Maria Pilar Aoki

Myeloid-derived suppressor cells are key players in the resolution of inflammation during a model of acute infection

Myeloid‐derived suppressor cells (MDSCs) are key players in the immune suppressive network. During acute infection with the causative agent of Chagas disease, Trypanosoma cruzi, BALB/c mice show less inflammation and better survival than C57BL/6 (B6) mice. In this comparative study, we found a higher number of MDSCs in the spleens and livers of infected BALB/c mice compared with infected B6 mice. An analysis of the two major MDSCs subsets revealed a greater number of granulocytic cells in the spleens and livers of BALB/c mice when compared with that in B6 mice. Moreover, splenic MDSCs purified from infected BALB/c mice inhibited ConA‐induced splenocyte proliferation. Mechanistic studies demonstrated that ROS and nitric oxide were involved in the suppressive activity of MDSCs, with a higher number of infected CD8+ T cells suffering surface‐nitration compared to uninfected controls. An upregulation of NADPH oxidase p47 phox subunit and p‐STAT3 occurred in MDSCs and infected IL‐6 KO mice showed less recruitment of MDSCs and impaired survival. Remarkably, in vivo depletion of MDSCs led to increased production of IL‐6, IFN‐γ, and a Th17 response with very high parasitemia and mortality. These findings demonstrate a new facet of MDSCs as crucial regulators of inflammation during T. cruzi infection.

Apoptosis induction by glucuronoxylomannan of Cryptococcus neoformans

We studied the ability of glucuronoxylomannan (GXM), the major constituent of Cryptococcus neoformans capsular polysaccharide, to induce apoptosis in lymphocytes from normal rats. Spleen mononuclear cells (Smc) from normal rats treated with GXM for 24 h exhibited, in comparison with controls, an increased hypodiploidy in the DNA profile after staining with propidium iodide, as well as increased ladder-type DNA fragmentation in agarose gel electrophoresis and a high number of positive cells in the terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL) reaction. Furthermore, increased hypodiploidy in the DNA profile was also observed in Smc expressing T-cell receptor (TCR +). We also studied the induction of apoptosis in lungs and spleens from rats in the immunosuppressor period of disseminated cryptococcosis. TUNEL labeling of lungs and spleens from rats obtained 14 days after infection with C. neoformans showed a large number of apoptotic cells. Our results provide strong cytometric, molecular and morphological evidence that apoptosis could be a previously unrecognized immunosuppressive property of GXM in vitro. This programmed cell death may be involved in the immunosuppression observed during C. neoformans infection.

Importance of TLR2 on hepatic immune and non-immune cells to attenuate the strong inflammatory liver response during Trypanosoma cruzi acute infection.

Background Toll-like receptors (TLR) and cytokines play a central role in the pathogen clearance as well as in pathological processes. Recently, we reported that TLR2, TLR4 and TLR9 are differentially modulated in injured livers from BALB/c and C57BL/6 (B6) mice during Trypanosoma cruzi infection. However, the molecular and cellular mechanisms involved in local immune response remain unclear. Methodology/Principal Findings In this study, we demonstrate that hepatic leukocytes from infected B6 mice produced higher amounts of pro-inflammatory cytokines than BALB/c mice, whereas IL10 and TGFβ were only released by hepatic leukocytes from BALB/c. Strikingly, a higher expression of TLR2 and TLR4 was observed in hepatocytes of infected BALB/c mice. However, in infected B6 mice, the strong pro-inflammatory response was associated with a high and sustained expression of TLR9 and iNOS in leukocytes and hepatic tissue respectively. Additionally, co-expression of gp91- and p47-phox NADPH oxidase subunits were detected in liver tissue of infected B6 mice. Notably, the pre-treatment previous to infection with Pam3CSK4, TLR2-agonist, induced a significant reduction of transaminase activity levels and inflammatory foci number in livers of infected B6 mice. Moreover, lower pro-inflammatory cytokines and increased TGFβ levels were detected in purified hepatic leukocytes from TLR2-agonist pre-treated B6 mice. Conclusions/Significance Our results describe some of the main injurious signals involved in liver immune response during the T. cruzi acute infection. Additionally we show that the administration of Pam3CSk4, previous to infection, can attenuate the exacerbated inflammatory response of livers in B6 mice. These results could be useful to understand and design novel immune strategies in controlling liver pathologies.

Immunosuppression, interleukin-10 synthesis and apoptosis are induced in rats inoculated with Cryptococcus neoformans glucuronoxylomannan

Glucuronoxylomannan (GXM) is the major Cryptococcus neoformans capsular polysaccharide and represents the main virulence factor of this fungus. In in vitro studies we have demonstrated previously that this acidic and high‐molecular‐weight polysaccharide suppresses lymphoproliferation, modulates cytokine production and promotes apoptosis in spleen mononuclear (Spm) cells from rats. In this study we demonstrate that these phenomena also occur in vivo after the intracardiac inoculation of GXM into normal Wistar rats. The results of this study show suppression of the proliferative response Spm cells to concanavalin A (Con A) or heat‐killed C. neoformans (HKCn) in the first 2 weeks after polysaccharide administration. In addition, increased levels of interleukin (IL)‐10 were produced by Con A‐stimulated Spm cells, coinciding with immunohistochemical GXM detection in the white pulp of spleen. In particular, high production of IL‐10 with diminution of IL‐2, interferon (IFN)‐γ and tumour necrosis factor (TNF)‐α synthesis were detected 14 days after GXM administration. In situ cell death detection by TdT‐mediated biotin–dUTP nick‐end labelling (TUNEL) reaction in sections of spleen, lung and liver demonstrates apoptosis in tissues with deposits of GXM. These data demonstrate the in vivo ability of GXM to modify cytokine synthesis by Spm cells and to promote host cell apoptosis.

Toll-like receptor-2 and interleukin-6 mediate cardiomyocyte protection from apoptosis during Trypanosoma cruzi murine infection

Local innate immunity plays a key role in initiating and coordinating homeostatic and defense responses in the heart. We have previously reported that the cardiotropic parasite Trypanosoma cruzi, the etiological agent of Chagas disease, protects cardiomyocytes against growth factor deprivation-induced apoptosis. In this study, we investigated cardiomyocyte innate immune response to T. cruzi infection and its role in cellular protection from apoptosis. We found that Toll-like receptor (TLR) 2-expressing cells were strongly increased by the parasite in BALB/c neonatal mouse cardiomyocyte cultures. Using a dominant-negative system, we showed that TLR2 mediated cardiomyocyte survival and the secretion of interleukin (IL) 6, which acted as an essential anti-apoptotic factor. Moreover, IL6 released by infected cells, as well as the recombinant bioactive cytokine, induced the phosphorylation of the signal transducers and activators of transcription-3 (STAT3) in cultured cardiomyocytes. In accord with the in vitro results, during the acute phase of the infection, TLR2 expression increased 2.9-fold and the anti-apoptotic factor Bcl-2 increased 4.5-fold in the cardiac tissue. We have clearly shown a cross-talk between the intrinsic innate response of cardiomyocytes and the pro-survival effect evoked by the parasite.

Sexual dimorphism of apoptosis in lactotrophs induced by bromocryptine

Abstract. Interruption of lactation provokes a massive degeneration of surplus lactotrophs in the rat pituitary gland. This process was determined to be non-apoptotic in nature, and this observation raised many questions as cell death by apoptosis has been described in several tissues after withdrawal of trophic hormones. In this study we explored various experimental conditions and gathered new information leading to a comprehensive interpretation of the factors involved in the induction of apoptosis in lactotrophs. With this aim, we investigated the apoptogenic role of bromocryptine on lactotrophs in several experimental models involving male and female rats. Even though bromocryptine increased the expression of P53 in all experimental models, apoptosis was only triggered in male and ovariectomised females. In both conditions the oestrogen stimulation is low or nil, and the occurrence of apoptosis can be correlated with the appearance of atypical lactotrophs and the level of P53 expression. The existence of apoptosis was validated with the observation of DNA laddering in electrophoresis. By contrast, in intact females the majority of lactotrophs present signs of an increased prolactin secretion and no DNA fragmentation was found. Endogenous oestrogens probably prevent the deep inhibitory effect of a dopamine agonist and thus block apoptosis. Besides, the morphological analysis of regressing pituitary revealed the coexistence of lactotrophs to be an important factor responsible for tissue remodelling in functional pituitary glands undergoing apoptotic and non-apoptotic cell deaths. The non-apoptotic cell death appeared to be an important factor responsible for tissue remodelling in functional pituitary glands. The present results suggest that the occurrence of apoptosis in regressing lactotrophs caused by bromocryptine is sexually dimorphic and probably associated with the survival effect of endogenous oestrogens in intact females.

Apoptotic and non-apoptotic cell death in hormone-dependent glands

Abstract The proliferation of cells and cell death are involved in the maintenance of appropriate tissue homeostasis. In the present study, two different mechanisms of cell death were identified in the prostate and pituitary glands when morphological data, fragmentation of DNA, and TUNEL labelling of apoptotic nuclei were compared. Typical cell death by apoptosis was identified by morphological and molecular approaches in the prostate after orchidectomy. By contrast, neither DNA fragmentation nor TUNEL labelling were found in dead cells occurring in the pituitary gland after interruption of lactation. Regressing lactotrophs were characterised by condensation and disruption of the cytoplasmic matrix, but preserved intact nuclei until advanced stages of regression. Degenerating “dark” cells comparable to those described in the pituitary were also seen coexisting with typical apoptosis in the prostate epithelial lining of orchidectomised rats. Both forms of cell death could be clearly differentiated, because dark cells suffer severe alterations of cytoplasmic organelles while maintaining the integrity of the nucleus. In contrast, apoptotic cells present well-preserved cytoplasmic organelles, but grossly disrupted nuclei with fragmentation and condensation of chromatin.

Nonimmune Cells Contribute to Crosstalk between Immune Cells and Inflammatory Mediators in the Innate Response to Trypanosoma cruzi Infection

Chagas myocarditis, which is caused by infection with the intracellular parasite Trypanosoma cruzi, remains the major infectious heart disease worldwide. Innate recognition through toll-like receptors (TLRs) on immune cells has not only been revealed to be critical for defense against T. cruzi but has also been involved in triggering the pathology. Subsequent studies revealed that this parasite activates nucleotide-binding oligomerization domain- (NOD-)like receptors and several particular transcription factors in TLR-independent manner. In addition to professional immune cells, T. cruzi infects and resides in different parenchyma cells. The innate receptors in nonimmune target tissues could also have an impact on host response. Thus, the outcome of the myocarditis or the inflamed liver relies on an intricate network of inflammatory mediators and signals given by immune and nonimmune cells. In this paper, we discuss the evidence of innate immunity to the parasite developed by the host, with emphasis on the crosstalk between immune and nonimmune cell responses.

IL-6 promotes M2 macrophage polarization by modulating purinergic signaling and regulates the lethal release of nitric oxide during Trypanosoma cruzi infection

The production of nitric oxide (NO) is a key defense mechanism against intracellular pathogens but it must be tightly controlled in order to avoid excessive detrimental oxidative stress. In this study we described a novel mechanism through which interleukin (IL)-6 mediates the regulation of NO release induced in response to Trypanosoma cruzi infection. Using a murine model of Chagas disease, we found that, in contrast to C57BL/6 wild type (WT) mice, IL-6-deficient (IL6KO) mice exhibited a dramatic increase in plasma NO levels concomitant with a significantly higher amount of circulating IL-1β and inflammatory monocytes. Studies on mouse macrophages and human monocytes, revealed that IL-6 decreased LPS-induced NO production but this effect was abrogated in the presence of anti-IL-1β and in macrophages deficient in the NLRP3 inflammasome. In accordance, while infected WT myocardium exhibited an early shift from microbicidal/M1 to anti-inflammatory/M2 macrophage phenotype, IL6KO cardiac tissue never displayed a dominant M2 macrophage profile that correlated with decreased expression of ATP metabolic machinery and a lower cardiac parasite burden. The deleterious effects of high NO production-induced oxidative stress were evidenced by enhanced cardiac malondialdehyde levels, myocardial cell death and mortality. The survival rate was improved by the treatment of IL-6-deficient mice with a NO production-specific inhibitor. Our data revealed that IL-6 regulates the excessive release of NO through IL-1β inhibition and determines the establishment of an M2 macrophage profile within infected heart tissue.

Chronic Trypanosoma cruzi infection potentiates adipose tissue macrophage polarization toward an anti-inflammatory M2 phenotype and contributes to diabetes progression in a diet-induced obesity model

Chronic obesity and Chagas disease (caused by the protozoan Trypanosoma cruzi) represent serious public health concerns. The interrelation between parasite infection, adipose tissue, immune system and metabolism in an obesogenic context, has not been entirely explored. A novel diet-induced obesity model (DIO) was developed in C57BL/6 wild type mice to examine the effect of chronic infection (DIO+I) on metabolic parameters and on obesity-related disorders. Dyslipidemia, hyperleptinemia, and cardiac/hepatic steatosis were strongly developed in DIO mice. Strikingly, although these metabolic alterations were collectively improved by infection, plasmatic apoB100 levels remain significantly increased in DIO+I, suggesting the presence of pro-atherogenic small and dense LDL particles. Moreover, acute insulin resistance followed by chronic hyperglycemia with hypoinsulinemia was found, evidencing an infection-related-diabetes progression. These lipid and glucose metabolic changes seemed to be highly dependent on TLR4 expression since TLR4−/− mice were protected from obesity and its complications. Notably, chronic infection promoted a strong increase in MCP-1 producing macrophages with a M2 (F4/80+CD11c-CD206+) phenotype associated to oxidative stress in visceral adipose tissue of DIO+I mice. Importantly, infection reduced lipid content but intensified inflammatory infiltrates in target tissues. Thus, parasite persistence in an obesogenic environment and the resulting host immunometabolic dysregulation may contribute to diabetes/atherosclerosis progression.