Interferon-alpha secretion

Abstract: OBJECTIVES CXCL16 is a member of CXC chemokine, which is synthesised in plasmacytoid dendritic cell as a transmembrane molecule. Transmembrane CXCL16 on plasmacytoid dendritic cell plays a role in binding, uptaking and accumulation of CpG D ODN in early endosomes rather then lysosomal vesicles, thereby causing a high level of interferon-alpha secretion. Previously, we disclosed pronounced interferon-alpha production from these cells in patients with Behcet's disease. The aim of this study was to investigate the relation between the secretion of IFN-α and the expression of CXCL16 on surface of plasmacytoid dendritic cell from patients with Behcet's disease, and compare it with patients with ankylosing spondylitis and healthy controls. METHODS The study population consisted of 73 cases (35 with Behcet's disease, 19 with ankylosing spondylitis and 19 controls). We investigated the expression of CXCL16 on surface of plasmacytoid dendritic cells by flow cytometry, and the serum levels of IFN-α and CXCL16 with ELISA. RESULTS Serum levels of IFN-α in patients with Behcet's disease were significantly higher than the controls (p=0.009), and than patients with ankylosing spondylitis, but not statistically significant (p=0.124). Serum levels of CXCL16 in patients with Behcet's disease and patients with ankylosing spondylitis were significantly higher than controls (p=0.009, p=0.003, respectively). We found no difference in the percentage and MFI of plasmacytoid dendritic cells and CD123+CXCL16+ cells determined by flow cytometry among the study and control groups. In patients with Behcet's disease, a positive correlation was found between the percentage of plasmacytoid dendritic cells and CD123+CXCL16+ cells (p<0.001). Furthermore, there was also a positive correlation between the percentage of plasmacytoid dendritic cells and serum levels of CXCL16 in patients with ankylosing spondylitis (p=0.001). In addition, there was a positive correlation between the percentage of CD123+CXCL16+ cells and serum levels of IFN-α in Behcet's disease group (p=0.034). We could not find any significant difference in other comparisons. CONCLUSIONS We suggested that the expression of transmembrane CXCL16 on surface of plasmacytoid dendritic cell might contribute to high serum IFN-α levels seen in patients with BD.

Abstract: Type I interferons play a key role in systemic lupus erythematosus (SLE) pathogenesis as an "IFN signature" is found in the majority of patients with active SLE. Immune complexes are internalized by plasmacytoid dendritic cells (DC) via Fc-[gamma] ReceptorIIA, reach the endosomal compartment and activate IFN-[alpha] secretion through TLR7/9-dependent pathways. Naturally occurring differences in expression of the TLR7/9 gene as well as factors that modulate TLR7/9 expression, including CD154 could therefore contribute to SLE pathogenesis. Although its origin is not elucidated CD154 is hyperexpressed in SLE patients, and is important for the differentiation of autoantibody-secreting cells. We hypothesized that platelets which are an abundant source of CD154, and which can mediate proinflammatory effects could be an actor involved in SLE pathogenesis. Platelets from SLE patients are activated in vivo by circulating immune complexes which are abundant in SLE sera, via a CD32-dependent mechanism. Activated platelets formed aggregates with antigen-presenting cells in SLE patients and enhanced interferon-[alpha] secretion induced by immune-complexes stimulated plasmacytoid DCs. Finally, in vivo depletion of platelets and megakaryocytes in NZBxNZW(F1) lupus prone mice improved all parameters assessing disease activity, whereas transfusion of activated platelets worsened the disease course. Altogether, these data identify platelets as a mediator of SLE pathogenesis and a new therapeutical target.

Abstract: In HIV-1 infection, plasmacytoid dendritic cell (PDC) numbers and function are decreased. No detailed comparisons of PDC responses to various stimuli in HIV-1-infected patients are available. Using for the first time purified PDCs, we compared PDC responses [interferon (IFN)-α production/cell] to various stimuli in a large number (n=48) of HIV-1-infected patients and healthy volunteers (n=19). Toll-like receptor (TLR)7- and TLR9-induced expression of PDC surface activation and maturation markers was also compared in the two populations. We have confirmed that PDC number coincides with CD4(+) T cell counts and clinical state. Notably, we have shown that a direct association of PDC function in terms of IFN-α production/cell exists with PDC numbers and CD4(+) cell counts when PDCs are exposed to a TLR9 ligand and HIV-infected cells, but not with a TLR7 ligand. Moreover, in the HIV-infected subjects but not the healthy controls, the magnitude of IFN-α release per PDC in response to the TLR7 ligand is significantly (p<0.01) lower than that to the TLR9 ligand. However, in both study populations, the TLR7 stimulation in comparison to TLR9 stimulation induced higher expression of PDC surface activation and maturation markers and significantly (p<0.05) decreased the expression of BDCA-2, a negative regulator of interferon. Furthermore, the cross-ligation of BDCA-2 significantly (p<0.05) inhibited TLR9- but not TLR7-induced IFN-α production by PDCs from both clinical groups. These findings suggest that differences exist in TLR7- and TLR9-induced IFN-α production by PDCs in HIV-infected individuals that are not directly related to BDCA-2 down-modulation.

Abstract: To improve the secretion and expression of human interferon alpha 2b (IFN) in Lactococcus lactis, a synthetic pro-peptide, LEISSTCDA (LEISS), was fused to the N-terminus of IFN. This gave a higher secretion efficiency (12% vs. 5%) and yield (approximately 2.8-fold) of IFN. The signal peptide, SP(SlpA) (SlpA, an S-layer protein of Lactobacillus brevis), was also tested to secrete IFN instead of SP(Usp45) (Usp45, the main secreted protein in L. lactis). This gave increased IFN secretion (approximately 3-fold) but lower total production. All the recombinant IFN had appropriate bioactivities in an antiviral assay.