The antitumor effect of metformin has been demonstrated in several types of cancer; however, the mechanisms involved are incompletely understood. In this study, we showed that metformin acts directly on melanoma cells as well as on the tumor microenvironment, particularly in the context of the immune response. In vitro, metformin induces a complex interplay between apoptosis and autophagy in melanoma cells. The anti-metastatic activity of metformin in vivo was assessed in several mouse models challenged with B16F10 cells. Metformin's activity was, in part, immune system-dependent, whereas its antitumor properties were abrogated in immunodeficient (NSG) mice. Metformin treatment increased the number of lung CD8-effector-memory T and CD4+Foxp3+IL-10+ T cells in B16F10-transplanted mice. It also decreased the levels of Gr-1+CD11b+ and RORγ+ IL17+CD4+ cells in B16F10-injected mice and the anti-metastatic effect was impaired in RAG-1−/− mice challenged with B16F10 cells, suggesting an important role for T cells in the protection induced by metformin. Finally, metformin in combination with the clinical metabolic agents rapamycin and sitagliptin showed a higher antitumor effect. The metformin/sitagliptin combination was effective in a BRAFV600E/PTEN tamoxifen-inducible murine melanoma model. Taken together, these results suggest that metformin has a pronounced effect on melanoma cells, including the induction of a strong protective immune response in the tumor microenvironment, leading to tumor growth control, and the combination with other metabolic agents may increase this effect.

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.

The increased incidence, high rates of mortality and few effective means of treatment of malignant melanoma, stimulate the search for new anti-tumor agents and therapeutic targets to control this deadly metastatic disease. In the present work the antitumor effect of arazyme, a natural bacterial-derived metalloprotease secreted by Serratia proteomaculans, was investigated. Arazyme significantly reduced the number of pulmonary metastatic nodules after intravenous inoculation of B16F10 melanoma cells in syngeneic mice. In vitro, the enzyme showed a dose-dependent cytostatic effect in human and murine tumor cells, and this effect was associated to the proteolytic activity of arazyme, reducing the CD44 expression at the cell surface, and also reducing in vitro adhesion and in vitro/in vivo invasion of these cells. Arazyme treatment or immunization induced the production of protease-specific IgG that cross-reacted with melanoma MMP-8. In vitro, this antibody was cytotoxic to tumor cells, an effect increased by complement. In vivo, arazyme-specific IgG inhibited melanoma lung metastasis. We suggest that the antitumor activity of arazyme in a preclinical model may be due to a direct cytostatic activity of the protease in combination with the elicited anti-protease antibody, which cross-reacts with MMP-8 produced by tumor cells. Our results show that the bacterial metalloprotease arazyme is a promising novel antitumor chemotherapeutic agent.

NLRP3 inflammasome [NLR (nucleotide-binding domain, leucine-rich repeat containing protein) Pyrin-domain-containing 3 ] functions as an innate sensor of several PAMPs and DAMPs (pathogen- and damage-associated molecular patterns). It has been also reported as a transcription factor related to Th2 pattern, although its role in the adaptive immunity has been controversial, mainly because the studies were performed using gene deletion approaches. In the present study, we have investigated the NLRP3 gain-of-function in the context of encephalomyelitis autoimmune disease (EAE), considered to be a Th1- and Th17-mediated disease. We took advantage of an animal model with NLRP3 gain-of-function exclusively to T CD4+ lymphocytes (CD4CreNLRP3fl/fl). These mice presented reduced clinical score, accompanied by less infiltrating T CD4+ cells expressing both IFN-γ and IL-17 at the central nervous system (CNS) during the peak of the disease. However, besides NLRP3 gain-of-function in lymphocytes, these mice lack NLRP3 expression in non-T CD4+ cells. Therefore, in order to circumvent this deficiency, we transferred naive CD4+ T cells from WT, NLRP3−/− or CD4CreNLRP3fl/fl into Rag-1−/− mice and immunized them with MOG35–55. Likewise, the animals repopulated with CD4CreNLRP3fl/fl T CD4+ cells presented reduced clinical score and decreased IFN-γ production at the peak of the disease. Additionally, primary effector CD4+ T cells derived from these mice presented reduced glycolytic profile, a metabolic profile compatible with Th2 cells. Finally, naive CD4+ T cells from CD4CreNLRP3fl/fl mice under a Th2-related cytokine milieu cocktail exhibited in vitro an increased IL-4 and IL-13 production. Conversely, naive CD4+ T cells from CD4CreNLRP3fl/fl mice under Th1 differentiation produced less IFN-γ and T-bet. Altogether, our data evidence that the NLRP3 gain-of-function promotes a Th2-related response, a pathway that could be better explored in the treatment of multiple sclerosis.

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BACKGROUND: Host-microbiota interactions shape T-cell differentiation and promote tumour immunity. Although IL-9-producing T cells have been described as potent antitumour effectors, their role in microbiota-mediated tumour control remains unclear. METHODS: We analysed the impact of the intestinal microbiota on the differentiation of colonic lamina propria IL-9-producing T cells in germ-free and dysbiotic mice. Systemic effects of the intestinal microbiota on IL-9-producing T cells and the antitumour role of IL-9 were analysed in a model of melanoma-challenged dysbiotic mice. RESULTS: We show that germ-free mice have lower frequency of colonic lamina propria IL-9-producing T cells when compared with conventional mice, and that intestinal microbiota reconstitution restores cell frequencies. Long-term antibiotic treatment promotes host dysbiosis, diminishes intestinal IL-4 and TGF-β gene expression, decreases the frequency of colonic lamina propria IL-9-producing T cells, increases the susceptibility to tumour development and reduces the frequency of IL-9-producing T cells in the tumour microenvironment. Faecal transplant restores intestinal microbiota diversity, and the frequency of IL-9-producing T cells in the lungs of dysbiotic animals, restraining tumour burden. Finally, recombinant IL-9 injection enhances tumour control in dysbiotic mice. CONCLUSIONS: Host-microbiota interactions are required for adequate differentiation and antitumour function of IL-9-producing T cells.

Conclusions: Multiplex-PCR proved to be more effective than culture for plague diagnosis, both for old and recent samples. This technique would be a valuable tool for the plague control programme 1 . Significance and Impact of the Study: The multiplex-PCR technique can be useful for the detection and characterization of Y. pestis even when the bacteria are no longer viable and when culture diagnosis has been hampered by the growth of contaminants.

Despite the recent approval of new agents for metastatic melanoma, its treatment remains challenging. Moreover, few available immunotherapies induce a strong cellular immune response, and selection of the correct immunoadjuvant is crucial for overcoming this obstacle. Here, we studied the immunomodulatory properties of arazyme, a bacterial metalloprotease, which was previously shown to control metastasis in a murine melanoma B16F10-Nex2 model. The antitumor activity of arazyme was independent of its proteolytic activity, since heat-inactivated protease showed comparable properties to the active enzyme; however, the effect was dependent on an intact immune system, as antitumor properties were lost in immunodeficient mice. The protective response was IFNγ-dependent, and CD8(+) T lymphocytes were the main effector antitumor population, although B and CD4(+) T lymphocytes were also induced. Macrophages and dendritic cells were involved in the induction of the antitumor response, as arazyme activation of these cells increased both the expression of surface activation markers and proinflammatory cytokine secretion through TLR4-MyD88-TRIF-dependent, but also MAPK-dependent pathways. Arazyme was also effective in the murine breast adenocarcinoma 4T1 model, reducing primary and metastatic tumor development, and prolonging survival. To our knowledge, this is the first report of a bacterial metalloprotease interaction with TLR4 and subsequent receptor activation that promotes a proinflammatory and tumor protective response. Our results show that arazyme has immunomodulatory properties, and could be a promising novel alternative for metastatic melanoma treatment.