Interleukin 27, Similar to Interferons, Modulates Gene Expression of Tripartite Motif (TRIM) Family Members and Interferes with Mayaro Virus Replication in Human Macrophages.

BACKGROUND: The Tripartite motif (TRIM) family includes more than 80 distinct human genes. Their function has been implicated in regulating important cellular processes, including intracellular signaling, transcription, autophagy, and innate immunity. During viral infections, macrophages are key components of innate immunity that produce interferons (IFNs) and IL27. We recently published that IL27 and IFNs induce transcriptional changes in various genes, including those involved in JAK-STAT signaling. Furthermore, IL27 and IFNs share proinflammatory and antiviral pathways in monocyte-derived macrophages (MDMs), resulting in both common and unique expression of inflammatory factors and IFN-stimulated genes (ISGs) encoding antiviral proteins. Interestingly, many TRIM proteins have been recognized as ISGs in recent years. Although it is already very well described that TRIM expression is induced by IFNs, it is not fully understood whether TRIM genes are induced in macrophages by IL27. Therefore, in this study, we examined the effect of stimulation with IL27 and type I, II, and III IFNs on the mRNA expression profiles of TRIM genes in MDMs. METHODS: We used bulk RNA-seq to examine the TRIM expression profile of MDMs treated with IFNs or IL27. Initially, we characterized the expression patterns of different TRIM subfamilies using a heatmap. Subsequently, a volcano plot was employed to identify commonly differentially expressed TRIM genes. Additionally, we conducted gene ontology analysis with ClueGO to explore the biological processes of the regulated TRIMs, created a gene-gene interaction network using GeneMANIA, and examined protein-protein interactions with the STRING database. Finally, RNA-seq data was validated using RT-qPCR. Furthermore, the effect of IL27 on Mayaro virus replication was also evaluated. RESULTS: We found that IL27, similar to IFNs, upregulates several TRIM genes' expression in human macrophages. Specifically, we identified three common TRIM genes (TRIM19, 21, and 22) induced by IL27 and all types of human IFNs. Additionally, we performed the first report of transcriptional regulation of TRIM19, 21, 22, and 69 genes in response to IL27. The TRIMs involved a broad range of biological processes, including defense response to viruses, viral life cycle regulation, and negative regulation of viral processes. In addition, we observed a decrease in Mayaro virus replication in MDMs previously treated with IL27. CONCLUSIONS: Our results show that IL27, like IFNs, modulates the transcriptional expression of different TRIM-family members involved in the induction of innate immunity and an antiviral response. In addition, the functional analysis demonstrated that, like IFN, IL27 reduced Mayaro virus replication in MDMs. This implies that IL27 and IFNs share many similarities at a functional level. Moreover, identifying distinct TRIM groups and their differential expressions in response to IL27 provides new insights into the regulatory mechanisms underlying the antiviral response in human macrophages.

Interleukin 27, like interferons, activates JAK-STAT signaling and promotes pro-inflammatory and antiviral states that interfere with dengue and chikungunya viruses replication in human macrophages.

Interferons (IFNs) are a family of cytokines that activate the JAK-STAT signaling pathway to induce an antiviral state in cells. Interleukin 27 (IL-27) is a member of the IL-6 and/or IL-12 family that elicits both pro- and anti-inflammatory responses. Recent studies have reported that IL-27 also induces a robust antiviral response against diverse viruses, both in vitro and in vivo, suggesting that IFNs and IL-27 share many similarities at the functional level. However, it is still unknown how similar or different IFN- and IL-27-dependent signaling pathways are. To address this question, we conducted a comparative analysis of the transcriptomic profiles of human monocyte-derived macrophages (MDMs) exposed to IL-27 and those exposed to recombinant human IFN-α, IFN-γ, and IFN-λ. We utilized bioinformatics approaches to identify common differentially expressed genes between the different transcriptomes. To verify the accuracy of this approach, we used RT-qPCR, ELISA, flow cytometry, and microarrays data. We found that IFNs and IL-27 induce transcriptional changes in several genes, including those involved in JAK-STAT signaling, and induce shared pro-inflammatory and antiviral pathways in MDMs, leading to the common and unique expression of inflammatory factors and IFN-stimulated genes (ISGs)Importantly, the ability of IL-27 to induce those responses is independent of IFN induction and cellular lineage. Additionally, functional analysis demonstrated that like IFNs, IL-27-mediated response reduced chikungunya and dengue viruses replication in MDMs. In summary, IL-27 exhibits properties similar to those of all three types of human IFN, including the ability to stimulate a protective antiviral response. Given this similarity, we propose that IL-27 could be classified as a distinct type of IFN, possibly categorized as IFN-pi (IFN-π), the type V IFN (IFN-V).

Mayaro virus infection elicits a robust pro-inflammatory and antiviral response in human macrophages.

Mayaro virus (MAYV), the etiological agent of Mayaro fever (MAYF), is an emergent arbovirus pathogen belonging to Togaviridae family. MAYF is characterized by high inflammatory component that can cause long-lasting arthralgia that persists for months. Macrophages are viral targets and reservoirs, key components of innate immunity and host response. Given the importance of this pathogen, our aim was to determine the inflammatory and antiviral response of human monocyte-derived macrophages (MDMs) infected with MAYV. First, we established the replication kinetics of the virus. Thereafter, we determined the expression of pattern recognition receptors, NF-ĸB complex, interferons (IFNs), two interleukin 27 (IL27) subunits, IFN-stimulated genes (ISGs), and the production of cytokines/chemokines. We found that human MDMs are susceptible to MAYV infection in vitro, with a peak of viral particles released between 24- and 48-hours post-infection (h.p.i) at MOI 0.5, and between 12 and 24 h.p.i at MOI 1. Interestingly, we observed a significant decline in the production of infectious viral particles at 72 h.p.i that was associated with the induction of antiviral response and high cytotoxic effect of MAYV infection in MDMs. We observed modulation of several genes after MAYV infection, as well, we noted the activation of antiviral detection and response pathways (Toll-like receptors, RIG-I/MDA5, and PKR) at 48 h.p.i but not at 6 h.p.i. Furthermore, MAYV-infected macrophages express high levels of the three types of IFNs and the two IL27 subunits at 48 h.p.i. Moreover, we found higher production of IL6, IL1ß, CXCL8/IL8, CCL2, and CCL5 at 48 h.p.i as compared to 6 h.p.i. A robust antiviral response (ISG15, APOBEC3A, IFITM1, and MX2) was observed at 48 but not at 6 h.p.i. The innate and antiviral responses of MAYV-infected MDMs differ at 6 and 48 h.p.i. We conclude that MAYV infection induces robust pro-inflammatory and antiviral responses in human primary macrophages.

Multitranscript analysis reveals an effect of 2-deoxy-d-glucose on gene expression linked to unfolded protein response and integrated stress response in primary human monocytes and monocyte-derived macrophages.

BACKGROUND: Glycolytic inhibitor 2-deoxy-d-glucose (2-DG) binds to hexokinase in a non-competitive manner and phosphoglucose isomerase in a competitive manner, blocking the initial steps of the glycolytic pathway. Although 2-DG stimulates endoplasmic reticulum (ER) stress, activating the unfolded protein response to restore protein homeostasis, it is unclear which ER stress-related genes are modulated in response to 2-DG treatment in human primary cells. Here, we aimed to determine whether the treatment of monocytes and monocyte-derived macrophages (MDMs) with 2-DG leads to a transcriptional profile specific to ER stress. METHODS: We performed bioinformatics analysis to identify differentially expressed genes (DEGs) in previously reported RNA-seq datasets of 2-DG treated cells. RT-qPCR was performed to verify the sequencing data on cultured MDMs. RESULTS: A total of 95 common DEGs were found by transcriptional analysis of monocytes and MDMs treated with 2-DG. Among these, 74 were up-regulated and 21 were down-regulated. Multitranscript analysis showed that DEGs are linked to integrated stress response (GRP78/BiP, PERK, ATF4, CHOP, GADD34, IRE1α, XBP1, SESN2, ASNS, PHGDH), hexosamine biosynthetic pathway (GFAT1, GNA1, PGM3, UAP1), and mannose metabolism (GMPPA and GMPPB). CONCLUSIONS: Results reveal that 2-DG triggers a gene expression program that might be involved in restoring protein homeostasis in primary cells. GENERAL SIGNIFICANCE: 2-DG is known to inhibit glycolysis and induce ER stress; however, its effect on gene expression in primary cells is not well understood. This work shows that 2-DG is a stress inducer shifting the metabolic state of monocytes and macrophages.

Vitamin D modulates expression of antimicrobial peptides and proinflammatory cytokines to restrict Zika virus infection in macrophages.

Although the impact of Zika virus (ZIKV) infection on human health has been well documented, we still have no vaccine or effective treatment. This fact highlights the importance of searching for alternative therapy for treating ZIKV. To search for ZIKV antivirals, we examined the effect of vitamin D in monocyte-derived macrophages (MDMs) differentiated in the presence of vitamin D (D3-MDM) and explored the molecular mechanisms by analyzing transcriptional profiles. Our data show the restriction of ZIKV infection in D3-MDMs as compared to MDMs. Transcriptional profiles show that vitamin D alters about 19% of Zika response genes (8.2% diminished and 10.8% potentiated). Among the genes with diminished expression levels, we found proinflammatory cytokines and chemokines such as IL6, TNF, IL1A, IL1B, and IL12B, CCL1, CCL4, CCL7, CXCL3, CXCL6, and CXCL8. On the other hand, genes with potentiated expression were related to degranulation such as Lysozyme, cathelicidin (CAMP), and Serglycin. Since the CAMP gene encodes the antimicrobial peptide LL-37, we treated MDMs with LL-37 and infected them with ZIKV. The results showed a decrease in the proportion of infected cells. Our data provide new insights into the role of vitamin D in restricting ZIKV infection in macrophages that are mediated by induction of cathelicidin/LL-37 expression and downregulation of proinflammatory genes. Results highlight the biological relevance of vitamin D-inducible peptides as an antiviral treatment for Zika fever.

Vitamin D modulates inflammatory response of DENV-2-infected macrophages by inhibiting the expression of inflammatory-liked miRNAs.

Dengue disease caused by dengue virus (DENV) infection is the most common vector-borne viral disease worldwide. Currently, no treatment is available to fight dengue symptoms. We and others have demonstrated the antiviral and immunomodulatory properties of VitD3 as a possible therapy for DENV infection. MicroRNAs (miRNAs) are small non-coding RNAs responsible for the regulation of cell processes including antiviral defense. Previous transcriptomic analysis showed that VitD3 regulates the expression of genes involved in stress and immune response by inducing specific miRNAs. Here, we focus on the effects of VitD3 supplementation in the regulation of the expression of inflammatory-liked miR-182-5p, miR-130a-3p, miR125b-5p, miR146a-5p, and miR-155-5p during DENV-2 infection of monocyte-derived macrophages (MDMs). Further, we evaluated the effects of inhibition of these miRNAs in the innate immune response. Our results showed that supplementation with VitD3 differentially regulated the expression of these inflammatory miRNAs. We also observed that inhibition of miR-182-5p, miR-130a-3p, miR-125b-5p, and miR-155-5p, led to decreased production of TNF-α and TLR9 expression, while increased the expression of SOCS-1, IFN-ß, and OAS1, without affecting DENV replication. By contrast, over-expression of miR-182-5p, miR-130a-3p, miR-125b-5p, and miR-155-5p significantly decreased DENV-2 infection rates and also DENV-2 replication in MDMs. Our results suggest that VitD3 immunomodulatory effects involve regulation of inflammation-linked miRNAs expression, which might play a key role in the inflammatory response during DENV infection.

Transcriptional and post-transcriptional mechanisms that regulate the genetic program in Zika virus-infected macrophages.

Besides our understanding of the effects of ZIKA virus (ZIKV) infection on neural progenitors' cells the pathogenesis of this RNA virus also involves antigen-presenting cells, including macrophages. However, the molecular mechanisms that control gene activation and repression associated with the macrophage response to acute ZIKV infection are not fully understood. We approached the issue by RNA-seq and miRNA-seq datasets to understand the genetic program of ZIKV-infected macrophages. Results indicate that macrophage activates a regulatory program, involving 1067 differentially expressed genes. These genetic programs induced an inflammatory response mediated by chemokines as well as an interferon-independent anti-viral response, presumptively activated by IL-27. Additionally, the pathogenetic process involves changes in other signaling pathways such as cellular stress, cell signaling, metabolism, and cell differentiation. Furthermore, transcriptional control analysis revealed regulatory functions of key transcription factors principally, NFκB and STAT1, as well as HIF1A, ETV7, and PRMD1 that are associated with metabolic reprogramming during viral infection. We also noted six long-noncoding RNAs (lncRNAs) that may act in the regulation of gene expression, including MROCKI and ZC2HC1A-2, that are involved in the inflammatory response and expression of the cytokines, respectively. On the other hand, post-transcriptional control by miRNAs, including miR-155-5p and miR-146a-5p, are associated with modulation of genes related to inflammatory and antiviral responses. Relevant to the post-transcriptional control, our data unveiled the role of RNA binding proteins that have diverse functions such as ribonucleases (PNPT1, ZC3H12A, and ZC3HAV1), splicing factors (SSB, RBM11, and RAVER2), and RNA modifiers (PARP10 and PARP14). Overall, the results establish an unbiased approach to discerning the wiring of a regulatory mechanism controlling the genetic program in ZIKV-infected macrophages.

Vitamin D boosts immune response of macrophages through a regulatory network of microRNAs and mRNAs.

Vitamin D is associated with the stimulation of innate immunity, inflammation, and host defense against pathogens. Macrophages express receptors of Vitamin D, regulating the transcription of genes related to immune processes. However, the transcriptional and post-transcriptional strategies controlling gene expression in differentiated macrophages, and how they are influenced by Vitamin D are not well understood. We studied whether Vitamin D enhances immune response by regulating the expression of microRNAs and mRNAs. Analysis of the transcriptome showed differences in expression of 199 genes, of which 68% were up-regulated, revealing the cell state of monocyte-derived macrophages differentiated with Vitamin D (D3-MDMs) as compared to monocyte-derived macrophages (MDMs). The differentially expressed genes appear to be associated with pathophysiological processes, including inflammatory responses, and cellular stress. Transcriptional motifs in promoter regions of up- or down-regulated genes showed enrichment of VDR motifs, suggesting possible roles of transcriptional activator or repressor in gene expression. Further, the microRNA-Seq analysis indicated that there were 17 differentially expressed miRNAs, of which, seven were up-regulated and 10 down-regulated, suggesting that Vitamin D plays a critical role in the regulation of miRNA expression during macrophages differentiation. The miR-6501-3p, miR-1273h-5p, miR-665, miR-1972, miR-1183, miR-619-5p were down-regulated in D3-MDMs compared to MDMs. The integrative analysis of miRNA and mRNA expression profiles predicts that miR-1972, miR-1273h-5p, and miR-665 regulate genes PDCD1LG2, IL-1B, and CD274, which are related to the inflammatory response. Results suggest an essential role of Vitamin D in macrophage differentiation that modulates host response against pathogens, inflammation, and cellular stress.

Vitamin D modulates the expression of Toll-like receptors and pro-inflammatory cytokines without affecting Chikungunya virus replication, in monocytes and macrophages.

Chikungunya virus (CHIKV) is a zoonotic arthropod-borne virus that causes Chikungunya fever (CHIKF), a self-limiting disease characterized by myalgia and acute or chronic arthralgia. CHIKF pathogenesis has an important immunological component since higher levels of pro-inflammatory factors, including cytokines and chemokines, are detected in CHIKV-infected patients. In vitro studies, using monocytes and macrophages have shown that CHIKV infection promotes elevated production of pro-inflammatory cytokines and antiviral response factors. Vitamin D3 (VD3) has been described as an important modulator of immune response and as an antiviral factor for several viruses. Here, we aimed to study the effects of VD3 treatment on viral replication and pro-inflammatory response in CHIKV-infected human monocytes (VD3-Mon) and monocyte-derived macrophages differentiated in the absence (MDMs) or the presence of VD3 (VD3-MDMs). We found that VD3 treatment did not suppress CHIKV replication in either VD3-Mon or VD3-MDMs. However, the expression of VDR, CAMP and CYP24A1 mRNAs was altered by CHIKV infection. Furthermore, VD3 treatment alters TLRs mRNA expression and production of pro-inflammatory cytokines, including TNFα and CXCL8/IL8, but not IL1ß and IL6, in response to CHIKV infection in both VD3-Mon and VD3-MDMs. While a significant decrease in CXCL8/IL8 production was observed in CHIKV-infected VD3-Mon, significantly higher production of CXCL8/IL8 was observed in CHIKV-infected VD3-MDM at 24 hpi. Altogether, our results suggest that vitamin D3 may play an important role in ameliorating pro-inflammatory response during CHIKV infection in human Mon, but not in MDMs. Although further studies are needed to evaluate the efficacy of VD3; nevertheless, this study provides novel insights into its benefits in modulating the inflammatory response elicited by CHIKV infection in humans.

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.

Functional Profile of CD8+ T-Cells in Response to HLA-A*02:01-Restricted Mutated Epitopes Derived from the Gag Protein of Circulating HIV-1 Strains from Medellín, Colombia.

CD8+ T-cells play a crucial role in the control of HIV replication. HIV-specific CD8+ T-cell responses rapidly expand since the acute phase of the infection, and it has been observed that HIV controllers harbor CD8+ T-cells with potent anti-HIV capacity. The development of CD8+ T-cell-based vaccine against HIV-1 has focused on searching for immunodominant epitopes. However, the strong immune pressure of CD8+ T-cells causes the selection of viral variants with mutations in immunodominant epitopes. Since HIV-1 mutations are selected under the context of a specific HLA-I, the circulation of viral variants with these mutations is highly predictable based on the most prevalent HLA-I within a population. We previously demonstrated the adaptation of circulating strains of HIV-1 to the HLA-A*02 molecule by identifying mutations under positive selection located in GC9 and SL9 epitopes derived from the Gag protein. Also, we used an in silico prediction approach and evaluated whether the mutations found had a higher or lower affinity to the HLA-A*02. Although this strategy allowed predicting the interaction between mutated peptides and HLA-I, the functional response of CD8+ T-cells that these peptides induce is unknown. In the present work, peripheral blood mononuclear cells from 12 HIV-1+ HLA-A*02:01+ individuals were stimulated with the mutated and wild-type peptides derived from the GC9 and SL9 epitopes. The functional profile of CD8+ T-cells was evaluated using flow cytometry, and the frequency of subpopulations was determined according to their number of functions and the polyfunctionality index. The results suggest that the quality of the response (polyfunctionality) could be associated with the binding affinity of the peptide to the HLA molecule, and the functional profile of specific CD8+ T-cells to mutated epitopes in individuals under cART is maintained.

Implicaciones de los virus Zika y Chikungunya en el semen durante la transmisión sexual / Implications of Zika and Chikungunya viruses in semen during sexual transmission

Introducción: Los brotes de enfermedades causados por los virus Zika (VZIK) y Chikungunya (VCHIK) representan un problema de salud pública para muchos países tropicales y subtropicales. Objetivo: Discutir las implicaciones del hallazgo del VZIK y del VCHIK en el semen, y su relación con la transmisión sexual y la fertilidad masculina. Métodos: Se realizó una revisión narrativa de la literatura usando artículos indexados en PubMed (Medline), Embase y Scopus. Información, análisis y síntesis: Si bien los mosquitos del género Aedes son el vector principal y transmiten ambos virus, la transmisión sexual es una vía de infección significativa del VZIK y una posible ruta alterna para el VCHIK. La diseminación de estas arbovirosis vía linfática y sanguínea contribuye a la infección de diversos tejidos, incluyendo el tracto reproductivo masculino, donde el VZIK puede persistir. La infección de los testículos y quizás también de las glándulas accesorias del sistema reproductor masculino, se asocia con síntomas genitourinarios o alteraciones espermáticas, relacionadas con la detección del virus por largos periodos. Aunque no hay evidencia contundente sobre la presencia del VCHIK en el tracto genital masculino, se ha hallado en orina y semen. Además, se ha sugerido una posible persistencia en macrófagos que pueden infiltrar diferentes tejidos periféricos y cumplir una función de reservorio. Conclusiones: Hay presencia y persistencia de los virus Zika y Chikungunya en el tracto reproductor masculino. La infección en el semen se asocia con la transmisión sexual del virus, y con la alteración en la producción y calidad de los espermatozoides, con consecuencias clínicas graves en la salud sexual y reproductiva de los hombres infectados(AU)

Introduction: Disease outbreaks caused by Zika (ZIKV) and Chikungunya (CHIKV) viruses represent a public health problem for many tropical and subtropical countries. Objective: To discuss the implications of finding ZIKV and CHIKV in semen, and their relationship to sexual transmission and male fertility. Methods: A narrative review of the literature was carried out using articles indexed in PubMed (Medline), Embase and Scopus. Information, Analysis and Synthesis: Although Aedes mosquitoes are the primary vector and transmit both viruses, sexual transmission is a significant route of infection for ZIKV and a possible alternate route for CHIKV. Spread of these arboviruses via lymphatic and blood routes contributes to infection of various tissues, including the male reproductive tract, where ZIKV may persist. Infection of the testes and probably of the accessory glands of the male reproductive system is associated with genitourinary symptoms or sperm alterations, related to the detection of the virus for long periods. Although there is no conclusive evidence of the presence of CHIKV in the male genital tract, it has been found in urine and semen. In addition, a possible persistence in macrophages that can infiltrate different peripheral tissues and function as reservoir has been suggested. Conclusions: Zika and Chikungunya viruses can be present and persist in the male reproductive tract. Infection in semen is associated with sexual transmission of the virus and with alterations in the production and quality of spermatozoa, with serious clinical consequences in the sexual and reproductive health of infected men(AU)

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.

Regulation of innate immune responses in macrophages differentiated in the presence of vitamin D and infected with dengue virus 2.

A dysregulated or exacerbated inflammatory response is thought to be the key driver of the pathogenesis of severe disease caused by the mosquito-borne dengue virus (DENV). Compounds that restrict virus replication and modulate the inflammatory response could thus serve as promising therapeutics mitigating the disease pathogenesis. We and others have previously shown that macrophages, which are important cellular targets for DENV replication, differentiated in the presence of bioactive vitamin D (VitD3) are less permissive to viral replication, and produce lower levels of pro-inflammatory cytokines. Therefore, we here evaluated the extent and kinetics of innate immune responses of DENV-2 infected monocytes differentiated into macrophages in the presence (D3-MDMs) or absence of VitD3 (MDMs). We found that D3-MDMs expressed lower levels of RIG I, Toll-like receptor (TLR)3, and TLR7, as well as higher levels of SOCS-1 in response to DENV-2 infection. D3-MDMs produced lower levels of reactive oxygen species, related to a lower expression of TLR9. Moreover, although VitD3 treatment did not modulate either the expression of IFN-α or IFN-ß, higher expression of protein kinase R (PKR) and 2'-5'-oligoadenylate synthetase 1 (OAS1) mRNA were found in D3-MDMs. Importantly, the observed effects were independent of reduced infection, highlighting the intrinsic differences between D3-MDMs and MDMs. Taken together, our results suggest that differentiation of MDMs in the presence of VitD3 modulates innate immunity in responses to DENV-2 infection.

Interleukin 27 as an inducer of antiviral response against chikungunya virus infection in human macrophages.

Chikungunya virus (CHIKV) is known to have a wide range of tropism in human cell types throughout infection, including keratinocytes, fibroblasts, endothelial cells, monocytes, and macrophages. We reported that human monocytes-derived macrophages (MDMs) are permissive to CHIKV infection in vitro. We found that the peak of CHIKV replication was at 24 hpi; however, at 48 hpi, a significant reduction in viral titer was observed that correlated with high expression levels of genes encoding antiviral proteins (AVPs) in an IFN-independent manner. To explore the molecular mechanisms involved in the induction of antiviral response in CHIKV-infected MDMs, we performed transcriptomic analysis by RNA-sequencing. Differential expression of genes at 24 hpi showed that CHIKV infection abrogated the expression of all types of IFNs in MDMs. However, we observed that CHIKV-infected MDMs activated the JAK-STAT signaling and induced a robust antiviral response associated with control of CHIKV replication. We identified that the IL27 pathway is activated in CHIKV-infected MDMs and that kinetics of IL27p28 mRNA expression and IL27 protein production correlated with the expression of AVPs in CHIKV-infected MDMs. Furthermore, we showed that stimulation of THP-1-derived macrophages with recombinant-human IL27 induced the activation of the JAK-STAT signaling and induced a robust pro-inflammatory and antiviral response, comparable to CHIKV-infected MDMs. Furthermore, pre-treatment of MDMs with recombinant-human IL27 inhibits CHIKV replication in a dose-dependently manner (IC50 = 1.83 ng/mL). Altogether, results show that IL27 is highly expressed in CHIKV-infected MDMs, leading to activation of JAK-STAT signaling and stimulation of pro-inflammatory and antiviral response to control CHIKV replication in an IFN-independent manner.

Inflammatory status and severity of disease in dengue patients are associated with lipoprotein alterations.

INTRODUCTION: The triggering of severe dengue has been associated with an exacerbated inflammatory process characterized by the production of pro-inflammatory cytokines such as IL-1ß/IL-18, which are the product of inflammasome activation. Furthermore, alteration in the levels of high-density (HDL) and low-density lipoproteins (LDL) has been observed; and HDL are known to have immunomodulatory properties, including the regulation of inflammasomes. While HDL would be expected to counteract hyperactivation of the inflammasome, the relationship between HDL and dengue severity, has not previously been explored. METHODOLOGY: We conducted a cross-sectional study of 30 patients with dengue and 39 healthy controls matched by sex and age. Lipid profile and levels of C-reactive protein were quantified. Serum levels of IL-1ß, IL-6, IL-10, IL-18, and TNF-α, were assessed by ELISA. Expression of inflammasome-related genes in PBMC was quantified by qPCR. RESULTS: Dengue patients presented an alteration in the parameters of the lipid profile, with a significant decrease in HDL levels, which was more pronounced in dengue patients with warning signs. Moreover, a decrease in the expression of the inflammasome-related genes NLRP1, NLRC4, caspase-1, IL-1ß and IL-18 was observed, as well as an increase in serum levels of C-reactive protein and IL-10 in dengue patients versus healthy donors. Significant positive correlations between LDL levels and the relative expression of NLRP3, NLRC4, IL-1ß and IL-18, were found. CONCLUSION: The results suggest that there is a relationship between the alteration of LDL and HDL with the imbalance in the inflammatory response, which could be associated with the severity of dengue.

Vitamin D-mediated attenuation of miR-155 in human macrophages infected with dengue virus: Implications for the cytokine response.

Clinical manifestations of dengue disease rely on complex interactions between dengue virus (DENV) and host factors that drive altered immune responses, including excessive inflammation. We have recently established that vitamin D can modulate DENV-induced cytokine responses and restrict infection in human macrophages. Cytokine responses are finely regulated by several homeostatic mechanisms, including microRNAs (miRNAs) that can rapidly target specific genes involved in the control of immune signaling pathways. However, the modulation of miRNAs by vitamin D during DENV infection is still unknown. Here, using a qPCR miRNA array we profiled immune-related miRNAs induced by DENV infection in human monocyte-derived macrophages (MDM) differentiated in absence or presence of vitamin D (D3-MDM). We found several miRNAs differentially expressed in both MDM and D3-MDM upon DENV infection. Interestingly, from these, a set of 11 miRNAs were attenuated in D3-MDM as compared to MDM. Gene set enrichment analysis of the predicted mRNA targets of these attenuated miRNAs suggested a predominant role of miR-155-5p in the TLR-induced cytokine responses. Indeed, validation of miR-155-5p attenuation in D3-MDM was linked to increased expression of its target gene SOCS-1, a key component for TLR4 signaling regulation. Likewise, TLR4 activation with LPS further corroborated the same miR-155-5p/SOCS-1 negative correlation observed in D3-MDM upon DENV exposure. Moreover, D3-MDM differentiation induced down-regulation of surface TLR4 that was linked to less TLR4/NF-κB-derived secretion of IL-1ß. These data suggest a key role of vitamin D in the control of inflammatory cytokine responses during DENV infection of human macrophages via the TLR4/NF-κB/miR-155-5p/SOCS-1 axis.

Mechanisms of monocyte cell death triggered by dengue virus infection.

Arthropod-borne viral diseases caused by dengue virus (DENV) are major re-emerging public health problem worldwide. In spite of intense research, DENV pathogenesis is not fully understood and remains enigmatic; however, current evidence suggests that dengue progression is associated with an inflammatory response, mainly in patients suffering from a second DENV infection. Monocytes are one of the main target cells of DENV infection and play an important role in pathogenesis since they are known to produce several inflammatory cytokines that can lead to endothelial dysfunction and therefore vascular leak. In addition, monocytes play an important role in antibody dependent enhancement, infection with consequences in viral load and immune response. Despite the physiological functions of monocytes in immune response, their life span in the bloodstream is very short, and activation of monocytes by DENV infection can trigger different types of cell death. For example, DENV can induce apoptosis in monocytes related with the production of Tumor necrosis factor alpha (TNF-α). Additionally, recent studies have shown that DENV-infected monocytes also exhibit a cell death process mediated by caspase-1 activation together with IL-1 production, referred to as pyroptosis. Taken together, the aforementioned studies strongly depict that multiple cell death pathways may be occurring in monocytes upon DENV-2 infection. This review provides insight into mechanisms of DENV-induced death of both monocytes and other cell types for a better understanding of this process. Further knowledge in cell death induced by DENV will help in the developing novel strategies to prevent disease progression.

[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.