Med EPZ-5676 biological activity synovial lining strongly suppresses joint inflammation during AIA. A: Knee joint swelling as measured by 99MTc-uptake is strongly suppressed after a single injection of Lip-PLP. B: Photomicrographs of frontal knee joint sections of ?mice with AIA at day 5 after treatment and naive mice. Note that the inflammatory infiltrate is reduced in mice treated with Lip-PLP. Original magnification 6100, Asterisks points to synovial infiltrate, hash sign points to inflammatory exudates. C: Histological scoring of synovial infiltration at day 1 and day 5 after systemic treatment with Lip-PLP or saline. D: Silver staining of frontal knee joint sections of mice with AIA, treated by intravenous injection with gold-containing liposomes. Note that the silver staining of the gold particles is mostly observed within the synovial lining cells (arrows). Mice were treated at day 3 after induction of AIA. Values are the mean of 8 mice per group. Original magnification 6100; insert 6400. F = femur, JS = joint space. Statistical significance was determined by Student’s t-test. * = P,0.05 compared to saline treatment. doi:10.1371/journal.pone.0054016.gGlucocorticoid liposomes suppress M1 activation but do not induce polarization of synovial lining macrophages towards MNext, we investigated whether intra-articular injection of LipPLP was able to alter M1 into an M2 signature in the synovial lining of the murine knee joint. We first induced an M1 signature in the lining macrophages by injection of LPS (1 mg) and IFN-c (100 ng). At 24 hours thereafter, no significant cellular infiltrate of the synovium was found (Fig. 4A). However, synovial biopsies which included the intima layer showed high expression of mRNA levels of M1 type cytokines TNF-a, IL-1b and IL-6 (13-, 16- and 16-fold, respectively) and of M1 markers iNOS, Ciita and CD86 ?(10-, 8- and 12-fold, respectively) when compared to naive synovium (Fig. 4B). Injection of Lip-PLP (50 mg) into the M1 knee joint strongly suppressed all the upregulated M1 type genes to ?levels not significantly Desoxyepothilone B different from those in naive mice when measured at 24 hours thereafter (with the exception of iNOS). In contrast, expression of M2 markers IL-10, IL-1RII, CD163, CD206 and FIZZ1 was hardly changed by Lip-PLP treatment (with the exception of Arg1) (Fig. 4B). These results suggest thatlocal injection of Lip-PLP inhibits M1 macrophages but does not induce polarization towards M2 macrophages.Systemic delivery of Lip-PLP during antigen induced arthritis suppresses the M1 synovial macrophage without altering the M2 phenotype within the inflamed synoviumTo determine whether the M1 phenotype is suppressed in favor of M2 by systemic treatment with PLP-liposomes during experimental arthritis, we measured gene expression of various M1 and M2 markers in the synovium at day 1 and day 5 after systemic treatment with Lip-PLP of established AIA (day 3). Treatment with PLP-liposomes resulted in a rapid and strong down regulation of mRNA levels of M1 type cytokines TNF-a (8fold), IL-1b (55-fold), IL-6 (94-fold) and IL-12 (levels not detected) at day 5 after treatment in the synovium (Fig. 5A). Additional genes reflecting M1 activation like FccRI, Ciita and CD86, were also significantly suppressed at day 5 after treatment (9-, 10- and 6fold, respectively), indicating a silencing of the M1 pattern by LipPLP (Fig. 5B). In contrast to M1, expression of M2 markers IL-10, IL-1RII, CD206, Arg1, CD163, FIZZ1 and Ym1 was not significantly downreg.Med synovial lining strongly suppresses joint inflammation during AIA. A: Knee joint swelling as measured by 99MTc-uptake is strongly suppressed after a single injection of Lip-PLP. B: Photomicrographs of frontal knee joint sections of ?mice with AIA at day 5 after treatment and naive mice. Note that the inflammatory infiltrate is reduced in mice treated with Lip-PLP. Original magnification 6100, Asterisks points to synovial infiltrate, hash sign points to inflammatory exudates. C: Histological scoring of synovial infiltration at day 1 and day 5 after systemic treatment with Lip-PLP or saline. D: Silver staining of frontal knee joint sections of mice with AIA, treated by intravenous injection with gold-containing liposomes. Note that the silver staining of the gold particles is mostly observed within the synovial lining cells (arrows). Mice were treated at day 3 after induction of AIA. Values are the mean of 8 mice per group. Original magnification 6100; insert 6400. F = femur, JS = joint space. Statistical significance was determined by Student’s t-test. * = P,0.05 compared to saline treatment. doi:10.1371/journal.pone.0054016.gGlucocorticoid liposomes suppress M1 activation but do not induce polarization of synovial lining macrophages towards MNext, we investigated whether intra-articular injection of LipPLP was able to alter M1 into an M2 signature in the synovial lining of the murine knee joint. We first induced an M1 signature in the lining macrophages by injection of LPS (1 mg) and IFN-c (100 ng). At 24 hours thereafter, no significant cellular infiltrate of the synovium was found (Fig. 4A). However, synovial biopsies which included the intima layer showed high expression of mRNA levels of M1 type cytokines TNF-a, IL-1b and IL-6 (13-, 16- and 16-fold, respectively) and of M1 markers iNOS, Ciita and CD86 ?(10-, 8- and 12-fold, respectively) when compared to naive synovium (Fig. 4B). Injection of Lip-PLP (50 mg) into the M1 knee joint strongly suppressed all the upregulated M1 type genes to ?levels not significantly different from those in naive mice when measured at 24 hours thereafter (with the exception of iNOS). In contrast, expression of M2 markers IL-10, IL-1RII, CD163, CD206 and FIZZ1 was hardly changed by Lip-PLP treatment (with the exception of Arg1) (Fig. 4B). These results suggest thatlocal injection of Lip-PLP inhibits M1 macrophages but does not induce polarization towards M2 macrophages.Systemic delivery of Lip-PLP during antigen induced arthritis suppresses the M1 synovial macrophage without altering the M2 phenotype within the inflamed synoviumTo determine whether the M1 phenotype is suppressed in favor of M2 by systemic treatment with PLP-liposomes during experimental arthritis, we measured gene expression of various M1 and M2 markers in the synovium at day 1 and day 5 after systemic treatment with Lip-PLP of established AIA (day 3). Treatment with PLP-liposomes resulted in a rapid and strong down regulation of mRNA levels of M1 type cytokines TNF-a (8fold), IL-1b (55-fold), IL-6 (94-fold) and IL-12 (levels not detected) at day 5 after treatment in the synovium (Fig. 5A). Additional genes reflecting M1 activation like FccRI, Ciita and CD86, were also significantly suppressed at day 5 after treatment (9-, 10- and 6fold, respectively), indicating a silencing of the M1 pattern by LipPLP (Fig. 5B). In contrast to M1, expression of M2 markers IL-10, IL-1RII, CD206, Arg1, CD163, FIZZ1 and Ym1 was not significantly downreg.
