Background/Purpose:

Macrophages and synovial fibroblasts function as key drivers of inflammation in rheumatoid arthritis (RA). We have developed a co-culture system that aims to define how these two cell types crossregulate within an inflammatory setting. Previously, we have shown that soluble synovial fibroblast factors suppress the macrophage TNF-induced Type I Interferon response. Here we aimed to understand the impact of macrophages on synovial fibroblasts and have identified a novel crossregulatory epidermal growth factor (EGF) pathway that is also expressed in synovial macrophages from RA patients. Through studying this EGF pathway we aim to identify potential therapeutic targets to resolve long-term inflammation in the RA synovium.

Methods:

Primary RA synovial fibroblast cell lines were plated in permeable transwell culture inserts and placed above wells with or without human blood-derived macrophages. The co-cultures were then incubated for 2 days with or without exogenous TNF. PolyA selected RNA was processed for paired-end RNAseq using the Tru-seq kits (Illumina) and Illumina HiSeq 2500. The Ingenuity Pathway Analysis and Gene Set Enrichment Analysis programs provided pathway analytics. Synovial fibroblasts were plated in Matrigel transwell invasion assays with TNF and macrophages in the chamber below. The number of invading fibroblasts was scored in the absence or presence of EGFR inhibitors erlotinib and gefitinib.

Results:

Upon TNF exposure, macrophages responding in close proximity to synovial fibroblasts altered the expression of ~1,200 fibroblast genes.  Pathway analysis identified an activated EGF pathway that contributed to this altered fibroblast transcriptome.  While fibroblasts are commonly considered a predominant source of growth factors, further work determined it was the macrophages that directly produced the EGF ligands responsible for the fibroblast EGF response.  The rare non-canonical EGF ligands produced by the fibroblast-trained macrophages are related to a recently described macrophage activation state found in chronic inflammation, which is distinct from both the prototypical pro-inflammatory (M1) and alternative (M2) polarization states.  We have detailed the mechanism by which signalling crosstalk induced massive amounts of fibroblast-generated prostaglandins that drives this unique macrophage activation state and non-canonical EGF ligand production.  Synovial macrophages from RA patients exhibited increased production of non-canonical EGF ligands, suggesting this crossregulatory EGF response is present in RA synovium and supporting the disease-relevance of these results.  Ultimately, the fibroblast-trained macrophages increased the invasive potential of synovial fibroblasts via the EGF response, thereby increasing pathologic fibroblast activity found in RA joints.

Conclusion:

We have identified a novel functional cross-coupling between macrophages and synovial fibroblasts that modulates TNF responses to drive production of rare non-canonical EGF ligands, which in turn induces the invasiveness of synovial fibroblasts. Our data support targeting of the EGF pathway as a possible therapeutic approach in the treatment of inflammatory diseases such as RA.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/crossregulatory-mechanisms-between-synovial-fibroblasts-and-macrophages-relevant-in-ra-pathogenesis/