Progesterone treatment enhances the expansion of placental immature myeloid cells in a mouse model of premature labor.

INTRODUCTION: immature-myeloid cells (IMCs) are proangiogenic bone marrow (BM)-derived cells that normally differentiate into inflammatory cells such as neutrophils, monocytes and dendritic cells (DCs). We characterized placental IMCs comparing their gene expression and subpopulations to tumor IMCs, and tested our hypothesis that progesterone that inhibits preterm labor, may affect their abundance and differentiation. METHODS: differences between IMC-subpopulations in subcutaneous tumors versus placentas in C57BL/6 or ICR (CD-1) mice were analyzed by flow cytometry and gene expression was detected by microarrays. BM- and placental cells were incubated with or without progesterone and IMC subpopulations were analyzed. For preterm labor induction pregnant mice pretreated or not with progesterone were or were not treated with Lipopolysaccharide (LPS). RESULTS: we detected enrichment of granulocytic-IMCs in placentas compared to tumors, paralleled by a decrease in monocytic-IMCs. mRNA expression of placenta- versus tumor IMCs revealed profound transcriptional alterations. Progesterone treated BM-CD11b+ cells ex-vivo induced enrichment of granulocytic-IMCs and a decrease in monocytic-IMCs and DCs. LPS treatment in-vivo led to an increase in BM-IMCs in both progesterone pretreated or non-pretreated mice. In the placenta LPS decreased the IMC population while progesterone led to complete abrogation of this effect. DISCUSSION: placental IMCs differ from tumor-IMCs in both subpopulations and gene expression. Progesterone enhances the proliferation of placenta-specific granulocytic IMCs ex-vivo and LPS induced labor is accompanied by a decrease in placental IMCs only in progesterone non-pretreated mice. We thus speculate that the protective effect of progesterone in preventing preterm labor may be explained at least in part by this specific anti-inflammatory effect.

ATF3 Regulates the Expression of AChE During Stress.

Acetylcholinesterase (AChE) expresses in non-cholinergic cells, but its role(s) there remain unknown. We have previously attributed a pro-apoptotic role for AChE in stressed retinal photoreceptors, though by unknown mechanism. Here, we examined its promoter only to find that it includes a binding sequence for the activating transcription factor 3 (ATF3); a prototypical mediator of apoptosis. This suggests that expression of AChE could be regulated by ATF3 in the retina. Indeed, ATF3 binds the AChE-promoter to down-regulate its expressions in vitro. Strikingly, retinas of "blinded" mice display hallmarks of apoptosis, almost exclusively in the outer nuclear layer (ONL); coinciding with elevated levels of AChE and absence of ATF3. A mirror image is observed in the inner nuclear layer (INL), namely prominent levels of ATF3 and lack of AChE as well as lack of apoptosis. We conclude that segregated patterns of expressions of ATF3 reflect its ability to repress apoptosis in different layers of the retina-a novel mechanism behind apoptosis.

N-acetyl ornithine deacetylase is a moonlighting protein and is involved in the adaptation of Entamoeba histolytica to nitrosative stress.

Adaptation of the Entamoeba histolytica parasite to toxic levels of nitric oxide (NO) that are produced by phagocytes may be essential for the establishment of chronic amebiasis and the parasite's survival in its host. In order to obtain insight into the mechanism of E. histolytica's adaptation to NO, E. histolytica trophozoites were progressively adapted to increasing concentrations of the NO donor drug, S-nitrosoglutathione (GSNO) up to a concentration of 110 µM. The transcriptome of NO adapted trophozoites (NAT) was investigated by RNA sequencing (RNA-seq). N-acetyl ornithine deacetylase (NAOD) was among the 208 genes that were upregulated in NAT. NAOD catalyzes the deacetylation of N-acetyl-L-ornithine to yield ornithine and acetate. Here, we report that NAOD contributes to the better adaptation of the parasite to nitrosative stress (NS) and that this function does not depend on NAOD catalytic activity. We also demonstrated that glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is detrimental to E. histolytica exposed to NS and that this detrimental effect is neutralized by NAOD or by a catalytically inactive NAOD (mNAOD). These results establish NAOD as a moonlighting protein, and highlight the unexpected role of this metabolic enzyme in the adaptation of the parasite to NS.

Proteomic Identification of Oxidized Proteins in Entamoeba histolytica by Resin-Assisted Capture: Insights into the Role of Arginase in Resistance to Oxidative Stress.

Entamoeba histolytica is an obligate protozoan parasite of humans, and amebiasis, an infectious disease which targets the intestine and/or liver, is the second most common cause of human death due to a protozoan after malaria. Although amebiasis is usually asymptomatic, E. histolytica has potent pathogenic potential. During host infection, the parasite is exposed to reactive oxygen species that are produced and released by cells of the innate immune system at the site of infection. The ability of the parasite to survive oxidative stress (OS) is essential for a successful invasion of the host. Although the effects of OS on the regulation of gene expression in E. histolytica and the characterization of some proteins whose function in the parasite's defense against OS have been previously studied, our knowledge of oxidized proteins in E. histolytica is lacking. In order to fill this knowledge gap, we performed a large-scale identification and quantification of the oxidized proteins in oxidatively stressed E. histolytica trophozoites using resin-assisted capture coupled to mass spectrometry. We detected 154 oxidized proteins (OXs) and the functions of some of these proteins were associated with antioxidant activity, maintaining the parasite's cytoskeleton, translation, catalysis, and transport. We also found that oxidation of the Gal/GalNAc impairs its function and contributes to the inhibition of E. histolytica adherence to host cells. We also provide evidence that arginase, an enzyme which converts L-arginine into L-ornithine and urea, is involved in the protection of the parasite against OS. Collectively, these results emphasize the importance of OS as a critical regulator of E. histolytica's functions and indicate a new role for arginase in E. histolytica's resistance to OS.

Proteomic identification of S-nitrosylated proteins in the parasite Entamoeba histolytica by resin-assisted capture: insights into the regulation of the Gal/GalNAc lectin by nitric oxide.

Entamoeba histolytica is a gastrointestinal protozoan parasite that causes amebiasis, a disease which has a worldwide distribution with substantial morbidity and mortality. Nitrosative stress, which is generated by innate immune cells, is one of the various environmental challenges that E. histolytica encounters during its life cycle. Although the effects of nitric oxide (NO) on the regulation of gene expression in this parasite have been previously investigated, our knowledge on S-nitrosylated proteins in E.histolytica is lacking. In order to fill this knowledge gap, we performed a large-scale detection of S-nitrosylated (SNO) proteins in E.histolytica trophozoites that were treated with the NO donor, S-nitrosocysteine by resin-assisted capture (RAC). We found that proteins involved in glycolysis, gluconeogenesis, translation, protein transport, and adherence to target cells such as the heavy subunit of Gal/GalNac lectin are among the S-nitrosylated proteins that were enriched by SNO-RAC. We also found that the S-nitrosylated cysteine residues in the carbohydrate recognition domain (CRD) of Gal/GalNAc lectin impairs its function and contributes to the inhibition of E.histolytica adherence to host cells. Collectively, these results advance our understanding of the mechanism of reduced E.histolytica adherence to mammalian cells by NO and emphasize the importance of NO as a regulator of key physiological functions in E.histolytica.

Glucose starvation boosts Entamoeba histolytica virulence.

The unicellular parasite, Entamoeba histolytica, is exposed to numerous adverse conditions, such as nutrient deprivation, during its life cycle stages in the human host. In the present study, we examined whether the parasite virulence could be influenced by glucose starvation (GS). The migratory behaviour of the parasite and its capability to kill mammalian cells and to lyse erythrocytes is strongly enhanced following GS. In order to gain insights into the mechanism underlying the GS boosting effects on virulence, we analyzed differences in protein expression levels in control and glucose-starved trophozoites, by quantitative proteomic analysis. We observed that upstream regulatory element 3-binding protein (URE3-BP), a transcription factor that modulates E.histolytica virulence, and the lysine-rich protein 1 (KRiP1) which is induced during liver abscess development, are upregulated by GS. We also analyzed E. histolytica membrane fractions and noticed that the Gal/GalNAc lectin light subunit LgL1 is up-regulated by GS. Surprisingly, amoebapore A (Ap-A) and cysteine proteinase A5 (CP-A5), two important E. histolytica virulence factors, were strongly down-regulated by GS. While the boosting effect of GS on E. histolytica virulence was conserved in strains silenced for Ap-A and CP-A5, it was lost in LgL1 and in KRiP1 down-regulated strains. These data emphasize the unexpected role of GS in the modulation of E.histolytica virulence and the involvement of KRiP1 and Lgl1 in this phenomenon.