Ghrelin effects on mitochondrial fitness modulates macrophage function.

Over the past years, systemic derived cues that regulate cellular metabolism have been implicated in the regulation of immune responses. Ghrelin is an orexigenic hormone produced by enteroendocrine cells in the gastric mucosa with known immunoregulatory roles. The mechanism behind the function of ghrelin in immune cells, such as macrophages, is still poorly understood. Here, we explored the hypothesis that ghrelin leads to alterations in macrophage metabolism thus modulating macrophage function. We demonstrated that ghrelin exerts an immunomodulatory effect over LPS-activated peritoneal macrophages, as evidenced by inhibition of TNF-α and IL-1ß secretion and increased IL-12 production. Concomitantly, ghrelin increased mitochondrial membrane potential and increased respiratory rate. In agreement, ghrelin prevented LPS-induced ultrastructural damage in the mitochondria. Ghrelin also blunted LPS-induced glycolysis. In LPS-activated macrophages, glucose deprivation did not affect ghrelin-induced IL-12 secretion, whereas the inhibition of pyruvate transport and mitochondria-derived ATP abolished ghrelin-induced IL-12 secretion, indicating a dependence on mitochondrial function. Ghrelin pre-treatment of metabolic activated macrophages inhibited the secretion of TNF-α and enhanced IL-12 levels. Moreover, ghrelin effects on IL-12, and not on TNF-α, are dependent on mitochondria elongation, since ghrelin did not enhance IL-12 secretion in metabolic activated mitofusin-2 deficient macrophages. Thus, ghrelin affects macrophage mitochondrial metabolism and the subsequent macrophage function.

Mitoimmunity-when mitochondria dictates macrophage function.

In the past decade, several reports have appointed the importance of mitochondria in the immune response. Our understanding of mitochondria evolved from a simple supplier of energy into a platform necessary for immunorregulation. Proinflammatory responses are associated with enhanced glycolytic activity and breakdown of the TCA cycle. Mitochondrial reactive species of oxygen (mROS) are key regulators of classically activated macrophages, with substantial impact in the anti-microbicidal activity and pro-inflammatory cytokine secretion of macrophages. The inflammasome activation in macrophages is dependent on mROS production and mitochondrial regulation and mitochondrial dynamics and functionality direct impact inflammatory responses. Alternative activated macrophage metabolism relies on fatty acid oxidation, and the mechanism responsible for this phenotype is not fully elucidated. Thus, cellular metabolism and mitochondria function is a key immunoregulatory feature of macrophage biology. In this review, we will provide insights into recently reported evidences of mitochondria-related metabolic nodes, which are important for macrophage physiology.

Standardization of a Continuous Assay for Glycosidases and Its Use for Screening Insect Gut Samples at Individual and Populational Levels.

Glycoside Hydrolases (GHs) are enzymes able to recognize and cleave glycosidic bonds. Insect GHs play decisive roles in digestion, in plant-herbivore, and host-pathogen interactions. GH activity is normally measured by the detection of a release from the substrate of products as sugars units, colored, or fluorescent groups. In most cases, the conditions for product release and detection differ, resulting in discontinuous assays. The current protocols result in using large amounts of reaction mixtures for the obtainment of time points in each experimental replica. These procedures restrain the analysis of biological materials with limited amounts of protein and, in the case of studies regarding small insects, implies in the pooling of samples from several individuals. In this respect, most studies do not assess the variability of GH activities across the population of individuals from the same species. The aim of this work is to approach this technical problem and have a deeper understanding of the variation of GH activities in insect populations, using as models the disease vectors Rhodnius prolixus (Hemiptera: Triatominae) and Lutzomyia longipalpis (Diptera: Phlebotominae). Here we standardized continuous assays using 4-methylumbelliferyl derived substrates for the detection of α-Glucosidase, ß-Glucosidase, α-Mannosidase, N-acetyl-hexosaminidase, ß-Galactosidase, and α-Fucosidase in the midgut of R. prolixus and L. longipalpis with results similar to the traditional discontinuous protocol. The continuous assays allowed us to measure GH activities using minimal sample amounts with a higher number of measurements, resulting in data that are more reliable and less time and reagent consumption. The continuous assay also allows the high-throughput screening of GH activities in small insect samples, which would be not applicable to the previous discontinuous protocol. We applied continuous GH measurements to 90 individual samples of R. prolixus anterior midgut homogenates using a high-throughput protocol. α-Glucosidase and α-Mannosidase activities showed the normal distribution in the population. ß-Glucosidase, ß-Galactosidase, N-acetyl-hexosaminidase, and α-Fucosidase activities showed non-normal distributions. These results indicate that GHs fluorescent-based high-throughput assays apply to insect samples and that the frequency distribution of digestive activities should be considered in data analysis, especially if a small number of samples is used.