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 (ECM)-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 adipocyte precursors 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 initially investigated the effect of myeloid depletion in the bleomycin (BLM)-induced mouse model of fibrosis using clodronate liposomes. At day -3 (prior to initiation of BLM), clodronate or control liposomes were given i.p., and then clodronate treatments were repeated every 3 days for the duration of the experiment (21 days). We also used a targeted immunotherapeutic approach using anti-CD206 CAR T cells to eliminate pathological MØs. Anti-CD206 CAR T cells were administered 7 days post-BLM initiation to assess effects on established fibrosis. To elucidate the potential mechanism by which activated MØs alter preadipocyte precursor differentiation, we developed an in vitro co-culture model.  Adipose derived stem cells (ADSCs) and bone marrow derived MØs (BMDMs) were isolated from C57BL/6J mice.  BMDMs were differentiated using skin conditioned media (SCM) generated by collecting supernatants from either naïve or fibrotic skin cultured in vitro for 36 hours. ADSCs were differentiated into pre-adipocytes using media containing dexamethasone, insulin, and rosiglitazone and were co-cultured with differentiated BMDMs for 96 hours in Transwells. BMDMs and pre-adipocytes were assessed for surface marker and gene expression analyses by flow cytometry and qRT-PCR. ELISA was used to quantify secreted mediators in cell supernatants. 

Results: MØ depletion using clodronate or anti-CD206 CAR T cells led to restoration of DWAT in BLM mice. Expression of CD206 was increased on BMDMs differentiated with fibrotic SCM. BMDMs differentiated with fibrotic SCM secreted increased levels of TGF-β1, IL-6 and CCL2.  Co-culture of BMDMs with ADSCs induced expression of profibrotic markers in preadipocytes. 

Conclusion: Our results provide insight into a mechanism by which MØs modulate skin fibrosis through regulation of DWAT deposition.  These findings suggest MØs play an important role in pathogenesis and progression of skin fibrosis and that 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/activated-macrophages-mediate-loss-of-dermal-white-adipose-tissue-in-fibrotic-skin/