Background/Purpose: An immune fibrotic axis consisting of activated macrophages (MØs) and fibroblasts has been identified in autoimmune systemic sclerosis (SSc) that drives disease across affected tissue types. In prior work, we have shown that SSc fibroblasts and MØs, which co-localize in lesional SSc skin, participate in reciprocal paracrine signaling mediated by SSc fibroblast-derived exosomes. The goal of this study was to identify the specific cargo carried by SSc fibroblast exosomes that mediates profibrotic MØ activation, and to elucidate the molecular mechanism responsible for this activation. Because MØ activation is intrinsically dependent on ATP availability, we hypothesized that SSc fibroblast-derived paracrine signals may induce profibrotic MØ activation in part by driving functional changes in MØ bioenergetics.

Methods: To identify candidate regulators responsible for inducing dermal SSc MØ activation, publicly available single cell RNAseq (scRNA-seq) data from SSc patient and non-SSc control skin were interrogated. Because MØ activation is driven by metabolic utilization, exosome-mediated effects on MØ bioenergetics were ability investigated using Single Cell ENergetIc metabolism by profilng Translation inhibition (SCENITH). Protein content of exosomes was analyzed by immunoblot. Inhibitor studies were performed to assess the contribution of noncanonical WNT signaling to exosome-mediated profibrotic MØ activation, and co-cultures were employed to determine the requirement for WNT signaling in the ability of SSc fibroblast exosome-stimulated MØs to induce reciprocal fibroblast activation.

Results: scRNA-seq analyses of SSc and control skin demonstrated that pathways related to lipid metabolism, nutrient sensing, and oxidative phosphorylation (OXPHOS) were significantly enriched in SSc dermal MØs. Using SCENITH, we demonstrated that stimulation of MØs with SSc fibroblast-derived exosomes resulted in enhanced OXPHOS reliance, consistent with scRNA-seq results, while healthy fibroblast-derived exosomes failed to increase OXPHOS utilization. Furthermore, intact OXPHOS was required for profibrotic activation of human MØs by SSc fibroblast-derived exosomes. WNT5A, a known regulator of myeloid metabolism, was overexpressed in SS fibroblast exosomes, and studies with the WNT5A inhibitor Box5 demonstrated that blockade of WNT5A signaling abrogates OXPHOS upregulation and significantly attenuates the ability of SSc fibroblast exosome-stimulated MØs to elicit SSc fibroblast activation.

Conclusion: Collectively, our data indicate that SSc fibroblast-derived paracrine signals promote profibrotic MØ activation by altering MØ bioenergetics through noncanonical WNT-mediated signaling. These findings suggest that MØs meet the increased energy demand of the SSc dermal fibrotic niche by upregulating both mitochondrial dependency and mitochondrial content, and that blocking either OXPHOS or the WNT5A receptor, FZD5, is sufficient to inhibit profibrotic SSc MØ activation. Thus, MØ-specific metabolism and/or WNT5A signaling may represent novel therapeutic targets in SSc.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/paracrine-wnt-signaling-modulates-profibrotic-macrophage-metabolic-activation-in-systemic-sclerosis/