Ulated, even at day 5 after treatment (withPLP Liposomes SC1 custom synthesis Inhibit M1 Macrophage ActivationFigure 3. Effect of Pentagastrin Lip-PLP on M1 macrophages in vitro. Cells and supernatant of bone-marrow macrophages (BMM) and M1 macrophages (stimulated with LPS and IFN-c for 24 hours) were obtained 24 hours after treatment with Lip-PLP or saline. A: Uptake of fluorescent liposomes by BMM and M1 macrophages as measured by flow cytometry. Note that uptake of liposomes is dependent on the amount of liposomes but not on PLP content. B: Protein levels of M1 cytokines TNF-a, IL-6 and IL-12 within the supernatant and surface expression of M1 marker CD86 as determined by flow cytometry. C: Gene expression of M2 markers. RE = Relative Expression compared to values of GAPDH. Data are expressed as mean +/2 S.D. UD = undetectable. Three independent experiments were performed. Statistical significance was determined by Student’s t-test. * = P,0.05, ** = P,0.01 compared to saline treatment. doi:10.1371/journal.pone.0054016.gthe exception of TGF-b), which does not point to a shift in phenotype from M1 to M2 after treatment with Lip-PLP (Fig. 5B).Systemic delivery of PLP-liposomes suppresses M1 activation during locally induced immune-complex arthritis (ICA)As antigen-induced arthritis is largely driven by T- and B-cells, intravenous delivery of PLP-liposomes may alter systemic immunity, which contributes to the rapid 26001275 and strong effects on joint inflammation. In order to investigate the direct effect of Lip-PLP accumulation in the lining on the joint 24272870 inflammation in more detail, we finally tested a locally induced immune-complex arthritis which is not dependent on T- or B-cell immunity. This arthritismodel is largely driven by macrophages in the knee joint in response to local application of antibody-complexes in the joint [19]. Similar to the AIA, systemic delivery of Lip-PLP inhibited synovial infiltration at day 1 after injection (Fig. 6A) and significantly suppressed M1 factors TNF-a (30-fold), IL-1b (230fold), IL-6 (116-fold), IL-12p40 (not detected anymore), FccRI (32fold), Ciita (18-fold) and CD86 (7-fold) (Fig. 6B). Treatment with Lip-PLP even suppressed M2 factors and only CD163 expression was somewhat upregulated by Lip-PLP (4-fold), suggesting that Lip-PLP inhibits joint inflammation in ICA largely through suppression of M1 macrophages.PLP Liposomes Inhibit M1 Macrophage ActivationFigure 4. Effect of Lip-PLP on M1 and M2 marker expression within the synovium after local M1 activation. Macrophages of the synovial lining in the knee joints were activated towards M1 by intra-articular injection of LPS and IFN-c for 24 hours and were subsequently treated by intra-articular injection of Lip-PLP or saline for 24 hours. A: Frontal knee joint sections of mice after local M1 activation and subsequent treatment ?with Lip-PLP or saline and naive mice. B+C: Expression of M1 (B) and M2 (C) markers in the synovium. RE = Relative Expression compared to values of GAPDH. Values represent the mean +/2 SD of eight mice. UD = undetectable. Statistical significance was determined by Student’s t-test. * = P,0.05, ** = P,0.01 compared to saline treatment. doi:10.1371/journal.pone.0054016.gPLP Liposomes Inhibit M1 Macrophage ActivationDiscussionIn earlier studies we found that a single systemic injection of PLP-containing liposomes very efficiently downregulated joint inflammation and destruction during AIA. In the present study we studied the mechanism of this treatment on macrophage p.Ulated, even at day 5 after treatment (withPLP Liposomes Inhibit M1 Macrophage ActivationFigure 3. Effect of Lip-PLP on M1 macrophages in vitro. Cells and supernatant of bone-marrow macrophages (BMM) and M1 macrophages (stimulated with LPS and IFN-c for 24 hours) were obtained 24 hours after treatment with Lip-PLP or saline. A: Uptake of fluorescent liposomes by BMM and M1 macrophages as measured by flow cytometry. Note that uptake of liposomes is dependent on the amount of liposomes but not on PLP content. B: Protein levels of M1 cytokines TNF-a, IL-6 and IL-12 within the supernatant and surface expression of M1 marker CD86 as determined by flow cytometry. C: Gene expression of M2 markers. RE = Relative Expression compared to values of GAPDH. Data are expressed as mean +/2 S.D. UD = undetectable. Three independent experiments were performed. Statistical significance was determined by Student’s t-test. * = P,0.05, ** = P,0.01 compared to saline treatment. doi:10.1371/journal.pone.0054016.gthe exception of TGF-b), which does not point to a shift in phenotype from M1 to M2 after treatment with Lip-PLP (Fig. 5B).Systemic delivery of PLP-liposomes suppresses M1 activation during locally induced immune-complex arthritis (ICA)As antigen-induced arthritis is largely driven by T- and B-cells, intravenous delivery of PLP-liposomes may alter systemic immunity, which contributes to the rapid 26001275 and strong effects on joint inflammation. In order to investigate the direct effect of Lip-PLP accumulation in the lining on the joint 24272870 inflammation in more detail, we finally tested a locally induced immune-complex arthritis which is not dependent on T- or B-cell immunity. This arthritismodel is largely driven by macrophages in the knee joint in response to local application of antibody-complexes in the joint [19]. Similar to the AIA, systemic delivery of Lip-PLP inhibited synovial infiltration at day 1 after injection (Fig. 6A) and significantly suppressed M1 factors TNF-a (30-fold), IL-1b (230fold), IL-6 (116-fold), IL-12p40 (not detected anymore), FccRI (32fold), Ciita (18-fold) and CD86 (7-fold) (Fig. 6B). Treatment with Lip-PLP even suppressed M2 factors and only CD163 expression was somewhat upregulated by Lip-PLP (4-fold), suggesting that Lip-PLP inhibits joint inflammation in ICA largely through suppression of M1 macrophages.PLP Liposomes Inhibit M1 Macrophage ActivationFigure 4. Effect of Lip-PLP on M1 and M2 marker expression within the synovium after local M1 activation. Macrophages of the synovial lining in the knee joints were activated towards M1 by intra-articular injection of LPS and IFN-c for 24 hours and were subsequently treated by intra-articular injection of Lip-PLP or saline for 24 hours. A: Frontal knee joint sections of mice after local M1 activation and subsequent treatment ?with Lip-PLP or saline and naive mice. B+C: Expression of M1 (B) and M2 (C) markers in the synovium. RE = Relative Expression compared to values of GAPDH. Values represent the mean +/2 SD of eight mice. UD = undetectable. Statistical significance was determined by Student’s t-test. * = P,0.05, ** = P,0.01 compared to saline treatment. doi:10.1371/journal.pone.0054016.gPLP Liposomes Inhibit M1 Macrophage ActivationDiscussionIn earlier studies we found that a single systemic injection of PLP-containing liposomes very efficiently downregulated joint inflammation and destruction during AIA. In the present study we studied the mechanism of this treatment on macrophage p.
