P231 Targeting the Rho/MRTF-A pathway inhibits growth factor and cytokine release but enhances efferocytosis in scleroderma macrophages
Authors: Garces Sandra Lopez , Abdi Bahja Ahmed , MacFadyen Claire , Smillie Robert , Nagib Lydia , Ghani Shahi , Shah Shivani , Abraham David , Denton Christopher , Stratton Richard J 
Published: April 2022

Abstract

Background/Aims
In the systemic sclerosis (SSc) microenvironment, accumulation of extracellular matrix (ECM) components induces changes in the matrix's mechanical properties, which triggers Rho/MRTFF-A activation and the downstream signalling cascades, inducing the expression of fibrotic and inflammatory genes in fibroblasts. However, macrophages also play a key role in tissue homeostasis and wound repair. It is possible that these cells are also influenced by ECM stiffness and therefore we investigated the effects of altering substrate stiffness and tested the effects of an inhibitor of the mechano-sensing Rho/MRTF-A pathway on the activation state of SSc macrophages.

Methods
SSc and healthy control macrophages (both n = 4 donors) were derived by culture of blood monocytes in the presence of M-CSF (4ng/ml) for 7 days. Soft substrates (4kPa) or stiff ECM substrates (50kPa) were used to model the effect of soft healthy skin tissue and stiff fibrotic SSc skin (Softwell, Matrigen, polymer gels, collagen I coated). Morphology, gene expression (qPCR for CD86 for M1-like, CD206 for M2-like, MerTK for efferocytosis), Multiplex assay, labelled E. Coli uptake assay and Rho/MRTF/SRF pathway inhibition (small molecule CCG-257081) were used to assess the polarisation and activation state of these cells.

Results
Morphology and CD86 did not vary with matrix stiffness, but CD206 expression was significantly lower in stiff compared to soft gels (Mean+/- SEM; soft 38.2+/- 4.2 vs stiff 16.2+/-3.7 relative expression, p value 0.0167). CCG-257081 strongly inhibited secretion of cytokines, growth factors and chemokines. Factors present at &gt; 50 pg/ml suppressed by CCG-257081 include TNFα, GM-CSF, IL-1RA, IP-10, RANTES, MIP-1α, MIP-1β, MCP-1, MCP-3, PDGF-BB (all p &lt; 0.05). SSc macrophages secreted at high levels on soft, and were attenuated by culture on stiff, whereas healthy control macrophages showed the opposite trend inducing more secretion on stiff, inhibited by CCG-257081. Although polarisation markers CD206 and CD86 did not alter with the CCG-257081 treatment, there was a significant increase in MerTK with treatment (p &lt; 0.0001). Endocytosis was moderately yet significantly suppressed by the CCG treatment (basal endocytosis 80.2+/- 4.0 vs CCG-257081 treated 62+/-2.5; % endocytosis; p &lt; 0.01), whereas cell viability was not significantly altered.

Conclusion
Although differences are observed amongst patients, this data indicates that stiffness of the matrices, as used in this study, did not greatly alter the morphology or polarisation of the macrophages. However, transfer to stiff matrices enhanced the secretion of certain cytokines by control macrophages. Strong effects were seen with the Rho/MRTF-A inhibitor, which appeared to block the secretion of multiple factors, including inflammatory cytokines, pro-fibrotic factors, as well as regulatory factors and to strongly induce MerTK, a marker of efferocytosis. One possibility is that the inhibitor converts these cells from secretory to resolving phenotype. Based on these findings, CCG-257081 has promising effects against activated macrophages, which could be utilised in inflammatory fibrotic diseases.

Disclosure
S. Lopez Garces: None. B. Ahmed Abdi: None. C. MacFadyen: None. R. Smillie: None. L. Nagib: None. S. Ghani: None. S. Shah: None. D. Abraham: None. C. Denton: None. R.J. Stratton: None.