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Liposomal targeting of prednisolone phosphate to synovial lining macrophages during experimental arthritis inhibits M1 activation but does not favor M2 differentiation.

Hofkens W, Schelbergen R, Storm G, van den Berg WB, van Lent PL - PLoS ONE (2013)

Bottom Line: In vitro studies showed that Lip-PLP phagocytosed by M1 macrophages resulted in a suppression of the M1 phenotype and induction of M2 markers (IL-10, TGF-β, IL-1RII, CD163, CD206 and Ym1).This study clearly shows that systemic treatment with PLP-liposomes selectively targets synovial lining macrophages and inhibits M1 activation.In contrast to in vitro findings, PLP-liposomes do not cause a shift of synovial lining macrophages towards M2.

View Article: PubMed Central - PubMed

Affiliation: Rheumatology Research and Advanced Therapeutics, Department of Rheumatology, Radboud University Medical Centre, Nijmegen, The Netherlands.

ABSTRACT

Background: To determine the effects of liposomal targeting of prednisolone phosphate (Lip-PLP) to synovial lining macrophages on M1 and M2 polarization in vitro and during experimental arthritis.

Material and methods: Experimental arthritis (antigen and immune complex induced) was elicited in mice and prednisolone containing liposomes were given systemically. Synovium was investigated using microarray analysis, RT-PCR and histology. Bone-marrow macrophages were stimulated towards M1 using LPS and IFNγ before treatment by PLP-liposomes. M1 and M2 markers were determined using RT-PCR.

Results: Microarray analysis of biopsies of inflamed synovium during antigen induced arthritis (AIA) showed an increased M1 signature characterized by upregulation of IL-1β, IL-6 and FcγRI starting from day 1 and lasting up until day 7 after arthritis induction. The M2 signature remained low throughout the 7 day course of arthritis. Treatment of AIA with intravenously delivered Lip-PLP strongly suppressed joint swelling and synovial infiltration whereas colloidal gold containing liposomes exclusively targeted the macrophages within the inflamed synovial intima layer. In vitro studies showed that Lip-PLP phagocytosed by M1 macrophages resulted in a suppression of the M1 phenotype and induction of M2 markers (IL-10, TGF-β, IL-1RII, CD163, CD206 and Ym1). In vivo, Lip-PLP treatment strongly suppressed M1 markers (TNF-α, IL-1β, IL-6, IL-12p40, iNOS, FcγRI, Ciita and CD86) after local M1 activation of lining macrophages with LPS and IFN-γ and during experimental AIA and immune complex arthritis (ICA). In contrast, M2 markers were not significantly upregulated in antigen-induced arthritis and down regulated in immune complex arthritis.

Conclusion: This study clearly shows that systemic treatment with PLP-liposomes selectively targets synovial lining macrophages and inhibits M1 activation. In contrast to in vitro findings, PLP-liposomes do not cause a shift of synovial lining macrophages towards M2.

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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-γ 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 naïve 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 +/− 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.
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pone-0054016-g004: 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-γ 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 naïve 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 +/− 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.

Mentions: Next, we investigated whether intra-articular injection of Lip-PLP 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 µg) and IFN-γ (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-α, IL-1β 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 naïve synovium (Fig. 4B). Injection of Lip-PLP (50 µg) into the M1 knee joint strongly suppressed all the upregulated M1 type genes to levels not significantly different from those in naïve 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 that local injection of Lip-PLP inhibits M1 macrophages but does not induce polarization towards M2 macrophages.


Liposomal targeting of prednisolone phosphate to synovial lining macrophages during experimental arthritis inhibits M1 activation but does not favor M2 differentiation.

Hofkens W, Schelbergen R, Storm G, van den Berg WB, van Lent PL - PLoS ONE (2013)

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-γ 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 naïve 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 +/− 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.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3585322&req=5

pone-0054016-g004: 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-γ 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 naïve 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 +/− 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.
Mentions: Next, we investigated whether intra-articular injection of Lip-PLP 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 µg) and IFN-γ (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-α, IL-1β 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 naïve synovium (Fig. 4B). Injection of Lip-PLP (50 µg) into the M1 knee joint strongly suppressed all the upregulated M1 type genes to levels not significantly different from those in naïve 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 that local injection of Lip-PLP inhibits M1 macrophages but does not induce polarization towards M2 macrophages.

Bottom Line: In vitro studies showed that Lip-PLP phagocytosed by M1 macrophages resulted in a suppression of the M1 phenotype and induction of M2 markers (IL-10, TGF-β, IL-1RII, CD163, CD206 and Ym1).This study clearly shows that systemic treatment with PLP-liposomes selectively targets synovial lining macrophages and inhibits M1 activation.In contrast to in vitro findings, PLP-liposomes do not cause a shift of synovial lining macrophages towards M2.

View Article: PubMed Central - PubMed

Affiliation: Rheumatology Research and Advanced Therapeutics, Department of Rheumatology, Radboud University Medical Centre, Nijmegen, The Netherlands.

ABSTRACT

Background: To determine the effects of liposomal targeting of prednisolone phosphate (Lip-PLP) to synovial lining macrophages on M1 and M2 polarization in vitro and during experimental arthritis.

Material and methods: Experimental arthritis (antigen and immune complex induced) was elicited in mice and prednisolone containing liposomes were given systemically. Synovium was investigated using microarray analysis, RT-PCR and histology. Bone-marrow macrophages were stimulated towards M1 using LPS and IFNγ before treatment by PLP-liposomes. M1 and M2 markers were determined using RT-PCR.

Results: Microarray analysis of biopsies of inflamed synovium during antigen induced arthritis (AIA) showed an increased M1 signature characterized by upregulation of IL-1β, IL-6 and FcγRI starting from day 1 and lasting up until day 7 after arthritis induction. The M2 signature remained low throughout the 7 day course of arthritis. Treatment of AIA with intravenously delivered Lip-PLP strongly suppressed joint swelling and synovial infiltration whereas colloidal gold containing liposomes exclusively targeted the macrophages within the inflamed synovial intima layer. In vitro studies showed that Lip-PLP phagocytosed by M1 macrophages resulted in a suppression of the M1 phenotype and induction of M2 markers (IL-10, TGF-β, IL-1RII, CD163, CD206 and Ym1). In vivo, Lip-PLP treatment strongly suppressed M1 markers (TNF-α, IL-1β, IL-6, IL-12p40, iNOS, FcγRI, Ciita and CD86) after local M1 activation of lining macrophages with LPS and IFN-γ and during experimental AIA and immune complex arthritis (ICA). In contrast, M2 markers were not significantly upregulated in antigen-induced arthritis and down regulated in immune complex arthritis.

Conclusion: This study clearly shows that systemic treatment with PLP-liposomes selectively targets synovial lining macrophages and inhibits M1 activation. In contrast to in vitro findings, PLP-liposomes do not cause a shift of synovial lining macrophages towards M2.

Show MeSH
Related in: MedlinePlus