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| Author(s): |
Djúlio César Zanin da Silva
Total Authors: 1
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| Document type: | Doctoral Thesis |
| Press: | Ribeirão Preto. |
| Institution: | Universidade de São Paulo (USP). Faculdade de Medicina de Ribeirão Preto (PCARP/BC) |
| Defense date: | 2025-04-15 |
| Examining board members: |
Maria Carolina de Oliveira Rodrigues;
Larissa Dias da Cunha;
Renê Donizeti Ribeiro de Oliveira;
Maira Galdino da Rocha Pitta
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| Advisor: | Maria Carolina de Oliveira Rodrigues; Kelen Cristina Ribeiro Malmegrim de Farias |
| Abstract | |
Systemic sclerosis (SSc) is an autoimmune disease characterized by three pathological axes of vascular damage, immunological dysregulation, and fibrosis of skin and internal organs. Auto-HSCT is an alternative for severe and progressive SSc forms, and despite promoting connective tissue remodeling, its effects on the epidermis remain unexplored. The complex interaction between cells involved in the inflammation-fibrosis axis requires experimental models incorporating these components, mimicking the disease microenvironment. In this two-phase study, we employed in vitro approaches using keratinocytes, (myo)fibroblasts, and monocytes/macrophages to elucidate the pathophysiological mechanisms in SSc. In the first phase, we focused on keratinocyte dysregulation in the epidermis and its potential association with clinical outcomes in patients undergoing autologous hematopoietic stem cell transplantation (auto-HSCT) (STUDY 1). Thus, in STUDY 1, we analyzed whether auto-HSCT alters the inflammatory and activation state in the epidermis. We integrated histopathological analyses of skin biopsies, quantification of serum molecules, and clinical data from patients (pre- and six months after auto-HSCT) with a keratinocyte lineage (HaCaT) exposed to patient serum from both time points. We hypothesized that auto-HSCT would reduce the inflammation and activation of keratinocytes in patients\' biopsies. In vitro, when exposed to post-transplant patient serum, we expected keratinocytes to show changes that reflect a microenvironment with reduced fibroinflammatory signals. Our results showed that, coupled with substantial improvement in clinical skin fibrosis, auto-HSCT significantly decreased epidermal thickness. The expression of S100A9, IL-1α, TNF-α, and Ki-67 was also changed in the skin post-auto-HSCT, indicating a visible decrease of inflammation and keratinocytes\' activation/proliferation. In SSc serum, auto-HSCT decreased epidermal growth factor (EGF) and interleukin (IL)-1α levels and normalized connective tissue growth factor (CTGF) concentrations. Exposure of HaCaT cells to post-transplant patient serum reduced CCL-5 release, which is associated with the NF-κB signaling pathway. Thus, we found that there was a partial improvement in the epidermal layer six months after auto-HSCT, justifying the development of new studies on this compartment in the future, to highlight more specific pathways and mechanisms. In the second phase (STUDY 2), we went further to a 3D co-culture model of monocytes/macrophages and fibroblasts in collagen hydrogels, analyzing mechanisms and signaling pathways involved in (myo)fibroblast activation and contraction, which reflect skin stiffness and fibrosis in SSc. The hypothesis was that monocytes would induce (mio)fibroblasts activation and contraction in the hydrogels. In the results, we identified that monocytes strongly promoted (myo)fibroblast contraction in the hydrogels (seen by decreased hydrogels\' area), coupled with an upregulation of activation-associated markers, such as alpha-smooth muscle actin (α-SMA) and fibroblast activation protein (FAP). Using reporter fibroblast constructs and pharmacological inhibitors (SB-505124 and tofacitinib), we demonstrated that monocyte-fibroblast communication was mediated by JAK/STAT and TGF-β/Smad signaling pathways. Flow cytometry analyses also revealed that fibroblast interaction led monocytes to differentiate into macrophages with a mixed M1/M2 polarization phenotype, expressing concomitantly CD163, CD206, CD86, and HLA-DR. Finally, adding M1- or M2-like macrophages to the plugs similarly resulted in (myo)fibroblast contraction. Hydrogels containing only fibroblasts exposed to M1- or M2-like cell supernatants strongly contracted, an effect abolished by TGF-β neutralization. Together, our work contributes to elucidating pathways and mechanisms associated with skin inflammation and fibrosis in systemic sclerosis and paves the way for developing new platforms for targeted therapy testing. (AU) | |
| FAPESP's process: | 22/01429-0 - PHENOTYPIC AND FUNCTIONAL STUDY OF KERATINOCYTES EXPOSED TO SERUM OF SYSTEMIC SCLEROSIS PATIENTS TREATED WITH AUTOLOGOUS HEMATOPOIETIC STEM CELL TRANSPLANTATION |
| Grantee: | Djúlio César Zanin da Silva |
| Support Opportunities: | Scholarships in Brazil - Doctorate |