The innate inflammatory immune response must be tightly controlled to avoid damage to the host. Here, we showed that the tuberous sclerosis complex-mammalian target of rapamycin (TSC-mTOR) pathway regulated inflammatory responses after bacterial stimulation in monocytes, macrophages, and primary dendritic cells. Inhibition of mTOR by rapamycin promoted production of proinflammatory cytokines via the transcription factor NF-kappaB but blocked the release of interleukin-10 via the transcription factor STAT3. Conversely, deletion of TSC2, the key negative regulator of mTOR, diminished NF-kappaB but enhanced STAT3 activity and reversed this proinflammatory cytokine shift. Rapamycin-hyperactivated monocytes displayed a strong T helper 1 (Th1) cell- and Th17 cell-polarizing potency. Inhibition of mTOR in vivo regulated the inflammatory response and protected genetically susceptible mice against lethal Listeria monocytogenes infection. These data identify the TSC2-mTOR pathway as a key regulator of innate immune homeostasis with broad clinical implications for infectious and autoimmune diseases, vaccination, cancer, and transplantation.

Aconitases are important cellular targets of nitric oxide (NO ⅐ ) toxicity, and NO ⅐ -derived species, rather than NO ⅐ per se, have been proposed to mediate their inactivation. NO ⅐ -mediated inactivation of the Escherichia coli aconitase and the porcine mitochondrial aconitase was investigated. In E. coli, aconitase activity decreased by ϳ70% during a 2-h exposure to an atmosphere containing 120 ppm NO ⅐ in N 2 . The NO ⅐ -inactivated aconitase reactivated poorly in E. coli under anaerobic or aerobic conditions. Elevated superoxide dismutase activity did not affect the aerobic inactivation of aconitase by NO ⅐ , thus indicating a limited role of the NO ⅐ -and superoxide-derived species peroxynitrite. Glutathionedeficient and glutathione-containing E. coli were comparably sensitive to NO ⅐ -mediated aconitase inactivation, thus excluding the participation of S-nitrosoglutathione or more oxidizing NO ⅐ -derived species. NO ⅐ progressively decreased aconitase activity in extracts in the presence of substrates, and inactivation was greatest at an acidic pH with cis-aconitate. The porcine mitochondrial aconitase was sensitive to NO ⅐ when exposed at pH 6.5, but not at pH 7. Nitric oxide (NO ⅐ ) is released as an intermediate of dissimilatory and assimilatory pathways of nitrite reduction in bacteria and fungi (1, 2). It is also produced by macrophages, neutrophils, endothelial cells, and epithelial cells to combat invading microorganisms and neoplastic tissue (3-5). In addition, NO ⅐ serves important functions as a second messenger for neurons and the vascular system (6, 7).NO ⅐ is cytotoxic. Mechanisms of NO ⅐ toxicity are complex and may include its direct reaction with heme, non-heme iron, and copper proteins (8). Other NO ⅐ -derived species including nitroxyl anion, nitrosonium, peroxynitrite (ONOO Ϫ ), nitrogen dioxide, and nitrosothiols may increase the spectrum of NO ⅐ -mediated damage to cells (9). The avid reactivity of NO ⅐ with superoxide radical (O 2 . ) to form the less discriminant oxidant ONOO Ϫ provides a potent mechanism for the deleterious actions of NO ⅐ (10, 11). Nevertheless, a thorough understanding of NO ⅐ toxicity continues to demand a greater knowledge of the NO ⅐ -sensitive targets and the NO ⅐ -derived species involved.The mammalian [4Fe-4S] cytoplasmic and mitochondrial aconitases are particularly sensitive to inactivation by NO ⅐ produced during various pathological conditions (12-18). In mammals, the cytoplasmic aconitase serves as an mRNA-binding regulator of iron homeostasis and may also participate with the mitochondrial aconitase as a catalyst of the energy-yielding reactions of the citric acid cycle (19,20). In addition, there is a growing family of homologous labile [4Fe-4S] (de)hydratases serving important biosynthetic and energy-yielding functions (21, 22), which may account for the toxicity of NO ⅐ toward a variety of organisms under a variety of conditions. Yet, it remains unclear how NO ⅐ inactivates aconitases in vivo. The NO ⅐ resistance of various isolated aconitases ha...

Recent studies have demonstrated that melatonin is a scavenger of oxyradicals and peroxynitrite and an inhibitor of nitric oxide (NO) production. NO, peroxynitrite (formed from NO and superoxide anion), and poly (ADP-Ribose) synthetase (PARS) have been implicated as mediators of neuronal damage following focal ischemia. In the present study, we have investigated the effects of melatonin treatment in Mongolian gerbils subjected to cerebral ischemia. Treatment of gerbils with melatonin (10 mg kg(-1), 30 min before reperfusion and 1, 2, and 6 hr after reperfusion) reduced the formation of post-ischemic brain edema, evaluated by water content. Melatonin also attenuated the increase in the brain levels malondialdehyde (MDA) and the increase in the hippocampus of myeloperoxidase (MPO) caused by cerebral ischemia. Positive staining for nitrotyrosine was found in the hippocampus of Mongolian gerbils subjected to cerebral ischemia. Hippocampus tissue sections, from Mongolian gerbils subjected to cerebral ischemia, also showed positive staining for PARS. The degrees of staining for nitrotyrosine and for PARS were markedly reduced in tissue sections obtained from animals that received melatonin. Melatonin treatment increased survival and reduced hyperactivity linked to neurodegeneration induced by cerebral ischemia and reperfusion. Histological observations of the pyramidal layer of CA-1 showed a reduction of neuronal loss in animals that received melatonin. These results show that melatonin improves brain injury induced by transient cerebral ischemia.

1 Peroxynitrite, a potent cytotoxic oxidant formed by the reaction of nitric oxide with superoxide anion, and hydroxyl radical, formed in the iron-catalysed Fenton reaction, are important mediators of reperfusion injury. In in vitro studies, DNA single strand breakage, triggered by peroxynitrite or by hydroxyl radical, activates the nuclear enzyme poly (ADP-ribose) synthetase (PARS), with consequent cytotoxic e ects. Using 3-aminobenzamide, an inhibitor of PARS, we investigated the role of PARS in the pathogenesis of splanchnic artery occlusion shock. 2 Splanchnic artery occlusion and reperfusion shock (SAO/R) was induced in rats by clamping both the superior mesenteric artery and the coeliac trunk for 45 min, followed by release of the clamp (reperfusion). At 60 min after reperfusion, animals were killed for histological examination and biochemical studies. 3 SAO/R rats developed a signi®cant fall in mean arterial blood pressure, signi®cant increase of tissue myeloperoxidase activity and marked histological injury to the distal ileum. SAO/R was also associated with a signi®cant mortality (0% survival at 2 h after reperfusion). 4 There was a marked increase in the oxidation of dihydrorhodamine 123 to rhodamine (a marker of peroxynitrite-induced oxidative processes) in the plasma of the SAO/R rats, starting early after reperfusion, but not during ischaemia alone. Immunohistochemical examination demonstrated a marked increase in the immunoreactivity to nitrotyrosine, a speci®c`footprint' of peroxynitrite, in the necrotic ileum in shocked rats, as measured at 60 min after the start of reperfusion. 5 In addition, in ex vivo studies in aortic rings from shocked rats, we found reduced contractions to noradrenaline and reduced responsiveness to a relaxant e ect to acetylcholine (vascular hyporeactivity and endothelial dysfunction, respectively). 6 In a separate set of studies, using a 4000 Dalton¯uorescent dextran tracer, we investigated the changes in epithelial permeability associated with SAO/R. Ten minutes of reperfusion, after 30 min of splanchnic artery ischaemia, resulted in a marked increase in epithelial permeability. 7 There was a signi®cant increase in PARS activity in the intestinal epithelial cells, as measured 10 min after reperfusion ex vivo. 3-Aminobenzamide, a pharmacological inhibitor of PARS (applied at 10 mg kg 71 , i.v., 5 min before reperfusion, followed by an infusion of 10 mg kg 71 h 71 ), signi®cantly reduced ischaemia/reperfusion injury in the bowel, as evaluated by histological examination. Also it signi®cantly improved mean arterial blood pressure, improved contractile responsiveness to noradrenaline, enhanced the endothelium-dependent relaxations and reduced the reperfusion-induced increase in epithelial permeability. 8 3-Aminobenzamide also prevented the in®ltration of neutrophils into the reperfused intestine, as evidenced by reduced myeloperoxidase activity. It improved the histological status of the reperfused tissues, reduced the production of peroxynitrite in the late phase of reperfu...