OSBPL10, RXRA and lipid metabolism confer African-ancestry protection against dengue haemorrhagic fever in admixed Cubans.

Ethnic groups can display differential genetic susceptibility to infectious diseases. The arthropod-born viral dengue disease is one such disease, with empirical and limited genetic evidence showing that African ancestry may be protective against the haemorrhagic phenotype. Global ancestry analysis based on high-throughput genotyping in admixed populations can be used to test this hypothesis, while admixture mapping can map candidate protective genes. A Cuban dengue fever cohort was genotyped using a 2.5 million SNP chip. Global ancestry was ascertained through ADMIXTURE and used in a fine-matched corrected association study, while local ancestry was inferred by the RFMix algorithm. The expression of candidate genes was evaluated by RT-PCR in a Cuban dengue patient cohort and gene set enrichment analysis was performed in a Thai dengue transcriptome. OSBPL10 and RXRA candidate genes were identified, with most significant SNPs placed in inferred weak enhancers, promoters and lncRNAs. OSBPL10 had significantly lower expression in Africans than Europeans, while for RXRA several SNPs may differentially regulate its transcription between Africans and Europeans. Their expression was confirmed to change through dengue disease progression in Cuban patients and to vary with disease severity in a Thai transcriptome dataset. These genes interact in the LXR/RXR activation pathway that integrates lipid metabolism and immune functions, being a key player in dengue virus entrance into cells, its replication therein and in cytokine production. Knockdown of OSBPL10 expression in THP-1 cells by two shRNAs followed by DENV2 infection tests led to a significant reduction in DENV replication, being a direct functional proof that the lower OSBPL10 expression profile in Africans protects this ancestry against dengue disease.

Secondary heterologous dengue infection risk: Disequilibrium between immune regulation and inflammation?

Increased serum levels of cytokines released by cells of the immune response have been detected in patients suffering from dengue disease. Likewise, secondary infections by a different dengue virus serotype result in a highest risk of development of the severe dengue disease. Both findings suggest that the memory immune response is one of the key players in the pathogenesis of this disease. Here we take advantage of the particular Cuban epidemiological situation in dengue to analyze a broad spectrum of cell-mediated immune response mediators at mRNA and protein level. Evidences for a regulatory immune pattern in homologous (TGF-beta, IL-10) vs. pro-inflammatory pattern (IFN-gamma, TNF-alpha) in heterologous dengue virus re-challenge were found, suggesting a possible association with the higher incidence of severe dengue cases in the latter case.

MCP-1 and MIP-1α expression in a model resembling early immune response to dengue.

Dengue virus has become endemic in most tropical urban areas throughout the world, and DHF has appeared concomitantly with this expansion. The intensity of dengue virus replication during the early stages of infection could determine clinical outcomes; therefore, it is important to understand the impact of dengue virus infection on the earliest immune defense against microbial infection, which also strongly regulates the adaptive immune responses. This study was aimed at evaluating the expression of the CC-chemokines MIP-1α/CCL3 and MCP-1/CCL2 in peripheral blood leukocytes using an ex vivo model resembling dengue infection in vivo, in subjects with a well characterized dengue immune background, due to the exceptional Cuban epidemiological situation in dengue. The expression of IFNγ, TNFα and IL10 was also evaluated, giving insight about the role of MCP-1 and MIP-1α in the interplay between innate and adaptive immunity. From individuals with different dengue immune background after dengue virus challenge, increased and different expression of the chemokines and cytokines studied was verified in peripheral blood mononuclear cells, thus demonstrating that the previous immunity to a dengue virus serotype has a strong influence on the early immune response after dengue re-infection.

HLA-A, -B, -C, and -DRB1 allele frequencies in Cuban individuals with antecedents of dengue 2 disease: advantages of the Cuban population for HLA studies of dengue virus infection.

Dengue virus infection has emerged as one of the most important arthropod-borne diseases. In some dengue-infected individual, the disease progresses to its severe, life-threatening form, dengue hemorrhagic fever (DHF). Host genetic factors may be relevant and predispose some individuals to the severe dengue disease. The unique history of dengue outbreaks in Cuba is extremely advantageous for genetic studies of dengue disease resistance or susceptibility. Consequently, samples collected from 120 healthy individuals that developed dengue fever (DF) and DHF during the 1997 dengue 2 outbreak in the Santiago de Cuba municipality were HLA genotyped using polymerase chain reaction-sequence-specific primers. Polymorphism at the human leukocyte antigen (HLA) class I loci was significantly associated with DHF disease susceptibility, but polymorphism in the HLA-DRB1 was associated with protection. Amino acid peptides present in the poly-protein of the dengue 2 Jamaica strain, which are able to bind to the HLA class I and class II allotypes associated with susceptibility to or protection against the dengue clinical disease, respectively, were predicted using the BIMAS and SYFPEITHI predictive algorithms of peptide/MHC interaction.

Role of CC chemokine receptor 1 and two of its ligands in human dengue infection. Three approaches under the Cuban situation.

Any of the four dengue serotypes can cause a severe disease, partly due to systemic inflammation orchestrated by mediators like cytokines and chemokines. We addressed the role of CCR1 and its ligands CCL3/MIP-1α and CCL5/RANTES in dengue infection using three different approaches: an ex vivo model exploring memory immune response in subjects with a well characterized dengue immune background, an in vivo study in patients with primary or secondary dengue infection, and an approach in fatal dengue. CCR1 and CCL3/MIP-1α gene expression showed differences after homotypic and heterotypic challenge according to dengue immune background of subjects, in correspondence with previous observations in Cuban dengue outbreaks. CCL5/RANTES gene expression was higher after homotypic challenge. CCR1 and CCL3/MIP-1α gene expression was higher in patients with secondary infection during critical days of the dengue disease, while the increase in RANTES expression started earlier than the observed for CCR1 and CCL3/MIP-1α. CCR1 and CCL3/MIP-1α gene expression was as high in brain as in spleen tissue from necropsy. Our results confirm the strong influence of previous immunity in subsequent dengue infections, and confer a possible pathogenic role to CCR1 and CCL3/MIP-1α in dengue disease and a possible protective role for CCL5/RANTES, probably through CCR5 interaction.

Variation in inflammatory/regulatory cytokines in secondary, tertiary, and quaternary challenges with dengue virus.

Secondary heterologous dengue infection is a risk factor for severe disease manifestations because of the immune-enhancement phenomenon. Succeeding clinical infections are seldom reported, and the clinical course of tertiary and quaternary dengue infections is not clear. Cuba represents a unique environment to study tertiary/quaternary dengue infections in a population with known clinical and serologic dengue markers and no dengue endemicity. We took advantage of this exceptional epidemiologic condition to study the effect of primary, secondary, tertiary, and quaternary dengue infection exposure on the expression of pro-inflammatory and regulatory cytokines, critical in dengue infection pathogenesis, by using a dengue infection ex vivo model. Whereas secondary exposure induced a high cytokine response, we found a significantly lower expression of tumor necrosis factor-α, interferon-γ, interleukin-10, and tumor growth factor-ß after tertiary and quaternary infectious challenge. Significant differences in expression of the cytokines were seen between the dengue immune profiles, suggesting that the sequence in which the immune system encounters serotypes may be important in determining the nature of the immune response to subsequent infections.

Association of MICA and MICB alleles with symptomatic dengue infection.

Dengue viruses (DV) are one of the most important arthropod-borne viral diseases in the developing world. DV can cause syndromes that are either self-limiting or severe. Allelic variants of human leukocyte antigen (HLA) genes have been demonstrated to be associated with disease susceptibility. Here we report the association of nonclassical HLA class I MICA-MICB genes with disease outcome during DV infection. A sequencing-based typing method and genotyping of MICA and MICB in a well-characterized group of Cuban individuals with dengue hemorrhagic fever (DHF), dengue fever (DF), or asymptomatic dengue infection (ADI) was performed. Statistical analysis revealed a tendency for MICA*008 and MICB*008 to associate with susceptibility to illness when symptomatic versus asymptomatic cases (odds ratio [OR] = 2.1, p(v) = 0.03, and OR = 10.4, p = 0.0096, respectively) were compared. Surprisingly, a stronger association of both allelic forms was observed for the DF patients compared with the ADI group (MICA*008, OR = 5.2, p = 0.0001; and MICB*008, OR = 13.2, p = 0.0025) rather than the severe cases. Major histocompatibility class I-related gene-related natural killer cells and/or γδ and αß T-cell activation might regulate the development of symptomatic DF and DHF.

Asymptomatic dengue infection in a Cuban population confirms the protective role of the RR variant of the FcgammaRIIa polymorphism.

The role of human Fcgamma receptors (FcgammaR) has been recognized considerably over the last years. These receptors vary in their affinity for IgG subclasses and the intracellular signals elicited by them. Allelic variants of FcgammaR genes may influence the biological phagocyte activity, accounting for an inherited pre-disposition to disease. The specific FcgammaRIIa (CD32) contains a polymorphic variant (H/R131) that has been associated to a reduced risk for developing dengue hemorrhagic fever (DHF). Here, we investigated the role of this polymorphism in a very well-characterized group of Cuban individuals with antecedents of DHF, dengue fever (DF), or subclinical dengue infection. The HH131 genotype was significantly associated with dengue disease, either DF (*P = 0.016; odds ratio = 4.425; 95% confidence interval = 1.10-20.52) or DHF (P = 0.00018; odds ratio = 10.56; 95% confidence interval = 2.33-54.64) with respect to the subclinical infection.