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.

Protective Effects of Caffeine on Chikungunya and Zika Virus Infections: An in Vitro and in Silico Study.

Infection by viruses Chikungunya (CHIKV) and Zika (ZIKV) continue to be serious problems in tropical and subtropical areas of the world. Here, we evaluated the antiviral and virucidal activity of caffeine against CHIKV and ZIKV in Vero, A549, and Huh-7 cell lines. Results showed that caffeine displays antiviral properties against both viruses. By pre-and post-infection treatment, caffeine significantly inhibited CHIKV and ZIKV replication in a dose-dependent manner. Furthermore, caffeine showed a virucidal effect against ZIKV. Molecular docking suggests the possible binding of caffeine with envelope protein and RNA-dependent RNA polymerase of CHIKV and ZIKV. This is the first study that showed an antiviral effect of caffeine against CHIKV and ZIKV. Although further studies are needed to better understand the mechanism of caffeine-mediated repression of viral replication, caffeine appears to be a promising compound that could be used for in vivo studies, perhaps in synergy with other compounds present in daily beverages.

Antiviral response and immunopathogenesis of interleukin 27 in COVID-19.

The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is associated with a high mortality rate. The clinical course is attributed to the severity of pneumonia and systemic complications. In COVID-19 patients and murine models of SARS-CoV-2 infection, the disease may be accompanied by excessive production of cytokines, leading to an accumulation of immune cells in affected organs such as lungs. Previous reports have shown that SARS-CoV-2 infection antagonizes interferon (IFN)-dependent antiviral response, thereby preventing the expression of IFN-stimulated genes (ISGs). Lower IFN levels have been linked to more-severe COVID-19. Interleukin 27 (IL27) is a heterodimeric cytokine composed of IL27p28 and EBI3 subunits, which induce both pro- and anti-inflammatory responses. Recently, we and others have reported that IL27 also induces a strong antiviral response in an IFN-independent manner. Here, we investigated transcription levels of both IL27 subunits in COVID-19 patients. The results show that SARS-CoV-2 infection modulates TLR1/2-MyD88 signaling in PBMCs and monocytes and induces NF-κB activation and expression of NF-κB-target genes that are dependent on a robust pro-inflammatory response, including EBI3; and activates IRF1 signaling which induces IL27p28 mRNA expression. The results suggest that IL27 induces a robust STAT1-dependent pro-inflammatory and antiviral response in an IFN-independent manner in COVID-derived PBMCs and monocytes as a function of a severe clinical course of COVID-19. Similar results were observed in macrophages stimulated with the SARS-CoV-2 spike protein. Thus, IL27 can trigger an antiviral response in the host, suggesting the possibility of novel therapeutics against SARS-CoV-2 infection in humans.

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.

American-Asian- and African lineages of Zika virus induce differential pro-inflammatory and Interleukin 27-dependent antiviral responses in human monocytes.

Zika virus (ZIKV) is an arbovirus that belongs to the Flaviviridae family and inflammatory responses play a critical role in ZIKV pathogenesis. As a first-line defense, monocytes are key components of innate immunity and host response to viruses. Monocytes are considered the earliest blood cell type to be infected by ZIKV and have been shown to be associated with ZIKV pathogenesis. The first ZIKV epidemic was reported in Africa and Asia although, it is less well known whether African- and Asian- lineages of ZIKV have different impacts on host immune response. We studied the pro-inflammatory and antiviral response of ZIKV-infected monocytes using publicly available RNA-seq analysis (GSE103114). We compared the transcriptomic profiles of human monocytes infected with ZIKV Puerto Rico strain (PRVABC59), American-Asian lineage, and ZIKV Nigeria strain (IBH30656), African lineage. We validated RNA-seq results by ELISA or RT-qPCR, in human monocytes infected with a clinical isolate of ZIKV from Colombia (American-Asian lineage), or with ZIKV from Dakar (African lineage). The transcriptomic analysis showed that ZIKV Puerto Rico strain promotes a higher pro-inflammatory response through TLR2 signaling and NF-kB activation and induces a strong IL27-dependent antiviral activity than ZIKV Nigeria strain. Furthermore, human monocytes are more susceptible to infection with ZIKV from Colombia than ZIKV from Dakar. Likewise, Colombian ZIKV isolate activated IL27 signaling and induced a robust antiviral response in an IFN-independent manner. Moreover, we show that treatment of monocytes with IL27 results in decreased release of ZIKV particles in a dose-dependent manner with an EC50 =2.870 ng/mL for ZIKV from Colombia and EC50 =10.23 ng/mL to ZIKV from Dakar. These findings highlight the differential inflammatory response and antiviral activity of monocytes infected with different lineages of ZIKV and may help better management of ZIKV-infected patients.

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)

Synergistic Effects of Toll-Like Receptor 1/2 and Toll-Like Receptor 3 Signaling Triggering Interleukin 27 Gene Expression in Chikungunya Virus-Infected Macrophages.

Chikungunya virus (CHIKV) is the etiological agent of chikungunya fever (CHIKF), a self-limiting disease characterized by myalgia and severe acute or chronic arthralgia. CHIKF is associated with immunopathology and high levels of pro-inflammatory factors. CHIKV is known to have a wide range of tropism in human cell types, including keratinocytes, fibroblasts, endothelial cells, monocytes, and macrophages. Previously, we reported that CHIKV-infected monocytes-derived macrophages (MDMs) express high levels of interleukin 27 (IL27), a heterodimeric cytokine consisting of IL27p28 and EBI3 subunits, that triggers JAK-STAT signaling and promotes pro-inflammatory and antiviral response, in interferon (IFN)-independent manner. Based on the transcriptomic analysis, we now report that induction of IL27-dependent pro-inflammatory and antiviral response in CHIKV-infected MDMs relies on two signaling pathways: an early signal dependent on recognition of CHIKV-PAMPs by TLR1/2-MyD88 to activate NF-κB-complex that induces the expression of EBI3 mRNA; and second signaling dependent on the recognition of intermediates of CHIKV replication (such as dsRNA) by TLR3-TRIF, to activate IRF1 and the induction of IL27p28 mRNA expression. Both signaling pathways were required to produce a functional IL27 protein involved in the induction of ISGs, including antiviral proteins, cytokines, CC- and CXC- chemokines in an IFN-independent manner in MDMs. Furthermore, we reported that activation of TLR4 by LPS, both in human MDMs and murine BMDM, results in the induction of both subunits of IL27 that trigger strong IL27-dependent pro-inflammatory and antiviral response independent of IFNs signaling. Our findings are a significant contribution to the understanding of molecular and cellular mechanisms of CHIKV infection.

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.

Effect of high doses of vitamin D supplementation on dengue virus replication, Toll-like receptor expression, and cytokine profiles on dendritic cells.

Dengue, caused by dengue virus (DENV) infection, is a public health problem worldwide. Although DENV pathogenesis has not yet been fully elucidated, the inflammatory response is a hallmark feature in severe DENV infection. Although vitamin D (vitD) can promote the innate immune response against virus infection, no studies have evaluated the effects of vitD on DENV infection, dendritic cells (DCs), and inflammatory response regulation. This study aimed to assess the impact of oral vitD supplementation on DENV-2 infection, Toll-like receptor (TLR) expression, and both pro- and anti-inflammatory cytokine production in monocyte-derived DCs (MDDCs). To accomplish this, 20 healthy donors were randomly divided into two groups and received either 1000 or 4000 international units (IU)/day of vitD for 10 days. During pre- and post-vitD supplementation, peripheral blood samples were taken to obtain MDDCs, which were challenged with DENV-2. We found that MDDCs from donors who received 4000 IU/day of vitD were less susceptible to DENV-2 infection than MDDCs from donors who received 1000 IU/day of vitD. Moreover, these cells showed decreased mRNA expression of TLR3, 7, and 9; downregulation of IL-12/IL-8 production; and increased IL-10 secretion in response to DENV-2 infection. In conclusion, the administration of 4000 IU/day of vitD decreased DENV-2 infection. Our findings support a possible role of vitD in improving the innate immune response against DENV. However, further studies are necessary to determine the role of vitD on DENV replication and its innate immune response modulation in MDDCs.

High-Density Lipoproteins Decrease Proinflammatory Activity and Modulate the Innate Immune Response.

Atherosclerosis, a chronic inflammatory disease of the arterial wall, is the leading cause of cardiac disorders and stroke. The onset and progression of these diseases are linked with the inflammatory response, especially NLRP3 inflammasome activation, inducing the production of proinflammatory cytokines, such as interleukin 1ß (IL-1ß). Because high-density lipoproteins (HDLs) have shown significant antiatherogenic and anti-inflammatory properties, we evaluated their immunomodulatory activity in response to cholesterol crystals and other innate immune activators. Human primary monocyte-derived macrophages, THP-1 cells, and murine macrophages were stimulated to activate NLRP3 inflammasome and other pattern recognition receptors, in the presence or absence of HDL. Then, HDL immunomodulatory effects were evaluated through IL-1ß and IL-6 production by enzyme-linked immunosorbent assay. Furthermore, in vivo HDL anti-inflammatory effects were evaluated in a murine model of peritoneal inflammatory infiltration. HDLs have an immunomodulatory effect on different cellular models, including peripheral blood mononuclear cells, THP-1 cells, and murine macrophages, by affecting the activity of innate immunity sensors, such as Toll-like receptors (TLRs), dectin-1, and inflammasomes. HDL reduces the proinflammatory role of cholesterol crystals, nigericin, and other NLRP3 and AIM2 inflammasome agonists, and several TLR agonists, leading to a decreased production of IL-1ß and IL-6. The results suggest that HDLs are highly important in the regulation of the innate immune response and may have a beneficial role in controlling diseases associated with the inflammatory response.