Vishwanath Venketaraman

Cytolytic P2X purinoceptors

Anedoctal evidence accumulated over almost 20 years has shown that many different cell types are killed by sustained exposure to high concentrations of extracellular ATP. The plasma membrane receptors involved have been pharmacologically characterized and cloned during the last 3 years, and named purinergic P2X. P2X receptors share an intriguing structural relatedness with Caenorhabditis elegans degenerins and mammalian amiloride-sensitive Na channels (ENaCs). Depending on the ATP dose, length of stimulation and receptor subtype, P2X receptor stimulation may cause necrosis or apoptosis. The intracellular pathways activated are poorly known, but the perturbation in intracellular ion homeostasis clearly plays a major role. ICE proteases (caspases) are also triggered, nonetheless their activation is not requested for ATP-dependent cell death. The physiological meaning of P2X receptor-dependent cytotoxicity is not understood, but an involvement in immune-mediated reactions is postulated.

Glutathione and infection.

BACKGROUND The tripeptide γ-glutamylcysteinylglycine or glutathione (GSH) has demonstrated protective abilities against the detrimental effects of oxidative stress within the human body, as well as protection against infection by exogenous microbial organisms. SCOPE OF REVIEW In this review we describe how GSH works to modulate the behavior of many cells including the cells of the immune system, augmenting the innate and the adaptive immunity as well as conferring protection against microbial, viral and parasitic infections. This article unveils the direct antimicrobial effects of GSH in controlling Mycobacterium tuberculosis (M. tb) infection within macrophages. In addition, we summarize the effects of GSH in enhancing the functional activity of various immune cells such as natural killer (NK) cells and T cells resulting in inhibition in the growth of M. tb inside monocytes and macrophages. Most importantly we correlate the decreased GSH levels previously observed in individuals with pulmonary tuberculosis (TB) with an increase in the levels of pro-inflammatory cytokines which aid in the growth of M. tb. MAJOR CONCLUSIONS In conclusion, this review provides detailed information on the protective integral effects of GSH along with its therapeutic effects as they relate to the human immune system and health. GENERAL SIGNIFICANCE It is important to note that the increases in the levels of pro-inflammatory cytokines are not only detrimental to the host due to the sequel that follow such as fever and cachexia, but also due to the alteration in the functions of immune cells. The additional protective effects of GSH are evident after sequel that follows the depletion of this antioxidant. This is evident in a condition such as Cystic Fibrosis (CF) where an increased oxidant burden inhibits the clearance of the affecting organism and results in oxidant-induced anti-protease inhibition. GSH has a similar protective effect in protozoans as it does in human cells. Thus GSH is integral to the survival of some of the protozoans because some protozoans utilize the compound trypanothione [T(SH)2] as their main antioxidant. T(SH)2 in turn requires GSH for its production. Hence a decrease in the levels of GSH (by a known inhibitor such as buthionine sulfoximine [BSO] can have adverse effects of the protozoan parasites. This article is part of a Special Issue entitled Cellular functions of glutathione.

Glutathione and Nitrosoglutathione in Macrophage Defense against Mycobacterium tuberculosis

ABSTRACT We demonstrate that Mycobacterium tuberculosis grown in vitro is sensitive to glutathione and its derivative S-nitrosoglutathione. Furthermore, our infection studies with J774.1 macrophages indicate that glutathione is essential for the control of the intracellular growth of M. tuberculosis. This study indicates the important role of glutathione in the control of macrophages by M. tuberculosis.

Role of Glutathione in Macrophage Control of Mycobacteria

ABSTRACT Reactive oxygen and nitrogen intermediates are important antimicrobial defense mechanisms of macrophages and other phagocytic cells. While reactive nitrogen intermediates have been shown to play an important role in tuberculosis control in the murine system, their role in human disease is not clearly established. Glutathione, a tripeptide and antioxidant, is synthesized at high levels by cells during reactive oxygen intermediate and nitrogen intermediate production. Glutathione has been recently shown to play an important role in apoptosis and to regulate antigen-presenting-cell functions. Glutathione also serves as a carrier molecule for nitric oxide, in the form of S-nitrosoglutathione. Previous work from this laboratory has shown that glutathione and S-nitrosoglutathione are directly toxic to mycobacteria. A mutant strain of Mycobacterium bovis BCG, defective in the transport of small peptides such as glutathione, is resistant to the toxic effect of glutathione and S-nitrosoglutathione. Using the peptide transport mutant as a tool, we investigated the role of glutathione and S-nitrosoglutathione in animal and human macrophages in controlling intracellular mycobacterial growth.

Characterization of a Glutathione Metabolic Mutant of Mycobacterium tuberculosis and Its Resistance to Glutathione and Nitrosoglutathione

Glutathione is a tripeptide and antioxidant, synthesized at high levels by cells during the production of reactive oxygen and nitrogen intermediates. Glutathione also serves as a carrier molecule for nitric oxide in the form of S-nitrosoglutathione. Previous studies from this laboratory have shown that glutathione and S-nitrosoglutathione are directly toxic to mycobacteria. Glutathione is not transported into the cells as a tripeptide. Extracellular glutathione is converted to a dipeptide due to the action of transpeptidase, and the dipeptide is then transported into the bacterial cells. The processing of glutathione and S-nitrosoglutathione is brought about by the action of the enzyme gamma-glutamyl transpeptidase. The function of gamma-glutamyl transpeptidase is to cleave glutathione and S-nitrosoglutathione to the dipeptide (Cys-Gly), which is then transported into the bacterium by the multicomponent ABC transporter dipeptide permease. We have created a mutant strain of Mycobacterium tuberculosis lacking this metabolic enzyme. We investigated the sensitivity of this strain to glutathione and S-nitrosoglutathione compared to that of the wild-type bacteria. In addition, we examined the role of glutathione and/or S-nitrosoglutathione in controlling the growth of intracellular M. tuberculosis inside mouse macrophages.

Control of Mycobacterium tuberculosis growth by activated natural killer cells.

We characterized the underlying mechanisms by which glutathione (GSH)‐enhanced natural killer (NK) cells inhibit the growth of Mycobacterium tuberculosis (M. tb) inside human monocytes. We observed that in healthy individuals, treatment of NK cells with N‐acetyl cysteine (NAC), a GSH prodrug in conjunction with cytokines such as interleukin (IL)‐2 + IL‐12, resulted in enhanced expression of NK cytotoxic ligands (FasL and CD40L) with concomitant stasis in the intracellular growth of M. tb. Neutralization of FasL and CD40L in IL‐2 + IL‐12 + NAC‐treated NK cells resulted in abrogation in the growth inhibition of M. tb inside monocytes. Importantly, we observed that the levels of GSH are decreased significantly in NK cells derived from individuals with HIV infection compared to healthy subjects, and this decrease correlated with a several‐fold increase in the growth of M. tb inside monocytes. This study describes a novel innate defence mechanism adopted by NK cells to control M. tb infection.

Glutathione and Adaptive Immune Responses against Mycobacterium tuberculosis Infection in Healthy and HIV Infected Individuals

Glutathione (GSH), a tripeptide antioxidant, is essential for cellular homeostasis and plays a vital role in diverse cellular functions. Individuals who are infected with Human immuno deficiency virus (HIV) are known to be susceptible to Mycobacterium tuberculosis (M. tb) infection. We report that by enhancing GSH levels, T-cells are able to inhibit the growth of M. tb inside macrophages. In addition, those GSH-replenished T cell cultures produced increased levels of Interleukin-2 (IL-2), Interleukin-12 (IL-12), and Interferon-gamma (IFN-γ), cytokines, which are known to be crucial for the control of intracellular pathogens. Our study reveals that T lymphocytes that are derived from HIV infected individuals are deficient in GSH, and that this deficiency correlates with decreased levels of Th1 cytokines and enhanced growth of M. tb inside human macrophages.

Unveiling the Mechanisms for Decreased Glutathione in Individuals with HIV Infection

We examined the causes for decreased glutathione (GSH) in individuals with HIV infection. We observed lower levels of intracellular GSH in macrophages from individuals with HIV compared to healthy subjects. Further, the GSH composition found in macrophages from HIV+ subjects heavily favors oxidized glutathione (GSSG) which lacks antioxidant activity, over free GSH which is responsible for GSHs antioxidant activity. This decrease correlated with an increase in the growth of Mycobacterium tuberculosis (M. tb) in macrophages from HIV+ individuals. In addition, we observed increased levels of free radicals, interleukin-1 (IL-1), interleukin-17 (IL-17) and transforming growth factor-β (TGF-β) in plasma samples derived from HIV+ individuals compared to healthy subjects. We observed decreased expression of the genes coding for enzymes responsible for de novo synthesis of GSH in macrophages derived from HIV+ subjects using quantitative PCR (qPCR). Our results indicate that overproduction of proinflammatory cytokines in HIV+ individuals lead to increased production of free radicals. This combined with the decreased expression of GSH synthesis enzymes leads to a depletion of free GSH and may lead in part to the loss of immune function observed in HIV patients.

Both leukotoxin and poly-N-acetylglucosamine surface polysaccharide protect Aggregatibacter actinomycetemcomitans cells from macrophage killing.

Two virulence factors produced by the periodontopathogen Aggregatibacter actinomycetemcomitans are leukotoxin, a secreted lipoprotein that kills human polymorphonuclear leukocytes and macrophages, and poly-N-acetylglucosamine (PGA), a surface polysaccharide that mediates intercellular adhesion, biofilm formation and detergent resistance. In this study we examined the roles of leukotoxin and PGA in protecting A. actinomycetemcomitans cells from killing by the human macrophage cell line THP-1. Monolayers of THP-1 cells were infected with single-cell suspensions of a wild-type A. actinomycetemcomitans strain, or of isogenic leukotoxin or PGA mutant strains. After 48h, viable bacteria were enumerated by dilution plating, macrophage morphology was evaluated microscopically, and macrophage viability was measured by a Trypan blue dye exclusion assay. The number of A. actinomycetemcomitans CFUs increased approximately twofold in wells infected with the wild-type strain, but decreased by approximately 70-90% in wells infected with the leukotoxin and PGA mutant strains. Infection with the wild-type or leukotoxin mutant strain caused a significant decrease in THP-1 cell viability, whereas infection with the PGA mutant strain did not result in any detectable changes in THP-1 viability. Pre-treatment of wild-type A. actinomycetemcomitans cells with the PGA-hydrolyzing enzyme dispersin B rendered them sensitive to killing by THP-1 cells. We concluded that both leukotoxin and PGA are necessary for evasion of macrophage killing by A. actinomycetemcomitans.

Arginine Homeostasis in J774.1 Macrophages in the Context of Mycobacterium bovis BCG Infection

The competition for L-arginine between the inducible nitric oxide synthase and arginase contributes to the outcome of several parasitic and bacterial infections. The acquisition of L-arginine, however, is important not only for the host cells but also for the intracellular pathogen. In this study we observe that strain AS-1, the Mycobacterium bovis BCG strain lacking the Rv0522 gene, which encodes an arginine permease, perturbs l-arginine metabolism in J774.1 murine macrophages. Infection with AS-1, but not with wild-type BCG, induced l-arginine uptake in J774.1 cells. This increase in L-arginine uptake was independent of activation with gamma interferon plus lipopolysaccharide and correlated with increased expression of the MCAT1 and MCAT2 cationic amino acid transport genes. AS-1 infection also enhanced arginase activity in resting J774.1 cells. Survival studies revealed that AS-1 survived better than BCG within resting J774.1 cells. Intracellular growth of AS-1 was further enhanced by inhibiting arginase and ornithine decarboxylase activities in J774.1 cells using L-norvaline and difluoromethylornithine treatment, respectively. These results suggest that the arginine-related activities of J774.1 macrophages are affected by the arginine transport capacity of the infecting BCG strain. The loss of Rv0522 gene-encoded arginine transport may have induced other cationic amino acid transport systems during intracellular growth of AS-1, allowing better survival within resting macrophages.