Spider venom administration impairs glioblastoma growth and modulates immune response in a non-clinical model.

Molecules from animal venoms are promising candidates for the development of new drugs. Previous in vitro studies have shown that the venom of the spider Phoneutria nigriventer (PnV) is a potential source of antineoplastic components with activity in glioblastoma (GB) cell lines. In the present work, the effects of PnV on tumor development were established in vivo using a xenogeneic model. Human GB (NG97, the most responsive line in the previous study) cells were inoculated (s.c.) on the back of RAG-/- mice. PnV (100 µg/Kg) was administrated every 48 h (i.p.) for 14 days and several endpoints were evaluated: tumor growth and metabolism (by microPET/CT, using 18F-FDG), tumor weight and volume, histopathology, blood analysis, percentage and profile of macrophages, neutrophils and NK cells isolated from the spleen (by flow cytometry) and the presence of macrophages (Iba-1 positive) within/surrounding the tumor. The effect of venom was also evaluated on macrophages in vitro. Tumors from PnV-treated animals were smaller and did not uptake detectable amounts of 18F-FDG, compared to control (untreated). PnV-tumor was necrotic, lacking the histopathological characteristics typical of GB. Since in classic chemotherapies it is observed a decrease in immune response, methotrexate (MTX) was used only to compare the PnV effects on innate immune cells with a highly immunosuppressive antineoplastic drug. The venom increased monocytes, neutrophils and NK cells, and this effect was the opposite of that observed in the animals treated with MTX. PnV increased the number of macrophages in the tumor, while did not increase in the spleen, suggesting that PnV-activated macrophages were led preferentially to the tumor. Macrophages were activated in vitro by the venom, becoming more phagocytic; these results confirm that this cell is a target of PnV components. Spleen and in vitro PnV-activated macrophages were different of M1, since they did not produce pro- and anti-inflammatory cytokines. Studies in progress are selecting the venom molecules with antitumor and immunomodulatory effects and trying to better understand their mechanisms. The identification, optimization and synthesis of antineoplastic drugs from PnV molecules may lead to a new multitarget chemotherapy. Glioblastoma is associated with high morbidity and mortality; therefore, research to develop new treatments has great social relevance. Natural products and their derivatives represent over one-third of all new molecular entities approved by FDA. However, arthropod venoms are underexploited, although they are a rich source of new molecules. A recent in vitro screening of the Phoneutria nigriventer spider venom (PnV) antitumor effects by our group has shown that the venom significantly affected glioblastoma cell lines. Therefore, it would be relevant to establish the effects of PnV on tumor development in vivo, considering the complex neoplastic microenvironment. The venom was effective at impairing tumor development in murine xenogeneic model, activating the innate immune response and increasing tumor infiltrating macrophages. In addition, PnV activated macrophages in vitro for a different profile of M1. These activated PnV-macrophages have potential to fight the tumor without promoting tumorigenesis. Studies in progress are selecting the venom molecules with antitumor and immunomodulatory effects and trying to better understand their mechanisms. We aim to synthesize and carry out a formulation with these antineoplastic molecules for clinical trials. Spider venom biomolecules induced smaller and necrotic xenogeneic GB; spider venom activated the innate immune system; venom increased blood monocytes and the migration of macrophages to the tumor; activated PnV-macrophages have a profile different of M1 and have a potential to fight the tumor without promote tumorigenesis

Violacein treatment modulates acute and chronic inflammation through the suppression of cytokine production and induction of regulatory T cells

FAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOCNPQ - CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICOInflammation is a necessary process to control infection. However, exacerbated inflammation, acute or chronic, promotes deleterious effects in the organism. Violacein (viola), a quorum sensing metabolite from the Gram-negative bacterium Chromobacterium violaceum, has been shown to protect mice from malaria and to have beneficial effects on tumors. However, it is not known whether this drug possesses anti-inflammatory activity. In this study, we investigated whether viola administration is able to reduce acute and chronic autoimmune inflammation. For that purpose, C57BL/6 mice were intraperitoneally injected with 1 mu g of LPS and were treated with viola (3.5mg/kg) via i.p. at the same time-point. Three hours later, the levels of inflammatory cytokines in the sera and phenotypical characterization of leukocytes were determined. Mice treated with viola presented a significant reduction in the production of inflammatory cytokines compared with untreated mice. Interestingly, although viola is a compound derived from bacteria, it did not induce inflammation upon administration to naive mice. To test whether viola would protect mice from an autoimmune inflammation, Experimental Autoimmune Encephalomyelitis (EAE)-inflicted mice were given viola i.p. at disease onset, at the 10th day from immunization. Viola-treated mice developed mild EAE disease in contrast with placebo-treated mice. The frequencies of dendritic cells and macrophages were unaltered in EAE mice treated with viola. However, the sole administration of viola augmented the levels of splenic regulatory T cells (CD4+ Foxp3+). We also found that adoptive transfer of viola-elicited regulatory T cells significantly reduced EAE. Our study shows, for the first time, that violacein is able to modulate acute and chronic inflammation. Amelioration relied in suppression of cytokine production (in acute inflammation) and stimulation of regulatory T cells (in chronic inflammation). New studies must be conducted in order to assess the possible use of viola in therapeutic approaches in human autoimmune diseases.Inflammation is a necessary process to control infection. However, exacerbated inflammation, acute or chronic, promotes deleterious effects in the organism. Violacein (viola), a quorum sensing metabolite from the Gram-negative bacteriumChromobacterium vio105116FAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOCNPQ - CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICOFAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOCNPQ - CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICOFAPESP [2011/17965-3]CNPq [471066/2012-5]FAPESP [2014/02631-0, 2011/23664-6, 2012/01892-0]2011/17965-3; 471066/2012-5; 2014/02631-0; 2011/23664-6; 2012/01892-0sem informaçã

The role of mTOR (mammalian target of rapamycin) signaling in the mechanism of chloroquine on dendritic cells

Orientadores: Liana Maria Cardoso Verinaud, Rodolfo ThoméDissertação (mestrado) - Universidade Estadual de Campinas, Instituto de BiologiaResumo: Células dendríticas (DCs) são células apresentadoras de antígenos com a capacidade de direcionar a resposta imune tanto para um contexto inflamatório como para um contexto anti-inflamatório dependendo de seu estado de maturação e/ou ativação. DCs imaturas são caracterizadas pela baixa expressão de moléculas de classe II do Complexo Maior de Histocompatibilidade (MHC-II) e moléculas coestimulatórias, como CD80 e CD86, e quando em contato com linfócitos T naïve estimulam sua diferenciação em células regulatórias. Por este motivo são também chamadas de DCs tolerogênicas. Já DCs maduras, por sua vez, expressam elevados níveis de MHC-II, CD80 e CD86, e induzem a geração de células T efetoras. Resultados anteriores do nosso grupo mostraram que o fármaco Cloroquina, mais conhecido por ser usado no tratamento da malária, possui efeitos tolerogênicos sobre as funções das DCs. Embora um número crescente de estudos tenha buscado avaliar o potencial terapêutico de geração e transferência de DCs tolerogênicas, pouco se conhece a respeito das vias de sinalização envolvidas em sua atividade supressora. Recentemente, foi verificado que a via de controle metabólico denominada mTOR (mammalian Target Of Rapamycin) desempenha importante papel na gênese de DCs tolerogênicas. Nesse contexto, o presente projeto tem o objetivo de avaliar a influência da via de sinalização mTOR sobre a atividade de Células Dendríticas tolerogências. Resultados apresentados nesta dissertação mostraram que a via de sinalização mTOR participa do mecanismo de ação da CQ sobre DCs uma vez que sua fosforilação se apresenta aumentada após o tratamento com este fármaco. Além disso, os efeitos moduladores da cloroquina sobre as DCs, visto por sua capacidade de inibir o completo amadurecendo destas células mesmo após um estímulo microbiano, foi revertido quando um inibidor da via mTOR foi adicionado na cultura. Embora parcial, este efeito inibidor foi demonstrado pela manutenção da morfologia das DCs após o estímulo, pela diminuição da expressão de moléculas apresentadoras de antígeno e de moléculas coestimulatórias, pela reduzida capacidade destas células de induzirem proliferação de linfócitos T e, ainda, por sua capacidade de induzir diferenciação de linfócitos T com perfil regulador. Em conjunto, estes resultados são sugestivos de um perfil tolerogênico das DCs. A via mTOR, entretanto, parece não estar envolvida no efeito da CQ sobre o perfil de liberação de citocinas inflamatórias e anti-inflamatórias pelas DCs. Em adição, o bloqueio da via mTOR inibiu a eficácia terapêutica das DCs tolerogênicas sobre o Sistema Nervoso Central de camundongos C57BL/6 portadores de Encefalomielite Autoimune Experimental, um modelo experimental da esclerose múltipla, quando estas células foram, adotivamente, transferidas para os animais. Estes dados sugerem fortemente a participação desta via de sinalização na indução de DCs com perfil tolerogênico e a manutenção de seu efeito mesmo após a transferência destas células para um sistema in vivo. Embora os efeitos da inibição da via mTOR tenham sido parciais, indicando a participação de outras vias de sinalização intracelular na indução do perfil tolerogênico das DCs, os resultados observados neste trabalho posicionam a via de sinalização celular mTOR como um dos mecanismos envolvidos na indução de tolerância imunológicaAbstract: Dendritic cells (DCs) are antigen-presenting cells capable of directing the immune response both to an context inflammatory and to an anti-inflammatory depending on their state of maturation and / or activation. Immature DCs are characterized by the low expression of Class II molecules of the Major Histocompatibility Complex (MHC-II) and costimulatory molecules, such as CD80 and CD86, and when in contact with naïve T lymphocytes stimulate their differentiation in regulatory cells. For this reason, they are also called tolerogenic dendritic cells. Mature DCs, in turn, express high levels of MHC-II, CD-80 and CD-86, and induce generation of effector T cells. Previous findings from our group showed that the drug Chloroquine (CQ), better known to be used in the treatment of malaria, has tolerogenic effects on the functions of DCs. Although a growing number of studies have sought to assess the therapeutic potential of generation and transfer of tolerogenic DCs, little is known about the signaling pathways involved in their suppressive activity. Recently, the metabolic control pathway named mTOR (mammalian Target Of Rapamycin) was shown to play an important role in the genesis of tolerogenic DCs. In this context, the present project aimed to evaluate the influence of the mTOR signaling pathway on the activity of tolerogenic DCs. Results presented in this dissertation showed that the mTOR signaling pathway participates in the mechanism of action of CQ on DCs since its expression is increased after treatment of DCs with that drug. In addition, the modulating effects of chloroquine on dendritic cells, seen by their ability to inhibit the full maturing of these cells even after a microbial stimulus, was reversed when an inhibitor of the mTOR pathway was added to the culture. Although partial, this inhibitory effect was demonstrated by the maintenance of dendritic cell morphology after stimulation, by the decrease in the expression of antigen-presenting molecules and costimulatory molecules, by the reduced ability of these cells to induce proliferation of T lymphocytes, and by their ability to induce differentiation of T lymphocytes with regulatory profile. Taken together, these results are suggestive of a tolerogenic profile of DCs. The mTOR pathway, however, does not appear to be involved in the effect of CQ on the release profile of inflammatory and anti-inflammatory cytokines by DCs. In addition, blocking the mTOR pathway inhibited the therapeutic efficacy of tolerogenic DCs on the Central Nervous System of C57BL/6 mice bearing Experimental Autoimmune Encephalomyelitis, an experimental model of multiple sclerosis, when these cells were transferred to the animals. These data strongly suggest the participation of this signaling pathway in the induction of DCs with a tolerogenic profile and the maintenance of their effect even after the transfer of these cells to an in vivo system. Although the effects of inhibition of the mTOR pathway have been partial, indicating the participation of other intracellular signaling pathways in inducing the tolerogenic profile of DCs, the results observed in this work position the mTOR cell signaling pathway as one of the mechanisms involved in the induction of immunological toleranceMestradoImunologiaMestra em Genética e Biologia Molecular148543/2016-2CAPESCNP

Severe Changes in Thymic Microenvironment in a Chronic Experimental Model of Paracoccidioidomycosis.

T cell maturation takes place within the thymus, a primary lymphoid organ that is commonly targeted during infections. Previous studies showed that acute infection with Paracoccidioides brasiliensis (Pb), the causative agent of paracoccidioidomycosis (PCM), promotes thymic atrophy that is associated with the presence of yeast cells in the organ. However, as human PCM is a chronic infection, it is imperative to investigate the consequences of Pb infection over the thymic structure and function in chronic infection. In this sense, we developed a new experimental model where Pb yeast cells are injected through the intraperitoneal route and mice are evaluated over 120 days of infection. Thymuses were analyzed in chronically infected mice and we found that the thymus underwent extensive morphological alterations and severe infiltration of P. brasiliensis yeast cells. Further analyses showed an altered phenotype and function of thymocytes that are commonly found in peripheral mature T lymphocytes. We also observed activation of the NLRP3 inflammasome in the thymus. Our data provide new information on the severe changes observed in the thymic microenvironment in a model of PCM that more closely mimics the human infection

Correction: Severe Changes in Thymic Microenvironment in a Chronic Experimental Model of Paracoccidioidomycosis.

[This corrects the article DOI: 10.1371/journal.pone.0164745.]

Severe changes in thymic microenvironment in a chronic experimental model of paracoccidioidomycosis

T cell maturation takes place within the thymus, a primary lymphoid organ that is commonly targeted during infections. Previous studies showed that acute infection with Paracoccidioides brasiliensis (Pb), the causative agent of paracoccidioidomycosis (PCM), promotes thymic atrophy that is associated with the presence of yeast cells in the organ. However, as human PCM is a chronic infection, it is imperative to investigate the consequences of Pb infection over the thymic structure and function in chronic infection. In this sense, we developed a new experimental model where Pb yeast cells are injected through the intraperitoneal route and mice are evaluated over 120 days of infection. Thymuses were analyzed in chronically infected mice and we found that the thymus underwent extensive morphological alterations and severe infiltration of P. brasiliensis yeast cells. Further analyses showed an altered phenotype and function of thymocytes that are commonly found in peripheral mature T lymphocytes. We also observed activation of the NLRP3 inflammasome in the thymus. Our data provide new information on the severe changes observed in the thymic microenvironment in a model of PCM that more closely mimics the human infection1110CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESPSem informação#2014/19492-3; 2013/08194-9; #2012/22131-7; #2013/01401-9; #2014/02631-0; #2012/03238-

Venom of the Phoneutria nigriventer spider alters the cell cycle, viability, and migration of cancer cells

The mechanisms of cancer involve changes in multiple biological pathways. Multitarget molecules, which are components of animal venoms, are therefore a potential strategy for treating tumors. The objective of this study was to screen the effects of Phoneutria nigriventer spider venom (PnV) on tumor cell lines. Cultured human glioma (NG97), glioblastoma (U-251) and cervix adenocarcinoma (HeLa) cells, and nontumor mouse fibroblasts (L929) were treated with low (14 mu g/ml) and high (280 mu g/ml) concentrations of PnV, and analyzed through assays for cell viability (thiazolyl blue tetrazolium blue), proliferation (carboxyfluorescein succinimidyl ester), death (annexin V/propidium iodide [Pi]), the cell cycle (Pi), and migration (wound healing and transwell assay). The venom decreased the viability of U-251 cells, primarily by inducing cell death, and reduced the viability of NG97 cells, primarily by inhibiting the cell cycle. The migration of all the tumor cell lines was delayed when treated with venom. The venom significantly affected all the tumor cell lines studied, with no cytotoxic effect on normal cells (L929), although the nonglial tumor cell (HeLa) was less sensitive to PnV. The results of the current study suggest that PnV may be composed of peptides that are highly specific for the multiple targets involved in the hallmarks of cancer. Experiments are underway to identify these molecules234213981415CNPQ - Conselho Nacional de Desenvolvimento Científico e TecnológicoFAPESP – Fundação de Amparo à Pesquisa Do Estado De São Paulo431465/2016-92017/16196-2; 2017/05402-0; 2015/04194-0; 2016/15827-

Nitric oxide plays a key role in the suppressive activity of tolerogenic dendritic cells

FAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO123384386FAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOFAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO2011/17965-3; 2013/08194-9; 2014/02631-

Increased inflammasome and caspase-1 activity in the thymus of infected mice.

<p>Male BALB/c mice (n = 5 mice/group for each analyses per experiment replicate) were inoculated with 5x10⁶ Pb18 yeasts contained in PBS intraperitoneally or with only PBS (control group). One hundred and twenty days after inoculation, mice were killed and the thymus was collected and processed individually for analysis. A) Increased initiator caspase-8 gene expression on 120dpi group compared to the naive group. Increased inflammatory caspase-1 gene expression on 120dpi group compared to the naive group. Increased NLRP3 inflammasome gene expression on 120dpi group compared to the naive group. B) Increased pro-caspase-1 production and increased caspase-1 activity on 120dpi group compared to the naive group. C) Increased NLRP3 inflammasome complex assembly on 120dpi compared to the naive group. Statistical analysis was carried out with Student’s t-test. **p<0.01, ****p<0.0001. Representative data from three independent experiments with similar results. Expression levels of genes were represented as a relative copy numbers by using the method of delta threshold (2^-ΔΔCt). Image J software (NIH, MD, USA) was used for the estimation of the pro-caspase-1, the active form of caspase-1 and NLRP3 inflammasome assembly, through a GAPDH ratio.</p