Research Report   THELEVEL DIFFERENCE OF MAP1LC3B AS MACROPHAGE AUTOPHAGY MARKER BETWEEN TUBERCULOSISPATIENTS WITH SENSITIVE AND RESISTANT RIFAMPICIN Dian Novita W1,Jusak Nugraha2, Soedarsono31PostGraduate Student of Master of Immunology, Faculty of Pasca Sarjana, AirlanggaUniversity2ClinicalPatology, dr. Soetomo Teaching Hospital3Pulmonology& Respiratory, dr. Soetomo Teaching HospitalCorrespondence:[email protected], [email protected] ABSTRACTM. tuberculosis (MTB)is an intracellular bacteria that live in the host macrophage cells.

Severalorgans can be affected by tuberculosis but most major illnesses are lungdiseases. Immediately after infection, MTB will be phagocytosed by the alveolarmacrophage cells and can survive in the phagosome. The macrophage plays a rolein innate immunity towards an infection using autophagy by removing the microbedirectly via phagocytosis. When bacteria phagocytosized, vacuole membrane formeddouble membranes called autophagosome, and followed by degradation by lysosome,which known as  autolysosome. Inductionof autophagy can be observed on the formation of microtubule-associatedproteins 1B lightchain 3B (MAP1LC3B/LC3). MAP1LC3B is protein that have role atautophagic way for selection autophagy substrate and biogenesis.

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In this studywe using serum from patients TB with rifampicin resistant and rifampicin sensitiveas control. Sample were divided using gene expert to differentiate betweenresistant and sensitive rifampicin.This research aims to compare MAP1LC3B levelsin resistant and sensitive rifampicin to study macrophages respond inautophagic way in tuberculosis patients, and give information for define therapyplan to improve therapy for MDR-TB patients. Type of this research is a casecontrol study design with cross sectional research with each groups sample is19 from age 18-65 years old. Result, MAP1LC3B serum levels on the rifampicinresistant group are lower compared to rifampicin sensitive group.

This occurbecause MTB is able to hide and evade innate immune defense mechanisms. MTB canmaintain intracellular growth inside the phagosome by inhibiting phagolysosomeformation in autophagy process especially inhibit MAP1LC3B formation by PDIM. Keywords:Mycobacterium tuberculosis, drugresistance, rifampicin, autophagy, MAP1LC3B ABSTRAKM. tuberculosis (MTB) adalah bakteriintraseluler yang hidup dalam makrofag pada sel inang. Beberapa organ dapatdipengaruhi oleh tuberkulosis tetapi yang paling utama adalah penyakit paru.

Segera setelah terjadi infeksi, kuman TB akan difagositosis oleh sel makrofagalveolar dan tetap bertahan hidup dalam fagosom. Makrofag mempunyai peranan pentingdalam respon imun bawaan terhadap infeksi melalui autofagi dengan mengeliminasibakteri secara langsung dengan cara fagositosis. Ketika bakteri di fagositosis membranvakuola membentuk dua lapisan membran yang disebut dengan autofagosom dandidegradasi oleh lisosom, yang biasa dikenal dengan autolisosom. Induksiautofagi dapat dipantau pada pembentukan formasi microtubule-associated protein1B light chain 3B ( MAP1LC3B/LC3). MAP1LC3B adalah protein yang mempunyaiperanan pada jalur autofagi untuk seleksi subrat dan biogenesis. Penelitian inimenggunakan serum darah pasien TB yang resisten dan sensitif rifampisin sebagaikontrol. Sampel resisten dan sensitive dibedakan menggunakan tes gen expert. Penelitianini bertujuan untuk membandingkan kadar MAP1LC3B pada resisten dan sensitifrifampisin untuk mempelajari autofagi makrofag pada pasien tuberkulosis danmemberikan informasi untuk meningkatkan terapi pada pasien MDR-TB.

Jenispenelitian ini adalah case control study dengan rancangan penelitian crosssectional dengan besar sampel tiap kelompok sebesar 19 dengan rentang umur18-65 tahun. Hasilnya, kadar MAP1LC3B pada kelompok resisten rifampisinmemiliki kadar lebih rendah dibandingkan dengan kelompok sensitif.Hal inidisebabkan karena MTB dapat menghindari sistem pertahanan respon imun bawaan.MTB dapat mempertahankan pertumbuhan intraseluler di dalam fagosom denganmenginhibisi formasi fagolisosom pada proses autofagi terutama menghambatpembentukan MAP1LC3B oleh PDIM. Kata Kunci: Mycobacterium tuberculosis, resisten obat, rifampisin,autofagi, MAP1LC3BINTRODUCTIONMycobacterium tuberculosis (MTB)can cause a dangerous disease called Tuberculosis (TB). This microbacteria canattack various organs, but mostly it attacks the lungs.

The TB infection canspread from coughing or sneezing which allows MTB to enter the body along withdusts or droplets19. There are 6 countries with the world’s largestTB disease spread: South Africa, Nigeria, China, Pakistan, India and Indonesia.MTB can evolve its resistance against antimicrobial drugs. There is a type ofTB called Multidrug-resistant TB (MDR-TB) which cannot be treated by at leastwith 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 furtherdevelopment is needed. There are 300,000 cases of MDR-TB patients that wereestimated in 2013. Around 45% cases from them were detected among all pulmonaryTB in the world while around 5% of cases of MDR-TB that are not detected or notmanaged outside the national TB programs were not reported25.Comparative genomic analyses drugresistance on MTB can be caused by 3 things, they are chromosonal mutationsthat required for the action of antibiotics, gene that encodes the proteintargets of drugs applied, or enzymes that are required to activate pro-drug.

The target of antibiotics is important to cell function. Resistant mutationsencodes gene target will affect pathogenesis15. In every 106to 108 replications, wild strains of MTB will undergo spontaneousmutations that confer resistance to a single drug, mutations variety toantibiotic shown at Table 1. Table 1.Mutationsin 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 RIFresistant strains are also resist to isoniazid. RIF resistant is used assubtitution marker for detecting MDR TB2. RIF resistant is caused bymutation of a single nucleotide-substitution on rpoB region. In this mutationprocess, the gene encodes the ?-subunit of RNA polymerase into DNA-dependent(RNAP)21. Transcription of the RNAP from the mutations of rpo in thegene has some effects toward physiology of the MTB. Mutations in this site cancause secondary mutations which lead resistance to another antibiotic9.

Autophagy is a complex processinvolving multiple protein that consist of complex formation and initiation ofdouble membrane development phagophore as nucleation, elongation of themembrane and completion of autophagosome vesicles surround the cargo, and thenthey will fuse with lysosom. Lysosom contain hydrolase that can degrade anddispose component18. MTB persist and multiply within infectedmacrophage, where it resides in host-derived phagosome which fails to fuse withlysosom10. Autophagy caused by metabolic and immune signals consistsof 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-associatedprotein 1A and 1B from rat brains. This protein is derived from 28% of aminoacids with Apg8/Aut7p who plays a role in autophagy in yeast, undergoes complexC-terminal proteolitic and lipid (phosphathydil ethanolamine) modifications,which is translocate from cytosol to the autophagosomal membrane12.Figure 1.

Autophagy processMAP1LC3B functions are for biogenesis, autophagyand substrate selection autophagosome26. If MTB resistance to rifampicin, it physiology change, MAP1LC3B cannot form autophagosome vesicles so elimination of bacteria with autophagyprocess not formed, result MTB survive inside body. This research was conductedto analyze the differences between the MAP3LC1B level in tuberculosis patientwith sensitive and resistant rifampicin where this protein used as autophagymarker from macrophage. MATERIALS AND METHODSA retrospective cross-sectional study was conducted from May 2017 toSeptember 2017 at the RSUD dr. Soetomo. Samples used are serum fromtuberculosis patients who visited RSUD dr. Soetomo during study period. Whenpatients coming they got blood tested and fill information for medical record.

Patients divided into sensitive and resistant rifampicin using gene experttest, and patients selection are patients that meet the inclusion and exclusioncriteria based on medical record. Based on the WHO (2013) theproportion value of TB with MDR was 4% of new TB cases, so the number of samplesobtained are 19 sensitive (as control) and 19 resistant. Normal groups were usedas MAP1LC3B baseline. After all samples collected,samples were processed by ELISA. These were diluted and decontaminated, andMAP1LC3B kit performed according to the manfacturer’s manual. Result wereanalyzed using one-way ANNOVA P<0.05, and comparisons between groups usingTukey. RESULTS AND DISCUSSIONResults            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 levelscomparison each groups (n normal=10; n sensitive=19; and n resistant=19) Based on the figure 2, anti-TB resistant group have MAP1LC3B level lowerthan sensitive group (Figure 1).

The highest mean value from highest to thelowest are the normal group (2.1486), sensitive group (1.6759), and resistantgroups (0.

9378).Macrophages are important fundamental for host defense system withphagocytic cells i.e neutrophil and monocyte which recognize and eradicatepathogenic bacteria. Pathogen destroyed by macrophages directly or indirectlythrough the innate and adaptive immune system13. Macrophages aretarget 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 vacuolecommonly known as the phagosome8. Autophagy isolate cargo  into the membrane with double structurecommonly referred by autophagosome5. Induction of autophagy can be monitored by MAP1LC3B (LC3) formation5.

To survive inside macrophages, intracellular bacteria develop avariety 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 tumorsuppressor in normal cells based on the efficiency of non-apoptotic cell deathfrom malignant cells and DNA damage by inhibiting ROS formation18. Antimicrobial activity andapoptosis of human macrophages can be triggered by cytosolic phospholipaseactivity through MTB which catalyze the release of arachidonic acid.Arachidonic acid is product of a second messenger of TNF which induce apoptosisand oxygen radicals, which are produced during arachidonic acid lipoxygenation,thus inducing the production of reactive oxidative species and are involved incell death4.Bacteria that are resistant todrugs is a threat to human health.

Resistant to antibiotics can be against twothings: bacterial survival ability and the ability to reproduce in the presenceof macrophages. When bacteria enters the macrophages, they will experienceenvironmental 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 tosurvive is manipulating the host cell death pathways in infected cells. One ofthe virulence factors are surface glycolipid PDIM (phthiocerol dimycocerosates)22. Lipid are not directlygenetically encoded and therefore are not amenable to traditional taggingmethods, also cell wall lipids have multiple overlapping functions16.

Multiple role functions fromPDIM on pathogenesis has been investigated before, includingthe invasion of macrophages, masking of pathogen-associated molecular pattern(PAMPS), resistance to death with nitric oxide, and the prevention of therecruitment 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 cellwall are likely to be bidirectional and change when infection25. PDIM in vivo22abundance depend on expression ofbisynthetic enzymes which decrease upon macrophage infection, shift metabolicflux which occur during host lipid catabolism10 and insertion of molecule intohost membranes1. There maybe variable amount of PDIM on MTB surface at different timepoints after infection25.

MTB initiated human infections indistal lung, and reside in upper respiratory tract. TLR signalling stimulatedby PAMPs from lung overrides PDIM and PGL-mediated immune evasion25.There is site named resistance-determining region (RRDR)22 that caused bymutations in MTB strains at 81-bp region of rpoB. This mutations result is highlevels of resistance to rifampicin.             Figure 3. MTB cell wall lipids atsites of infection25According to Comas7all laboratory-generated mutans of MTB with rifampicin-resistance  mutations in the RRDR reduced fitnesscompared to their respond for drug ancestors when without rifampicin8,MTB with RIF resistant caused by mutations in the rpoB gene, where the majorityis on codon 531 and 52612. According to Kawamura mutation in codon526 related to oxidative stress sensitivity14. In addition, some reports saythat just one gene mutations in the rpoB encodes in sub-unit of RNA polymerase? can cause interaction between the RNA polymerase and some promoter alsotranscription regulation that trigger changes in phenotype16.

The mechanism of the rpoB gene mutationcaused by resistant rifampicin indicates that specific lead to mutations in therpoB changes aspects of transcription. These transcription factors causingchanges in gene expression which encodes the protein secretion, and proteomicchanges produce some enzymes and lipid biosynthetic of intermediate in the pathof phthiocerol dymycocerosate (PDIM). To prove PDIM plays role in induction ofautophagy and necrosis on MTB, Quigley observed conversion of cytosolic LC3I toautophagosome-bound LC3II, using the expression of green fluorescentprotein-LC3 (GFP) and flow cytometry22. As a result, autophagy wasdecreased in cells infected by MTB. PDIM plays role in induction of autophagywith decreasing autophagy on infected cells by MTB22.Resistance to rifampicin causedby mutations in rpoB gene related with physiological and metabolic changes in bacterialsystems15. These RIF resistance might be under dual selection inMTB, combined benefit and physiological advantage of rpoB gene can fix rpoBmutants to infect in MTB populations. CONCLUSIONLevels of MAP1LC3B on groupsrifampicin resistant groups lower than on sensitive groups, that indicate no autophagyprocess 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 inhibitingphagolysosome formation especially inhibiting MAP1LC3B formation by PDIM.