Ich were especially high at day 1 after induction of AIA (Fig. 1B). PD-1/PD-L1 inhibitor 1 custom synthesis Altogether though, this suggests a shift towards a M1 signature in the inflamed synovium during AIA.Intravenous injection of gold-liposomes targets synovial lining macrophagesTo determine whether the Lip-PLP formulation is directly targeted to macrophages in the synovial intima layer, we injected liposomes containing colloidal gold intravenously into mice with day 3 AIA. Silver enhancement staining of frontal sections of the inflamed knee joint showed that at day 1 after injection, the goldladen liposomes were taken up by macrophages lying within the synovial intima (Fig. 2D) suggesting that these liposomes leave the bloodstream through the vessels lying just beneath the lining layer and then become directly engulfed by the intima macrophages. Type B synovial fibroblasts do not take up liposomes and may thus be less affected [23].Lip-PLP skews M1 macrophages towards an M2 phenotype in vitroTo study the direct effect of Lip-PLP on activated macrophages, we first investigated whether liposomal PLP may alter M1 macrophages into an M2 phenotype in vitro. Bone marrow-derived macrophages (BMMs) were stimulated towards an M1 type using IFN-c (10 ng/ml) and LPS (100 ng/ml) for 24 hours and subsequently treated with Lip-PLP for another 24 hours. Liposomes were directly engulfed by non-stimulated macrophages and M1 macrophages as measured by flow cytometry of fluorescently labeled empty and PLP-liposomes (10, 100 and 500 mg/ml, Fig. 3A). PLP-liposomes did not cause cell death as measured by trypan blue uptake and by counting living cells and flow cytometry of apoptotic cells with 7-AAD staining (data not shown). Cytokine and membrane markers reflecting the polarization status of M1 and M2 were measured by Luminex, flow cytometry and QPCR. Treatment of M1 macrophages with Lip-PLP strongly suppressed protein levels of M1 cytokines TNF-a (100 ), IL-6 (100 ), and IL-12 (91 ) (Fig. 3B). Furthermore, Lip-PLP significantly suppressed the M1 status as represented by surface expression of CD86 by 82 (Fig. 3B). To evaluate whether Lip-PLP treatment skews BMMs and M1 macrophages towards an M2 phenotype, we measured gene expression of various generally accepted M2 markers. In BMMs Lip-PLP treatment strongly upregulated mRNA levels of M2 associated genes IL-10 (7-fold) and CD163 (10-fold) (Fig. 3C). In M1 macrophages Lip-PLP treatment strongly upregulated mRNA levels of M2 associated genes IL-10 (3-fold), TGF-b (3-fold), IL1RII (14-fold), CD163 (undetected in M1 macrophages), CD206 (5-fold) and Ym1 (12-fold) (Fig. 3C), indicating that Lip-PLP is capable of skewing 
both BMMs as well as M1 macrophages towards an M2 phenotype.Figure 1. Expression of M1 and M2 markers within the inflamed synovium during AIA as determined by micro-array analysis. Gene expression was determined at day 1, 3,5 and 7 after induction of AIA. Fold 
increase of gene expression was compared to ?synovium of naive mice. A: Expression of M1 markers (IL-1b, IL-6, FccRI and CD86). B: Expression of M2 markers (IL-1RII, CD163, CD206, Arg1, FIZZ1 and Ym1). Note that expression of M1 markers is highly upregulated compared to M2 markers, with the exception of Arg1 and Ym1. Values are presented as the fold change in mean gene expression levels (relative to GAPDH) from mean gene expression levels ?of inflamed synovium (n = 8) compared to synovium drived from naive mice (n = 3). doi:10.1371/journal.pone.3PO chemical information 0054016.gPLP Liposomes Inhibit.Ich were especially high at day 1 after induction of AIA (Fig. 1B). Altogether though, this suggests a shift towards a M1 signature in the inflamed synovium during AIA.Intravenous injection of gold-liposomes targets synovial lining macrophagesTo determine whether the Lip-PLP formulation is directly targeted to macrophages in the synovial intima layer, we injected liposomes containing colloidal gold intravenously into mice with day 3 AIA. Silver enhancement staining of frontal sections of the inflamed knee joint showed that at day 1 after injection, the goldladen liposomes were taken up by macrophages lying within the synovial intima (Fig. 2D) suggesting that these liposomes leave the bloodstream through the vessels lying just beneath the lining layer and then become directly engulfed by the intima macrophages. Type B synovial fibroblasts do not take up liposomes and may thus be less affected [23].Lip-PLP skews M1 macrophages towards an M2 phenotype in vitroTo study the direct effect of Lip-PLP on activated macrophages, we first investigated whether liposomal PLP may alter M1 macrophages into an M2 phenotype in vitro. Bone marrow-derived macrophages (BMMs) were stimulated towards an M1 type using IFN-c (10 ng/ml) and LPS (100 ng/ml) for 24 hours and subsequently treated with Lip-PLP for another 24 hours. Liposomes were directly engulfed by non-stimulated macrophages and M1 macrophages as measured by flow cytometry of fluorescently labeled empty and PLP-liposomes (10, 100 and 500 mg/ml, Fig. 3A). PLP-liposomes did not cause cell death as measured by trypan blue uptake and by counting living cells and flow cytometry of apoptotic cells with 7-AAD staining (data not shown). Cytokine and membrane markers reflecting the polarization status of M1 and M2 were measured by Luminex, flow cytometry and QPCR. Treatment of M1 macrophages with Lip-PLP strongly suppressed protein levels of M1 cytokines TNF-a (100 ), IL-6 (100 ), and IL-12 (91 ) (Fig. 3B). Furthermore, Lip-PLP significantly suppressed the M1 status as represented by surface expression of CD86 by 82 (Fig. 3B). To evaluate whether Lip-PLP treatment skews BMMs and M1 macrophages towards an M2 phenotype, we measured gene expression of various generally accepted M2 markers. In BMMs Lip-PLP treatment strongly upregulated mRNA levels of M2 associated genes IL-10 (7-fold) and CD163 (10-fold) (Fig. 3C). In M1 macrophages Lip-PLP treatment strongly upregulated mRNA levels of M2 associated genes IL-10 (3-fold), TGF-b (3-fold), IL1RII (14-fold), CD163 (undetected in M1 macrophages), CD206 (5-fold) and Ym1 (12-fold) (Fig. 3C), indicating that Lip-PLP is capable of skewing both BMMs as well as M1 macrophages towards an M2 phenotype.Figure 1. Expression of M1 and M2 markers within the inflamed synovium during AIA as determined by micro-array analysis. Gene expression was determined at day 1, 3,5 and 7 after induction of AIA. Fold increase of gene expression was compared to ?synovium of naive mice. A: Expression of M1 markers (IL-1b, IL-6, FccRI and CD86). B: Expression of M2 markers (IL-1RII, CD163, CD206, Arg1, FIZZ1 and Ym1). Note that expression of M1 markers is highly upregulated compared to M2 markers, with the exception of Arg1 and Ym1. Values are presented as the fold change in mean gene expression levels (relative to GAPDH) from mean gene expression levels ?of inflamed synovium (n = 8) compared to synovium drived from naive mice (n = 3). doi:10.1371/journal.pone.0054016.gPLP Liposomes Inhibit.
