[Caloric restriction suppresses endothelial cells senescence via down-regulation of NOX4 induced by HNF3γ].

The aim of current study is to investigate the molecular mechanism that caloric restriction (CR) suppresses endothelial cells senescence. Human aortic endothelial cells (HAECs) were divided into 5 groups: control group, high caloric group (about 1.5 times caloric intake of control group), low caloric group (about 0.5 times caloric intake of control group), siRNA plus low caloric group (low caloric treatment pretreated with special siRNA targeting hepatocyte nuclear factor 3γ (HNF3γ)), and siRNA plus high caloric group (high caloric treatment pretreated with special siRNA targeting HNF3γ). The gene and protein expressions of HNF3γ and NADPH oxidase 4 (NOX4) were quantified by real-time quantitative PCR (RT-qPCR) and Western blotting, respectively. Intracellular reactive oxygen species (ROS) production was measured by flow cytometry. Endothelial cells senescence was assayed by senescence associated ß-galactosidase staining. After verifying the binding of HNF3γ to NOX4 promoter region by chromatin immunoprecipitation assay (ChIP), NOX4 promoter activity was assayed by dual-luciferase reporter system. Compared with the control group, the mRNA and protein expression levels of HNF3γ,and the ratio of phosphorylated HNF3γ protein increased significantly (P<0.05) in low caloric group, and decreased significantly (P<0.05) in high caloric group and siRNA plus low or high caloric group; whereas the mRNA and protein levels of NOX4 intracellular ROS and endothelial cells senescence decreased significantly (P<0.05) in low caloric group and increased significantly (P<0.05) in high caloric group and siRNA plus low or high caloric group. ChIP result showed there were four HNF3γ binding sites in NOX4 gene promoter region (-6, -76, -249 and -954 bp) and HNF3γ could bind to all 4 predicted sites. According to the results of dual-luciferase reporter system, HNF3γ binding to 1 site (-6 bp), 2 sites (-6 and -76 bp), 3 sites (-6, -76 and -249 bp) and 4 sites(-6, -76, -249 and -954 bp) could suppress NOX4 promoter activity to 80.15±4.64%, 40.02.±2.15%, 16.46±2.24% and 12.13±1.46% compared with that of baseline, respectively ( P<0.05). In a word, low caloric intake decreases the production of intracellular ROS and suppresses endothelial cells senescence through promoting HNF3γ binging to NOX4 promoter region and inhibiting NOX4 gene expression induced by up-regulated HNF3γ.

Porcine reproductive and respiratory syndrome virus induces CD4+CD8+CD25+Foxp3+ regulatory T cells (Tregs).

The aim of this study was to analyze the regulatory T cells (Tregs) induced by the porcine reproductive and respiratory syndrome virus (PRRSV) in pigs. Serum, blood, tonsil, and mediastinal lymph nodes' samples were obtained at different time post-infection (dpi). The frequencies of CD4(+)CD8(-)CD25(+)Foxp3(+), CD4(+)CD8(+)CD25(+)Foxp3(+), or CD4(-)CD8(+)CD25(+)Foxp3(+) phenotypes were determined in PBMC and lymph node cells, and cells producing IL-10 or TGF-ß were analyzed. PRRSV increased the number of CD4(+)CD8(+)CD25(+)Foxp3(+) cells at 14 dpi, whereas CD4(+)CD8(-)CD25(+)Foxp3(+) remained constant until 28 dpi. Positive correlation exists between viremia and induced regulatory cells. CD4(+)CD8(+)CD25(+)Foxp3(+)-induced Treg cells were consistently observed in lymphoid tissues. Analysis of IL-10- and TGF-ß-producing cell demonstrated that in response to PRRSV, CD4(+)CD8(-)Foxp3(low) and CD4(+)CD8(+)Foxp3(high) cells increase moderately the proportion of IL-10(+) cells. TGF-ß was only observed in the CD4(+)CD8(+)Foxp3(high) population after PRRSV stimulation. In conclusion, PRRSV infection increases the frequency of Tregs with the phenotype CD4(+)CD8(+)CD25(+)Foxp3(high) and produces TGF-ß.

Infection of mouse bone marrow-derived dendritic cells by live attenuated Japanese encephalitis virus induces cells maturation and triggers T cells activation.

An attenuated Japanese encephalitis virus (JEV) strain SA14-14-2, generated from the wild strain SA14, is an effective live vaccine against JEV infection. It has led to a significant decrease in JEV infection around the world. Although it is highly effective, the mechanism for its robust immunity was not well investigated. In this study, the interaction of SA14-14-2 with bone marrow-derived dendritic cells (bmDCs) was investigated. Our results showed that the infection of bmDCs with SA14-14-2 resulted in viral replication and upregulation of bmDC maturation marker molecules (CD40, CD80, CD83 and MHC I). SA14-14-2 infection also stimulated the production of interferon-α (IFN-α), monocyte chemoattractant protein-1 (MCP-1/CCL2), tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) of bmDC. Both MLR and ELISPOT assay showed an enhanced allostimulatory capacity of SA14-14-2-infected bmDCs. Furthermore, the SA14-14-2-infected bmDCs impaired the expansion of Foxp3+ regulatory T (Treg) cells with immunosuppressive potential, suggesting that SA14-14-2 infection induced antiviral immunity rather than immunosuppression. Taken together, our results indicated that SA14-14-2 infection caused bmDC maturation, changed the expression profiles of several cytokines, and triggered T cell activation. This offered an insight in the immunologic mechanisms associated with the high efficiency of the SA14-14-2 vaccine.

Systemic and local characterization of regulatory T cells in a chronic fungal infection in humans.

The long-term persistence of pathogens in a host is a hallmark of certain infectious diseases, including schistosomiasis, leishmaniasis, and paracoccidioidomycosis (PCM). Natural regulatory T (Treg) cells are involved in control of the immune responses, including response to pathogens. Because CTLA-4 is constitutively expressed in Treg cells and it acts as a negative regulator of T cell activation in patients with PCM, here we investigated the involvement of Treg cells in the control of systemic and local immune response in patients with PCM. We found that the leukocyte subsets were similar in patients and controls, except for CD11c+CD1a+ cells. However, a higher frequency of CD4+CD25+ T cells expressing CTLA-4, glucorticoid-inducible TNFR, membrane-bound TGF-beta, and forkhead-box 3 were observed in PBMC of patients. In accordance, these cells exhibited stronger suppressive activity when compared with those from controls (94.0 vs 67.5% of inhibition of allogeneic T cell proliferation). In addition, the data showed that CD4+CD25+ T cells expressing CTLA-4+, glucocorticoid-inducible TNFR positive, CD103+, CD45RO+, membrane-bound TGF-beta, forkhead-box 3 positive, and the chemokines receptors CCR4 and CCR5 accumulate in the Paracoccidioides brasiliensis-induced lesions. Indeed, the secreted CCL17 and CCL22, both associated with the migration of Treg cells to peripheral tissues, were also detected in the biopsies. Moreover, the CD4+CD25+ T cell derived from lesions, most of them TGF-beta+, also exhibited functional activity in vitro. Altogether, these data provide the first evidence that Treg cells play a role in controlling local and systemic immune response in patients with a fungal-induced granulomatous disease advancing our understanding about the immune regulation in human chronic diseases.