Of the experiment, showed a significant increase in the amount of IL-10 per cell compared to control mice, measured as mean fluorescence intensity (MFI), though there was no difference in the number of IL-10-producing cells (data not shown). Gating on different cell populations demonstrated that IL-10 was in particular produced by B cells, and by non-B antigen presenting cells (APC) (Figure 2 B, C and 2F). The proportion and expression (MFI) of IL-10 in B cells and non-B cell APCs in spleen were similar between theDisease-Dependent IL-10 Ameliorates CIAFigure 1. Lentiviral gene constructs and clinical development of arthritis. (A) Lentiviral constructs: LNT-GFP and LNT-IL-10. LTR; long terminal repeat, cPPT; central polypurine tract, pA; polyadenylic acid tail, WPRE; Woodchuck post-transcriptional regulatory element, IL-1E; Interleukin-1 enhancer, IL-6 promoter. (B) Severity of arthritis (mean arthritis score 6 SEM). LNT-GFP (day 0?2 n = 18, day 44?9 n = 10) and LNT-IL-10 (day 0?2 n = 25, day 44?9 n = 14)). (C) Histopathological severity of synovitis and cartilage and bone erosivity measured as histological severity score (Y-axis) ranging from 0?. Data in figure 1B and C were analysed by Mann-Whitney U-test. Closed circles represents LNT-GFP and open circles LNT-IL-10 mice. Bars in 1C represent the median. doi:10.1371/journal.pone.0049731.gDisease-Dependent IL-10 Ameliorates CIAFigure 2. Levels of IL-10 mRNA, intracellular IL-10 production and SOCS expression (A). Levels of IL-10 mRNA expression in lymph nodes at day 42 in LNT-GFP or LNT-IL-10 mice. (B) The amount of IL-10/cell measured as geometric mean flourescent intensity (MFI) in lymph node CD19+MHC II+B cells, (C) in lymph node CD19-MHC II+non-B APCs (D) in splenic B cells, (E) in splenic non-B APCs. (F) Typical gating for intracellular cytokine staining showing one sample from an LNT-GFP mouse and an LNT-IL-10 mouse (G) Levels of mRNA SOCS1 and 3 expression in draining lymph nodes at day 42. 1676428 In figure 2A and G data were analysed by Mann-Whitney U-test. Closed circles represents LNT-GFP and open circles LNT-IL10 mice. doi:10.1371/journal.pone.0049731.gDiscussionOur report shows that increased local, 24272870 but not systemic, levels of IL-10 conferred by disease-driven gene therapy delays the progression of CIA in mice. A buy GNF-7 precise and restricted increase in IL-10, produced by B cells and other APCs, ameliorates the course and severity of arthritis. Based on our data, a possible scenario would be that the increase in IL-10 upregulates SOCS1 resulting in a decrease in serum levels of IL-6. This in turn results in a decrease in both frequency and number of B cells and anti-CII antibody levels, accompanied by reduced severity of arthritis. IL-10 is a potent pleiotropic cytokine that is produced e.g. by monocytes, macrophages, T and B cells. This cytokine has the capacity to inhibit synthesis of pro-inflammatory 194423-15-9 web cytokines such as IL-2, IFN-c, TNF-a and importantly IL-6 [4]. It has earlier been shown that systemically increased IL-10 levels suppresses the frequency and severity of CIA [17,18,19,20,21]. The inflammation-dependent IL-1/IL-6 promoter has low basal activity, which significantly increases during acute inflammatory conditions [13]. We found that this promoter, driving the IL-10 gene expression, does not induce increased systemic (serum) levels of IL-10 during the course of arthritis in vivo, but a locally increased IL-expression in lymph nodes; particularly in B cells and other APCs.Of the experiment, showed a significant increase in the amount of IL-10 per cell compared to control mice, measured as mean fluorescence intensity (MFI), though there was no difference in the number of IL-10-producing cells (data not shown). Gating on different cell populations demonstrated that IL-10 was in particular produced by B cells, and by non-B antigen presenting cells (APC) (Figure 2 B, C and 2F). The proportion and expression (MFI) of IL-10 in B cells and non-B cell APCs in spleen were similar between theDisease-Dependent IL-10 Ameliorates CIAFigure 1. Lentiviral gene constructs and clinical development of arthritis. (A) Lentiviral constructs: LNT-GFP and LNT-IL-10. LTR; long terminal repeat, cPPT; central polypurine tract, pA; polyadenylic acid tail, WPRE; Woodchuck post-transcriptional regulatory element, IL-1E; Interleukin-1 enhancer, IL-6 promoter. (B) Severity of arthritis (mean arthritis score 6 SEM). LNT-GFP (day 0?2 n = 18, day 44?9 n = 10) and LNT-IL-10 (day 0?2 n = 25, day 44?9 n = 14)). (C) Histopathological severity of synovitis and cartilage and bone erosivity measured as histological severity score (Y-axis) ranging from 0?. Data in figure 1B and C were analysed by Mann-Whitney U-test. Closed circles represents LNT-GFP and open circles LNT-IL-10 mice. Bars in 1C represent the median. doi:10.1371/journal.pone.0049731.gDisease-Dependent IL-10 Ameliorates CIAFigure 2. Levels of IL-10 mRNA, intracellular IL-10 production and SOCS expression (A). Levels of IL-10 mRNA expression in lymph nodes at day 42 in LNT-GFP or LNT-IL-10 mice. (B) The amount of IL-10/cell measured as geometric mean flourescent intensity (MFI) in lymph node CD19+MHC II+B cells, (C) in lymph node CD19-MHC II+non-B APCs (D) in splenic B cells, (E) in splenic non-B APCs. (F) Typical gating for intracellular cytokine staining showing one sample from an LNT-GFP mouse and an LNT-IL-10 mouse (G) Levels of mRNA SOCS1 and 3 expression in draining lymph nodes at day 42. 1676428 In figure 2A and G data were analysed by Mann-Whitney U-test. Closed circles represents LNT-GFP and open circles LNT-IL10 mice. doi:10.1371/journal.pone.0049731.gDiscussionOur report shows that increased local, 24272870 but not systemic, levels of IL-10 conferred by disease-driven gene therapy delays the progression of CIA in mice. A precise and restricted increase in IL-10, produced by B cells and other APCs, ameliorates the course and severity of arthritis. Based on our data, a possible scenario would be that the increase in IL-10 upregulates SOCS1 resulting in a decrease in serum levels of IL-6. This in turn results in a decrease in both frequency and number of B cells and anti-CII antibody levels, accompanied by reduced severity of arthritis. IL-10 is a potent pleiotropic cytokine that is produced e.g. by monocytes, macrophages, T and B cells. This cytokine has the capacity to inhibit synthesis of pro-inflammatory cytokines such as IL-2, IFN-c, TNF-a and importantly IL-6 [4]. It has earlier been shown that systemically increased IL-10 levels suppresses the frequency and severity of CIA [17,18,19,20,21]. The inflammation-dependent IL-1/IL-6 promoter has low basal activity, which significantly increases during acute inflammatory conditions [13]. We found that this promoter, driving the IL-10 gene expression, does not induce increased systemic (serum) levels of IL-10 during the course of arthritis in vivo, but a locally increased IL-expression in lymph nodes; particularly in B cells and other APCs.
