The rapid and sustained responses of dendritic cells to influenza virus infection in a non-human primate model

Dendritic cells (DCs) are readily infected by influenza viruses and play a crucial role in regulating host innate and adaptive immune responses to viral infection. The aims of this study are to characterize the dynamic changes in the numbers and maturation status of dendritic cells present in the lung and lung-associated lymph nodes (LALNs) in the model of a non-human primate (NHP) infected by influenza A virus (IAV). Cynomolgus macaques were infected with influenza A virus (H3N2) via bronchoscopy. Flow cytometry was used to analyze the DC numbers, maturation status and subsets during the time of acute infection (days 1, 2, 3, 4, 7) and the resolution phase (day 30). A dramatic increase in the numbers of influenza A virus-infected CD11c+CD14- myeloid dendritic cells (mDCs) and CD11c-CD123+ plasmacytoid dendritic cells (pDCs) were observed from day 1 to day 4 and peak up from day 7 post-infection. In lung and lung-associated lymph nodes, the numbers and maturation status of myeloid dendritic cells and plasmacytoid dendritic cells increased more slowly than those in the lung tissues. On day 30 post-infection, influenza A virus challenge increased the number of myeloid dendritic cells, but not plasmacytoid dendritic cells, compared with baseline. These findings indicate that dendritic cells are susceptible to influenza A virus infection, with the likely purpose of increasing mature myeloid dendritic cells numbers in the lung and lung and lung-associated lymph nodes, which provides important new insights into the regulation of dendritic cells in a non-human primate model.

Effects of Epstein-Barr virus on the development of dendritic cells derived from cord blood monocytes: an essential role for apoptosis

OBJECTIVE: Epstein-Barr virus (EBV) is a ubiquitous human γ-herpes virus, which can adapt and evade host immune defense. Dendritic cells (DCs) play a pivotal role in the initiation and maintenance of immune responses. This study investigated the effects of EBV on cord blood monocytes derived DCs (CBDC). METHODS: Monocytes were isolated from cord blood and cultured in medium containing recombinant IL-4 and GM-CSF to induce DCs development. B95-8 supernatant was added in monocytes culture medium for EBV infection at day 0. Phenotypic characterization of DCs, apoptotic cells, and mitochondrial membrane potential (MMP) were detected by flow cytometry. The morphology was observed by Hoechst 33258 staining and TUNEL staining, the expression of X-linked inhibitor of apoptosis protein (XIAP) was detected by Western blotting assay and caspase 3, 8 and 9 activity was measured. RESULTS: Phenotypic characterization of DCs was changed in EBV-treated group. Chromatin condensation and DNA fragmentation were observed in EBV induced CBDC apoptosis. In addition, caspase 3, caspase 8, and caspase 9 activation were enhanced in the EBV-treated group. This was accompanied by the loss of MMP. Furthermore, XIAP expression was down-regulated in the EBV-treated group and compared to mock-infected group. CONCLUSION: These results suggested that EBV could inhibit CBDC phenotypic differentiation, and induce CBDC apoptosis in caspase-dependent manner with involvement of the mitochondrial pathway. This might help EBV to evade host immune responses to establish persistent infection.

Morphometric analysis of dendritic cells from anal mucosa of HIV-positive patients and the relation to intraepithelial lesions and cancer seen at a tertiary health institution in Brazil / Análise morfométrica das células dendríticas da mucosa anal de pacientes HIV-positivos e relação com as lesões intraepiteliais e o câncer numa instituição de saúde terciária no Brasil

PURPOSE: To morphometrically quantify CD1a+ dentritic cells and DC-SIGN+ dendritic cells in HIV-positive patients with anal squamous intraepithelial neoplasia and to evaluate the effects of HIV infection, antiretroviral therapy and HPV infection on epithelial and subepithelial dendritic cells. METHODS: A prospective study was performed to morphometrically analyze the relative volume of the dendritic cells and the relationship between anal intraepithelial neoplasia and cancer in HIV-positive patients from the Tropical Medicine Foundation of Amazonas, Brazil. All patients were submitted to biopsies of anorectal mucosa to perform a classic histopathological and immunohistochemical analysis, employing antibodies against CD1a and DC-SIGN for the morphometric quantification of dendritic cells. RESULTS: HIV-negative patients displayed a CD1a DC density significantly higher than that of HIV-positives patients (3.75 versus 2.54) (p=0.018), and in patients with severe anal intraepithelial neoplasia had correlated between DC CD1a density with levels of CD4 + cells (p: 0.04) as well as the viral load of HIV-1 (p: 0.035). A not significant rise in the median density of CD1a+ DC was observed in the HIV positive/ HAART positive subgroup compared to the HIV positive/ HAART negative subgroup. The CD1a+ DC were also significantly increased in HIV-negative patients with anorectal condyloma (2.33 to 3.53; p=0.05), with an opposite effect in HIV-positive patients. CONCLUSIONS: Our data support an enhancement of the synergistic action caused by HIV-HPV co-infection on the anal epithelium, weakening the DC for its major role in immune surveillance. Notoriously in patients with severe anal intraepithelial neoplasia, the density of CD1a+ epithelial dendritic cells was influenced by the viral load of HIV-1. Our study describes for the first time the density of subepithelial DC-SIGN+ dendritic cells in patients with anal severe anal intraepithelial neoplasia and points to the possibility that a specific therapy for HIV induces the recovery of the density of epithelial DC.

OBJETIVO: Quantificar morfometricamente as células dendríticas DC CD1a+ e DC DC-SIGN+ em pacientes HIV positivos portadores de neoplasia escamosa intraepitelial anal e avaliar os efeitos da infecção pelo HIV, da terapia antirretroviral e da infecção pelo HPV sobre as células dendríticas epiteliais e subepiteliais. MÉTODOS: Um estudo prospectivo foi realizado para analisar morfometricamente o volume relativo das células dendríticas e as relações entre neoplasia intraepitelial anal e o câncer em pacientes HIV positivos da Fundação de Medicina Tropical do Amazonas, Brasil.Todos os pacientes foram submetidos a biópsia da mucosa retal para realizar uma análise clássica histopatológica e imunohistoquímica utilizando anticorpos contra anti-CD1a e anti-DC-SIGN, para a quantificação morfométrica das células dendríticas. RESULTADOS: Os pacientes HIV negativos apresentaram densidade das DC CD1a+ significativamente maior do que a dos pacientes HIV positivos (3,75 versus 2,54) (p:0,018), e os pacientes com severa apresentaram correlação das DC CD1a com os níveis de células TCD4(p:0,04) assim como a carga viral do HIV-1 (p:0,035). Observamos no subgrupo HIV-positivo/HAART positivo elevação não significativa na mediana da densidade das DC CD1a+ em relação ao grupo HIV-positivo/HAART negativo. As DC CD1a+ também se elevaram nos pacientes HIV negativo portadores de condiloma anorretal(2,33 para 3,53; p:0,05), com efeito inverso nos pacientes HIV positivos. CONCLUSÕES: Nossos dados confirmam a potencialização da ação sinérgica representada pela coinfecção HIV-HPV sobre o epitélio anal, fragilizando as DC em sua função primordial de vigilância imune. Notoriamente nos pacientes com neoplasia intraepithelial anal grave, a densidade das DC CD1a+ epiteliais sofreu influência da carga viral do HIV-1. Nosso estudo descreveu pela primeira vez a densidade das DC subepiteliais DC-SIGN+ em pacientes com neoplasia intraepithelial anal severa e apontamos para a possibilidade de que a terapia específica para o HIV induza a recuperação da densidade das DC epiteliais.

Construction of a recombinant adenovirus co-expressing truncated human prostate-specific membrane antigen and mouse 4-1BBL genes and its effect on dendritic cells

Our aim was to construct a recombinant adenovirus co-expressing truncated human prostate-specific membrane antigen (tPSMA) and mouse 4-1BBL genes and to determine its effect on dendritic cells (DCs) generated from bone marrow suspensions harvested from C57BL/6 mice for which the effect of 4-1BBL on DCs is not clear, especially during DCs processing tumor-associated antigen. Replication deficient adenovirus AdMaxTM Expression System was used to construct recombinant adenovirus Ad-tPSMA-internal ribosome entry site-mouse 4-1BBL (Ad-tPSMA-IRES-m4-1BBL) and Ad-enhanced green fluorescent protein. Day 7 proliferating DC aggregates generated from C57BL/6 mice were collected as immature DCs and further mature DCs were obtained by lipopolysaccharide activated immature DCs. After DCs were exposed to the recombinant adenovirus with 250 multiplicity of infection, the expression of tPSMA and m4-1BBL proteins were detected by Western blot, and the apoptosis and phenotype of DCs were analyzed by flow cytometry. Cytokines (IL-6 and IL-12) in the supernatant were detected by enzyme-linked immunosorbent assay (ELISA). Proliferation of T cells was detected by allogeneic mixed lymphocyte reactions. The tPSMA and m4-1BBL proteins were expressed correctly. The apoptosis rate of DCs transfected with Ad-tPSMA-IRES-m4-1BBL was 14.6 percent, lower than that of control DCs. The expression of co-stimulatory molecules [CD80 (81.6 ± 5.4 percent) and CD86 (80.13 ± 2.81 percent)] up-regulated in Ad-tPSMA-IRES-m4-1BBL-pulsed DCs, and the level of IL-6 (3960.2 ± 50.54 pg/mL) and IL-12 (249.57 ± 12.51 pg/mL) production in Ad-tPSMA-IRES-m4-1BBL-transduced DCs were significantly higher (P < 0.05) than those in control DCs. Ad-tPSMA-IRES-m4-1BBL induced higher T-cell proliferation (OD450 = 0.614 ± 0.018), indicating that this recombinant adenovirus can effectively enhance the activity of DCs.