Apoptosis and Phagocytosis Activity of Macrophages Infected by Mycobacterium tuberculosis Resistant and Sensitive Isoniazid Clinical Isolates

Mycobacterium tuberculosis (M.tb) is the main causative pathogen that cause the pulmonary tuberculosis. Intracellular M.tb was reported able to induce macrophages apoptosis, which may have crucial role in the regulation of immun response against M.tb infection. As an intracellular bacteria, M.tb able to live and replicate within macrophages. Phagocytosis is the first step to achieved this condition. The induction of macrophages apoptosis by INH resistant and sensitive M.tb clinical isolates, and H37Rv was studied. The macrophages apoptosis level were plus measured using an Ag-capture ELISA for histone and fragmented DNA (Cell Death Detection ELISA , Roche TM Diagnostic GmBH). Phagocytosis activity also analyzed, after staining using fluorescence dye (AcriFluor , Scientific Device Lab.). The results showed that there was no significantly different between INH resistant and sensitive M.tb clinical isolates in respect their ability to induce apoptosis. The phagocytosis activity among the clinical isolates was shown to be strain dependent, and undistinguishable between the Mtb clinical isolates. There was no association between macrophages apoptosis level and the phagocytosis activity. These data suggested that among the virulent Mtb clinical isolates, the ability to induce macrophages apoptosis and phagocytosis were consistently in comparable


Introduction
Mycobacterium tuberculosis (M.tb), the main causative pathogen of tuberculosis (TB), is responsible for eight million incidences of TB and killing more than 1.7 million peoples per year worldwide (WHO, 2005).TB as world's health problem becomes more complicated as the multi drug resistant (MDR) TB occurs, especially to isoniazid able to live and replicate in the macrophages, which was defined its pathogenocity.These process is start from the phagocytosis event.
A mannose-containing lipoglycan of the M.tb cell wall, the terminal mannose-capped lipoarabinomannan (ManLAM), has been implicated in the regulation of several of these processes.The presence of ManLAM on the mycobacterial surface places this molecule in an ideal position to mediate the initial interactions between M.tb and macrophages, which in turn facilitates the phagocytosis (Hunter and Brennan, 1990;Kang et al., 2005) M.tb infection could induce human alveolar macrophages and monocytesmacrophages apoptosis (Keane et al., 1997;Klingler et al., 1997;Placido et al., 1997).
There is an inverse correlation between mycobacterial virulence with infected macrophages apoptosis level.Virulent M.tb induces little apoptosis as compare to attenuated strains (Keane et al., 2000).Macrophages apoptosis has direct correlation with killing of intracellular bacilli (Molloy et al., 1994;Oddo et al., 1998).Apoptosis of the macrophages is crucial for innate immunity against M.tb and eradication of intracellular M.tb.
Previous reports showed that the M.tb cell wall is very important in its p a t h o g e n i c i t i e s .F u r t h e r m o r e , t h e accumulated data suggested that INH target molecule reside in the cell wall, apoptosis were induced by component of the cell wall, and the phagocytosis also was facilitated by the component of cell wall.It is also reported that the disturbance of mycolic acid biosynthesis may resulted in the disturbance of M.tb cell wall structure.The aim of this work is to study the induction of macrophages apoptosis by M.tb resistant and sensitive INH clinical isolates.Furthermore, the association between the apoptosis level and phagocytosis activity of the macrophages was explored.

Mycobacterium tuberculosis clinical isolates.
M.tb isolates were obtained from patients attend to the primary health care centers in Yogyakarta.The M.tb were cultured on Lõwenstein-Jensen (LJ) medium.Sensitivity of the M.tb clinical isolates to INH were tested using the agar proportional method on LJ medium (Freixo et al., 2002).INH sensitivity were tested with concentrations of 0.1 mg/ml and 1 mg/ml.The colonies of the M.tb were only observed after 3 weeks incubation on LJ medium.Two INH resistant M.tb clinical isolates (R1 and R2) and two INH sensitive M.tb clinical isolates (S1 and S2) were chosen for further analysis in this work.

Primary culture of macrophages derived from peripheral blood
Peripheral blood mononuclear cells were isolated using standard gradient ® centrifugation method of Histopaque (Sigma Diagnostic Inc.) from heparintreated blood of healthy and non-smoker donors after informed consent were granted.Mononuclear cells were suspended with complete medium which was consist of RPMI 1640 supplemented with L-glutamine, without sodium bicarbonate (GIBCO), 10% fetal bovine serum (Invitrogen Corp.), antifungal, and antibiotics.The cells were plated in polystyrene tissue culture disc TM (Nunc ) and then incubated for 2 h at 37°C with 5% CO .After incubation, non-adherent 2 cells were removed by three times washing with RPMI 1640.Adherent monocytes were collected by vigorous pippeting and subjected for viability test and cell counting after trypan blue staining.Freshly isolated monocytes were suspended in complete medium and re-plated to the 24 wells polystyrene tissue culture disc at a 5 concentration of 5 X 10 /ml and incubated at 37°C with 5% CO .The cells were ready to be 2 infected at the fourth Intracellular M.tb counting was performed at 200 microscope fields (400 time magnificence).The data were collected from five independent experiments and showed as means with standard error means (SEM).
Previously, significantly different phagocytosis level were reported by Schlesinger (1993) between virulent and avirulent mycobacteria.Our data showed t h a t p h a g o c y t o s i s l e v e l w e r e undistinguishable between virulent M.tb strains.
The association between macrophages apoptosis level and the phagocytosis activity were measured using Spearman's test.It was observed a negative association between the two variables.The M.tb clinical isolates that phagocytosed easier (more intracellular M.tb) seemed induce less apoptosis to the macrophages, though this phenomenon was not statistically significant (P>0.05).

Conclusion
M.tb clinical isolates were able to induce the macrophages apoptosis in vitro.The ability of M.tb virulent strains to induce macrophages apoptosis was not attributed with its sensitivity to isoniazid.There was also no significantly difference between INH-resistant and sensitive M.tb in respect with its phagocytosis into the macrophages.Negative association between level of apoptosis and level of phagocytosis activity was not significantly observed in this work.