Macrophage Activity Test of Pulmonary Tuberculosis Patients with Diabetes Mellitus (TB-DM)

Background: Control of pulmonary TB is getting more and more complicated as the number of patients with diabetes mellitus (DM) is increasing. The increasing prevalence of DM is followed by the increasing prevalence of pulmonary TB. Diabetes Mellitus patients have 4,7 times higher risk to develop pulmonary TB compared to patients without DM, since DM can increase the frequency and severity of an infection, including pulmonary TB.Aim: To analyze macrophage activity (phagocytosis, intracellular killing, and TNF-α synthesis) of TB-DM patients.Method: The research is an experimental study using a PBMC cultured sample from TB-DM patient's which undergo observation of macrophage activity (phagocytic, intracellular killing and TNF-α synthesis). The data were taken from microscopic observation of TB-DM patients, colony growth of viable Mycobacterium tuberculosis and the TNF-α level secreted by macrophages.Result: Microscopic observation showed that there are less amount of phagocytosed M. tuberculosis (in macrophage/intracellular level) and there is a little amount of formed vacuoles and giant cells. Furthermore, macrophages in TB-DM patients secrete low level of TNF- α, and there are more viable M. tuberculosis from this macrophage.Conclusions: Macrophages of TB-DM patients are less activated, with reduced phagocytic activity (due to the intrinsic defect of PMN) and reduced antigen presenting activity of phagocytes toward M. tuberculosis. Therefore, there is a need for a further study focused on macrophage activity enhancement on TB-DM patients against M. tuberculosis infection in DM patients.


INTRODUCTION
Tuberculosis (TB) is a major cause of death among bacterial infection diseases. TB affects 9,4 million people and kills 1,7 million of world population annually. The etiology is Mycobacterium tuberculosis bacteria which has a rod shape, aerobe characteristic, and acid-fast feature. Indonesia rank 4th worldwide for its high number of TB patients 1,2,3 World Health Organization expects that TB control will become more difficult with the increasing number of diabetes mellitus (DM) patients, due to DM is one of the risk factors for TB deterioration.
Correlation between TB and DM have been reported since 1000 AD, though it is still difficult to be defined. 4,5,6 The increasing cases of TB-DM are associated with an increase in morbidity and mortality of TB and DM. DM patients have 4,7 times higher risk to develop pulmonary TB. 4 This is due to the treatment of MDR-DM cases, which one of its aim is to restore the function of the immune system, i.e. immunostimulant. 7 Less activated alveolar macrophage of pulmonary TB patients with DM reduces the interaction between T lymphocyte and macrophage, resulting in defect of M. tuberculosis elimination.
The entry of M. tuberculosis into the macrophage and its ability to survive are the key lement of the pathogenesis of tuberculosis. 8 9,10 Immune response by macrophage in the form of phagocytosis and intracellular killing which contributes to the immune defense is expected to be the first in line to eliminate M. tuberculosis and lower the incidence of TB. However, M. tuberculosisis able to multiply within macrophage thereby causing tuberculosis.
The increase in cases of TB-DM that associated with the increased of morbidity and mortality due to TB-DM, which is DM patients have an immune respond disorder, thereby facilitati erculosis infection. Thus, it is important to know more about the treatment of cases of TB-DM, one of which emphasizes the restoration of immune abnormalities. Therefore, to be able to find out, it is necessary to do the initial research with emphasis on the activity of macrophages from TB-DM patients.
This study is aimed to analyze macrophage activity (phagocytic, intracellular killing and TNFα synthesis) from TB-DM patients, by identifying peripheral blood -DM patients.

METHODS
DM patients were obtained from the TB patients who hav patients were t have BTA (+) and chest X-ray (+). Healthy individu were the individu who have BTA (-) and chest X-ray (-). There were 18 samples of DM patients, TB patients, and healthy individu were recruited for this study, and all of the samples was signed the informed consents.
Ethical clearance was provided by Gadjah Mada University (EC numb. 36/EC/2.08.16). Most of the laboratory works were performed in Faculty of Medicine GadjahMada Univ

PBMC's Isolation (Peripheral Blood Mononuclear Cells)
A taken from TB-DM patients and continued with defibrination.

Opsonization of Mycobacterium tuberculosis
An ose 6 (derived from cultures of tuberculosis laborat pical Disease Centre Institute, Airlangga University)were inserted aseptically into screw cap tube containing 4000µL of Middlebrook 7H9 liquid medium and ± 6 -7 bead glass and homogenous vortex, then 4000 µL suspension was added and centrifuged. Supernatant was discarded, the pellet was set aside and rinsed with 5000 µl sterile PBS for 3 times. Pellet again was set aside and 4000µl RPMI 1640 (-) medium and 4000µl PHS/Pooled Human Serum were added. Next step is suction spray approximately 10 times with 26G tuberculin syringe and incubated at 37°C containing 5% CO 2 for 20 minutes then centrifuged. The supernatant layer was discarded, the pellet was rinsed with 5000mL of sterile PBS for 3 times, then 4000mL RPMI 1640 medium (+) were added.

Co-Culture of Macrophage and M. tuberculosis
On the 7 th day, the macrophage cells culture opsonized-M. tuberculosis strain H37Rv ATCC 294T, and then it was incubated at 37°C with air containing 5% CO 2 for 24 hours, 48 hours, 7 times 24 hours and 14 times 24 hours. 13,15,17   and healthy dividuals can be seen in Table 2.
ndividu can be see -20% buffy coat is consisted of monocytes, the rest were lymphocytes. 19 Buffy etween the upper layer (plasma) and a to inv plate was aseptically rinsed with sterile PBS for 5 times.
Sixty µl supernatant was taken from 24-well tissue culture plate aseptically, then test by used an ELISA !,eBioscience, DTA00C). TNF-α levels was measured by ELISA reader.
Coverslip base of a 24-well tissue culture plate was scraped to harvest the macrophage, then shaken well by mix pipettin d transferred into eppendorf tubes. An 800mL sterile PBS were added and centrifuged. Supernatant layer was discarded, the pellet was set aside, added with 1000mL of sterile distilled water, incubated for 30 minutes at 4°C and then vortex for 5 minutes macrophage are lysis and intracellular M. tuberculosis free from macrophage.
Thirty µl was taken from, dropped on a solid Middlebrook 10 agar medium, and incubated at 37 O C with 5% days to determine the number of bacteria surviving, not digested by macrophages; with counting colonies grown per ml (CFU / ml). 18

CFU Measurement
The number of M. tuberculosis colonies w medium are counted d 14th day.

Data Analysis
The data DM patients, wth, and the TNF-α levels secreted by macrophages by used statistic test.

RESULTS
The study results showed that th blood of nocytes and lymphocytes. Monocytes of TB-DM patients which have matured into macrophages within 7 days were co-cultured with M. tuberculosis ( in vitro). The results of microscopic observation from macrophages of TB-DM patients showed that there is less number of ingested M. tuberculosis(red) and less formation of vacuoles and giant cell macrophages (blue) ) ( Figure 1).

TNF-α levels secreted by macrophages of TB-DM patients, TB patients non-DM, in
Based on Table 2, the average levels of TNF-α secreted by macrophages of TB-DM patients, TB patients without DM, and healthy i n as shown in Figure 2 .
Average of viable M. tuberculosis colonies calculated as CFU/ml on day 7th, 10th, and 14 th can be seen in Figure 3.

DISCUSSIONS
Approximately 10 coat is located b the bottom layer (erythrocytes and granulocytes sediment) from PBMCs of TB-DM patients, consisting monocytes and lymphocytes. TB-DM patients' monocytes which had matured into macrophages within 7 days appears as large rounded cells with regularly spherical nucleus resembling kidney/horseshoe (horseshoe-shaped), have cell wall protrusions and large cytoplasm, and also macrophages showing some presence of giant cells. While lymphocytes appeared as having large nuclei, round shape, heterochromatin features with a large nucleus-cytoplasm ratio. 11,12,19,20 This study eliminated the presence of the lymphocytes using the PBS rinsing method in order to avoid the bias results from the process of intracellular killing because the lymphocytes have a role in producing IFN-γ as a mediator of macrophage activation.
Additionally, this study carried out co-culture between macrophages and M. tuberculosis (in vitro). This was intended to provide the bacteri ade macrophages in order to allow the phagocytosis process to take place. M. tuberculosis The process of ingestion/ engulfment begins with the introduction of M. tuberculosis by macrophages receptors, memb crophages will surround the bacteria in a circle (zipper mechanism), thus the bacteria are in the phagosome. 21 Phagosome then undergoes maturation and then fuse with lysosomes to form phagolysosome, 21 an organelle with antimicrobial component and acidic pH (pH ~ 6,2).
Macrophages from the activated TB patients are more effective in performing the phagosome and lysosome fusion. In addition, macroph re effective in producing oxygen radicals, NO, and various antimicrobial molecules [26][27][28] and may increase respiratory burst, ROI production, RNI, and TNF-α releasing. 29,30 Macrophages of TB-DM patients are activated macrophages, but because the TB-DM patients have defects in their crophages become less activated. Furthermore, macrophages in DM patients have disruption of chemotaxis, phagocytosis, and antigen presenting phagocytes against M. tuberculosis. This defect cannot be resolved with insulin therapy. 14 Patients with poorly controlled DM will upset the phagocytosis, especially if it has been in an acidosis state. This phagocytosis disruption is due to the intrinsic defect of the PMN. 12 This results showed that there were decreased production of TNF-α in pulmonary tuberculosis patients with DM rather than non tients. It is according to the literature mentioned that Th-1 response levels, IFN-γ, as well as the production of IL-1 β and IL-6 are more profound in pulmonary tuberculosis patients with DM rather than non-diabetic TB patients. Decreased production of IFN-γ was more significant in patients with pulmonary tuberculosis with uncontrolled diabetes than in patients with controlled DM TB. This IFN-γ production will return to normal within six months, either in patients with pulmonary TB alone or pulmonary TB patients with uncontrolled DM, but will continue to decline in pulmonary TB patients with uncontrolled DM. Also, there were changes in pulmonary vascular and alveolar oxygen tension that aggravated the patients' condition. 4,23,33 TNF-α proved to be effective in eliminat erculosis, this cytokine will increase the phagocytosis capability and induces apoptosis of infected macrophages. Both TNF-α and IFN-γ will increase the intracellular killing macrophages process by producing reactive NO. TNF-α also plays a role in maintaining granuloma integrity, which formed when macrophages infected by M. tuberculosis. Macrophages that are unable or only slightly expresses TNF-α would be very easily infected and difficult to eliminate M. tuberculosis.
The presence of viable M. tuberculosis showed t this bacteria is able to escape and survive the macrophage phagocytosis and intracellular killing macrophage. Immune cell defects, fewer macrophages activation, and decreased in phagocytic capability in patients with TB-DM can support the viability of M. tuberculosis. In accordance with these results, it is known that there are more viable M. tuberculosis in TB-DM patients' macrophages as shown in Figure 3.
M. tuberculosis can also pr oarabinomannan (LAM), which can inhibit the activation of macrophages and is an important virulence determinant of these bacteria. LAM can inhibit the functions induced IFN-γ thus disrupts the microbicidal function of macrophages, inhibiting the activity of protein kinase C, and TNF-α evocation. 34

NC
Macrophages o ivated macrophages, where there are disturbances in its phagocytosis capability (due to the intrinsic defect of the PMN) and its phagocytes antigen presenting toward M. tuberculosis. These are shown in the results, that the TB-DM macrophages secreted low levels of TNF-α and there are more numbers of viable M. tuberculosis.    .