Background/Purpose: Autoimmune systemic sclerosis (SSc) is characterized by inflammation, vasculopathy, and dermal and internal organ fibrosis.  A widely-reported but poorly understood aspect of SSc skin disease is the loss of hypodermal cutaneous fat or dermal white adipose tissue (DWAT), which precedes the onset of dermal fibrosis. While recent work suggests that reprogramming of adipocytic cells into activated, extracellular matrix-secreting myofibroblasts may account for the loss of DWAT and resulting fibrosis observed in SSc patient skin, the molecular mechanism that regulates this process has not been elucidated. Because we have shown that macrophages (MØs) from SSc patients secrete active TGF-b, which has been implicated in adipocyte-myofibroblast transition, we hypothesized that activated MØs in SSc modulate the differentiation and proliferation of adipocytes through release of secreted mediators, including TGF-b, leading to loss of DWAT and increased ECM deposition.

Methods: To assess the effect of MØs on DWAT deposition in vivo, we employed the bleomycin-induced mouse model of fibrosis and used a cellular immunotherapeutic approach to eliminate dermal MØs. To elucidate the potential mechanism by which activated MØs alter adipocyte differentiation, we developed an in vitro co-culture model.  Adipose derived stem cells (ADSC) and bone marrow derived MØs (BMDM) were isolated from C57BL/6J mice.  BMDMs were differentiated using GM-CSF, M-CSF, IL-4 and IL-13 or skin conditioned media (SCM). SCM was generated by subcutaneously implanting osmotic pumps with BLM for 7 days, harvesting the fibrotic skin, incubating the skin for 48 hours in media followed by collection of skin supernatant.  ADSCs were differentiated into pre-adipocytes using media containing IBMX, dexamethasone, insulin, and rosigalitazone and co-cultured with differentiated BMDMs for 36 hours in Transwells. BMDMs and pre-adipocytes were collected for surface marker and gene expression analyses by flow cytometry and RT-qPCR. Cell supernatants were collected for analysis of secreted mediators by ELISA. 

Results: Targeted elimination of dermal pro-fibrotic MØs in the bleomycin mouse model of fibrosis led to a restoration of DWAT. In vitro results demonstrated that expression of CD206 was increased on BMDMs differentiated with M-CSF, IL-4 and IL-13 and SCM, in contrast with GM-CSF. However, only BMDMs differentiated with SCM showed increased expression of IL-6, M-CSF, and IFNγ, which we have shown in prior studies are characteristic of SSc MØs. Furthermore, SCM-differentiated BMDMs secrete increased levels of TGF-β1 and inhibit adipogenesis. 

Conclusion: Our results provide insight into a mechanism by which MØs modulate skin fibrosis through regulation of DWAT deposition.  These findings suggest MØ-targeted therapies may be effective in ameliorating subcutaneous fat loss and potentially limiting excessive ECM deposition, particularly in combination with anti-fibrotic therapies.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/macrophages-regulate-adipocyte-differentiation-and-proliferation-in-skin-fibrosis/