Research Report

 

THE
LEVEL DIFFERENCE OF MAP1LC3B AS MACROPHAGE AUTOPHAGY MARKER BETWEEN TUBERCULOSIS
PATIENTS WITH SENSITIVE AND RESISTANT RIFAMPICIN

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Dian Novita W1,
Jusak Nugraha2, Soedarsono3

1Post
Graduate Student of Master of Immunology, Faculty of Pasca Sarjana, Airlangga
University

2Clinical
Patology, dr. Soetomo Teaching Hospital

3Pulmonology
& Respiratory, dr. Soetomo Teaching Hospital

Correspondence:
[email protected], [email protected]

 

ABSTRACT

M. tuberculosis (MTB)
is an intracellular bacteria that live in the host macrophage cells. Several
organs can be affected by tuberculosis but most major illnesses are lung
diseases. Immediately after infection, MTB will be phagocytosed by the alveolar
macrophage cells and can survive in the phagosome. The macrophage plays a role
in innate immunity towards an infection using autophagy by removing the microbe
directly via phagocytosis. When bacteria phagocytosized, vacuole membrane formed
double membranes called autophagosome, and followed by degradation by lysosome,
which known as  autolysosome. Induction
of autophagy can be observed on the formation of microtubule-associated
proteins 1B lightchain 3B (MAP1LC3B/LC3). MAP1LC3B is protein that have role at
autophagic way for selection autophagy substrate and biogenesis. In this study
we using serum from patients TB with rifampicin resistant and rifampicin sensitive
as control. Sample were divided using gene expert to differentiate between
resistant and sensitive rifampicin.This research aims to compare MAP1LC3B levels
in resistant and sensitive rifampicin to study macrophages respond in
autophagic way in tuberculosis patients, and give information for define therapy
plan to improve therapy for MDR-TB patients. Type of this research is a case
control study design with cross sectional research with each groups sample is
19 from age 18-65 years old. Result, MAP1LC3B serum levels on the rifampicin
resistant group are lower compared to rifampicin sensitive group. This occur
because MTB is able to hide and evade innate immune defense mechanisms. MTB can
maintain intracellular growth inside the phagosome by inhibiting phagolysosome
formation in autophagy process especially inhibit MAP1LC3B formation by PDIM.

 

Keywords:
Mycobacterium tuberculosis, drug
resistance, rifampicin, autophagy, MAP1LC3B

 

ABSTRAK

M. tuberculosis (MTB) adalah bakteri
intraseluler yang hidup dalam makrofag pada sel inang. Beberapa organ dapat
dipengaruhi oleh tuberkulosis tetapi yang paling utama adalah penyakit paru.
Segera setelah terjadi infeksi, kuman TB akan difagositosis oleh sel makrofag
alveolar dan tetap bertahan hidup dalam fagosom. Makrofag mempunyai peranan penting
dalam respon imun bawaan terhadap infeksi melalui autofagi dengan mengeliminasi
bakteri secara langsung dengan cara fagositosis. Ketika bakteri di fagositosis membran
vakuola membentuk dua lapisan membran yang disebut dengan autofagosom dan
didegradasi oleh lisosom, yang biasa dikenal dengan autolisosom. Induksi
autofagi dapat dipantau pada pembentukan formasi microtubule-associated protein
1B light chain 3B ( MAP1LC3B/LC3). MAP1LC3B adalah protein yang mempunyai
peranan pada jalur autofagi untuk seleksi subrat dan biogenesis. Penelitian ini
menggunakan serum darah pasien TB yang resisten dan sensitif rifampisin sebagai
kontrol. Sampel resisten dan sensitive dibedakan menggunakan tes gen expert. Penelitian
ini bertujuan untuk membandingkan kadar MAP1LC3B pada resisten dan sensitif
rifampisin untuk mempelajari autofagi makrofag pada pasien tuberkulosis dan
memberikan informasi untuk meningkatkan terapi pada pasien MDR-TB. Jenis
penelitian ini adalah case control study dengan rancangan penelitian cross
sectional dengan besar sampel tiap kelompok sebesar 19 dengan rentang umur
18-65 tahun. Hasilnya, kadar MAP1LC3B pada kelompok resisten rifampisin
memiliki kadar lebih rendah dibandingkan dengan kelompok sensitif.Hal ini
disebabkan karena MTB dapat menghindari sistem pertahanan respon imun bawaan.
MTB dapat mempertahankan pertumbuhan intraseluler di dalam fagosom dengan
menginhibisi formasi fagolisosom pada proses autofagi terutama menghambat
pembentukan MAP1LC3B oleh PDIM.

 

Kata Kunci: Mycobacterium tuberculosis, resisten obat, rifampisin,
autofagi, MAP1LC3B

INTRODUCTION

Mycobacterium tuberculosis (MTB)
can cause a dangerous disease called Tuberculosis (TB). This microbacteria can
attack various organs, but mostly it attacks the lungs. The TB infection can
spread from coughing or sneezing which allows MTB to enter the body along with
dusts or droplets19. There are 6 countries with the world’s largest
TB disease spread: South Africa, Nigeria, China, Pakistan, India and Indonesia.
MTB can evolve its resistance against antimicrobial drugs. There is a type of
TB called Multidrug-resistant TB (MDR-TB) which cannot be treated by at least
with two of the potent first line anti-TB drugs like isoniazid and rifampicin.
To improve detection of the case and treatment for MDR-TB, any further
development is needed. There are 300,000 cases of MDR-TB patients that were
estimated in 2013. Around 45% cases from them were detected among all pulmonary
TB in the world while around 5% of cases of MDR-TB that are not detected or not
managed outside the national TB programs were not reported25.

Comparative genomic analyses drug
resistance on MTB can be caused by 3 things, they are chromosonal mutations
that required for the action of antibiotics, gene that encodes the protein
targets of drugs applied, or enzymes that are required to activate pro-drug.
The target of antibiotics is important to cell function. Resistant mutations
encodes gene target will affect pathogenesis15. In every 106
to 108 replications, wild strains of MTB will undergo spontaneous
mutations that confer resistance to a single drug, mutations variety to
antibiotic shown at Table 1.

 

Table 1.Mutations
in antibiotic19

Drug

Average
mutation rate

Isoniazid

2.56 x 10-6

Rifampicin

2.25x 10-10

Ethambutol

1 x 10-7

Streptomycin

2.95 x 10-8

Pyrazinamide

1 x 10-3

 

TB therapy with fast onset needs Rifampicin (RIF)
as critical component of first-line therapy3. Almost 90% of RIF
resistant strains are also resist to isoniazid. RIF resistant is used as
subtitution marker for detecting MDR TB2. RIF resistant is caused by
mutation of a single nucleotide-substitution on rpoB region. In this mutation
process, the gene encodes the ?-subunit of RNA polymerase into DNA-dependent
(RNAP)21. Transcription of the RNAP from the mutations of rpo in the
gene has some effects toward physiology of the MTB. Mutations in this site can
cause secondary mutations which lead resistance to another antibiotic9.

Autophagy is a complex process
involving multiple protein that consist of complex formation and initiation of
double membrane development phagophore as nucleation, elongation of the
membrane and completion of autophagosome vesicles surround the cargo, and then
they will fuse with lysosom. Lysosom contain hydrolase that can degrade and
dispose component18. MTB persist and multiply within infected
macrophage, where it resides in host-derived phagosome which fails to fuse with
lysosom10. Autophagy caused by metabolic and immune signals consists
of recognition of pathogen and stimulation by pro-inflammatory cytokines.
Autophagy trigger microtubule-associated proteins 1B light chain 3B
(MAP1LC3B/LC3), a protein encoded by the gene MAP1LC3B in humans26.
LC3 was first identified as a protein co-purified with microtubule-associated
protein 1A and 1B from rat brains. This protein is derived from 28% of amino
acids with Apg8/Aut7p who plays a role in autophagy in yeast, undergoes complex
C-terminal proteolitic and lipid (phosphathydil ethanolamine) modifications,
which is translocate from cytosol to the autophagosomal membrane12.

Figure 1. Autophagy process

MAP1LC3B functions are for biogenesis, autophagy
and substrate selection autophagosome26. If MTB resistance to rifampicin, it physiology change, MAP1LC3B can
not form autophagosome vesicles so elimination of bacteria with autophagy
process not formed, result MTB survive inside body. This research was conducted
to analyze the differences between the MAP3LC1B level in tuberculosis patient
with sensitive and resistant rifampicin where this protein used as autophagy
marker from macrophage.

 

MATERIALS AND METHODS

A retrospective cross-sectional study was conducted from May 2017 to
September 2017 at the RSUD dr. Soetomo. Samples used are serum from
tuberculosis patients who visited RSUD dr. Soetomo during study period. When
patients coming they got blood tested and fill information for medical record.
Patients divided into sensitive and resistant rifampicin using gene expert
test, and patients selection are patients that meet the inclusion and exclusion
criteria based on medical record.

Based on the WHO (2013) the
proportion value of TB with MDR was 4% of new TB cases, so the number of samples
obtained are 19 sensitive (as control) and 19 resistant. Normal groups were used
as MAP1LC3B baseline.

After all samples collected,
samples were processed by ELISA. These were diluted and decontaminated, and
MAP1LC3B kit performed according to the manfacturer’s manual. Result were
analyzed using one-way ANNOVA P<0.05, and comparisons between groups using Tukey.   RESULTS AND DISCUSSION Results            analyzed and obtained significant results with a value of P<0.05. Table 2. MAP1LC3B Concentration (ng/ml) Normal Sensitive Resistance 1.061 2.291 0.136 1.418 0.866 0.475 1.537 0.983 0.321 1.753 3.268 3.473 1.978 0.482 1.067 2.435 2.268 0.435 2.45 0.684 0.402 2.504 1.072 0.776 2.538 3.012 0.796 3.812 0.526 0.949 0.345 0.512 1.432 1.637 0.381 1.828 1.584 1.435 2.35 0.954 4.137 0.529 2.033 1.505 1.973 0.59 2.156 0                             Table 3. Comparison between groups (ng/ml) Groups Mean Significant P val. Normal vs. Sensitive 0.4727 No 0.3908 Normal vs. Resistant 1.211 Yes 0.0042 Sensitive vs. Resistant 0.7381 Yes 0.0434               Figure 2. MAP1LC3B levels comparison each groups (n normal=10; n sensitive=19; and n resistant=19)   Based on the figure 2, anti-TB resistant group have MAP1LC3B level lower than sensitive group (Figure 1). The highest mean value from highest to the lowest are the normal group (2.1486), sensitive group (1.6759), and resistant groups (0.9378). Macrophages are important fundamental for host defense system with phagocytic cells i.e neutrophil and monocyte which recognize and eradicate pathogenic bacteria. Pathogen destroyed by macrophages directly or indirectly through the innate and adaptive immune system13. Macrophages are target for bacterial pathogens that also can give an advantage for  bacteria to evade the immune system25. Phagocytosis is an ingestion of antigens that are large into membrane vacuole commonly known as the phagosome8. Autophagy isolate cargo  into the membrane with double structure commonly referred by autophagosome5. Induction of autophagy can be monitored by MAP1LC3B (LC3) formation5. To survive inside macrophages, intracellular bacteria develop a variety of strategies to avoid or fight the host defense system13. In this case, MTB has the ability to hold phagosome maturation20. Autophagy can act as a tumor suppressor in normal cells based on the efficiency of non-apoptotic cell death from malignant cells and DNA damage by inhibiting ROS formation18. Antimicrobial activity and apoptosis of human macrophages can be triggered by cytosolic phospholipase activity through MTB which catalyze the release of arachidonic acid. Arachidonic acid is product of a second messenger of TNF which induce apoptosis and oxygen radicals, which are produced during arachidonic acid lipoxygenation, thus inducing the production of reactive oxidative species and are involved in cell death4. Bacteria that are resistant to drugs is a threat to human health. Resistant to antibiotics can be against two things: bacterial survival ability and the ability to reproduce in the presence of macrophages. When bacteria enters the macrophages, they will experience environmental stress such as nutritional restriction induced by the host, acidification, toxic peptides, osmotic stress, and reactive oxygen species (ROS), which later became the biggest cause the death of the bacteria16. To survive inside macrophages, MTB developed a variety of strategies to avoid or fight the host defense system13. One of the mechanisms of MTB to survive is manipulating the host cell death pathways in infected cells. One of the virulence factors are surface glycolipid PDIM (phthiocerol dimycocerosates)22. Lipid are not directly genetically encoded and therefore are not amenable to traditional tagging methods, also cell wall lipids have multiple overlapping functions16. Multiple role functions from PDIM on pathogenesis has been investigated before, including the invasion of macrophages, masking of pathogen-associated molecular pattern (PAMPS), resistance to death with nitric oxide, and the prevention of the recruitment of active macrophages to infected area19. PDIM suppress recruitment of microbicidal, iNOS positive macrophages by inhibiting TLR signaling (Figure 3)25.  Interactions between host and bacterial cell wall are likely to be bidirectional and change when infection25. PDIM in vivo22abundance depend on expression of bisynthetic enzymes which decrease upon macrophage infection, shift metabolic flux which occur during host lipid catabolism10 and insertion of molecule into host membranes1. There maybe variable amount of PDIM on MTB surface at different time points after infection25. MTB initiated human infections in distal lung, and reside in upper respiratory tract. TLR signalling stimulated by PAMPs from lung overrides PDIM and PGL-mediated immune evasion25. There is site named resistance-determining region (RRDR)22 that caused by mutations in MTB strains at 81-bp region of rpoB. This mutations result is high levels of resistance to rifampicin.                           Figure 3. MTB cell wall lipids at sites of infection25 According to Comas7 all laboratory-generated mutans of MTB with rifampicin-resistance  mutations in the RRDR reduced fitness compared to their respond for drug ancestors when without rifampicin8, MTB with RIF resistant caused by mutations in the rpoB gene, where the majority is on codon 531 and 52612. According to Kawamura mutation in codon 526 related to oxidative stress sensitivity14. In addition, some reports say that just one gene mutations in the rpoB encodes in sub-unit of RNA polymerase ? can cause interaction between the RNA polymerase and some promoter also transcription regulation that trigger changes in phenotype16. The mechanism of the rpoB gene mutation caused by resistant rifampicin indicates that specific lead to mutations in the rpoB changes aspects of transcription. These transcription factors causing changes in gene expression which encodes the protein secretion, and proteomic changes produce some enzymes and lipid biosynthetic of intermediate in the path of phthiocerol dymycocerosate (PDIM). To prove PDIM plays role in induction of autophagy and necrosis on MTB, Quigley observed conversion of cytosolic LC3I to autophagosome-bound LC3II, using the expression of green fluorescent protein-LC3 (GFP) and flow cytometry22. As a result, autophagy was decreased in cells infected by MTB. PDIM plays role in induction of autophagy with decreasing autophagy on infected cells by MTB22. Resistance to rifampicin caused by mutations in rpoB gene related with physiological and metabolic changes in bacterial systems15. These RIF resistance might be under dual selection in MTB, combined benefit and physiological advantage of rpoB gene can fix rpoB mutants to infect in MTB populations.   CONCLUSION Levels of MAP1LC3B on groups rifampicin resistant groups lower than on sensitive groups, that indicate no autophagy process or only few at macrophage on resistant groups than sensitive groups. This occur because MTB successfully evade host defense by innate immune mechanisms. MTB can maintain intracellular growth inside the phagosome by inhibiting phagolysosome formation especially inhibiting MAP1LC3B formation by PDIM.