Vitamin D-induced LL-37 modulates innate immune responses of human primary macrophages during DENV-2 infection.

Epidemics of dengue, an acute and potentially severe disease caused by mosquito-borne dengue virus (DENV), pose a major challenge to clinicians and health care services across the sub(tropics). Severe disease onset is associated with a dysregulated inflammatory response to the virus, and there are currently no drugs to alleviate disease symptoms. LL-37 is a potent antimicrobial peptide with a wide range of immunoregulatory properties. In this study, we assessed the effect of LL-37 on DENV-2-induced responses in human monocyte-derived macrophages (MDMs). We show that simultaneous exposure of exogenous LL-37 and DENV-2 resulted in reduced replication of the virus in MDMs, while the addition of LL-37 postexposure to DENV-2 did not. Interestingly, the latter condition reduced the production of IL-6 and increased the expression of genes involved in virus sensing and antiviral response. Finally, we demonstrate that low endogenous levels and limited production of LL-37 in MDMs in response to DENV-2 infection can be increased by differentiating MDMs in the presence of Vitamin D (VitD3). Taken together, this study demonstrates that in addition to its antimicrobial properties, LL-37 has immunomodulatory properties in the curse of DENV infection and its production can be increased by VitD3.

Vitamin D Regulates the Expression of Immune and Stress Response Genes in Dengue Virus-infected Macrophages by Inducing Specific MicroRNAs.

BACKGROUND: The pathogenesis associated with Dengue virus (DENV) infection is marked by the impairment of host immune response. Consequently, the modulation of immune response has emerged as an important therapeutic target for the control of DENV infection. Vitamin D has been shown to regulate the immune response in DENV infection, although the molecular mechanism remains poorly understood. Post-transcriptional regulation of mRNA by miRNAs offers an opportunity to gain insight into the immunomodulation mediated by vitamin D. OBJECTIVE: Previously, it has been observed that a high dose of vitamin D (4000 IU) decreased DENV-2 infection and inflammatory response in monocyte-derived macrophages (MDMs). Here, we examine whether high or low doses of vitamin D supplements exert differential effect on miRNA expression in DENV-infected macrophages. METHODS: We analyzed miRNA expression profiles in MDMs isolated from healthy individuals who were given either 1000 or 4000 IU/day of vitamin D for 10 days. MDMs before or after vitamin D supplementation were challenged with DENV-2, and miRNAs profiles were analyzed by qPCR arrays. RESULTS: DENV-2 infected MDMs supplemented with 4000 IU, showed up-regulation of miR-374a-5p, miR-363-3p, miR-101-3p, miR-9-5p, miR-34a-5p, miR-200a-3p, and the family of miRNAs miR-21-5p, and miR-590-p. The miRNA profile and predicted target mRNAs suggested regulatory pathways in MDMs obtained from healthy donors who received higher doses of vitamin D. These DENV-2 infected MDMs expressed a unique set of miRNAs that target immune and cellular stress response genes. CONCLUSION: The results suggest vitamin D dose-dependent differential expression of miRNAs target key signaling pathways of the pathogenesis of dengue disease.

[Modulation of high-density lipoprotein and cytokine IL-1ß and IL-6 levels in patients with dengue]. / Modulación de los niveles de lipoproteínas de alta densidad y las citoquinas IL-1ß e IL-6 en pacientes con dengue.

OBJECTIVES: To assess High-Density Lipoprotein (HDL) and pro-inflammatory cytokine levels in patients with dengue classified according to the severity of the clinical presentation. MATERIALS AND METHODS: A descriptive study was carried out with 70 individuals with dengue, classified as no alarm signs (DSSA), with signs of alarm (DCSA), and severe dengue (DG); 15 healthy donors were included as controls (CS). The serum levels of IL-1ß and IL-6 were quantified through ELISA, and the HDL levels through serum by a colorimetric test. In addition, the platelet count and the hematocrit percentage were determined. RESULTS: Levels of IL-1ß and IL-6 were found to be increased in patients with DENV vs. the healthy controls. Said increase was more evident in patients with dengue and in line with the severity of the clinical presentation. The HDL levels were found to be decreased in patients with dengue versus healthy controls. The correlation analysis showed statistically significant associations between HDL and the platelet number, as well as between the hematocrit percentage and the levels of IL-1ß. CONCLUSIONS: The increased IL-1ß and IL-6 and the decreased HDL levels in the most severe clinical stages suggest that both factors are involved in the development of the pathogenesis and severity. The correlations allow the observation of a potential association between HDL and platelet count, which allows us to provide an approach to the possible role of HDL in the pathogenesis of dengue, but it is still necessary to perform additional studies that will allow to state their importance in the course of the DENV infection.

OBJETIVOS: Evaluar los niveles de lipoproteínas de alta densidad (HDL) y citoquinas proinflamatorias en pacientes con dengue clasificados según la gravedad del cuadro clínico. MATERIALES Y MÉTODOS: Se realizó un estudio descriptivo, incluyendo 70 individuos con dengue, clasificados como: sin signos de alarma (DSSA), con signos de alarma (DCSA) y dengue grave (DG); 15 donantes sanos fueron incluidos como controles (CS). Los niveles séricos de IL-1ß e IL-6 fueron cuantificados por ELISA, y los de HDL en suero por ensayo colorimétrico; además se determinó el recuento de plaquetas y el porcentaje de hematocrito. RESULTADOS: Los niveles de IL-1ß e IL-6 se encontraron aumentados en los pacientes con DENV respecto a los controles sanos. Dicho aumento fue más evidente en los pacientes con dengue, de acuerdo con la gravedad del cuadro clínico. Los niveles de HDL se hallaron disminuidos en los pacientes con dengue comparados con los controles sanos. Los análisis de correlación mostraron asociaciones estadísticamente significativas entre HDL y el número de plaquetas; así como entre el porcentaje de hematocrito y los niveles de IL-1ß. CONCLUSIONES: El aumento de IL-1ß e IL-6 y la disminución de HDL en los estadios clínicos más graves sugieren que ambos factores están implicados en el desarrollo de la patogénesis y severidad. Las correlaciones permiten observar una posible asociación entre HDL y el número de plaquetas que permite dar una aproximación al posible papel de las HDL en la patogénesis del dengue, pero aún es necesario realizar estudios adicionales que permitan elucidar su importancia en el curso de la infección por DENV.

Modulación de los niveles de lipoproteínas de alta densidad y las citoquinas IL-1β e IL-6 en pacientes con dengue / Modulation of high-density lipoprotein and cytokine IL-1β and IL-6 levels in patients with dengue

RESUMEN Objetivos. Evaluar los niveles de lipoproteínas de alta densidad (HDL) y citoquinas proinflamatorias en pacientes con dengue clasificados según la gravedad del cuadro clínico. Materiales y métodos. Se realizó un estudio descriptivo, incluyendo 70 individuos con dengue, clasificados como: sin signos de alarma (DSSA), con signos de alarma (DCSA) y dengue grave (DG); 15 donantes sanos fueron incluidos como controles (CS). Los niveles séricos de IL-1β e IL-6 fueron cuantificados por ELISA, y los de HDL en suero por ensayo colorimétrico; además se determinó el recuento de plaquetas y el porcentaje de hematocrito. Resultados. Los niveles de IL-1β e IL-6 se encontraron aumentados en los pacientes con DENV respecto a los controles sanos. Dicho aumento fue más evidente en los pacientes con dengue, de acuerdo con la gravedad del cuadro clínico. Los niveles de HDL se hallaron disminuidos en los pacientes con dengue comparados con los controles sanos. Los análisis de correlación mostraron asociaciones estadísticamente significativas entre HDL y el número de plaquetas; así como entre el porcentaje de hematocrito y los niveles de IL-1β. Conclusiones. El aumento de IL-1β e IL-6 y la disminución de HDL en los estadios clínicos más graves sugieren que ambos factores están implicados en el desarrollo de la patogénesis y severidad. Las correlaciones permiten observar una posible asociación entre HDL y el número de plaquetas que permite dar una aproximación al posible papel de las HDL en la patogénesis del dengue, pero aún es necesario realizar estudios adicionales que permitan elucidar su importancia en el curso de la infección por DENV.

ABSTRACT Objectives. To assess High-Density Lipoprotein (HDL) and pro-inflammatory cytokine levels in patients with dengue classified according to the severity of the clinical presentation. Materials and Methods. A descriptive study was carried out with 70 individuals with dengue, classified as no alarm signs (DSSA), with signs of alarm (DCSA), and severe dengue (DG); 15 healthy donors were included as controls (CS). The serum levels of IL-1β and IL-6 were quantified through ELISA, and the HDL levels through serum by a colorimetric test. In addition, the platelet count and the hematocrit percentage were determined. Results. Levels of IL-1β and IL-6 were found to be increased in patients with DENV vs. the healthy controls. Said increase was more evident in patients with dengue and in line with the severity of the clinical presentation. The HDL levels were found to be decreased in patients with dengue versus healthy controls. The correlation analysis showed statistically significant associations between HDL and the platelet number, as well as between the hematocrit percentage and the levels of IL-1β. Conclusions. The increased IL-1β and IL-6 and the decreased HDL levels in the most severe clinical stages suggest that both factors are involved in the development of the pathogenesis and severity. The correlations allow the observation of a potential association between HDL and platelet count, which allows us to provide an approach to the possible role of HDL in the pathogenesis of dengue, but it is still necessary to perform additional studies that will allow to state their importance in the course of the DENV infection.

High-dose of vitamin D supplement is associated with reduced susceptibility of monocyte-derived macrophages to dengue virus infection and pro-inflammatory cytokine production: An exploratory study.

BACKGROUND: Dengue, one the most important public health problems in tropical and subtropical areas, is the most important mosquito-borne viral infection in humans. In the absence of effective treatment and vaccine against dengue, the active form of vitamin D could play a central role in protection against dengue virus (DENV), the causal agent of dengue. Recently we reported that monocyte-derived macrophages (MDMs) differentiated in the presence of vitamin D, in addition to expressing lower levels of mannose receptor, are less susceptible to DENV infection and produce low levels of pro-inflammatory cytokines, compared to MDMs differentiated in the absence of vitamin D. OBJECTIVE: The aim of this study was to determine that oral vitamin D supplementation exerts an effect on DENV susceptibility and pro-inflammatory cytokine production in MDMs. METHODS: Healthy individuals were supplemented with 1000 or 4000 international units (IU)/day of vitamin D during 10days. Before and after vitamin D supplementation, a peripheral blood (PB) sample was taken and the monocytes recovered were used to obtain MDMs and were challenged with DENV-2. Furthermore, the expression of genes encoding vitamin D receptor (VDR), CYP24A1 and CAMP were analyzed using real-time quantitative PCR. RESULTS: The data indicate that macrophages differentiated from monocytes obtained from healthy donors who received higher doses of vitamin D (4000IU/day), exhibited higher resistance to DENV-2 infection and produced a significant decrease of pro-inflammatory cytokines and high production of interleukin-10 (IL-10). Furthermore, a significant decrease in intracellular toll-like receptor (TLR) and CAMP mRNA was observed. CONCLUSION: A supplement of 4000IU/day of vitamin D may represent an adequate dose to control dengue progression and DENV replication. Although the results of our study suggest that the vitamin D status can influence the immune response, further studies are needed to determine the feasibility of vitamin D as anti-DENV agent and immune modulator.

HIV-1-neutrophil interactions trigger neutrophil activation and Toll-like receptor expression.

Although neutrophils are the first-line of host defense against infection and express a wide number of pattern recognition receptors (PRRs), the function of these PRRs, including Toll-like receptors (TLRs), in HIV-1 infection remains unclear. TLRs play an important role in innate immunity, and while their involvement in viral immune pathogenesis was recently proposed, little is known about their expression and function during the neutrophil response to HIV-1 exposure. Here, we have shown that freshly isolated human neutrophils from healthy donors exhibited altered TLR expression, which may affect their function, after being challenged with HIV-1, alone or in the presence of TLR agonists. TLRs may promote neutrophil activation, pro-inflammatory cytokine secretion, and the production of reactive oxygen species. To our knowledge, this study is the first demonstration of functional TLR expression on neutrophils in response to HIV-1 treatment, suggesting a possible neutrophil/HIV-1 interaction through TLRs. Although additional studies are required to confirm the function of TLRs in neutrophils, our data clearly suggest that they play a role in the regulation of innate immunity by neutrophils, which could be engaged in HIV-1 pathogenesis or host defense.

Impact of in vitro costimulation with TLR2, TLR4 and TLR9 agonists and HIV-1 on antigen-presenting cell activation.

OBJECTIVE: HIV-1 infects several immune cells including dendritic cells (DCs) and monocytes, which contributes in both to dissemination of HIV-1 infection and induction of antiviral immunity. These cells produce high amounts of type I IFN and proinflammatory cytokines upon Toll-like receptor (TLR) stimulation. During HIV-1 infection, an altered production of proinflammatory cytokines has been reported. However, the mechanisms underlying cytokine modulation have not been well described. Here, we evaluated the production of proinflammatory cytokines and activation of myeloid and plasmacytoid DCs and monocytes costimulated in vitro with TLR agonists and HIV-1. METHODS: Changes in cytokine expression by real-time PCR and activation of DCs and monocytes by flow cytometry were evaluated after costimulation with HIV-1 and TLR agonists. RESULTS: We observed an upregulation of TNF-α expression after TLR4 stimulation, but a downregulation of IL-6 when TLR2/TLR9 were stimulated. Interestingly, the expression of CD80 and CD86 costimulatory molecules in monocytes and DCs were significantly increased in cells challenged with HIV-1 and TLR2/TLR4/TLR9 agonists. CONCLUSION: This regulation of TNF-α and IL-6 production and changes in the expression of costimulatory molecules can be critical in the context of HIV-1 infection, by favoring the antigen-presenting cell activation through the stimulation of TLRs.