Early Leishmania infectivity depends on miR-372/373/520d family-mediated reprogramming of polyamines metabolism in THP-1-derived macrophages.

Leishmania amazonensis is a protozoan that primarily causes cutaneous leishmaniasis in humans. The parasite relies on the amino acid arginine to survive within macrophages and establish infection, since it is a precursor for producing polyamines. On the other hand, arginine can be metabolized via nitric oxide synthase 2 (NOS2) to produce the microbicidal molecule nitric oxide (NO), although this mechanism does not apply to human macrophages since they lack NOS2 activity. MicroRNAs (miRNAs) are small noncoding RNAs that regulate gene expression at posttranscriptional levels. Our previous work showed that mmu-miR-294 targets Nos2 favoring Leishmania survival in murine macrophages. Here, we demonstrate that human macrophages upregulate the hsa-miR-372, hsa-miR-373, and hsa-miR-520d, which present the same seed sequence as the murine mmu-miR-294. Inhibition of the miR-372 impaired Leishmania survival in THP-1 macrophages and the effect was further enhanced with combinatorial inhibition of the miR-372/373/520d family, pointing to a cooperative mechanism. However, this reduction in survival is not caused by miRNA-targeting of NOS2, since the seed-binding motif found in mice is not conserved in the human 3'UTR. Instead, we showed the miR-372/373/520d family targeting the macrophage's main arginine transporter SLC7A2/CAT2 during infection. Arginine-related metabolism was markedly altered in response to infection and miRNA inhibition, as measured by Mass Spectrometry-based metabolomics. We found that Leishmania infection upregulates polyamines production in macrophages, as opposed to simultaneous inhibition of miR-372/373/520d, which decreased putrescine and spermine levels compared to the negative control. Overall, our study demonstrates miRNA-dependent modulation of polyamines production, establishing permissive conditions for intracellular parasite survival. Although the effector mechanisms causing host cell immunometabolic adaptations involve various parasite and host-derived signals, our findings suggest that the miR-372/373/520d family may represent a potential target for the development of new therapeutic strategies against cutaneous leishmaniasis.

Lipopolysaccharide Regulates Pro- and Anti-Inflammatory Cytokines, Corticosterone, and Melatonin in Toads.

Glucocorticoids and melatonin (MEL) show integrated and complex immunomodulatory effects, mostly described for endotherms, yet underexplored in amphibians. In this context, the RT-qPCR of molecules mediating inflammatory processes in amphibians is a valuable tool to explore the relationships among molecular biology, endocrine mediators, and immune response in these animals. In this study, toads (Rhinella diptycha) received an intraperitoneal saline injection or lipopolysaccharide (LPS; 2 mg/kg). Six hours post-injection, we analyzed plasma corticosterone (CORT) and MEL levels and pro- and anti-inflammatory molecules (IL-1ß, IL-6, IL-10, IFN-γ, and C1s). We found increased CORT and decreased MEL levels in response to LPS. Also, IL-1ß, IL-6, and IL-10 were upregulated in LPS-injected toads compared with saline-injected toads. Overall, our results demonstrate an LPS-induced inflammatory response with endocrine and immune modulation in R. diptycha toads, exhibiting expected patterns for an inflammatory stimulus within this time frame (6 h post-injection). Toads were responsive to LPS by secreting different cytokines, such as proinflammatory cytokines IL-1ß and IL-6, related to immune cell attraction to inflammatory sites and the anti-inflammatory cytokine IL-10, which limits the rate of leukocyte infiltration, inflammation, and downregulates the expression of proinflammatory cytokines. Increased circulating CORT levels are probably associated with the activation of the hypothalamus-pituitary-interrenal axis by the LPS and the endocrine actions of IL-6. Furthermore, decreased circulating MEL levels are likely due to inhibited MEL secretion by the pineal gland by inflammatory stimuli, indicating the activation/existence of the immune-pineal axis in amphibians.

Os glicocorticoides e a melatonina (MEL) apresentam efeitos imunomoduladores integrados e complexos, principalmente descritos para endotérmicos, porém pouco explorados em anfíbios. Nesse contexto, o RT-qPCR de moléculas mediadoras de processos inflamatórios em anfíbios é uma ferramenta valiosa para explorar as relações entre biologia molecular, mediadores endócrinos e resposta imune nesses animais. Neste estudo, sapos (Rhinella diptycha) receberam uma injeção intraperitoneal de solução salina ou lipopolissacarídeo (LPS; 2 mg/kg). Seis horas após a injeção, analisamos as concentrações plasmáticas de corticosterona (CORT) e MEL e moléculas pró e anti-inflamatórias (IL-1ß, IL-6, IL-10, IFN-γ e C1s). Encontramos aumento de CORT e diminuição da concentração de MEL em resposta ao LPS. Além disso, IL-1ß, IL-6 e IL-10 foram reguladas positivamente em sapos injetados com LPS em comparação com os sapos injetados com solução salina. No geral, nossos resultados demonstram uma resposta inflamatória induzida por LPS com modulação endócrina e imunológica em sapos R. diptycha, exibindo padrões esperados para um estímulo inflamatório dentro deste período (6 h pós-injeção). Os sapos foram responsivos ao LPS secretando diferentes citocinas, como as citocinas pró-inflamatórias IL-1ß e IL-6, relacionadas à atração de células imunes para os sítios inflamatórios e a citocina anti-inflamatória IL-10, que limita a taxa de infiltração leucocitária, inflamação, e diminui a expressão de citocinas pró-inflamatórias. As concentrações elevadas de CORT circulante estão provavelmente associados à ativação do eixo hipotálamo-hipófise-interrenal pelo LPS e às ações endócrinas da IL-6. Além disso, a diminuição da concentração circulante de MEL é provavelmente devida à inibição da secreção de MEL pela glândula pineal por estímulos inflamatórios, indicando a ativação/existência do eixo imune-pineal em anfíbios.

MT1 and MT2 melatonin receptors play opposite roles in brain cancer progression.

Primary brain tumors remain among the deadliest of all cancers. Glioma grade IV (glioblastoma), the most common and malignant type of brain cancer, is associated with a 5-year survival rate of < 5%. Melatonin has been widely reported as an anticancer molecule, and we have recently demonstrated that the ability of gliomas to synthesize and accumulate this indolamine in the surrounding microenvironment negatively correlates with tumor malignancy. However, our understanding of the specific effects mediated through the activation of melatonin membrane receptors remains limited. Thus, here we investigated the specific roles of MT1 and MT2 in gliomas and medulloblastomas. Using the MT2 antagonist DH97, we showed that MT1 activation has a negative impact on the proliferation of human glioma and medulloblastoma cell lines, while MT2 activation has an opposite effect. Accordingly, gliomas have a decreased mRNA expression of MT1 (also known as MTNR1A) and an increased mRNA expression of MT2 (also known as MTNR1B) compared to the normal brain cortex. The MT1/MT2 expression ratio negatively correlates with the expression of cell cycle-related genes and is a positive prognostic factor in gliomas. Notably, we showed that functional selective drugs that simultaneously activate MT1 and inhibit MT2 exert robust anti-tumor effects in vitro and in vivo, downregulating the expression of cell cycle and energy metabolism genes in glioma stem-like cells. Overall, we provided the first evidence regarding the differential roles of MT1 and MT2 in brain tumor progression, highlighting their relevance as druggable targets. KEY MESSAGES: • MT1 impairs while MT2 promotes the proliferation of glioma and medulloblastoma cell lines. • Gliomas have a decreased expression of MT1 and an increased expression of MT2 compared to normal brain cortex. • Tumors with a high MT1/MT2 expression ratio have significantly better survival rates. • Functional selective drugs that simultaneously activate MT1 and inhibit MT2 downregulate the expression of cell cycle and energy metabolism genes in glioma stem-like cells and exert robust anti-tumor effects in vivo.