Evidence for a direct action of IL-10-activated STAT3 in modulating TNF-a, CXCL8 and IL-1ra gene transcription in LPS-treated neutrophils by protein synthesis-independent and dependent mechanisms

The anti-inflammatory action of IL-10 primarily consists in limiting the production of proinflammatory cytokines and chemokines and promoting the release of anti-inflammatory molecules from phagocytes activated by agonists of Toll-like receptors. We have recently shown that the ability of IL-10 to rapidly exert its full array of anti-inflammatory effects on human neutrophils is dependent upon exposure of these cells to LPS for at least 3-4 hours. Here, we demonstrate that, in neutrophils \u201cpreconditioned\u201d by LPS, IL-10 primarily targets the transcription of TNF-\uf061, CXCL8 and IL-1ra genes in a rapid and protein synthesis-independent manner, as revealed by Primary Transcript (PT) real-time RT-PCR. We also show that the ability of IL-10 to maintain repressed, but not to initiate, LPS-induced TNF-\uf061 and CXCL8 transcription results impaired if neutrophils are exposed to the protein synthesis inhibitor cycloheximide (CHX), in coincidence with a reduced ability of IL-10 to induce STAT3 tyrosine phosphorylation. Importantly, inhibition of IL-10-induced STAT3 activation and IL-10-suppression of LPS-induced TNF-\uf061 and CXCL8 mRNA expression by a prolonged exposure to CHX was observed to occur also in human monocytes and was caused by a defective IL-10-mediated activation of Jak1 and Tyk2 kinases, downstream of the IL-10R. Taken together, our findings demonstrate that protein synthesis inhibition reverts the suppressive effects of IL-10 on LPS-induced cytokine gene expression by interrupting the signaling pathway upstream of STAT3 activation. These data challenge the concept of the requirement of an IL-10-induced mediator to execute IL-10 anti-inflammatory program

IL-10 primarly targets transcription of LPS-induced genes in human polymorphonuclear neutrophils

The mechanisms through which the anti-inflammatory cytokine IL-10 modulates the production of multiple mediators from activated immune cells have been extensively investigated over the last years, nevertheless several feature of IL-10 signalling are still controversial. In the present study, we analyzed the mechanism of IL-10-mediated modulation of cytokine expression in primary human neutrophils, given the crucial role that these cells play in directing and shaping the subsequent inflammatory and immune responses. We have recently shown that, differently from mononuclear cells, responsiveness of human neutrophils to IL-10 is strictly dependent on the levels of IL-10R1 expression, which is undetectable in freshly isolated neutrophils and upregulated upon exposure to LPS. Based on this knowledge, IL-10 effects on cytokine and chemokine expression has been investigated on neutrophils that have been \u201cpre-conditioned\u201d by LPS for few hours, and were readily responsive to this cytokine. The same analysis has been conducted in parallel on human monocytes purified from the same donors and immediately challanged with LPS with or without IL-10. Under these circumstances, we found that IL-10 targets LPS-induced gene expression at the level of transcription, both in human neutrophils and monocytes. Indeed, by using \u201cPrimary Transcript\u201d RT Q-PCR, we were able to estimate that the accumulation of newly transcribed genes triggered by LPS was inhibited (in the case of TNF-\uf061\uf020and CXCL8) or enhanced (in the case of IL-1ra and SOCS-3) by IL-10. We then addresses the controversial question of the role of new protein synthesis in IL-10 anti-inflammatory effects. In the presence of cycloheximide IL-10 effects on LPS-induced genes were no longer observed in monocytes. In contrast, in neutrophils IL-10 continued to modulate the expression of LPS-induced genes independently of the presence of a protein synthesis inhibitor, indicating that IL-10 anti-inflammatory effects are mediated through a direct action of STAT3 on the transcriptional rate of the target genes. Moreover, in contrast to studies proposing that IL-10 inhibitory activity proceed via inhibition of NF-kB activation, we show that in neutrophils a different mechanism should be envisioned. In fact, IL-10 did not modify LPS-activated NF-kB nuclear translocation nor it modified LPS-induced transcription of IkB\uf061\uf02c an early NF-kB-target gene

Fertility, Gestation Outcome and Parasite Congenital Transmissibility in Mice Infected with TcI, TcII and TcVI Genotypes of Trypanosoma cruzi

This work aims to compare the effects of acute or chronic infections with the T. cruzi genotypes TcI (X10 strain), TcII (Y strain) and TcVI (Tulahuen strain) on fertility, gestation, pup growth and the possible vertical transmission of parasites in BALB/c mice. The occurrence of congenital infection was evaluated by microscopic examination of blood and/or qPCR on blood and heart in newborn pups and/or older offspring submitted to cyclophosphamide-induced immunosuppression in order to detect possible cryptic congenital infection. Altogether, the results show that: i) for the three strains tested, acute infection occurring after the embryo implantation in the uterus (parasite inoculation 4 days before mating), or close to delivery (parasite inoculation on day 13 of gestation), prevents or severely jeopardizes gestation outcome (inducing pup mortality and intra-uterine growth retardation); ii) for the three strains tested, gestation during chronic infection results in intra-uterine growth retardation, whereas re-inoculation of TcVI parasites during gestation in such chronically infected mice, in addition, strongly increases pup mortality; iii) congenital infection remains a rare consequence of infection (occurring in approximately 4% of living pups born to acutely infected dams); iv) PCR, detecting parasitic DNA and not living parasites, is not convenient to detect congenial infection close to delivery; v) transmission of parasites by breast milk is unlikely. This study should encourage further investigations using other parasite strains and genotypes to explore the role of virulence and other factors, as well as the mechanisms of such effects on gestation and on the establishment of congenital infection

Looking for combination of benznidazole and Trypanosoma cruzi-triosephosphate isomerase inhibitors for Chagas disease treatment

BACKGROUND The current chemotherapy for Chagas disease is based on monopharmacology with low efficacy and drug tolerance. Polypharmacology is one of the strategies to overcome these limitations. OBJECTIVES Study the anti-Trypanosoma cruzi activity of associations of benznidazole (Bnz) with three new synthetic T. cruzi-triosephosphate isomerase inhibitors, 2, 3, and 4, in order to potentiate their actions. METHODS The in vitro effect of the drug combinations were determined constructing the corresponding isobolograms. In vivo activities were assessed using an acute murine model of Chagas disease evaluating parasitaemias, mortalities and IgG anti-T. cruzi antibodies. FINDINGS The effect of Bnz combined with each of these compounds, on the growth of epimastigotes, indicated an additive action or a synergic action, when combining it with 2 or 3, respectively, and an antagonic action when combining it with 4. In vivo studies, for the two chosen combinations, 2 or 3 plus one fifth equivalent of Bnz, showed that Bnz can also potentiate the in vivo therapeutic effects. For both combinations a decrease in the number of trypomastigote and lower levels of anti-T. cruzi IgG-antibodies were detected, as well clear protection against death. MAIN CONCLUSIONS These results suggest the studied combinations could be used in the treatment of Chagas disease

Parasitic loads in tissues of mice infected with Trypanosoma cruzi and treated with AmBisome.

Chagas disease is one of the most important public health problems and a leading cause of cardiac failure in Latin America. The currently available drugs to treat T. cruzi infection (benznidazole and nifurtimox) are effective in humans when administered during months. AmBisome (liposomal amphotericin B), already shown efficient after administration for some days in human and experimental infection with Leishmania, has been scarcely studied in T. cruzi infection.Journal ArticleResearch Support, Non-U.S. Gov'tSCOPUS: ar.jinfo:eu-repo/semantics/publishe