BackgroundTo 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 MethodsExperimental 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.ResultsMicroarray 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.ConclusionThis 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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Background and objective Experimental antigen-induced arthritis (AIA) is highly driven by pro-infl ammatory 'M1' macrophages which express cytokines such as tumour necrosis factor α (TNFα), interleukin 1β (IL-1β), IL-6 and IL-12. However, macrophages can also be differentiated towards an anti-infl ammatory 'M2' phenotype in vitro with glucocorticoids, thereby expressing IL-10 and membrane markers IL-1 receptor type II (IL-1RII) and CD163. The authors now explored whether liposomal targeting of synovial lining macrophages with glucocorticoids shifts the M1/M2 macrophage balance. Materials and methods Mice with established AIA were treated with a single intravenous injection of 10 mg/kg liposomal prednisolone phosphate (Lip-PLP) or phosphate-buffered saline (PBS). Naïve mice were injected intra-articularly with interferon γ (IFNγ)/LPS to induce M1 phenotype and 24 h thereafter with Lip-PLP or PBS. Mice were killed at day 1 after Lip-PLP treatment and whole knee joints were isolated for histology and synovial biopsies were isolated for RNA analysis. In vitro, murine bone marrow macrophages were differentiated towards M1 phenotype with LPS and IFNγ, treated with Lip-PLP and M1/M2 markers characterised by quantitative RT-PCR. Results Lip-PLP strongly suppressed joint infl ammation within 1 day from control treated mice during AIA. Liposomes were primarily targeted to the synovial lining macrophages as demonstrated with gold-laden liposomes. One day after treatment, synovial gene expression was strongly downregulated for the M1 cytokines TNFα IL-1β (10.6-fold), IL-6 (5.6-fold) and IL-12p40 (3-, 11-, 6-and 6-fold respectively) by Lip-PLP compared to PBS treated mice. Expression of M2 markers was only slightly downregulated (IL-10: twofold and IL-1RII: onefold) or even upregulated (CD163: twofold). To evaluate whether Lip-PLP had a direct effect on M1 macrophages, these cells were induced in vitro by IFNγ and LPS and subsequently incubated with Lip-PLP for 24 h. Lip-PLP signifi cantly downregulated M1 markers IL-1β, IL-6 and IL-12p40 (40-, 11-and 5-fold) and increased expression of IL-1RII, CD163 and IL-10 (34-, 11-and 5-fold). Next, the synovial lining was activated in vivo towards M1 by local injection of IFNγ/LPS into normal knee joint, followed by local Lip-PLP treatment 24 h thereafter. Strikingly, signifi cant downregulation of M1 markers IL-1β, IL-6 and IL-12p40 (17-, 9-and 36-fold) was found without signifi cant change in M2 markers IL-1RII, CD163 and IL-10 (one-, one-and twofold change), providing cogent evidence for selective skewing of M1 macrophages in favour of the M2 phenotype. Conclusion Systemic delivery of liposomal glucocorticoids during experimental arthritis inhibits M1 macrophages in favour of M2 phenotype in the lining layer and may drive their strong suppressive effect.on 12 May 2018 by guest. Protected by copyright.

Objective. Scavenger receptor class A type I (SR-AI) and SR-AII are expressed by macrophages in particular and bind and internalize a broad range of molecules (including endotoxins, apoptotic bodies, and oxidized low-density lipoprotein). This study was undertaken to investigate the role of SR-AI/II in mediating severe cartilage destruction in antigen-induced arthritis (AIA).Methods. AIA was induced in the knee joints of SR-AI/II ؊/؊ mice and wild-type (WT) controls. Joint inflammation and cartilage destruction (chondrocyte death) were measured by examining the histology of total knee joints. Matrix metalloproteinase (MMP)-mediated neoepitopes were measured by immunolocalization using anti-VDIPEN antibodies and chondrocyte activation with anti-S100A8 antibodies. Messenger RNA (mRNA) levels were determined in inflamed synovium using microarray analysis and quantitative reverse transcriptase-polymerase chain reaction. In synovial washouts, cytokines (interleukin-1␤ [IL-1␤], IL-10, and tumor necrosis factor ␣) and S100A8/S100A9 were measured using Luminex and enzyme-linked immunosorbent assay.Results. Levels of SR-AI/II mRNA were strongly elevated in inflamed synovium in AIA. On days 2, 8, and 14 after AIA induction, joint inflammation (exudates/ infiltrate) was similar between the 2 groups. In WT mice, severe cartilage destruction was found in multiple cartilage surfaces of the inflamed knee joint on day 14 after AIA induction. MMP-mediated matrix destruction ranged between 40% and 60%, and chondrocyte death was prominent in 40-75% of the cartilage surfaces. In striking contrast, in SR-AI/II ؊/؊ mice, despite comparable joint inflammation, pronounced cartilage destruction was almost completely absent. Levels of IL-1␤ and S100A8/S100A9 were significantly lower on days 7 and 14 after AIA induction, but levels of mRNA for various MMPs (MMP-2, MMP-3, MMP-9, and MMP-13) were comparable.Conclusion. Our findings indicate that SR-AI and SR-AII are crucial receptors involved in mediating severe cartilage destruction in AIA.

Objective: To investigate whether macrophages in the synovial lining can be selectively eliminated by local administration of an improved boron-10 ( 10 B) containing liposome formulation combined with neutron irradiation (boron neutron capture synovectomy [BNCS]). Methods: Disodium dodecahydrododecaborate (Na 2 10 B 12 H 12 ) was encapsulated into unilamellar liposomes ( 10 Bliposomes). 10 B-liposomes were injected into the mouse knee joint. Amounts of 10 B in synovial tissue were measured over time using inductively coupled plasma-optical emission spectrometry (ICP-OES). Arthritis was induced in knee joints of mice. Joint inflammation and cartilage destruction was measured using histology. Results: When a 10 ml 10 B-liposome solution (containing 40 mg 10 B) was injected into the murine knee joint, high concentrations of 10 B were measured in macrophages in the synovial lining (At 24 h 306+226 mg . g 71 macrophages). Completing the BNCS by neutron irradiation of the legs 24 h after 10 B-liposome injection showed a clear selective depletion of macrophages in synovial lining of the knee joints. An estimated total physical dose of 13+9 Gy was given to the macrophages. When arthritis was induced in the macrophage-depleted joints, swelling of the knee was significantly lower as compared to the controls (53% and 79% lower at days 1 and 3, respectively). Histology confirmed the influx of inflammatory cells was strongly decreased and severe cartilage destruction was almost completely prevented. Conclusion: BNCS using an improved 10 B-containing liposome formulation can cause selective depletion of macrophages in the synovial lining of murine knee joints. As a result of this proof-of principle, future applications are recommended.