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Inhibition of M1 macrophage activation in favour of M2 differentiation by liposomal targeting of glucocorticoids to the synovial lining during experimental arthritis
  1. Wouter Hofkens1,
  2. Gert Storm2,
  3. Wim van den Berg1,
  4. Peter van Lent1
  1. 1Rheumatology Research and Advanced Therapeutics, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands
  2. 2Department of Pharmaceutics, Utrecht University, Utrecht, The Netherlands

Abstract

Background and objective Experimental antigen-induced arthritis (AIA) is highly driven by pro-inflammatory ‘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-inflammatory ‘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 inflammation 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 significantly 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, significant downregulation of M1 markers IL-1β, IL-6 and IL-12p40 (17-, 9- and 36-fold) was found without significant 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.

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