Resistance of Leishmania (Viannia) braziliensis to nitric oxide: correlation with antimony therapy and TNF-α production

<p>Abstract</p> <p>Background</p> <p>Nitric oxide (NO) produced in macrophages plays a pivotal role as a leishmanicidal agent. A previous study has demonstrated that 20% of the <it>L. (V.) braziliensis </it>isolated from initial cutaneous lesions of patients from the endemic area of Corte de Pedra, Bahia, Brazil, were NO resistant. Additionally, 5 to 11% of the patients did not respond to three or more antimony treatments" (refractory patients). The aim of this study is to investigate if there is an association between the resistance of <it>L. (V.) braziliensis </it>to NO and nonresponsiveness to antimony therapy and cytokine production.</p> <p>Methods</p> <p>We evaluated the <it>in vitro </it>toxicity of NO against the promastigotes stages of <it>L. (V.) braziliensis </it>isolated from responsive and refractory patients, and the infectivity of the amastigote forms of these isolates against human macrophages. The supernatants from <it>Leishmania </it>infected macrophage were used to measure TNF-α and IL-10 levels.</p> <p>Results</p> <p>Using NaNO₂(pH 5.0) as the NO source, <it>L. (V.) braziliensis </it>isolated from refractory patients were more NO resistant (IC50 = 5.8 ± 4.8) than <it>L. (V.) braziliensis </it>isolated from responsive patients (IC50 = 2.0 ± 1.4). Four isolates were selected to infect human macrophages: NO-susceptible and NO-resistant <it>L. (V.) braziliensis </it>isolated from responsive and refractory patients. NO-resistant <it>L. (V.) braziliensis </it>isolated from refractory patients infected more macrophages stimulated with LPS and IFN-γ at 120 hours than NO-susceptible <it>L. (V.) braziliensis </it>isolated from refractory patients. Also, lower levels of TNF-α were detected in supernatants of macrophages infected with NO-resistant <it>L. (V.) braziliensis </it>as compared to macrophages infected with NO-susceptible <it>L. (V.) braziliensis </it>(p < 0.05 at 2, 24 and 120 hours), while no differences were detected in IL-10 levels.</p> <p>Conclusion</p> <p>These data suggest that NO resistance could be related to the nonresponsiveness to antimony therapy seen in American Tegumentary Leishmaniasis.</p

Impaired Chromatin Remodelling at STAT1-Regulated Promoters Leads to Global Unresponsiveness of Toxoplasma gondii-Infected Macrophages to IFN-γ

Intracellular pathogens including the apicomplexan and opportunistic parasite Toxoplasma gondii profoundly modify their host cells in order to establish infection. We have shown previously that intracellular T. gondii inhibit up-regulation of regulatory and effector functions in murine macrophages (MΦ) stimulated with interferon (IFN)-γ, which is the cytokine crucial for controlling the parasites' replication. Using genome-wide transcriptome analysis we show herein that infection with T. gondii leads to global unresponsiveness of murine macrophages to IFN-γ. More than 61% and 89% of the transcripts, which were induced or repressed by IFN-γ in non-infected MΦ, respectively, were not altered after stimulation of T. gondii-infected cells with IFN-γ. These genes are involved in a variety of biological processes, which are mostly but not exclusively related to immune responses. Analyses of the underlying mechanisms revealed that IFN-γ-triggered nuclear translocation of STAT1 still occurred in Toxoplasma-infected MΦ. However, STAT1 bound aberrantly to oligonucleotides containing the IFN-γ-responsive gamma-activated site (GAS) consensus sequence. Conversely, IFN-γ did not induce formation of active GAS-STAT1 complexes in nuclear extracts from infected MΦ. Mass spectrometry of protein complexes bound to GAS oligonucleotides showed that T. gondii-infected MΦ are unable to recruit non-muscle actin to IFN-γ-responsive DNA sequences, which appeared to be independent of stimulation with IFN-γ and of STAT1 binding. IFN-γ-induced recruitment of BRG-1 and acetylation of core histones at the IFN-γ-regulated CIITA promoter IV, but not β-actin was diminished by >90% in Toxoplasma-infected MΦ as compared to non-infected control cells. Remarkably, treatment with histone deacetylase inhibitors restored the ability of infected macrophages to express the IFN-γ regulated genes H2-A/E and CIITA. Taken together, these results indicate that Toxoplasma-infected MΦ are unable to respond to IFN-γ due to disturbed chromatin remodelling, but can be rescued using histone deacetylase inhibitors

Evidence for involvement of peptidoglycan in the triggering of an oxidative burst by Listeria monocytogenes in phagocytes

We have shown previously that in listeric encephalitis of cattle and rats, nitrotyrosine was produced in microabscesses, implying that both superoxide anion (O(2)(–)) and nitric oxide (NO) are present and react with each other. Evidence of local synthesis of NO by macrophages was provided, but the source of O(2)(–) remained unknown. Here we have examined whether phagocytes exposed to viable and heat-killed Listeria monocytogenes (LMΔ) produce O(2)(–) and, if so, whether this results from direct interaction of phagocytes with the bacterial surface of L. monocytogenes or whether prior opsonization is required. Using lucigenin-enhanced chemiluminescence (LCL) for the measurement of O(2)(–), we show that LMΔ induces an oxidative burst in human neutrophils, monocytes and monocyte-derived macrophages (Mφ). Viability is not required, and opsonization by antibodies and/or complement does not enhance the LCL signal. As Toll-like receptors (TLR) were shown recently to mediate an oxidative burst, TLR agonists representative for pathogen-associated molecular patterns (PAMPs) were tested for their ability to elicit an oxidative burst. These included lipoteichoic acid (LTA), bacterial peptidoglycan (PGN), recombinant flagellin, CpG-containing DNA and double-stranded RNA. Only PGN and flagellin consistently elicited an LCL signal resembling that induced by LMΔ with regard to the kinetics and cell spectrum stimulated. However, flagellin was unlikely to be responsible for the LMΔ-mediated burst, as a flagellin-deficient mutant showed no decrease in LCL. We therefore assume that in LMΔ, core PGN acts as a PAMP and directly induces an oxidative burst in all phagocyte populations. We conclude that in cerebral lesions superoxide anion is generated locally by phagocytes recognizing bacterial PGN