Single-cell epigenomic dysregulation of Systemic Sclerosis fibroblasts via CREB1/EGR1 axis in self-assembled human skin equivalents.

Systemic sclerosis (SSc) is an autoimmune disease characterized by skin fibrosis, internal organ involvement and vascular dropout. We previously developed and phenotypically characterized an in vitro 3D skin-like tissue model of SSc, and now analyze the transcriptomic (scRNA-seq) and epigenetic (scATAC-seq) characteristics of this model at single-cell resolution. SSc 3D skin-like tissues were fabricated using autologous fibroblasts, macrophages, and plasma from SSc patients or healthy control (HC) donors. SSc tissues displayed increased dermal thickness and contractility, as well as increased α-SMA staining. Single-cell transcriptomic and epigenomic analyses identified keratinocytes, macrophages, and five populations of fibroblasts (labeled FB1 - 5). Notably, FB1 APOE-expressing fibroblasts were 12-fold enriched in SSc tissues and were characterized by high EGR1 motif accessibility. Pseudotime analysis suggests that FB1 fibroblasts differentiate from a TGF-ß1-responsive fibroblast population and ligand-receptor analysis indicates that the FB1 fibroblasts are active in macrophage crosstalk via soluble ligands including FGF2 and APP. These findings provide characterization of the 3D skin-like model at single cell resolution and establish that it recapitulates subsets of fibroblasts and macrophage phenotypes observed in skin biopsies.

CDDO-Methyl Ester Inhibits BRAF Inhibitor Resistance and Remodels the Myeloid Compartment in BRAF-mutant Melanoma.

Approximately 50% of advanced melanomas harbor activating BRAF V600E mutations that are sensitive to BRAF inhibition. However, the duration of the response to BRAF inhibitors (BRAFi) has been limited due to the development of acquired resistance, which is preceded by recruitment of immunosuppressive myeloid cells and regulatory T cells (T regs ). While the addition of MAPK/ERK kinase 1 inhibitors (MEKi) prolongs therapeutic response to BRAF inhibition, most patients still develop resistance. Using a Braf V600E/+ /Pten -/- graft mouse model of melanoma, we now show that the addition of the methyl ester of the synthetic triterpenoid 2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oic acid (C-Me) to the BRAFi vemurafenib analog PLX4720 at resistance significantly reduces tumor burden. Dual treatment remodels the BRAFi resistant-tumor microenvironment (TME), reducing infiltration of T regs and tumor associated macrophages (TAMs), and attenuates immunosuppressive cytokine production. For the first time, we characterize myeloid populations using scRNA-seq in BRAFi-resistant tumors and demonstrate that restoration of therapeutic response is associated with significant changes in immune-activated myeloid subset representation. Collectively, these studies suggest that C-Me inhibits acquired resistance to BRAFi. Use of C-Me in combination with other therapies may both inhibit melanoma growth and enhance therapeutic responsiveness more broadly.

Human dermal fibroblast-derived exosomes induce macrophage activation in systemic sclerosis.

OBJECTIVES: Prior work demonstrates that co-cultured macrophages and fibroblasts from patients with SSc engage in reciprocal activation. However, the mechanism by which these cell types communicate and contribute to fibrosis and inflammation in SSc is unknown. METHODS: Fibroblasts were isolated from skin biopsies obtained from 7 SSc patients or 6 healthy age and gender-matched control subjects following written informed consent. Human donor-derived macrophages were cultured with exosomes isolated from control or SSc fibroblasts for an additional 48 h. Macrophages were immunophenotyped using flow cytometry, qRT-PCR and multiplex. For mutual activation studies, exosome-activated macrophages were co-cultured with SSc or healthy fibroblasts using Transwells. RESULTS: Macrophages activated with dermal fibroblast-derived exosomes from SSc patients upregulated surface expression of CD163, CD206, MHC Class II and CD16 and secreted increased levels of IL-6, IL-10, IL-12p40 and TNF compared with macrophages incubated with healthy control fibroblasts (n = 7, P < 0.05). Exosome-stimulated macrophages and SSc fibroblasts engaged in reciprocal activation, as production of collagen and fibronectin was significantly increased in SSc fibroblasts receiving signals from SSc exosome-stimulated macrophages (n = 7, P < 0.05). CONCLUSION: In this work, we demonstrate for the first time that human SSc dermal fibroblasts mediate macrophage activation through exosomes. Our findings suggest that macrophages and fibroblasts engage in cross-talk in SSc skin, resulting in mutual activation, inflammation, and extracellular matrix (ECM) deposition. Collectively, these studies implicate macrophages and fibroblasts as cooperative mediators of fibrosis in SSc and suggest therapeutic targeting of both cell types may provide maximal benefit in ameliorating disease in SSc patients.

T Cells and CDDO-Me Attenuate Immunosuppressive Activation of Human Melanoma-Conditioned Macrophages.

Melanoma tumors are highly immunogenic, making them an attractive target for immunotherapy. However, many patients do not mount robust clinical responses to targeted therapies, which is attributable, at least in part, to suppression of immune responses by tumor-associated macrophages (TAMs) in the tumor microenvironment (TME). Using a human in vitro tri-culture system of macrophages with activated autologous T cells and BRAFV600E mutant melanoma cells, we now show that activated T cells and the synthetic triterpenoid the methyl ester of 2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oic acid (CDDO-Me) attenuate immune suppression. Surface expression of CD206, CD16 and CD163 on melanoma-conditioned macrophages was inhibited by the addition of T cells, suggesting relief of immuno-suppressive macrophage activation. We also demonstrated that addition of CDDO-Me to tri-cultures enhanced T cell-mediated reductions in CCL2, VEGF and IL-6 production in a contact-independent manner. Because these results suggest CDDO-Me alters melanoma-conditioned macrophage activation, we interrogated CDDO-Me-mediated changes in macrophage signaling pathway activation. Our results indicated that CDDO-Me inhibited phosphorylation of STAT3, a known inducer of TAM activation. Collectively, our studies suggest that activated T cells and CDDO-Me synergistically relieve immune suppression in melanoma cultures and implicate the potential utility of CDDO-Me in the treatment of melanoma.