter the respiratory tract and causing

active infection. (Salgame, 2011)

The damage to collagen is an early onset of

immunopathology in tuberculosis, causing necrosis

and building an immune response, revealing the role

of extracellular matrix in regulating host and

pathogen interactions (Al Shammari et al., 2015)

Type I collagen is a fibril structure in the lung

and is highly resistant to enzymatic degradation. In

addition to biomechanical properties, type I collagen

has an important role in cell survival, adhesion,

proliferation, and migration (Brilha et al., 2017).

This study reports on the effect of collagen

administration on the structure of granuloma and the

secretion of Tumor Necrosis Factor (TNF) -α in

granuloma tuberculosis in vitro models.

2 MATERIALS AND METHODES

2.1 RPMI

Roswell Park Memorial Institute (RPMI) 1640.

RPMI 1640 media is a medium used for cell and

tissue culture, usually used for the growth of human

lymphoid cells. RPMI 1640 uses the bicarbonate

buffer system so that it enables the growth of several

types of cells, especially T lymphocytes,

hybridomas.

2.2 PBMCs

Peripheral Blood Mononuclear Cells = PBMCs are

cells made from human blood which are then

processed for the PBMC cell capture. The

concentrations of PBMC used in this study was 10

in each well.

2.3 Mycobacterium Tuberculosis

This study used bacterial isolates Mycobacterium

tuberculosis strain H37Rv with concentration 10

each well.

2.4 Extracellular Matrix

The extracellular matrix used collagen. The collagen

used has a number Cat # 04902 as a solution. In this

study, 950 μL of collagen was added with 50 μL

PBS 10 × and add 10uL NaOH 1N, then in PH

check, with neutral PH result.

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.

Figure 1: Direct observation with infection (400x

magnification)

The figure 2 seen there is aggregate it indicates a

response to infection.

The Effect Collagen to Granuloma Structure annd Immune Response on Granuloma Tuberculosis Invitro Models

357

3.2 Calculation Count of Cells on the

Structure Granuloma

Figure 2: HE staining (400x magnification)

Figure 2 is the result of HE staining, the

measurement of lymphocyte cell distance and

monocyte / macrophage cells can be identified and

calculated on the granuloma structure.

Table 1. Average amount of cells constituent of granuloma

structure

Treatment Cells Day

1 3 5 7

With

Collagen

lymphocyte 14.5 11.33 5.17 2

monocyte /

macrophage

1.33 1.16 0.83 0.5

Without

Collagen

lymphocyte 10.5 9.83 3.83 2.333

monocyte /

macrophage

1.33 1 0.67 0.33

Figure 3: Average amount of lymphocytes

Figure 4:Average amount of monocytes/macrophages

Average number of cells of lymphocytes and

monocytes/macrophages shown in Figure 3 and

Figure 4. With paired t test P value of lymphocyte

cell count = 0.1662 and monocyte cell number the

value P = 0057 so there is no effect of collagen on

the amount lymphocyte and

monocytes/macrophages.

3.3 Examination the Levels of TNF-α

Table 2. Average levels of TNF-α

Treatment Day (pg/ml)

1 3 5 7

With

Colla

en 321.051 808.725 299.990 249.786

Without

Collagen 340.997 496.227 272.972 245.512

Figure 5: average levels of TNF-α based on variations day

With Paired t test P-value = 0.3744 or P > 0.05 so there is

no effect of collagen on the TNF-α.

3.4 Discussion

The formation of a granuloma is a dynamic process.

Formation of granuloma can be divided into three

phases, first phase is the stage where the innate

granuloma formed from macrophages and

neutrophils. The second phase immune granuloma

ICPS 2018 - 2nd International Conference Postgraduate School

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formed after the emergence of specific antigen of T

cells. The third phase chronic granuloma comes

from the difference in the morphology and the

change of the structure of the granuloma. After the

M. Tb root is infected with Alveolar Macrophages

(MA) then it goes earlier against the inflammatory

response. In the meantime, it strengthens the

immune response of the host then the recruitment of

innate immune cells against the new target M. Tb

and contribute to spreading M.Tb. The

mycobacterium species is inhibited by the fusion

process fagolysosome. It is associated with

virulence, the strain of relativity M.Tb inhibits

fusion. Infected macrophages produce a number of

pro-inflammatory cytokines and chemoatractant

cytokines TNF-α, IL-6 and IL-8, which facilitate the

recruitment of macrophages and granulocytes into

new infections and lead to the formation of

congenital granulomas (Shaler et al., 2013).

The chronic Granuloma phase causes significant

changes in morphology and granuloma function. In

infected individuals a spectrum of granuloma

structures, the classification of either bacterial or

non-bacterial lesions and fibrotic necrotic

granulomas, suggests that granuloma evolution is a

highly dynamic process (Shaler et al., 2013).

The pathologic infection of tuberculosis in

humans is an organized aggregate granuloma that is

organized from immune cells consisting of

macrophages, lymphocytes and immune cells

present in the host (Cadena A.M, et al, 2017).

Formation of granuloma phase of immune

granuloma will produce continuous chemokine by

APC in infected lung and efficiently recruit T cells.

Then, T cells will surround and close the infected

macrophages by M.Tb bacteria. T cell activation

serves as bactericide and limits bacterial mobility

thus it prevents the spread of bacteria to other cells.

The arrival of T cells and the formation of immune

granulomas are associated with the growth of stable

bacteria (Mogues et al., 2001).

Monocyte / macrophage cells will clump in

response to Mtb infection and form a structure such

as granuloma and initiate granuloma formation. It is

defined as a grouping of monocytes / macrophages

during inflammation, an initial occurrence during

mycobacterial infection (Parasa et al, 2014).

Macrophages will secrete IL-8 as a strong

chemoatractant for T lymphocytes that will surround

the granuloma structure. T lymphocytes then secrete

IFN-ɣ to activate additional macrophages.

Macrophages will produce TNF-α and play an

important role in the accumulation of macrophages

and other cells (Fitzgerald et al., 2014).

Tumor Necrosis Factor Alpha (TNF-α) is a

cytokine that emerges since early inflammation

plays an important role in the mechanism of the

innate immune response. TNF-α is an autocrine

cytokine produced by macrophages, dendritic cells,

lymphocytes, neutrophils, mast cells, and endothelial

cells and performs functions such as chemotaxis

with the formation of granulomas (Sasindran and

Torrelles, 2011).

4 CONCLUSIONS

The invitro is a risky method, since the

contamination is likely to occur. Therefore, it is

suggested to absolutely ensure the sterile condition

before conducting the isolation, the tools, materials,

specimens.

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