LAUSR

Background Functional heterogeneity is a hallmark of macrophages, which can classified into 2 major phenotypes with opposite role in inflammation termed M1 (inflammatory or classically activated) macrophages and M2 (alternatively activated) macrophages. In addition, M1-M2 polarisation of macrophages is a highly dynamic process and the phenotype of polarised macrophages can be switched under physiological and pathological conditions. Progranulin (PGRN), a multiple functional growth factor, binds to TNF receptor 2 (TNFR2) and activates the protective and anti-inflammatory pathway in inflammatory arthritis. In addition, 14–3–3e was identified as a component of PGRN/TNFR2 complexes in Raw264.7 macrophages. Objectives In this study, we examined whether 14–3–3e regulated macrophage polarisation and if so, whether this was important for PGRN’s anti-inflammatory action in inflammatory arthritis. Methods LysMCre and14–3–3ε F/F mouse line was obtained from Jackson Laboratory. Results : 14–3–3e regulates macrophage polarisationin vitro . We found that 14–3–3e deficiency enhanced M1 while inhibited M2 polarisation (figure 1a, b). Interestingly, PGRN showed reverse effects on macrophage polarisation. In addition, PGRN’s effects were largely lost in 14–3–3e deficient BMDMs (figure 1a, b). Together, these data indicate that 14–3–3e is a critical downstream mediator of PGRN regulation of macrophage polarisation. Macrophage-specific 14–3–3e contributes to control of inflammatory arthritis and is critical for PGRN’s anti-inflammatory action. We then explored the role of macrophage-specific 14–3–3e in inflammatory arthritis and whether PGRN’s anti-inflammatory activity depended on 14–3–3e in vivo. We established CIA in 14–3–3eF/F (serve as WT) and 14–3–3eΔ/Δ mice, followed by i.p. injection of recombinant PGRN. The clinical arthritis score demonstrated that 14–3–3eΔ/Δ mice displayed increased severity of CIA compared with WT CIA. In addition, PGRN’s protective effects against inflammatory arthritis was compromised in 14–3–3eΔ/Δ mice (figure 1c), suggesting that 14–3–3e is critical downstream mediator of PGRN’s anti-inflammatory effects. In addition, FACS analysis showed that total numbers of F4/80 cells were not different in all WT and knockout CIA mice. However, analysis of CD45 +CD11b+cell population in spleen demonstrated a significant increase in mean fluorescence intensity (MFI) of iNOS and a significant decrease of CD206 +cells in PBS treated 14–3–3eΔ/Δ CIA mice as compared with PBS treated WT CIA mice; moreover, there was a significant decrease of iNOS MFI while a dramatic increase of CD206 +cells in PGRN-treated WT mice compared to that in PBS-treated mice. Further PGRN-mediated effects on macrophage polarisation was lost in 14–3–3eΔ/Δ CIA mice (Fig 1d, e). Cellectively, these results indicate that PGRN skews macrophage toward M2 polarisation to resolve inflammation and this effect depends on 14–3–3ε.Abstract THU0056 – Figure 1 14–3–3ε is a critical mediator of PGRN’ regulation of macrophage polarisation and contributes to PGRN’s anti-inflammation action. (a, b) qPCR analysis of Il1b and Nos2 (a), or Arg1 and Mgl1 (b) mRNA expression in WT, or 14–3–3ε-/- macrophages which are polarised to M1 (a) or M2 (b) in the the absence or presence of PGRN (200ng/ml). (c) Clinical arthritis score of WT or 14–3–3εΔ/Δ CIA mice treated with or without PGRN. n=8 (d, e) CD45+CD11b+ cells were analysed for MFL of iNOS (d) and percentage of CD206+ cells (e). * p<0.05, ** p<0.01, NS=no significance Conclusions Both in vitro and in vivo results indicate that 14–3–3ε is a key molecule regulating macrophage polarisation which plays an important role in inflammatory arthritis, and it is an essential component for PGRN/TNFR2 mediated protective effect against inflammatory arthritis. Disclosure of Interest None declared