Dengue e imunologia inata: estudo dos fatores antivirais e citotóxicos em células dendríticas plasmacitoides e células NK durante a infecção pelo vírus Dengue / Dengue and innate immunology: study of antiviral and cytotoxic fators in plasmacytoid dendritic cells and nk cells during dengue virus infection

O vírus dengue é um flavivírus causador de síndrome febril hemorrágica, sendoalguns casos graves evidenciados pelo acúmulo de plasma nas cavidades. Sugereseque a intensa replicação viral na fase aguda resultaria na intensa produção demediadores inflamatórios, levando ao extravasamento plasmático. A imunidade inataé uma importante barreira na limitação da dispersão viral. As células dendríticasplasmacitoides (PDCs) e as células NK são fundamentais durante a fase inicial dasinfecções por suas características antivirais e citotóxicas contra células infectadas. Ofenótipo Killer das PDCs (IKPDCs) resultaria da ativação viral e é caracterizadopela expressão membranar do ligante indutor de apoptose relacionado ao fator denecrose tumoral (TRAIL) e pela produção de interferons do tipo I. As células NKpodem ser diretamente ativadas reconhecendo as células alvo ou, indiretamenteativadas pelas citocinas (como os interferons) tornando-se citotóxica, expressandoTRAIL membranar. Apesar do papel antiviral, pouco se sabe sobre os mecanismosde ação dessas populações celulares durante a febre do dengue (FD). Nossoobjetivo foi estudar o envolvimento das PDCs e células NK ativadas naimunopatologia da FD. Analisando as células de pacientes ex vivo, observamosmaior frequência de IKPDCs nos casos brandos de FD, assim como de células NKTRAIL+. Outros marcadores de ativação como o marcador de degranulação CD107ae o receptor de reconhecimento padrão TLR3 foram expressos em maiores níveisnos pacientes comparando aos controles saudáveis...

Dengue virus is a flavivirus that can cause a hemorrhagic febrile syndrome, in whichsome severe cases are characterized by plasma accumulation in cavities. It issuggested that a high viral burden in the acute phase would lead to an enhancedproduction of inflammatory mediators, leading to plasma leakage. Innate immunity isan important barrier limiting viral spread. Plasmacytoid dendritic cells (PDCs) and NKcells are main actors in the beginning of infection because of their antiviral andcytotoxic features against infected cells. PDC killer phenotype (IKPDCs) is inducedby viral activation and main profile is membrane expression of TNF-relatedapoptosis-inducer ligand (TRAIL) and type I interferon production. NK cells can beactivated directly, by target recognition, or indirectly, by cytokines (like interferons)and become cytotoxic, expressing membrane TRAIL. Despite its antiviral role, thefunction of those cell populations during dengue fever (DF) is not largely known. Ouraim was to study activated PDCs and NK cells involvement in DF immunopathology.DF patients’ cells analysis ex vivo presented higher frequency of IKPDCs and alsohigher frequency of TRAIL+NK cells in mild DF cases. NK activation markers such asCD107a degranulation marker and pattern recognition receptor TLR3 wereupregulated in patients’ cells compared to healthy donors...

Profile of circulating levels of IL-1Ra, CXCL10/IP-10, CCL4/MIP-1β and CCL2/MCP-1 in dengue fever and parvovirosis

Dengue virus (DENV) and parvovirus B19 (B19V) infections are acute exanthematic febrile illnesses that are not easily differentiated on clinical grounds and affect the paediatric population. Patients with these acute exanthematic diseases were studied. Fever was more frequent in DENV than in B19V-infected patients. Arthritis/arthralgias with DENV infection were shown to be significantly more frequent in adults than in children. The circulating levels of interleukin (IL)-1 receptor antagonist (Ra), CXCL10/inducible protein-10 (IP-10), CCL4/macrophage inflammatory protein-1 beta and CCL2/monocyte chemotactic protein-1 (MCP-1) were determined by multiplex immunoassay in serum samples obtained from B19V (37) and DENV-infected (36) patients and from healthy individuals (7). Forward stepwise logistic regression analysis revealed that circulating CXCL10/IP-10 tends to be associated with DENV infection and that IL-1Ra was significantly associated with DENV infection. Similar analysis showed that circulating CCL2/MCP-1 tends to be associated with B19V infection. In dengue fever, increased circulating IL-1Ra may exert antipyretic actions in an effort to counteract the already increased concentrations of IL-1β, while CXCL10/IP-10 was confirmed as a strong pro-inflammatory marker. Recruitment of monocytes/macrophages and upregulation of the humoral immune response by CCL2/MCP-1 by B19V may be involved in the persistence of the infection. Children with B19V or DENV infections had levels of these cytokines similar to those of adult patients.

Dengue-2 and yellow fever 17DD viruses infect human dendritic cells, resulting in an induction of activation markers, cytokines and chemokines and secretion of different TNF-α and IFN-α profiles

Flaviviruses cause severe acute febrile and haemorrhagic infections, including dengue and yellow fever and the pathogenesis of these infections is caused by an exacerbated immune response. Dendritic cells (DCs) are targets for dengue virus (DENV) and yellow fever virus (YF) replication and are the first cell population to interact with these viruses during a natural infection, which leads to an induction of protective immunity in humans. We studied the infectivity of DENV2 (strain 16681), a YF vaccine (YF17DD) and a chimeric YF17D/DENV2 vaccine in monocyte-derived DCs in vitro with regard to cell maturation, activation and cytokine production. Higher viral antigen positive cell frequencies were observed for DENV2 when compared with both vaccine viruses. Flavivirus-infected cultures exhibited dendritic cell activation and maturation molecules. CD38 expression on DCs was enhanced for both DENV2 and YF17DD, whereas OX40L expression was decreased as compared to mock-stimulated cells, suggesting that a T helper 1 profile is favoured. Tumor necrosis factor (TNF)-α production in cell cultures was significantly higher in DENV2-infected cultures than in cultures infected with YF17DD or YF17D/DENV. In contrast, the vaccines induced higher IFN-α levels than DENV2. The differential cytokine production indicates that DENV2 results in TNF induction, which discriminates it from vaccine viruses that preferentially stimulate interferon expression. These differential response profiles may influence the pathogenic infection outcome.

Dengue-2 infection and the induction of apoptosis in human primary monocytes

Monocytes/macrophages are important targets for dengue virus (DENV) replication; they induce inflammatory mediators and are sources of viral dissemination in the initial phase of the disease. Apoptosis is an active process of cellular destruction genetically regulated, in which a complex enzymatic pathway is activated and may be trigged by many viral infections. Since the mechanisms of apoptotic induction in DENV-infected target cells are not yet defined, we investigated the virus-cell interaction using a model of primary human monocyte infection with DENV-2 with the aim of identifying apoptotic markers. Cultures analyzed by flow cytometry and confocal microscopy yielded DENV antigen positive cells with rates that peaked at the second day post infection (p.i.), decayed afterwards and produced the apoptosis-related cytokines TNF-á and IL-10. Phosphatidylserine, an early marker for apoptosis, was increased at the cell surface and the Fas death receptor was upregulated at the second day p.i. at significantly higher rates in DENV infected cell cultures than controls. However, no detectable changes were observed in the expression of the anti-apoptotic protein Bcl-2 in infected cultures. Our data support virus modulation of extrinsic apoptotic factors in the in vitro model of human monocyte DENV-2 infection. DENV may be interfering in activation and death mechanisms by inducing apoptosis in target cells.

An in vitro model for dengue virus infection that exhibits human monocyte infection, multiple cytokine production and dexamethasone immunomodulation

An important cytokine role in dengue fever pathogenesis has been described. These molecules can be associated with haemorrhagic manifestations, coagulation disorders, hypotension and shock, all symptoms implicated in vascular permeability and disease worsening conditions. Several immunological diseases have been treated by cytokine modulation and dexamethasone is utilized clinically to treat pathologies with inflammatory and autoimmune ethiologies. We established an in vitro model with human monocytes infected by dengue virus-2 for evaluating immunomodulatory and antiviral activities of potential pharmaceutical products. Flow cytometry analysis demonstrated significant dengue antigen detection in target cells two days after infection. TNF-alpha, IFN-alpha, IL-6 and IL-10 are produced by in vitro infected monocytes and are significantly detected in cell culture supernatants by multiplex microbead immunoassay. Dexamethasone action was tested for the first time for its modulation in dengue infection, presenting optimistic results in both decreasing cell infection rates and inhibiting TNF-alpha, IFN-alpha and IL-10 production. This model is proposed for novel drug trials yet to be applyed for dengue fever.