Nitric Oxide Dependent Killing of Mycobacterium Tuberculosis by Human Mononuclear Phagocytes from Patients with Active Tuberculosis

Abstract

In this study we have demonstrated that nitric oxide, the product of the arginine dependent pathway of human mononuclear phagocytes effectively kills the M.tuberculosis in-vitro. The release of reactive nitrogen intermediates was triggered by incubation with various proinflammatory cytokines namely IFN gamma,TNF-alpha and IL-1 R. We have earlier shown that human mononuclear phagocytes can be induced to release nitric,oxide (NO) radicals which can kill tumour cells. In the present communication, by using colony forming assays we demonstrated that human mononuclear phagocytes can effectively kill M.tuberculosis by using a NO dependent pathway. Treatment of mononuclear phagocytes with L-arginine resulted in markedly increased killing activity whereas, by using NGMMA, an analogue of L-arginine, the cidal activity could be brought down to the basal level. These results clearly suggest that cytokines, particularly IFN-gamma, induced NO release and its reactive product with oxygen radical, peroxynitrite, could play an important role in the killing of M. tuberculosis by human mononuclear phagocytes. A significant production of interleukin-4 and interleukin-10, by the ex-vivo matured, untreated macrophages from the active tuberculosis patients indicate that regulation of cytokine network to encourage in situ/local production of nitric oxide may be useful in the management of pulmonary tuberculosis.

Induction of nitric oxide release from the human alveolar epithelial cell line A549: an in vitro correlate of innate immune response to Mycobacterium tuberculosis

“…It was necessary to stimulate the alveolar epithelial cells by cytokines to bring down the CFU (Table 2). Our results confirm earlier reports 4,27 that cytokine mixture leads to release of very high concentration of NO. We further demonstrated that the reduction in CFU occurred in the same wells.…”

“…The pellet was resuspended in DMEM supplemented with 10% FCS and passed through an 8‐µm filter to prepare a single cell suspension. The dispersed bacterial concentration was measured by taking absorbance at 600 nm using MacFarland's nephelometric standards 27 . The bacterial concentration was adjusted to a density of 10 8 bacteria/ml, aliquoted and kept as a single lot at 4°.…”

“…To determine the effect of nitric oxide produced by the infected A549 cell, A CFU assay was performed for each set of experiments. Infected A549 cells were harvested at 48 hr according to the experimental design described above, and lysed with 0·5 ml of 1% Triton‐X‐100 for 15 min followed by repeated passage through a 21‐gauge needle to release intracellular mycobacteria 4,27 . Serial 10‐fold dilutions of the lysate were prepared in DMEM as 10 −1 , 10 −2 ,10 −3 ,10 −4 and then plated on LJ medium slants.…”

The Role of Airway Epithelial Cells in Response to Mycobacteria Infection

“…Bose et al demonstrated that in a murine TB model NO production played an essential role in Mtb killing by mononuclear phagocytes, particularly during the early phase of the infection. Additionally, it may also play a role in tissue damage during the late phases of the disease [88]. In vitro studies using A549 cells showed an induction of NO in response to Mtb infection, which is consistent with the finding that NO has antimicrobial effects against Mtb infection [89].…”

Nitric oxide production and nitric oxide synthase type 2 expression by cotton rat (Sigmodon hispidus) macrophages reflect the same pattern as human macrophages

“…Interestingly enough, NO secretion by human macrophages is enhanced when alveolar macrophages are obtained from patients with lung injury, chronic lung inflammation [3] or patients with tuberculosis [44,45]. In the latter case, killing of mycobacteria in vitro by these macrophages was inhibited by NO inhibitors, thus indicating an antimicrobial role for NO, possibly also in humans during mycobacterial infection [46]. Currently, the mouse is an often-used animal model for the study of tuberculosis, and whether the differences in the antibacterial properties of human and mouse macrophages are relevant to study outcomes in the mouse has not been determined.…”