Juliana Terzi Maricato

Beta2‐adrenergic receptor signaling in CD4+ Foxp3+ regulatory T cells enhances their suppressive function in a PKA‐dependent manner

Beta2‐adrenergic receptor (B2AR) signaling is known to impair Th1‐cell differentiation and function in a cAMP‐dependent way, leading to inhibition of cell proliferation and decreased production of IL‐2 and IFN‐γ. CD4+ Foxp3+ Treg cells play a key role in the regulation of immune responses and are essential for maintenance of self‐tolerance. Nevertheless, very little is known about adrenergic receptor expression in Treg cells or the influence of noradrenaline on their function. Here we show that Foxp3+ Treg cells express functional B2AR. B2AR activation in Treg cells leads to increased intracellular cAMP levels and to protein kinase A (PKA)‐dependent CREB phosphorylation. We also found that signaling via B2AR enhances the in vitro suppressive activity of Treg cells. B2AR‐mediated increase in Treg‐cell suppressive function was associated with decreased IL‐2 mRNA levels in responder CD4+ T cells and improved Treg‐cell‐induced conversion of CD4+ Foxp3− cells into Foxp3+ induced Treg cells. Moreover, B2AR signaling increased CTLA‐4 expression in Treg cells in a PKA‐dependent way. Finally, we found that PKA inhibition totally prevented the B2AR‐mediated increase in Treg‐cell suppressive function. Our data suggest that sympathetic fibers are able to regulate Treg‐cell suppressive activity in a positive manner through B2AR signaling.

Peptides from Paracoccidioides brasiliensis GP43 inhibit macrophage functions and inflammatory response

Paracoccidioidomycosis (PCM) is a systemic granulomatous disease caused by Paracoccidioides brasiliensis (Pb), a thermal dimorphic fungus. Its major antigen is a 43-kDa glycoprotein. Gp43 embodies different functions: it participates in evasion mechanisms during the installation of primary infection, stimulates granuloma-like formation in vitro and presents T-cell epitopes that induce protective response against the fungus. Here, we investigated epitopes from gp43 inhibitory of both, macrophage functions and inflammatory reaction. Different gp43 peptides, spanning the entire sequence of the molecule, were added to cultures of bone marrow-derived macrophages. After challenge with zymosan or Pb cells, phagocytic indexes were measured. Peptides expressed on the molecule surface were determined by graphic analysis using the Protean module; DNAstar Inc. Two peptides which decreased phagocytic index and were expressed at the surface of the molecule, P4 and P23, were selected for further studies. It was shown that both inhibited the release of NO by zymosan stimulated macrophages while enhanced release of H(2)O(2). The release of TNF-alpha in culture supernatants from in vitro phagocytic tests showed different response depending of P4 concentration (data not shown). In vivo assays with Mycobacterium bovis - bacillus Calmette-Guérin (BCG) or Pb cells demonstrated that these peptides presented non-specific and specific anti-inflammatory properties.

GCN2 kinase plays an important role triggering the remission phase of experimental autoimmune encephalomyelitis (EAE) in mice

Experimental autoimmune encephalomyelitis (EAE) has been widely employed as a model to study multiple sclerosis (MS) and indeed has allowed some important advances in our comprehension of MS pathogenesis. Several pieces of evidence suggest that infiltrating Th1 and Th17 lymphocytes are important players leading to CNS demyelination and lesion during the peak of murine EAE. Subsequently, effector T cell responses rapidly decline and the recovery phase of the disease strongly correlates with the expression of anti-inflammatory cytokines and the enrichment of Foxp3+ regulatory T (Treg) cells within the target organ. However, the mechanisms leading to the increased presence of Treg cells and to the remission phase of the disease are still poorly understood. Recent researches demonstrated that chemically induced amino-acid starvation response might suppress CNS immune activity. Here we verified an important participation of the general control nonrepressible 2 (GCN2), a key regulator kinase of the amino-acid starvation response, in the development of the remission phase of EAE in C57BL/6 mice. By immunizing wild type C57BL/6 (WT) and GCN2 knock-out mice (GCN2 KO) with myelin oligodendrocyte glycoprotein peptide (MOG35-55), it was noticed that GCN2 KO mice did not develop the remission phase of the disease and this was associated with higher levels of CNS inflammation and increased presence of effector T cells (Th1/Th17). These animals also showed lower frequency of Treg cells within the CNS as compared to WT animals. Higher expression of indoleamine 2,3-dioxygenase (IDO) and higher frequency of plasmacytoid dendritic cells (pDCs) were found at the peak of the disease in the CNS of WT animals. Our results suggest that the GCN2 kinase-dependent sensing of IDO activity represents an important trigger to the EAE remission phase. The IDO-mediated immunoregulatory events may include the arresting of effector T cell responses and the differentiation/expansion of Treg cells within the target organ.

Epigenetic Modulations in Activated Cells Early after HIV-1 Infection and Their Possible Functional Consequences

Epigenetic modifications refer to a number of biological processes which alter the structure of chromatin and its transcriptional activity such as DNA methylation and histone post-translational processing. Studies have tried to elucidate how the viral genome and its products are affected by epigenetic modifications imposed by cell machinery and how it affects the ability of the virus to either, replicate and produce a viable progeny or be driven to latency. The purpose of this study was to evaluate epigenetic modifications in PBMCs and CD4+ cells after HIV-1 infection analyzing three approaches: (i) global DNA- methylation; (ii) qPCR array and (iii) western blot. HIV-1 infection led to methylation increases in the cellular DNA regardless the activation status of PBMCs. The analysis of H3K9me3 and H3K27me3 suggested a trend towards transcriptional repression in activated cells after HIV-1 infection. Using a qPCR array, we detected genes related to epigenetic processes highly modulated in activated HIV-1 infected cells. SETDB2 and RSK2 transcripts showed highest up-regulation levels. SETDB2 signaling is related to transcriptional silencing while RSK2 is related to either silencing or activation of gene expression depending on the signaling pathway triggered down-stream. In addition, activated cells infected by HIV-1 showed lower CD69 expression and a decrease of IL-2, IFN-γ and metabolism-related factors transcripts indicating a possible functional consequence towards global transcriptional repression found in HIV-1 infected cells. Conversely, based on epigenetic markers studied here, non-stimulated cells infected by HIV-1, showed signs of global transcriptional activation. Our results suggest that HIV-1 infection exerts epigenetic modulations in activated cells that may lead these cells to transcriptional repression with important functional consequences. Moreover, non-stimulated cells seem to increase gene transcription after HIV-1 infection. Based on these observations, it is possible to speculate that the outcome of viral infections may be influenced by the cellular activation status at the moment of infection.

In Vitro and In Vivo Phagocytic Ability of Mouse B-1 Cells

B-1 cells are a peculiar subpopulation of B cells found in the peritoneal and pleural cavities in mice. These cells are typically IgM+ and CD11b+. B-1 cells are able to migrate from the peritoneal cavity to non-specific inflammatory sites in mice. In addition, they can differentiate into mononuclear phagocyte-like cells in vitro; however, it is still unknown whether B-1 cells are capable of performing phagocytosis in vivo. Here we further characterized B-1 cells as phagocytes in vitro, and we investigated their ability to phagocytose apoptotic cells and bacteria in vivo. Our results demonstrate that B-1 phagocytes are able to uptake apoptotic thymocytes and Escherichia coli bacteria, both in vitro and in vivo. These findings indicate that along with macrophages, B-1 phagocytic cells might play a role in fundamental processes such as tissue remodeling, resolution of inflammation and pathogen clearance.

Norepinephrine Controls Effector T Cell Differentiation through β2-Adrenergic Receptor–Mediated Inhibition of NF-κB and AP-1 in Dendritic Cells

Despite accumulating evidence indicating that neurotransmitters released by the sympathetic nervous system can modulate the activity of innate immune cells, we still know very little about how norepinephrine impacts signaling pathways in dendritic cells (DC) and the consequence of that in DC-driven T cell differentiation. In this article, we demonstrate that β2-adrenergic receptor (β2AR) activation in LPS-stimulated DC does not impair their ability to promote T cell proliferation; however, it diminishes IL-12p70 secretion, leading to a shift in the IL-12p70/IL-23 ratio. Although β2AR stimulation in DC induces protein kinase A–dependent cAMP-responsive element–binding protein phosphorylation, the effect of changing the profile of cytokines produced upon LPS challenge occurs in a protein kinase A–independent manner and, rather, is associated with inhibition of the NF-κB and AP-1 signaling pathways. Moreover, as a consequence of the inverted IL-12p70/IL-23 ratio following β2AR stimulation, LPS-stimulated DC promoted the generation of CD4+ T cells that, upon TCR engagement, produced lower amounts of IFN-γ and higher levels of IL-17. These findings provide new insights into molecular and cellular mechanisms by which β2AR stimulation in murine DC can influence the generation of adaptive immune responses and may explain some aspects of how sympathetic nervous system activity can modulate immune function.

Anti-metastatic immunotherapy based on mucosal administration of flagellin and immunomodulatory P10

Current therapies against malignant melanoma generally fail to increase survival in most patients, and immunotherapy is a promising approach as it could reduce the dosage of toxic therapeutic drugs. In the present study, we show that an immunotherapeutic approach based on the use of the Toll‐like receptor (TLR)‐5 ligand flagellin (Salmonella Typhimurium FliCi) combined with the major histocompatibility complex class II‐restricted P10 peptide, derived from the Paracoccidioides brasiliensis gp43 major surface protein, reduced the number of lung metastasis in a murine melanoma model. Compounds were administered intranasally into C57Bl/6 mice intravenously challenged with syngeneic B16F10‐Nex2 melanoma cells, aiming at the local (pulmonary) immune response modulation. Along with a marked reduction in the number of lung nodules, a significant increase in survival was observed. The immunization regimen induced both local and systemic proinflammatory responses. Lung macrophages were polarized towards a M1 phenotype, lymph node cells, and splenocytes secreted higher interleukin‐12p40 and interferon (IFN)‐γ levels when re‐stimulated with tumor antigens. The protective effect of the FliCi+P10 formulation required TLR‐5, myeloid differentiation primary response gene 88 and IFN‐γ expression, but caspase‐1 knockout mice were only partially protected, suggesting that intracellular flagellin receptors are not involved with the anti‐tumor effect. The immune therapy resulted in the activation of tumor‐specific CD4+ T lymphocytes, which conferred protection to metastatic melanoma growth after adoptive transfer. Taken together, our results report a new immunotherapeutic approach based on TLR‐5 activation and IFN‐γ production capable to control the metastatic growth of B16F10‐Nex2 melanoma, being a promising alternative to be associated with chemotherapeutic drugs for an effective anti‐tumor responses.

A Recombinant Protein Based on Trypanosoma cruzi P21 Enhances Phagocytosis

Background P21 is a secreted protein expressed in all developmental stages of Trypanosoma cruzi. The aim of this study was to determine the effect of the recombinant protein based on P21 (P21-His6) on inflammatory macrophages during phagocytosis. Findings Our results showed that P21-His6 acts as a phagocytosis inducer by binding to CXCR4 chemokine receptor and activating actin polymerization in a way dependent onthe PI3-kinase signaling pathway. Conclusions Thus, our results shed light on the notion that native P21 is a component related to T. cruzi evasion from the immune response and that CXCR4 may be involved in phagocytosis. P21-His6 represents an important experimental control tool to study phagocytosis signaling pathways of different intracellular parasites and particles.

Low Concentrations of Hydrogen Peroxide or Nitrite Induced of Paracoccidioides brasiliensis Cell Proliferation in a Ras-Dependent Manner

Paracoccidioides brasiliensis, a causative agent of paracoccidioidomycosis (PCM), should be able to adapt to dramatic environmental changes inside the infected host after inhalation of air-borne conidia and transition to pathogenic yeasts. Proteins with antioxidant functions may protect fungal cells against reactive oxygen (ROS) and nitrogen (RNS) species generated by phagocytic cells, thus acting as potential virulence factors. Ras GTPases are involved in stress responses, cell morphology, and differentiation in a range of organisms. Ras, in its activated form, interacts with effector proteins and can initiate a kinase cascade. In lower eukaryotes, Byr2 kinase represents a Ras target. The present study investigated the role of Ras in P. brasiliensis after in vitro stimulus with ROS or RNS. We have demonstrated that low concentrations of H2O2 (0.1 mM) or NO2 (0.1–0.25 µM) stimulated P. brasiliensis yeast cell proliferation and that was not observed when yeast cells were pre-incubated with farnesyltransferase inhibitor. We constructed an expression plasmid containing the Byr2 Ras-binding domain (RBD) fused with GST (RBD-Byr2-GST) to detect the Ras active form. After stimulation with low concentrations of H2O2 or NO2, the Ras active form was observed in fungal extracts. Besides, NO2 induced a rapid increase in S-nitrosylated Ras levels. This alternative posttranslational modification of Ras, probably in residue Cys123, would lead to an exchange of GDP for GTP and consequent GTPase activation in P. brasiliensis. In conclusion, low concentrations of H2O2 or NO2 stimulated P. brasiliensis proliferation through Ras activation.

Paracoccidioides brasiliensis GP43-derived peptides are potent modulators of local and systemic inflammatory response.

Paracoccidioidomycosis is a systemic granulomatous disease caused by the dimorphic fungus Paracoccidioides brasiliensis. Its major antigen is a 43 kDa glycoprotein whose peptides embody different functions: P10 peptide, a T-cell epitope, induces protective response while P4 and P23 peptides inhibit both, macrophage functions and inflammatory reaction, thus facilitating infection. Here we investigated the modulating mechanisms of the immune response exerted by P4 and P23 involved in the latter inhibitory effect on macrophages. Moreover we analyzed the peptides effects in different models in vivo. While evaluating whether P4 and P23 present systemic anti-inflammatory effects in vivo, we showed that their intraperitonial administration decreased footpad swelling in mice infected with either P. brasiliensis or Mycobacterium bovis. Both, qPCR and ELISA assays suggested that this anti-inflammatory effect depended on alterations in the kinetics of production of innate immunity modulators such as TNF-α, IL6, IL10 and TLR2. IL10 seems to be early produced than TNF-α and IL6, produced later in presence of peptides. Higher doses or intravenously given P4 and P23 resulted in earlier and more prolonged anti-inflammatory effects. Moreover, continuous treatment with P4 and P23 sustained the anti-inflammatory activity throughout.