Activation of the Innate Immune Response against DENV in Normal Non-Transformed Human Fibroblasts

In this work, we demonstrate that that both human whole skin and freshly isolated skin fibroblasts are productively infected with Dengue virus (DENV). In addition, primary skin fibroblast cultures were established and subsequently infected with DENV-2; we showed in these cells the presence of the viral antigen NS3, and we found productive viral infection by a conventional plaque assay. Of note, the infectivity rate was almost the same in all the primary cultures analyzed from different donors. The skin fibroblasts infected with DENV-2 underwent signaling through both TLR3 and RIG-1, but not Mda5, triggering up-regulation of IFNβ, TNFα, defensin 5 (HB5) and β defensin 2 (HβD2). In addition, DENV infected fibroblasts showed increased nuclear translocation of interferon (IFN) regulatory factor 3 (IRF3), but not interferon regulatory factor 7 IRF7, when compared with mock-infected fibroblasts. Our data suggest that fibroblasts might even participate producing mediators involved in innate immunity that activate and contribute to the orchestration of the local innate responses. This work is the first evaluating primary skin fibroblast cultures obtained from different humans, assessing both their susceptibility to DENV infection as well as their ability to produce molecules crucial for innate immunity

Construct and expression of recombinant domains I/II of dengue virus- 2 and its efficacy to evaluate immune response in endemic area: possible use in prognosis

The envelope (E) protein from DENV, contain three functional and structural domains (DI, DII and DIII). Some studies suggest that neutralizing antibodies during natural DENV infection are predominantly against DI and DII, in contrast, low proportion of the antibodies were against DIII. Thus it is necessary to establish the proportion of human antibodies against DENV E protein that bind to DI and DII during the normal course of infection; as an indicator of the quality of the antibody response and to further design new vaccine candidates for DENV. The aim of this study was to express recombinant proteins harboring a 240-aminoacid fragment of the E protein from DI and DII of DENV serotypes 2 and 3 in a eukaryotic S2 system. Further, we evaluate the antibodies against these antigens in samples from patients in acute phase of DF or DHF and compare it with the response of samples from healthy individuals from the same endemic areas and samples from healthy individuals from a non-endemic area (EA and NEA, respectively). These results suggest that the presence of antibodies against rEDI/DII might be used to identify patients at risk for severe disease

Competitive suppression of dengue virus replication occurs in chikungunya and dengue co-infected Mexican infants

Abstract Background Co-circulation of dengue virus (DENV) and chikungunya virus (CHIKV) is increasing worldwide but information on the viral dynamics and immune response to DENV-CHIKV co-infection, particularly in young infants, is scant. Methods Blood samples were collected from 24 patients, aged 2 months to 82 years, during a CHIKV outbreak in Mexico. DENV and CHIKV were identified by RT-PCR; ELISA was used to detect IgM and IgG antibodies. CHIKV PCR products were cloned, sequenced and subjected to BLAST analysis. To address serological findings, HMEC-1 and Vero cells were inoculated with DENV-1, DENV-2 and CHIKV alone and in combination (DENV-2-CHIKV and DENV-1-CHIKV); viral titers were measured at 24, 48 and 72 h. Results Nine patients (38%) presented co-infection, of who eight were children. None of the patients presented severe illness. Sequence analysis showed that the circulating CHIKV virus belonged to the Asian lineage. Seroconversion to both viruses was only observed in the four patients five years or older, while the five infants under two years of age only seroconverted to CHIKV. Viral titers in the CHIKV mono-infected cells were greater than in the DENV-1 and DENV-2 mono-infected cells. Furthermore, we observed significantly increased CHIKV progeny and reduction of DENV progeny in the co-infected cells. Conclusions In our population, DENV-CHIKV co-infection was not associated with increased clinical severity. Our in vitro assay findings strongly suggest that the lack of DENV IgG conversion in the co-infected infants is due to suppression of DENV replication by the Asian lineage CHIKV. The presence of maternal antibody and immature immune responses in the young infants may also play a role

Human platelets and megakaryocytes express defensin alpha 1

Platelets are anucleate cells that have a role in several innate immune functions, including the secretion of proteins with antimicrobial activity. Several studies have demonstrated the ability of platelets to secrete thrombin-induced platelet microbicidal proteins and antimicrobial peptides, like hBD-1. However, the expression and secretion of defensins of the alpha family by platelets have not been fully elucidated. The aim of this study was to characterize the expression of defensin alpha 1 (DEFA1) in human platelets and megakaryocytes. Our data indicate that DEFA1 mRNA and protein are present in peripheral blood platelets and in the megakaryoblastic leukemia cell line (MEG-01). DEFA1 co-localize with α-granules of platelets and MEG-01 cells, and was also detected in cytoplasm of MEG-01 cells. The assay of our in vitro model of platelet-like particles (PLPs) revealed that MEG-01 cells could transfer DEFA1 mRNA to their differentiated PLPs. Furthermore, platelets secreted DEFA1 into the culture medium when activated with thrombin, adenosine diphosphate, and lipopolysaccharide; meanwhile, MEG-01 cells secreted DEFA1 when activated with thrombopoietin. Platelet’s secreted DEFA1 can rebind to platelet’s surface and have antibacterial activity against the gram-negative bacteria Escherichia coli. In summary, our data indicate that both, human platelets and megakaryocytes, can express and secrete DEFA1. These results suggest a new role of platelets and megakaryocytes in the innate immune response

Crosstalk between dermal fibroblasts and dendritic cells during dengue virus infection

Dengue virus infection (DENV-2) is transmitted by infected mosquitoes via the skin, where many dermal and epidermal cells are potentially susceptible to infection. Most of the cells in an area of infection will establish an antiviral microenvironment to control viral replication. Although cumulative studies report permissive DENV-2 infection in dendritic cells, keratinocytes, and fibroblasts, among other cells also infected, little information is available regarding cell-to-cell crosstalk and the effect of this on the outcome of the infection. Therefore, our study focused on understanding the contribution of fibroblast and dendritic cell crosstalk to the control or promotion of dengue. Our results suggest that dendritic cells promote an antiviral state over fibroblasts by enhancing the production of type I interferon, but not proinflammatory cytokines. Infected and non-infected fibroblasts promoted partial dendritic cell maturation, and the fibroblast-matured cells were less permissive to infection and showed enhanced type I interferon production. We also observed that the soluble mediators produced by non-infected or Poly (I:C) transfected fibroblasts induced allogenic T cell proliferation, but mediators produced by DENV-2 infected fibroblasts inhibited this phenomenon. Additionally, the effects of fibroblast soluble mediators on CD14+ monocytes were analyzed to assess whether they affected the differentiation of monocyte derived dendritic cells (moDC). Our data showed that mediators produced by infected fibroblasts induced variable levels of monocyte differentiation into dendritic cells, even in the presence of recombinant GM-CSF and IL-4. Cells with dendritic cell-like morphology appeared in the culture; however, flow cytometry analysis showed that the mediators did not fully downregulate CD14 nor did they upregulate CD1a. Our data revealed that fibroblast-dendritic cell crosstalk promoted an antiviral response mediated manly by type I interferons over fibroblasts. Furthermore, the maturation of dendritic cells and T cell proliferation were promoted, which was inhibited by DENV-2-induced mediators. Together, our results suggest that activation of the adaptive immune response is influenced by the crosstalk of skin resident cells and the intensity of innate immune responses established in the microenvironment of the infected skin

Human Platelets Contain, Translate, and Secrete Azurocidin; A Novel Effect on Hemostasis

Platelets play a significant role in hemostasis and perform essential immune functions, evidenced by the extensive repertoire of antimicrobial molecules. Currently, there is no clear description of the presence of azurocidin in human platelets. Azurocidin is a 37 kDa cationic protein abundant in neutrophils, with microbicidal, opsonizing, and vascular permeability-inducing activity. Therefore, this work aimed to characterize the content, secretion, translation, and functions of azurocidin in platelets. Our results show the presence of azurocidin mRNA and protein in α-granules of platelet and megakaryoblasts, and stimulation with thrombin, ADP, and LPS leads to the secretion of free azurocidin as well as within extracellular vesicles. In addition, platelets can translate azurocidin in a basal or thrombin-induced manner. Finally, we found that the addition of low concentrations of azurocidin prevents platelet aggregation and activation. In conclusion, we demonstrate that platelets contain, secrete, and translate azurocidin, and this protein may have important implications for hemostasis

Additional file 1: of Competitive suppression of dengue virus replication occurs in chikungunya and dengue co-infected Mexican infants

Figure S1. Co-infection of DENV-2 and CHIKV in Vero cells, analyzed by flow cytometry: The Vero cells were infected in a 6-well plate with DENV-2 or CHIKV at MOI of 0.25 in both mono-infection and co-infection assays and then evaluated at 48 h. The cells were harvested from each plate, then double stained assay and flow cytometry analysis were performed to confirm cells infected with DENV anti-NS5 (X) and infected cells with CHIKV anti-E2 (Y). (TIFF 917 kb