CD14 signaling mediates lung immunopathology and mice mortality induced by Achromobacter xylosoxidans.

OBJECTIVE AND DESIGN: Our research aimed to investigate the role of CD14 in pulmonary infection by Achromobacter xylosoxidans in an experimental murine model. METHODS: C57Bl/6 or CD14-deficient mice were infected intratracheally with non-lethal inoculum of A. xylosoxidans. At times 1, 3 and 7 days after infection, lungs, bronchoalveolar lavage and blood were collected. CD14 gene expression was determined by RT-PCR. The bacterial load in the lungs was assessed by counting colony forming units (CFU). Cytokines, chemokines, lipocalin-2 and sCD14 were quantified by the ELISA method. Inflammatory infiltrate was observed on histological sections stained with HE, and leukocyte subtypes were assessed by flow cytometry. In another set of experiments, C57Bl/6 or CD14-deficient mice were inoculated with lethal inoculum and the survival rate determined. RESULTS: CD14-deficient mice are protected from A. xylosoxidans-induced death, which is unrelated to bacterial load. The lungs of CD14-deficient mice presented a smaller area of tissue damage, less neutrophil and macrophage infiltration, less pulmonary edema, and a lower concentration of IL-6, TNF-α, CXCL1, CCL2 and CCL3 when compared with lungs of C57Bl/6 mice. We also observed that A. xylosoxidans infection increases the number of leukocytes expressing mCD14 and the levels of sCD14 in BALF and serum of C57Bl/6-infected mice. CONCLUSIONS: In summary, our data show that in A. xylosoxidans infection, the activation of CD14 induces intense pulmonary inflammatory response resulting in mice death.

Targeted analysis of eicosanoids derived from cytochrome P450 pathway by high-resolution multiple-reaction monitoring mass spectrometry.

Cytochrome P450 (CYP450) pathway is one of the critical enzymatic via eicosanoid biosynthesis. Nevertheless, their metabolites are far less explored. This pathway plays a crucial role in converting arachidonic acid to hydroxyeicosatetraenoic (HETEs), epoxyeicosatrienoic (EETs), dihydroxyeicosatetraenoic acids (DiHETEs), and dihydroxyeicosatrienoic acids (DiHETrEs), which mediate several physiological and pathological functions. However, CYP450-derived eicosanoids are structurally complex, making those analyses a challenge in lipidomics studies. Herein, a high-resolution multiple-reaction monitoring (MRMHR ) method has been proposed as a powerful tool for the simultaneous analysis of CYP450-eicosanoids on different biological samples. The developed liquid chromatography (LC)-MRMHR method was partially validated according to the Food and Drug Administration (FDA) criteria, demonstrating adequate specificity, linearity, precision, and accuracy. Besides, several biological samples were analyzed to guarantee the feasibility of the method. The proposed strategy may improve the understanding of CYP450-derived eicosanoids in biological systems, which could be fundamental to reveal new aspects of those in physiologic and pathologic conditions.

The influence of dehydroepiandrosterone on effector functions of neutrophils

Dehydroepiandrosterone (DHEA) is a steroid hormone secreted by the adrenal glands, gonads and brain. It is a precursor to sex hormones and also is known to have immune modulatory activity. However, little is known about the relationship between DHEA and neutrophils and thus our study evaluates the influence of DHEA in the effector functions of neutrophils. Human neutrophils were treated in vitro with DHEA and further infected with Salmonella enterica serovar Typhimurium. The treatment of neutrophils with 0.01 µM of DHEA increased the phagocytosis of Salmonella independent of TLR4 as the treatment did not modulate the TLR4 expression. Additionally, DHEA caused a decrease in ROS (reactive oxygen species) production and did not influence the formation of the neutrophil extracellular trap (NET). Steroid treated neutrophils, infected or stimulated with LPS (lipopolysaccharide), showed reduced production of IL-8, compared to untreated cells. Also, the protein levels of p-NFκB were decreased in neutrophils treated with DHEA, and this reduction could explain the reduced levels of IL-8. These results led us to conclude that the steroid hormone DHEA has important modulatory functions in neutrophils

High-resolution multiple reaction monitoring method for quantification of steroidal hormones in plasma.

Multiple reaction monitoring (MRM) is one of the most powerful modes of analysis in liquid chromatographic tandem mass spectrometry for quantification of low-concentration metabolites in biological samples. The advances in mass spectrometry enabled the development of high-resolution multiple reaction monitoring (MRMHR ) and became suitable for the more specific analysis of target analytes. This is important for lipidomic studies and contributes in the medical and pharmaceutical fields, primarily in investigating alterations in cells or fluids relevant to various diseases. Therefore, this work proposes the development of the MRMHR method for quantification of circulating steroids. We focused on the determination of corticosterone, 11-dehydrocorticosterone (11-DHC), cortisol, cortisone, aldosterone, and progesterone concentration in serum, by using 129sv male mice exposed to chronic unpredictable stress to validate the quantification. The method was conducted according to the ANVISA normative, adopting a coefficient of variation, as well as relative standard deviation and relative error lower than 15% in linearity, intraday and interday precision, and accuracy. For cortisol, corticosterone, and their inert metabolites (cortisone and 11-DHC), the lower limit of quantification was 3.9 ng· mL-1 , while that for progesterone and aldosterone was 7.8 and 15.6 ng· mL-1 , respectively. MRMHR analysis showed that animals submitted to stressors have 4.5 times more corticosterone in their serum than nonstressed mice. However, 11-DHC concentration does not vary significantly in response to stress for these animals. The results indicate that the method can be applied for quantification of steroids in several biological samples, such as human plasma.

Protective Effect of Galectin-1 during Histoplasma capsulatum Infection Is Associated with Prostaglandin E2 and Nitric Oxide Modulation.

Histoplasma capsulatum is a dimorphic fungus that develops a yeast-like morphology in host's tissue, responsible for the pulmonary disease histoplasmosis. The recent increase in the incidence of histoplasmosis in immunocompromised patients highlights the need of understanding immunological controls of fungal infections. Here, we describe our discovery of the role of endogenous galectin-1 (Gal-1) in the immune pathophysiology of experimental histoplasmosis. All infected wild-type (WT) mice survived while only 1/3 of Lgals1-/- mice genetically deficient in Gal-1 survived 30 days after infection. Although infected Lgals1-/- mice had increased proinflammatory cytokines, nitric oxide (NO), and elevations in neutrophil pulmonary infiltration, they presented higher fungal load in lungs and spleen. Infected lung and infected macrophages from Lgals1-/- mice exhibited elevated levels of prostaglandin E2 (PGE2, a prostanoid regulator of macrophage activation) and prostaglandin E synthase 2 (Ptgs2) mRNA. Gal-1 did not bind to cell surface of yeast phase of H. capsulatum, in vitro, suggesting that Gal-1 contributed to phagocytes response to infection rather than directly killing the yeast. The data provides the first demonstration of endogenous Gal-1 in the protective immune response against H. capsulatum associated with NO and PGE2 as an important lipid mediator in the pathogenesis of histoplasmosis.

Erythropoietin Exacerbates Inflammation and Increases the Mortality of Histoplasma capsulatum-Infected Mice.

Erythropoietin (EPO) is a key hormone involved in red blood cell formation, but its effects on nonerythroid cells, such as macrophages, have not been described. Macrophages are key cells in controlling histoplasmosis, a fungal infection caused by Histoplasma capsulatum (Hc). Considering that little is known about EPO's role during fungal infections and its capacity to activate macrophages, in this study we investigated the impact of EPO pretreatment on the alveolar immune response during Hc infection. The consequence of EPO pretreatment on fungal infection was determined by evaluating animal survival, fungal burden, activation of bronchoalveolar macrophages, inflammatory mediator release, and lung inflammation. Pretreatment with EPO diminished mononuclear cell numbers, increased the recruitment of F4/80(+)/CD80(+) and F4/80(+)/CD86(+) cells to the bronchoalveolar space, induced higher production of IFN-γ, IL-6, MIP-1α, MCP-1, and LTB4, reduced PGE2 concentration, and did not affect fungal burden. As a consequence, we observed an increase in lung inflammation with extensive tissue damage that might account for augmented mouse mortality after infection. Our results demonstrate for the first time that EPO treatment has a deleterious impact on lung immune responses during fungal infection.