Extract of Poster presented in ACIBICON, Dec. 2007, New Delhi
Jawed Iqbal, Ajay Kumar and Najmul Islam
(Group leader: Dr. Najmul Islam, Dept. of Biochemistry, J.N.Medical College, A.M.U., Aligarh 202002, U.P., India
Azadirachta indica induced suppression of Mycobacterium tuberculosis secreted proteins in human monocyte
Tuberculosis is the most important mycobacterial infection in humans, being the second major infectious cause of death and killing around two million people every year . With the emergence of multidrug-resistant TB (MDR-TB) as well as AIDS-associated TB, this killer parasite is spiraling out of control . Thus, in view of it, focus has now shifted in exploring new novel natural compounds that may combat tuberculosis [3, 4]. Therefore, in the present study, we probed the anti-tuberculosis effect of Azadirachta indica (Neem), which has attained worldwide prominence in recent years owing to its medicinal properties. Neem elaborates a vast array of biologically active compounds that are chemically diverse and structurally complex having wide range of pharmacological properties. It has been known to possess antioxidant and anti-inflammatory properties. It is hoped that the data of the present study may help in understanding the potentiality of neem extract in employing it as a valuable adjunct in TB treatment.
1. Preparation of Mycobacteria:
Virulent laboratory-adapted M. tuberculosis (H37Rv) were grown in Middlebrook 7H9 broth (HiMedia, India) supplemented with Middlebrook ADC enrichment fluid (HiMedia) or modified Souton medium  at 37°C in 5% CO2. Midlogarithmic mycobacterial cultures (14 days) were harvested and quantified by use of a colony-forming unit assay, as described elsewhere . Aliquots of the stock were kept at −70°C. The viability of the stock remained >99% at 1 year.
2. Preparation of PBMC, cell culture and infection:
Peripheral blood mononuclear cells (PBMCs) from blood were isolated as described by us previously . PBMC (0.5x106 cells/well) were added onto 12-well tissue culture plates in complete RPMI-1640 medium, and were subsequently incubated at 37°C, 5% CO2 for 1-2 hrs for adherence. Thereafter, non-adherent cells were removed and the adherent monocytes were rested overnight at 37°C, 5% CO2. Thereafter, monocytes were infected with M. tuberculosis at 1:1 (bacteria: cell) in 30% autologous unheated serum for 90 min. at 37°C, 5% CO2, and subsequently washed for 4 times complete medium. Cells harvested at this time point were considered as time zero after infection (t0). Other cultures received RPMI-1640 medium with 2% autologous serum. As per experimental design, cultures immediately after infection received varying concentrations of neem extract (0-1000 ng/ml)., NAC (10 mM), SN50 (100 ug/ml) or SN50M (100 ug/ml). Cultures were then harvested after 24 hrs and cell protein lysates were prepared as described earlier [3,4].
3. Measurement of secreted TNF-α and MTB 85B protein in culture supernatant by ELISA:
The amount of soluble secreted TNF-α protein in various culture supernatants were determined by use of a commercial ELISA Kit (R & D systems), according to the manufacturer’s specifications. The cut off or lower limit of sensitivity was 4.4 pg/ml. ELISA was carried out to determine secreted MTB 85B protein in culture supernatants as described by us previously .
4. Glutathione peroxidase assay:
The activity of glutathione peroxidase (GPx) was measured as described elsewhere . Briefly, MTB-infected monocytes were co-cultured for 24 h with varying concentrations of neem extract (0-1000 ng/ml). Thereafter, cells were scrapped, sonicated and centrifuged as described earlier [3 and references within], and the supernatants were subjected to GPx activity determination. The GPx activity was quantified in 100 μl of each sample, with continuous photometric monitoring of oxidized glutathione (GSSG) at 37oC. The conversion of NADPH to NADP was evaluated using UV absorbance at 340 nm . GPx activity was calculated after subtraction of the blank value, as lmol of NADPH oxidized/min/mg protein (U/mg protein).
Dose response effect of neem extract on soluble TNF-alpha and MTB Ag85B protein expressions in monocyte cultures infected with MTB
We observed neem extract-induced suppression in secreted TNF-α and MTB Ag85B protein expressions in MTB-infected monocyte cultures were dose dependent. Neem extract at a maximum dose of 1 ug/ml exhibited a down-regulation in secreted TNF-α and 85B proteins to the extent of 4.7-folds and 4.3-folds respectively.
Glutathione peroxidase activity:
Healthy control cultures showing appreciably high GPx activity was suppressed in MTB-infected monocytes (Fig. 3, P<0.001). Interestingly, with neem extract treatment of MTB infected monocytes, GPx activity ameliorated appreciably in a dose-dependent manner. Regulation studies by employing SN50 exhibited the above suppression to be mediated via NF-kB. These results clearly point to neem extract as a potent natural enhancer of GPx levels in monocytes infected with MTB.
1. The neem extract-induced suppression in secreted TNF-alpha and MTB Ag85B proteins in 24 hr culture supernatants of MTB-infected human monocytes was found to be dose-dependent.
2. Glutathione peroxidase activity was decreased after M. tuberculosis infection of monocytes.
3. Interestingly, glutathione peroxidase activity was augmented significantly with neem extract treatment in a dose-dependent manner.
4. The above-said suppression was mediated via NF-kB.
5. The results are indicative of neem extract- a natural antioxidant and an anti-inflammatory agent, may act as an adjunct in combating tuberculosis infection.
1. WHO (2001) Global Tuberculosis Control, Geneva, Switzerland, Report no. WHO/CDS/TB/2001.287.
2. Toossi, Z; Wu, M; Islam, N; Teixeira-Johnson, L; Hejal, R and Aung, H. (2004) Transactivation of human immunodeficiency virus-1 in T-cells by Mycobacterium tuberculosis-infected mononuclear phagocytes. J. Lab. Clin. Med., 144(2):108-15.
3. Hasan, N., Yusuf, N., Toossi, Z. and Islam, N. (2006) Suppression of Mycobacterium tuberculosis induced reactive oxygen species (ROS) and TNF- α mRNA expression in human monocytes by allicin. FEBS Letters 580: 2517-2522.
4. Hasan, N., Siddiqui, M. U., Toossi, Z., Khan, S., Iqbal, J. and Islam, N. (2007) Allicin-induced suppression of Mycobacterium tuberculosis 85B mRNA in human monocytes. Biochem. Biophys. Res. Comm., 355(2): 471-476.
5. Islam, N; Kanost, RA; Teixeira-Johnson, L; Hejal, R; Aung, H; Wilkinson, RJ; Hirsch, CS; and Toossi, Z. (2004) The role of cellular activation and tumor necrosis factor alpha (TNF-alpha) in the early expression of M. tuberculosis 85B mRNA in human alveolar macrophages. J. Infect. Dis. 15; 190:341-351.
6. Wilkinson, RJ; DesJardin, LE; Islam, N; Gibson, BM; Wilkinson, KA; Poelman, D; Kanost, RA; Eisenach, KD and Toossi, Z. (2001) An increase in expression of a M. tuberculosis mycolyl gene (fbpB) occurs early after infection of human monocytes. Mol. Microbiol, 39(3): 813-821.