Publicação
Dissecting the Mechanisms Governing Skin Wound Healing Induced by an Umbilical Cord Blood-Derived Product
| Resumo: | Skin diseases are among the most common human disease conditions where most skin lesions are immune-related. Controlling and balancing pathological factors in this system should be a priority in the treatment of these disorders. Over the last few years, investigation of extracellular vesicles (EVs)-based therapies has grown substantially, making them an extremely attractive and innovative product for medicine market. An innovative product enriched in small EVs (sEVs) derived from umbilical cord blood mononuclear cells (UCB-MNCs) have previously shown promising results in accelerating wound healing in vitro and in vivo. Yet, other potentialities of EVs derived from UCB-MNCs remain to be elucidated. Thus, this doctoral thesis aims to evaluate the potential of UCB-MNCs-EVs as a tool for the treatment of immune-related skin lesions, characterizing their ability to act on inflammation and/or as an immunomodulator. After optimizing a new methodology to isolate and purify the EVs, the biophysical and biochemical composition of UCB-MNCs-sEVs was characterized. We demonstrate that this new methodology results in a final product enriched in small EVs (~ 90%) with an average size of 102.7 ± 10.55nm. These vesicles have characteristics typical of sEVs such as the presence of classical (CD9 and CD63) and non-classical (CD15) markers and several annexins. Also, they have a content mostly composed of phosphatidylcholines and phosphatidylserines, free of genomic DNA and made up of several small RNA species. UCB-MNCs-sEVs obtained by this new methodology showed superior efficacy in wound healing when compared to the sEVs obtained by standard methodologies. On day 10, in mice with induced type I diabetes, wounds treated with sEVs isolated with the new methodology measured 37% less than the corresponding wounds treated with sEVs derived from the standard methodology. In addition, their inflammatory and immunomodulatory potential was tested. Mechanistically, these EVs were able to affect the polarization of macrophages, decreasing the M1/M2 ratio, in a physiological environment and more pronouncedly in an inflammatory environment (LPS stimulus). Its pattern of gene expression and cytokine secretion was also modulated. This impact on the macrophage microenvironment was able to induce the proliferation of fibroblasts as well as a less inflammatory phenotype, leading to decreased production of IL-8 and modulation of the genetic expression of growth factors and extracellular matrix molecules. UCB-MNCs-sEVs also mediate suppression of proliferation, differentiation, and activation of T lymphocytes in vitro. These vesicles enhance immunotolerance, increasing the abundance of regulatory T cells (Tregs) and decreasing auxiliary T cells 17 (TH17). This potential was further confirmed in vivo by sequencing the genetic profile of type I-induced diabetes mice wounds treated or not with UCB-MNCs-sEVs on day 3. Of the genes related to the inflammatory and the immune responses, approximately 70% of these were downregulated. The expression of many of these genes was validated, confirming the immunomodulatory potential of UCB-MNCs-sEVs in the skin. In a three-dimensional in vitro model of psoriatic disease, we demonstrated that UCB-MNCs-sEVs modulate the gene expression of cytokines (IL-6, IL-8 and CXCL10), antimicrobial peptides (psoriasin and beta-defensin 4), and other anti-inflammatory factors considered critical in the pathogenesis of psoriasis. In addition, these vesicles decrease the secretion of TNF-α and CCL20 by 40 and 30%, respectively. In vivo, in a mouse model of psoriasis induced by topical application of imiquimod, topical application of emulsified UCB-MNCs-sEVs in a hydrogel formulation decreased the thickness of the epidermis and increased the number of Tregs on the skin compared to untreated animals. When administered intravenously, UCB-MNCs-sEVs decreased the number of effector T cells observed and the expression of proinflammatory cytokines. In addition, in vitro stimulated lymph nodes and spleen cells treated with these vesicles decreased IFN-γ secretion and increased IL-10 expression. These results strongly support an immunomodulatory effect of UCB-MNCs-sEVs both in vitro (human) and in vivo (mouse). UCB-MNCs-sEVs probably act by stimulating differentiation of T lymphocytes in Tregs, which can lead to the activation of anti-inflammatory pathways in detriment of pro-inflammatory ones. The positive results observed in psoriasis open the door to future studies on the effect of UCB-MNCs-sEVs on other inflammatory skin diseases, where TH17/Treg dysfunction is thought to play a crucial role (e.g. vitiligo, alopecia areata). In general, they have shown to be a very promising therapeutic tool for inflammatory skin diseases. |
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| Autores principais: | Rodrigues, Sílvia Maria do Couto |
| Assunto: | Chronic Wounds Exosomes Mode of action Regeneration/Repair Wound healing |
| Ano: | 2021 |
| País: | Portugal |
| Tipo de documento: | tese de doutoramento |
| Tipo de acesso: | acesso aberto |
| Instituição associada: | Universidade de Coimbra |
| Idioma: | inglês |
| Origem: | Estudo Geral - Universidade de Coimbra |
| Resumo: | Skin diseases are among the most common human disease conditions where most skin lesions are immune-related. Controlling and balancing pathological factors in this system should be a priority in the treatment of these disorders. Over the last few years, investigation of extracellular vesicles (EVs)-based therapies has grown substantially, making them an extremely attractive and innovative product for medicine market. An innovative product enriched in small EVs (sEVs) derived from umbilical cord blood mononuclear cells (UCB-MNCs) have previously shown promising results in accelerating wound healing in vitro and in vivo. Yet, other potentialities of EVs derived from UCB-MNCs remain to be elucidated. Thus, this doctoral thesis aims to evaluate the potential of UCB-MNCs-EVs as a tool for the treatment of immune-related skin lesions, characterizing their ability to act on inflammation and/or as an immunomodulator. After optimizing a new methodology to isolate and purify the EVs, the biophysical and biochemical composition of UCB-MNCs-sEVs was characterized. We demonstrate that this new methodology results in a final product enriched in small EVs (~ 90%) with an average size of 102.7 ± 10.55nm. These vesicles have characteristics typical of sEVs such as the presence of classical (CD9 and CD63) and non-classical (CD15) markers and several annexins. Also, they have a content mostly composed of phosphatidylcholines and phosphatidylserines, free of genomic DNA and made up of several small RNA species. UCB-MNCs-sEVs obtained by this new methodology showed superior efficacy in wound healing when compared to the sEVs obtained by standard methodologies. On day 10, in mice with induced type I diabetes, wounds treated with sEVs isolated with the new methodology measured 37% less than the corresponding wounds treated with sEVs derived from the standard methodology. In addition, their inflammatory and immunomodulatory potential was tested. Mechanistically, these EVs were able to affect the polarization of macrophages, decreasing the M1/M2 ratio, in a physiological environment and more pronouncedly in an inflammatory environment (LPS stimulus). Its pattern of gene expression and cytokine secretion was also modulated. This impact on the macrophage microenvironment was able to induce the proliferation of fibroblasts as well as a less inflammatory phenotype, leading to decreased production of IL-8 and modulation of the genetic expression of growth factors and extracellular matrix molecules. UCB-MNCs-sEVs also mediate suppression of proliferation, differentiation, and activation of T lymphocytes in vitro. These vesicles enhance immunotolerance, increasing the abundance of regulatory T cells (Tregs) and decreasing auxiliary T cells 17 (TH17). This potential was further confirmed in vivo by sequencing the genetic profile of type I-induced diabetes mice wounds treated or not with UCB-MNCs-sEVs on day 3. Of the genes related to the inflammatory and the immune responses, approximately 70% of these were downregulated. The expression of many of these genes was validated, confirming the immunomodulatory potential of UCB-MNCs-sEVs in the skin. In a three-dimensional in vitro model of psoriatic disease, we demonstrated that UCB-MNCs-sEVs modulate the gene expression of cytokines (IL-6, IL-8 and CXCL10), antimicrobial peptides (psoriasin and beta-defensin 4), and other anti-inflammatory factors considered critical in the pathogenesis of psoriasis. In addition, these vesicles decrease the secretion of TNF-α and CCL20 by 40 and 30%, respectively. In vivo, in a mouse model of psoriasis induced by topical application of imiquimod, topical application of emulsified UCB-MNCs-sEVs in a hydrogel formulation decreased the thickness of the epidermis and increased the number of Tregs on the skin compared to untreated animals. When administered intravenously, UCB-MNCs-sEVs decreased the number of effector T cells observed and the expression of proinflammatory cytokines. In addition, in vitro stimulated lymph nodes and spleen cells treated with these vesicles decreased IFN-γ secretion and increased IL-10 expression. These results strongly support an immunomodulatory effect of UCB-MNCs-sEVs both in vitro (human) and in vivo (mouse). UCB-MNCs-sEVs probably act by stimulating differentiation of T lymphocytes in Tregs, which can lead to the activation of anti-inflammatory pathways in detriment of pro-inflammatory ones. The positive results observed in psoriasis open the door to future studies on the effect of UCB-MNCs-sEVs on other inflammatory skin diseases, where TH17/Treg dysfunction is thought to play a crucial role (e.g. vitiligo, alopecia areata). In general, they have shown to be a very promising therapeutic tool for inflammatory skin diseases. |
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Atividades financiadas
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