Endocrine Abstracts (2019) 65 OC5.4 | DOI: 10.1530/endoabs.65.OC5.4

Pre-receptor metabolism and signalling of glucocorticoids between macrophages and fibroblasts

Claire Martin1, Chloe Fenton1, Andrew Filer2, Martin Hewison1, Andy Clark2 & Rowan Hardy1,2

1Institute of Metabolism and Systems Research, University of Birmingham, Birmingham, UK; 2Institute of Inflammation and Ageing, University of Birmingham, Birmingham, UK

The glucocorticoid (GC) activating enzyme 11β-HSD1 is potently upregulated within macrophages and synovial fibroblasts of inflamed joints, where it increases therapeutic GC signalling and regulates their anti-inflammatory profiles. Whilst in vitro studies have demonstrated the importance of autocrine signalling in this context, the importance of paracrine signalling remains poorly defined. In this study, we examined the role of 11β-HSD1 in paracrine GC signalling between macrophages and fibroblasts in vitro. Conditioned media (CM) and transwell co-culture experiments were set up using fibroblast-like synoviocytes (FLS) and peritoneal macrophages from wild type (WT) or 11β-HSD1 knock-out (KO) mice. CM was generated by stimulating WT or KO macrophages and FLS with the inactive GC dehydrocorticosterone (DHC; 1000 nmol/l) for 24 h. KO FLS or macrophages were then cultured for 24 h with CM. Parallel transwell (0.4 μm) co-cultures were established using the same cell populations. GC responses in CM-treated or co-cultured KO macrophages and FLS were examined by RT-qPCR (Gilz, Il-6, Tnfa) and cytokine secretion measured using ELISA (IL-6). KO FLS significantly upregulated Gilz and suppressed Il-6 expression in response to CM from WT macrophages exposed to inactive DHC (GILZ 4.8 fold increase, Il-6 4.2 fold decrease). These responses were lost with CM from KO macrophages on KO fibroblasts. Similar responses were evident in KO macrophages exposed to CM from WT fibroblasts treated with DHC (Gilz 7.8 fold increase, Il-6 5.6 fold decrease, Tnfa 1.4 fold decrease). Similar results were apparent in co-culture experiments exposed to DHC, where KO macrophages and FLS responded to inactive DHC when cultured with WT but not KO counterparts. Co-culture ELISA data for IL-6 production matched mRNA results. This study demonstrates that 11β-HSD1 can mediate paracrine GC signalling between macrophages and fibroblasts in vitro. These data demonstrate that targeted delivery of GCs to either cell population will likely impact on both in vivo.

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