SFRP4 Expression Is Linked to Immune-Driven Fibrotic Conditions, Correlates with Skin and Lung Fibrosis in SSc and a Potential EMT Biomarker

Secreted Frizzled Receptor Protein 4 (SFRP4) has been shown to be increased in Scleroderma (SSc). To determine its role in immune-driven fibrosis, we analysed SSc and sclerotic Chronic Graft Versus Host Disease (sclGVHD) biosamples; skin biopsies (n = 24) from chronic GVHD patients (8 with and 5 without sclGVHD), 8 from SSc and 3 healthy controls (HC) were analysed by immunofluorescence (IF) and SSc patient sera (n = 77) assessed by ELISA. Epithelial cell lines used for in vitro Epithelial-Mesenchymal-Transition (EMT) assays and analysed by Western Blot, RT-PCR and immunofluorescence. SclGVHD skin biopsies resembled pathologic features of SSc. IF of fibrotic skin biopsies indicated the major source of SFRP4 expression were dermal fibroblasts, melanocytes and vimentin positive/caveolin-1 negative cells in the basal layer of the epidermis. In vitro studies showed increased vimentin and SFRP4 expression accompanied with decreased caveolin-1 expression during TGFβ-induced EMT. Additionally, SFRP4 serum concentration correlated with severity of lung and skin fibrosis in SSc. In conclusion, SFRP4 expression is increased during skin fibrosis in two different immune-driven conditions, and during an in vitro EMT model. Its serum levels correlate with skin and lung fibrosis in SSc and may function as biomarker of EMT. Further studies are warranted to elucidate the role of SFRP4 in EMT within the pathogenesis of tissue fibrosis.

Adapting the Scar-in-a-Jar to Skin Fibrosis and Screening Traditional and Contemporary Anti-Fibrotic Therapies

Skin fibrosis still constitutes an unmet clinical need. Although pharmacological strategies are at the forefront of scientific and technological research and innovation, their clinical translation is hindered by the poor predictive capacity of the currently available in vitro fibrosis models. Indeed, customarily utilised in vitro scarring models are conducted in a low extracellular matrix milieu, which constitutes an oxymoron for the in-hand pathophysiology. Herein, we coupled macromolecular crowding (enhances and accelerates extracellular matrix deposition) with transforming growth factor β1 (TGFβ1; induces trans-differentiation of fibroblasts to myofibroblasts) in human dermal fibroblast cultures to develop a skin fibrosis in vitro model and to screen a range of anti-fibrotic families (corticosteroids, inhibitors of histone deacetylases, inhibitors of collagen crosslinking, inhibitors of TGFβ1 and pleiotropic inhibitors of fibrotic activation). Data obtained demonstrated that macromolecular crowding combined with TGFβ1 significantly enhanced collagen deposition and myofibroblast transformation. Among the anti-fibrotic compounds assessed, trichostatin A (inhibitors of histone deacetylases); serelaxin and pirfenidone (pleiotropic inhibitors of fibrotic activation); and soluble TGFβ receptor trap (inhibitor of TGFβ signalling) resulted in the highest decrease of collagen type I deposition (even higher than triamcinolone acetonide, the gold standard in clinical practice). This study further advocates the potential of macromolecular crowding in the development of in vitro pathophysiology models.

CpG-activated Regulatory B-cell Progenitors Alleviate Murine Sclerodermatous Chronic GVHD

BackgroundDevelopment of chronic Graft Versus Host Disease (cGVHD) represents a major impediment in allogeneic hematopoietic stem cell transplantation (HSCT). This disorder is associated with severe impairment of B-cell homeostasis, regulatory B-cell function and distribution. Conversely, the presence of bone marrow and circulating hematogones is associated with reduced GVHD risks. These findings raised the question whether B-cell progenitors, which provide protection in various autoimmune disease models following activation with the TLR-9 agonist CpG (CpG-proBs), could likewise reduce disease symptoms in a sclerodermatous model of cGVHD. MethodsChronic sclerodermatous GVHD was induced in irradiated Balb/c recipients reconstituted with T- and B-cell-depleted bone marrow cells and splenocytes from C57BL/6J donors. CpG-proB-cell progenitors sorted from in vitro CpG-activated bone marrow cells were then adoptively transferred into GVHD recipients. Their effect on disease symptoms, such as diarrhea, skin fibrosis and survival was evaluated in the therapeutic window defined beforehand. Transferred progenitors were analyzed for migration, differentiation and cytokine expression using flow cytofluorimetric methods, which were also used to establish their impact on T-cell cytokine expression and follicular helper/regulatory T-cell ratios (Tfh/Tfr) in peripheral and mesenteric lymph nodes. Skin fibrosis was assessed by histology, identification of infiltrating cells and gene expression profiles of cytokines and molecules involved in the fibrotic process, using qRT-PCR microarrays in all tissue samples.ResultsWe found that CpG-proBs, adoptively transferred during the initial phase of disease, reduced the diarrhea score and mostly prevented cutaneous fibrosis. Progenitors migrated to the draining lymph nodes and to the skin where they mainly differentiated into follicular B cells. CpG activation and IFN-γ expression were required for the protective effect, which resulted in reduced CD4+ T-cell-derived production of cytokines critically involved in cGVHD, such as TGF-β, IL-13 and IL-21. Adoptive transfer increased the Tfr/Tfh ratio. Moreover, CpG-proBs privileged the accumulation of IL-10-positive CD8+ T cells, B cells and dendritic cells in the skin. ConclusionOur findings support the notion that adoptively transferred CpG-proBs provide an efficient strategy for alleviating sclerodermatous cGVHD either per se or as a beneficial adjunct to the HSC graft.