A key role for heme oxygenase-1 in nitric oxide resistance in murine motor neurons and glia

Nitric oxide is utilized at low levels for intercellular signaling, and at high levels as a cytotoxic weapon during inflammation. Cellular NO resistance can be increased by prior exposure to sublethal NO levels to induce defense gene expression (adaptive NO resistance), which has been correlated with increased expression of heme oxygenase-1 (HO1) and was blocked by a heme oxygenase inhibitor. However, the possibility remained that other activities were affected by the inhibitor. To address this question, we conducted a genetic study of the HO1 role. We show here that primary cultures of spinal motor neurons and glia from homozygous HO1-null mice are strikingly more sensitive to NO cytotoxicity than are cells expressing HO1. Following an exposure to NO, the HO1-deficient cells were much more prone to apoptosis than were HO1-expressing cells with either one or two copies of a functional HO1 gene. These results confirm the in vivo role of HO1 as a front-line defense against NO toxicity in neuronal cells.     

Examination of bacterial resistance to exogenous nitric oxide

While much research has been directed to harnessing the antimicrobial properties of exogenous NO, the possibility of bacteria developing resistance to such therapy has not been thoroughly studied. Herein, we evaluate potential NO resistance using spontaneous and serial passage mutagenesis assays. Specifically, Staphylococcus aureus, Methicillin-resistant S. aureus (MRSA), Staphylococcus epidermidis, Escherichia coli, and Pseudomonas aeruginosa were systematically exposed to NO-releasing 75mol% MPTMS-TEOS nitrosothiol particles at or below minimum inhibitory concentration (MIC) levels. In the spontaneous mutagenesis assay, bacteria that survived exposure to lethal concentrations of NO showed no increase in MIC. Similarly, no increase in MIC was observed in the serial passage mutagenesis assay after exposure of these species to sub-inhibitory concentrations of NO through 20 d.     

Hypothermia translocates nitric oxide synthase from cytosol to membrane in snail neurons

Neuronal nitric oxide (NO) levels are modulated through the control of catalytic activity of NO synthase (NOS). Although signals limiting excess NO synthesis are being extensively studied in the vertebrate nervous system, our knowledge is rather limited on the control of NOS in neurons of invertebrates. We have previously reported a transient inactivation of NOS in hibernating snails. In the present study, we aimed to understand the mechanism leading to blocked NO production during hypothermic periods of Helix pomatia. We have found that hypothermic challenge translocated NOS from the cytosol to the perinuclear endoplasmic reticulum, and that this cytosol to membrane trafficking was essential for inhibition of NO synthesis. Cold stress also downregulated NOS mRNA levels in snail neurons, although the amount of NOS protein remained unaffected in response to hypothermia. Our studies with cultured neurons and glia cells revealed that glia-neuron signaling may inhibit membrane binding and inactivation of NOS. We provide evidence that hypothermia keeps NO synthesis "hibernated" through subcellular redistribution of NOS.     

Role of nitric oxide and reactive oxide species in disease resistance to necrotrophic pathogens

Nitric oxide (NO) and reactive oxygen species (ROS) are important signaling molecules in plant immunity. However, roles of NO and ROS in disease resistance to necrotrophic pathogens are not fully understood. We have recently demonstrated that NO plays a pivotal role in basal defense against Botrytis cinerea and the expression of the salicylic acid (SA)-responsive gene PR-1 in Nicotiana benthamiana. By contrast, ROS function negatively in resistance or positively in expansion of disease lesions during B. cinerea-N. benthamiana interaction. Here, analysis in NahG-transgenic N. benthamiana showed that SA signaling is not involved in resistance to B. cinerea in N. benthamiana. We discuss how NO and ROS participate in disease resistance to necrotrophic pathogens on the basis of recent reports.Key words: NO burst, oxidative burst, necrotrophic pathogen, salicylic acid, plant immunity, MAPKNecrotrophs are pathogens that kill host cells by means of toxic molecules and lytic enzymes, and they feed on the remains for their own growth. If the toxic molecule shows differential activity to one or a few plant species, the pathogen has a limited host range and the metabolite is referred to as a host-selective toxin (HST).¹ Several well-studied necrotrophs, in particular Cochliobolus and Alternaria spp., produce HSTs required for the pathogenicity. There are also necrotrophic fungal pathogens with a broad host range, particularly those in the order of Helotiales, including Sclerotinia sclerotiorum and Botrytis cinerea.Rapid production of nitric oxide (NO) and reactive oxygen species (ROS), called NO burst and oxidative burst, respectively, is one of the earliest responses of plants to pathogen attacks. Our recent study showed that NO and oxidative bursts accompanied by activation of the mitogen-activated protein kinase (MAPK)² are induced after inoculation with B. cinerea, and that NO plays a key role, but ROS have an opposite effect in basal defense against B. cinerea in Nicotiana benthamiana.³ NO and ROS are believed to play key roles independently or coordinately in plant innate immunity.^4,5 NO signaling comprises complex processes including increases in cytosolic Ca²⁺ concentration, cyclic GMP (cGMP), cyclic ADP ribose and activation of protein kinases. NO also modulates protein activities directly by cysteine S-nitrosylation.⁶ In addition, NO appears to act as an antioxidant of ROS, because NO can react quickly with superoxide (O₂⁻) to form peroxynitrite (ONOO⁻) and then, reduces the amount of endogenous ROS. Actually, treatment with a mammalian NO synthase inhibitor and silencing NbNOA1 decreased endogenous NO levels and increased the levels of ROS after inoculation with B. cinerea.³ The suppression of NO burst induced high susceptibility to B. cinerea, and depletion of oxidative burst by an NADPH oxidase inhibitor or silencing NbRBOHB led to reduction in disease lesions by B. cinerea,³ suggesting that the growth of B. cinerea might be determined by endogenous levels of ROS which is an important component of virulence.⁷ However, depletion of both NO and oxidative bursts by double silencing NbNOA1/NbRBOHB resulted in expansion of disease lesions compared with reduction of oxidative burst alone by silencing NbRBOHB.³ Similarly, our most recent study showed that silencing NbRibA which compromises production of both NO and ROS do not affect basal resistance against B. cinerea.⁸ These findings suggest that NO positively functions in resistance to necrotrophic pathogens in the manner other than as an antioxidant of ROS.The relationship between NO and salicylic acid (SA) has been studied.⁹ SA signaling-deficient mutants of Arabidopsis thaliana show high susceptibility to B. cinerea.^10,11 We have suggested that reduced basal defense against B. cinerea in N. benthamiana resulting from compromised endogenous NO production may be due to depletion of SA signaling, because NbNOA1-silenced plants showed suppression of the SA-responsive gene NbPR-1 expression induced by inoculation with B. cinerea.³ To confirm the possibility, we used N. benthamiana expressing NahG that converts all SA to catechol. NahG and non-NahG (WT) leaves were inoculated with B. cinerea. NahG plants showed similar susceptibility to B. cinerea compared with WT plants (Fig. 1). We also evaluated effects of silencing NbNOA1 and NbRBOHB in NahG plants on susceptibility to B. cinerea. Like NbNOA1-silenced WT plants shown previusly,³ NbNOA1-silenced NahG leaves showed high susceptibility to B. cinerea. On the other hand, NbRBOHB-silenced NahG leaves showed marked reduction of disease lesions compared with silencing-control NahG leaves. NbNOA1/NbRBOHB-silenced NahG leaves showed expansion of disease lesions compared with NbRBOHB-silenced NahG leaves (Fig. 2). These results suggest that NO-mediated basal defense against B. cinerea is not due to SA signaling, and effects of ROS on disease lesions may not depend on SA in N. benthamiana.Open in a separate windowFigure 1Effects of NahG transgene on susceptibility to B. cinerea. NahG and non-NahG (WT) leaves were inoculated with B. cinerea conidial suspension (1 × 10⁵ conidia/ml). (A) Inoculated leaves were photographed at 4 days postinoculation (dpi). (B) Average diameter of lesions formed on the leaves at 3 and 4 dpi. Data are means ± SD from fourteen experiments.Open in a separate windowFigure 2Effects of silencing NbNOA1 (N), NbRBOHB (B) or NbNOA1/NbRBOHB (N/B) in NahG plants on susceptibility to B. cinerea. Silenced NahG leaves were inoculated with B. cinerea conidial suspension (1 × 10⁵ conidia/ml). (A) Inoculated leaves were photographed at 4 dpi. (B) Average diameter of lesions formed on the leaves at 3 and 4 dpi. Data are means ± SD from four experiments. Data were subjected to Student''s t-test. *p < 0.05 versus silencing-control plants (TRV). **p < 0.05 versus NbRBOHB-silenced plants.Recently, it has been reported that NO and ROS are involved in HSTs responses.^12–15 Victorin, an HST produced by Cochliobolus victoriae, elicits generation of NO and ROS in victorin-sensitive oat leaves.¹² Cell death induced by victorin is suppressed by treatment with ROS scavengers.¹³ Similarly, treatment with ToxA, an HST produced by Pyrenophora triticirepentis, induces oxidative burst, and scavenging ROS compromises ToxA-inducible cell death in ToxA-sensitive wheatleaves.^14,15 SA-induced MAPK, which regulates both NO and ROS production,² is activated by AAL-toxin produced by Alternaria alternata f. sp. lycopersici in AAL-toxin-sensitive tobacco (Mizuno et al. unpublished data). These findings indicate requirement of ROS for the HST-inducible cell death and participation of NO in HST responses.In conclusion, NO and ROS appear to play a contrasting role in disease resistance to necrotrophic pathogens as shown in Figure 3. However, how NO signaling participates in defense responses against necrotrophic pathogens has yet to be elucidated. Recently, several targets of protein S-nitrosylation during hypersensitive response have been characterized in A. thaliana.¹⁶ Evidence is also accumulating for cGMP as an important component of NO-related signal transduction.¹⁷ Further investigations of NO signaling will lead to our understanding of interactions between plants and necrotrophic pathogens.Open in a separate windowFigure 3Model showing role of NO and oxidative bursts in disease resistance to necrotrophic pathogens. After recognition of necrotrophs, plants immediately provoke activation of MAPK which could regulate production of both NO and ROS,² and then NO and oxidative bursts. NO burst plays an important role in disease resistance to necrotrophic pathogens, whereas oxidative burst has a negative role in resistance or has a positive role in expansion of disease lesions by necrotrophs.     

Neuronal nitric oxide synthase proteolysis limits the involvement of nitric oxide in kainate-induced neurotoxicity in hippocampal neurons

In this work, we investigated the role of nitric oxide (NO) in neurotoxicity triggered by alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptor activation in cultured hippocampal neurons. In the presence of cyclothiazide (CTZ), short-term exposures to kainate (KA; 5 and 15 min, followed by 24-h recovery) decreased cell viability. Both NBQX and d-AP-5 decreased the neurotoxicity caused by KA plus CTZ. Long-term exposures to KA plus CTZ (24 h) resulted in increased toxicity. In short-, but not in long-term exposures, the presence of NO synthase (NOS) inhibitors (l-NAME and 7-NI) decreased the toxicity induced by KA plus CTZ. We also found that KA plus CTZ (15-min exposure) significantly increased cGMP levels. Furthermore, short-term exposures lead to decreased intracellular ATP levels, which was prevented by NBQX, d-AP-5 and NOS inhibitors. Immunoblot analysis revealed that KA induced neuronal NOS (nNOS) proteolysis, gradually lowering the levels of nNOS according to the time of exposure. Calpain, but not caspase-3 inhibitors, prevented this effect. Overall, these results show that NO is involved in the neurotoxicity caused by activation of non-desensitizing AMPA receptors, although to a limited extent, since AMPA receptor activation triggers mechanisms that lead to nNOS proteolysis by calpains, preventing a further contribution of NO to the neurotoxic process.     

Projections of nitric oxide synthesizing neurons in the guinea-pig colon

The neuronal form of the enzyme nitric oxide synthase, which is an obligatory constituent of neurons that utilise nitric oxide as a transmitter, was revealed histochemically in this study by its ability to transfer a proton from reduced nicotinamide adenine dinucleotide phosphate to nitro-blue tetrazolium. In the guinea-pig colon, nitric oxide synthase was located in numerous irregularly-shaped myenteric neurons with single axons. In the submucosa, a small number of neurons had strong enzyme activity, whereas many were weakly stained. Nerve fibres were found in the longitudinal muscle, circular muscle, muscularis mucosae and ganglia of the two plexuses. No nerve fibres were found in the lamina propria of the mucosa. The same distribution of nerve cells and fibres was revealed using immunohistochemistry for nitric oxide synthase. Lesion studies showed that the axons of myenteric neurons all projected anally. Myenteric cells were the source of nerve fibres in the circular muscle and in more anally located myenteric ganglia. The sparse innervation of submucous ganglia was intrinsic to the submucous plexus. It is suggested that nitric oxide synthase is one of the transmitters of inhibitory neurons to the muscle and is also utilized by descending interneurons of the myenteric plexus.     

Cytochrome c' from Rhodobacter capsulatus confers increased resistance to nitric oxide

We report the cloning and sequencing of the gene containing cytochrome c' (cycP) from the photosynthetic purple bacterium Rhodobacter capsulatus and the regions flanking that gene. Mutant strains unable to synthesize cytochrome c' had increased sensitivity to nitrosothiols and to nitric oxide (which binds to the heme moiety of cytochrome c').     

Mechanism of strong resistance of Helicobacter pylori respiration to nitric oxide

The aim of the present work is to elucidate the mechanism by which the respiration of Helicobacter pylori but not of Escherichia coli shows a strong resistance to nitric oxide (NO). Nitric oxide strongly but reversibly inhibited the oxygen consumption by sonicated membranes from H. pylori and Triton X-100-treated cells. Although the sensitivity of the H. pylori respiration to cyanide was low, it also increased after the treatment with Triton X-100. Kinetic analyses revealed that NO was rapidly degraded by E. coli and the Triton X-100-treated H. pylori, but not by the intact H. pylori. Thus, the low sensitivity to NO might reflect the low affinity of the cytochrome c oxidase for this radical within the membrane/lipid bilayers of H. pylori. Such properties of the oxidase in H. pylori membranes may, at least in part, underlie the mechanism by which this bacterium thrives in NO-enriched gastric juice.     

Analysis of nitric oxide donor effectiveness in resistance vessels

Decreased nitric oxide (NO) bioavailability is associated with a number of pathological conditions. Administration of a supplemental source of NO can counter the pathological effects arising from decreased NO bioavailability. A class of NO-nucleophile adducts that spontaneously release NO (NONOates) has been developed, and its members show promise as therapeutic sources of NO. Because the NONOates release NO spontaneously, a significant portion of the NO may be consumed by the myriad of NO reactive species present in the body. Here we develop a model to analyze the efficacy of NO delivery, by membrane-impermeable NONOates, in the resistance arterioles. Our model identifies three features of blood vessels that will enhance NONOate efficacy: 1) the amount of NO delivered to the abluminal region increases with lumen radius; 2) the presence of a flow-induced red blood cell-free zone will augment NO delivery; and 3) extravasation of the NONOate into the interstitial space will increase abluminal NO delivery. These results suggest that NONOates may be more effective in larger vessels and that NONOate efficacy can be altered by modifying permeability to the interstitial space.     

Spatial organization of neurons in the dorsal motor nucleus of the vagus synapsing with intragastric cholinergic and nitric oxide/VIP neurons in the rat

The dorsal motor nucleus of the vagus (DMV) contains preganglionic neurons that control gastric motility and secretion. Stimulation of different parts of the DMV results in a decrease or an increase in gastric motor activities, suggesting a spatial organization of vagal preganglionic neurons in the DMV. Little is known about how these preganglionic neurons in the DMV synapse with different groups of intragastric motor neurons to mediate contraction or relaxation of the stomach. We used pharmacological and immunohistochemical methods to characterize intragastric neural pathways involved in mediating gastric contraction and relaxation in rats. Microinjections of L-glutamate (L-Glu) into the rostral or caudal DMV produced gastric contraction and relaxation, respectively, in a dose-related manner. Intravenous infusion of hexamethonium blocked these actions, suggesting mediation via preganglionic cholinergic pathways. Atropine inhibited gastric contraction by 85.5 +/- 4.5%. Gastric relaxation was reduced by intravenous administration of N(G)-nitro-L-arginine methyl ester (L-NAME; 52.5 +/- 11.9%) or VIP antagonist (56.3 +/- 14.9%). Combined administration of L-NAME and VIP antagonist inhibited gastric relaxation evoked by L-Glu (87.8 +/- 4.3%). Immunohistochemical studies demonstrated choline acetyltransferase immunoreactivity in response to L-Glu microinjection into the rostral DMV in 88% of c-Fos-positive intragastric myenteric neurons. Microinjection of L-Glu into the caudal DMV evoked expression of nitric oxide (NO) synthase and VIP immunoreactivity in 81 and 39%, respectively, of all c-Fos-positive intragastric myenteric neurons. These data indicate spatial organization of the DMV. Depending on the location, microinjection of L-Glu into the DMV may stimulate intragastric myenteric cholinergic neurons or NO/VIP neurons to mediate gastric contraction and relaxation.     

IFN-gamma-dependent nitric oxide production is not linked to resistance in experimental African trypanosomiasis

Resistance to African trypanosomes is dependent on B cell and Th1 cell responses to the variant surface glycoprotein (VSG). While B cell responses to VSG control levels of parasitemia, the cytokine responses of Th1 cells to VSG appear to be linked to the control of parasites in extravascular tissues. We have recently shown that IFN-gamma knockout (IFN-gamma KO) mice are highly susceptible to infection and have reduced levels of macrophage activation compared to the wild-type C57BL/6 (WT) parent strain, even though parasitemias were controlled by VSG-specific antibody responses in both strains. In the present work, we examine the role of IFN-gamma in the induction of nitric oxide (NO) production and host resistance and in the development of suppressor macrophage activity in mice infected with Trypanosoma brucei rhodesiense. In contrast to WT mice, susceptible IFN-gamma KO mice did not produce NO during infection and did not develop suppressor macrophage activity, suggesting that NO might be linked to resistance but that suppressor cell activity was not associated with resistance or susceptibility to trypanosome infection. To further examine the consequence of inducible NO production in infection, we monitored survival, parasitemia, and Th cell cytokine production in iNOS KO mice. While survival times and parasitemia of iNOS KO mice did not differ significantly from WT mice, VSG-specific Th1 cells from iNOS KO mice produced higher levels of IFN-gamma and IL-2 than cells from WT mice. Together, these results show for the first time that inducible NO production is not the central defect associated with susceptibility of IFN-gamma KO mice to African trypanosomes, that IFNgamma-induced factors other than iNOS may be important for resistance to the trypanosomes, and that suppressor macrophage activity is not linked to either the resistance or the susceptibility phenotypes.     

Inputs from intrinsic primary afferent neurons to nitric oxide synthase-immunoreactive neurons in the myenteric plexus of guinea pig ileum

Nitric oxide synthase (NOS) immunoreactivity occurs in two groups of neurons in the guinea pig small intestine: descending interneurons that are also immunoreactive for choline acetyltransferase (ChAT), and inhibitory motor neurons that lack ChAT immunoreactivity. Interneurons that are involved in local reflexes would be expected to have inputs from intrinsic primary afferent (sensory) neurons, most of which are calbindin-immunoreactive. We examined this possibility using triple staining for NOS, ChAT and calbindin immunoreactivity and investigated the relationships between calbindin-immunoreactive varicosities and the cell bodies of NOS-immunoreactive neurons, using high-resolution confocal microscopy and electron microscopy. By confocal microscopy, we found that the cell bodies of ChAT/NOS interneurons received 84 +/- 23 (mean +/- SD) direct appositions from calbindin-immunoreactive varicosities and that the cell bodies of NOS-inhibitory motor neurons received 82 +/- 20 appositions. Electron-microscopic examination of the relations of 265-calbindin-immunoreactive varicosities, at distances within the resolution of the confocal microscope (300 nm), to 30 NOS-immunoreactive nerve cells indicated that 84% formed close contacts or synapses and 16% were separated from neurons by thin glial cell processes. Thus, each NOS-immunoreactive nerve cell receives about 70 synaptic inputs or close contacts from the calbindin-immunoreactive varicosities of intrinsic primary afferent neurons. It is concluded that there are monosynaptic reflex connections in which intrinsic primary afferent neurons synapse directly with motor neurons and di- or poly-synaptic reflexes in which ChAT- and NOS-immunoreactive neurons are interneurons, interposed between intrinsic primary afferent neurons and NOS-inhibitory neurons.     

Src kinase activation by nitric oxide promotes resistance to anoikis in tumour cell lines

Tumour progression involves the establishment of tumour metastases at distant sites. Resistance to anoikis, a form of cell death that occurs when cells lose contact with the extracellular matrix and with neighbouring cells, is essential for metastases. NO has been associated with anoikis. NO treated HeLa cells and murine melanoma cells in suspension triggered a nitric oxide (NO)-Src kinase signalling circuitry that enabled resistance to anoikis. Two NO donors, sodium nitroprusside (SNP) (500 µM) and DETANO (125 µM), protected against cell death derived from detachment of a growth permissive surface (experimental anoikis). Under conditions of NO-mediated Src activation the following were observed: (a) down-regulation of the pro-apoptotic proteins Bim and cleaved caspase-3 and the cell surface protein, E-cadherin, (b) up-regulation of caveolin-1, and (c) the dissociation of cell aggregates formed when cells are detached from a growth permissive surface. Efficiency of reattachment of tumour cells in suspension and treated with different concentrations of an NO donor, was dependent on the NO concentration. These findings indicate that NO-activated Src kinase triggers a signalling circuitry that provides resistance to anoikis, and allows for metastases.     

Regulation of obesity and insulin resistance by nitric oxide

Obesity is a risk factor for developing type 2 diabetes and cardiovascular disease and has quickly become a worldwide pandemic with few tangible and safe treatment options. Although it is generally accepted that the primary cause of obesity is energy imbalance, i.e., the calories consumed are greater than are utilized, understanding how caloric balance is regulated has proven a challenge. Many “distal” causes of obesity, such as the structural environment, occupation, and social influences, are exceedingly difficult to change or manipulate. Hence, molecular processes and pathways more proximal to the origins of obesity—those that directly regulate energy metabolism or caloric intake—seem to be more feasible targets for therapy. In particular, nitric oxide (NO) is emerging as a central regulator of energy metabolism and body composition. NO bioavailability is decreased in animal models of diet-induced obesity and in obese and insulin-resistant patients, and increasing NO output has remarkable effects on obesity and insulin resistance. This review discusses the role of NO in regulating adiposity and insulin sensitivity and places its modes of action into context with the known causes and consequences of metabolic disease.     

Nitric oxide from neuronal nitric oxide synthase sensitises neurons to hypoxia-induced death via competitive inhibition of cytochrome oxidase

Hypoxia/ischaemia is known to trigger neuronal death, but the role of neuronal nitric oxide synthase (nNOS) in this process is controversial. Nitric oxide (NO) inhibits cytochrome oxidase in competition with oxygen. We tested whether NO derived from nNOS synergises with hypoxia to induce neuronal death by inhibiting mitochondrial cytochrome oxidase. Sixteen hours of hypoxia (2% oxygen) plus deoxyglucose (an inhibitor of glycolysis) caused extensive, excitotoxic death of neurons in rat cerebellar granule cell cultures. Three different nNOS inhibitors (including the selective inhibitor N-4S-4-amino-5-2-aminoethyl-aminopentyl-N'-nitroguanidine) decreased this neuronal death by half, indicating a contribution of nNOS to hypoxic death. The selective nNOS inhibitor did not, however, block neuronal death induced either by added glutamate or by added azide (an uncompetitive inhibitor of cytochrome oxidase), indicating that nNOS does not act downstream of glutamate or cytochrome oxidase. Hypoxia plus deoxyglucose-induced glutamate release and neuronal depolarisation, and the nNOS inhibitor decreased this. Hypoxia inhibited cytochrome oxidase activity in the cultures, but a selective nNOS inhibitor prevented this inhibition, indicating NO from nNOS was inhibiting cytochrome oxidase in competition with oxygen. These data indicate that hypoxia synergises with NO from nNOS to induce neuronal death via cytochrome oxidase inhibition causing neuronal depolarisation. This mechanism might contribute to ischaemia/stroke-induced neuronal death in vivo.     

Brucella abortusd-alanyl-D-alanine carboxypeptidase contributes to its intracellular replication and resistance against nitric oxide

Brucella spp. are facultative intracellular pathogens that have the ability to survive and multiply in professional and nonprofessional phagocytes, and cause abortion in domestic animals and undulant fever in humans. However, the mechanism and factors of virulence are not fully understood. In the present study, a D-alanyl-D-alanine carboxypeptidase (DAP) mutant of Brucella abortus failed to replicate in mouse macrophages and HeLa cells, and showed less virulence than the wild type in mice. Under nitric oxide (NO) stress, the growth of the DAP mutant in vitro decreased and it also had less capability to reduce NO than the wild type. Intracellular replication of the DAP mutant was partially restored by pretreatment of macrophages with the NO synthase inhibitor, 1-phenyl-imidazole, and the level of expression of the NO reductase gene, norB, in the DAP mutant was lower than that in the wild type. These results suggest that DAP contributes to resistance against NO and that it is required for the intracellular growth of the bacterium.     

Type I IFNs stimulate nitric oxide production and resistance to Trypanosoma cruzi infection

The participation of type I IFNs (IFN-I) in NO production and resistance to Trypanosoma cruzi infection was investigated. Adherent cells obtained from the peritoneal cavity of mice infected by the i.p. route produced NO and IFN-I. Synthesis of NO by these cells was partially inhibited by treatment with anti-IFN-alphabeta or anti-TNF-alpha Abs. Compared with susceptible BALB/c mice, peritoneal cells from parasite-infected resistant C57BL/6 mice produced more NO (2-fold), IFN-I (10-fold), and TNF-alpha (3.5-fold). Later in the infection, IFN-I levels measured in spleen cell (SC) cultures from 8-day infected mice were greater in C57BL/6 than in infected BALB/c mice, and treatment of the cultures with anti-IFN-alphabeta Ab reduced NO production. IFN-gamma or IL-10 production by SCs was not different between the two mouse strains; IL-4 was not detectable. Treatment of C57BL/6 mice with IFN-I reduced parasitemia levels in the acute phase of infection. Mice deprived of the IFN-alphabetaR gene developed 3-fold higher parasitemia levels in the acute phase in comparison with control 129Sv mice. Production of NO by peritoneal macrophages and SCs was reduced in mice that lacked signaling by IFN-alphabeta, whereas parasitism of macrophages was heavier than in control wild-type mice. We conclude that IFN-I costimulate NO synthesis early in T. cruzi infection, which contributes to a better control of the parasitemia in resistant mice.