FTY720 administration results in a M2 associated immunoregulatory effect that positively influences the outcome of alveolar bone repair outcome in mice.

The alveolar bone repair process may be influenced by multiple local and systemic factors, which include immune system cells and mediators. Macrophages allegedly play important roles in the repair process, and the transition of an initial inflammatory M1 profile into a pro-reparative M2 profile theoretically contributes to a favorable repair outcome. In this context, considering immunoregulatory molecules as potential targets for improving bone repair, this study evaluated the role of the immunoregulatory molecule FTY720, previously described to favor the development of the M2 phenotype, in the process of alveolar bone healing in C57Bl/6 (WT) mice. Experimental groups submitted to tooth extraction and maintained under control conditions or treated with FTY720 were evaluated by microtomographic (µCT), histomorphometric, immunohistochemical and molecular analysis to characterize healing and host response features at 0, 1, 3, 7 and 14 days. Our results demonstrated that the FTY720 group presented higher bone tissue density, higher bone tissue volume, greater tissue volume fraction, greater number and thickness of trabeculae and a higher number of osteoblasts and osteoclasts than the control group. Accordingly, the bone markers BMP2, BMP7, ALPL, SOST and RANK mRNA expressions increased in the FTY720 treated group. Furthermore, the levels of FIZZ, ARG2 and IL-10 mRNA increased in the FTY720 group together with the presence of CD206+ cells, suggesting that the boost of bone formation mediated by FTY720 involves an increased polarization and activity of M2 macrophages in healing sites. Thus, our results demonstrate that FTY720 favored the process of alveolar bone repair, probably trough a strengthened M2 response, associated with an increased expression of markers osteogenic differentiation and activity markers. Immunoregulatory strategies based in the modulation of macrophage polarization profile can comprise effective tools to improve the bone repair process.

Modulating Fingolimod (FTY720) Anti-SARS-CoV-2 Activity Using a PLGA-Based Drug Delivery System.

COVID-19 has resulted in more than 490 million people being infected worldwide, with over 6 million deaths by April 05th, 2022. Even though the development of safe vaccine options is an important step to reduce viral transmission and disease progression, COVID-19 cases will continue to occur, and for those cases, efficient treatment remains to be developed. Here, a drug repurposing strategy using nanotechnology is explored to develop a therapy for COVID-19 treatment. Nanoparticles (NPs) based on PLGA for fingolimod (FTY720) encapsulation show a size of ∼150 nm and high drug entrapment (∼90%). The NP (NP@FTY720) can control FTY720 release in a pH-dependent manner. Cytotoxicity assays using different cell lines show that NP@FTY720 displays less toxicity than the free drug. Flow cytometry and confocal microscopy reveal that NPs are actively internalized mostly through caveolin-mediated endocytosis and macropinocytosis pathways and co-localized with lysosomes. Finally, NP@FTY720 not only exhibits anti-SARS-CoV-2 activity at non-cytotoxic concentrations, but its biological potential for viral infection inhibition is nearly 70 times higher than that of free drug treatment. Based on these findings, the combination of drug repurposing and nanotechnology as NP@FTY720 is presented for the first time and represents a promising frontline in the fight against COVID-19.

Synergic effect of OP449 and FTY720 on oral squamous cell carcinoma.

As SET protein is overexpressed and PP2A activity is reduced in oral squamous cell carcinoma (OSCC), this study aimed to assess the effects induced by OP449, a PP2A activator/SET inhibitor, on OSCC cells in vitro, and its potential either isolated or combined with FTY720, a PP2A activator/sphingosine kinase 1 antagonist, as antitumoral therapy in vivo. SET protein was analyzed in cells by immunoblotting and cancer stem cells by aldehyde dehydrogenase 1 assay (ALDH1). The cytotoxicity of OP449 was determined in five OSCC lineages by resazurin assay. Molecular actions of OP449 in SET targets were determined by immunoblotting. The coefficient of drug interaction (CDI) was used to characterize the synergism of OP449 and FTY720. The xenograft HN12 tumor model in nude mice was used to assess the antitumoral effect of OP449 and/or FTY720. HN12 (metastatic) cells showed higher SET and ALDH1 levels, and together with SCC9 cells were selected for molecular analysis. OP449 altered several SET functions/targets, such as histone H3 acetylation and NFkB. A synergism in cytotoxicity was observed when HN12 and SCC9 cells were pre-treated with 2 µM OP449 in combination with 15 µM FTY720 (CDI = 0.27 ± 0.088). Nude mice bearing xenograft HN12 tumors treated with OP449 and FTY720 showed reduced tumor mass. Moreover, NFkB was reduced in tumors after treatment. OP449 targets several SET functions, not only PP2A inhibition. Besides, OP449 plus FTY720 has a synergistic antitumoral effect on OSCC. Our results suggest new combined therapies and highlight SET and NFκB signaling as targets for OSCC therapy.

Fingolimod (FTY720) reduces viability and survival and increases histone H3 acetylation in medulloblastoma cells.

Histone deacetylase inhibitors (HDACis) are epigenetic agents that display antitumor activities in experimental medulloblastoma (MB). Fingolimod (FTY720), an immunosuppressant agent currently used in the treatment of multiple sclerosis, also has anticancer actions and can act as an HDACi. Here we examined whether fingolimod can inhibit human MB cell viability and survival, and if the effects are accompanied by increased histone acetylation. D283 and DAOY MB cells were treated with different doses of fingolimod. Cell viability was assessed by cell counting in a hemocytometer, and cell survival was analyzed with a colony formation assay. Histone H3 acetylation was measured with an enzyme-linked immunosorbent assay (ELISA). Fingolimod at 7.5 or 10 µM, but not at 5 µM, induced a significant reduction in cell viability in D283 and DAOY cultures, and similar results were observed for inhibition of cell survival. In both cell lines, fingolimod also led to a significant increase in the levels of acetylated H3. These findings provide preliminary evidence indicating that fingolimod induces antitumor activities in MB, possibly through a mechanism which increases H3 histone acetylation.

S1P1 receptor phosphorylation, internalization, and interaction with Rab proteins: effects of sphingosine 1-phosphate, FTY720-P, phorbol esters, and paroxetine.

Sphingosine 1-phosphate (S1P) and FTY720-phosphate (FTYp) increased intracellular calcium in cells expressing S1P1 mCherry-tagged receptors; the synthetic agonist was considerably less potent. Activation of protein kinase C by phorbol myristate acetate (PMA) blocked these effects. The three agents induced receptor phosphorylation and internalization, with the action of FTYp being more intense. S1P1 receptor-Rab protein (GFP-tagged) interaction was studied using FRET. The three agents were able to induce S1P1 receptor-Rab5 interaction, although with different time courses. S1P1 receptor-Rab9 interaction was mainly increased by the phorbol ester, whereas S1P1 receptor-Rab7 interaction was only increased by FTYp and after a 30-min incubation. These actions were not observed using dominant negative (GDP-bound) Rab protein mutants. The data suggested that the three agents induce interaction with early endosomes, but that the natural agonist induced rapid receptor recycling, whereas activation of protein kinase C favored interaction with late endosome and slow recycling and FTYp triggered receptor interaction with vesicles associated with proteasomal/lysosomal degradation. The ability of bisindolylmaleimide I and paroxetine to block some of these actions suggested the activation of protein kinase C was associated mainly with the action of PMA, whereas G protein-coupled receptor kinase (GRK) 2 (GRK2) was involved in the action of the three agents.

FTY720 induces apoptosis in B16F10-NEX2 murine melanoma cells, limits metastatic development in vivo, and modulates the immune system

OBJECTIVE: Available chemotherapy presents poor control over the development of metastatic melanoma. FTY720 is a compound already approved by the Food and Drug Administration for the treatment of patients with multiple sclerosis. It has also been observed that FTY720 inhibits tumor growth in vivo (experimental models) and in vitro (animal and human tumor cells). The aim of this study was to evaluate the effects of FTY720 on a metastatic melanoma model and in tumor cell lines. METHODS: We analyzed FTY720 efficacy in vivo in a syngeneic murine metastatic melanoma model, in which we injected tumor cells intravenously into C57BL/6 mice and then treated the mice orally with the compound for 7 days. We also treated mice and human tumor cell lines with FTY720 in vitro, and cell viability and death pathways were analyzed. RESULTS: FTY720 treatment limited metastatic melanoma growth in vivo and promoted a dose-dependent decrease in the viability of murine and human tumor cells in vitro. Melanoma cells treated with FTY720 exhibited characteristics of programmed cell death, reactive oxygen species generation, and increased β-catenin expression. In addition, FTY720 treatment resulted in an immunomodulatory effect in vivo by decreasing the percentage of Foxp3+ cells, without interfering with CD8+ T cells or lymphocyte-producing interferon-gamma. CONCLUSION: Further studies are needed using FTY720 as a monotherapy or in combined therapy, as different types of cancer cells would require a variety of signaling pathways to be extinguished. .

The imbalance between Treg and Th17 cells caused by FTY720 treatment in skin allograft rejection

OBJECTIVES: FTY720 modulates CD4+T cells by the augmentation of regulatory T cell activity, secretion of suppressive cytokines and suppression of IL-17 secretion by Th17 cells. To further understand the process of graft rejection/acceptance, we evaluated skin allograft survival and associated events after FTY720 treatment. METHODS: F1 mice (C57BL/6xBALB/c) and C57BL/6 mice were used as donors for and recipients of skin transplantation, respectively. The recipients were transplanted and either not treated or treated with FTY720 by gavage for 21 days to evaluate the allograft survival. In another set of experiments, the immunological evaluation was performed five days post-transplantation. The spleens, axillary lymph nodes and skin allografts of the recipient mice were harvested for phenotyping (flow cytometry), gene expression (real-time PCR) and cytokine (Bio-Plex) analysis. RESULTS: The FTY720 treatment significantly increased skin allograft survival, reduced the number of cells in the lymph nodes and decreased the percentage of Tregs at this site in the C57BL/6 recipients. Moreover, the treatment reduced the number of graft-infiltrating cells and the percentage of CD4+ graft-infiltrating cells. The cytokine analysis (splenocytes) showed decreased levels of IL-10, IL-6 and IL-17 in the FTY720-treated mice. We also observed a decrease in the IL-10, IL-6 and IL-23 mRNA levels, as well as an increase in the IL-27 mRNA levels, in the splenocytes of the treated group. The FTY720-treated mice exhibited increased mRNA levels of IL-10, IL-27 and IL-23 in the skin graft. CONCLUSIONS: Our results demonstrated prolonged but not indefinite skin allograft survival by FTY720 treatment. This finding indicates that the drug did not prevent the imbalance between Tr1 and Th17 cells in the graft that led to rejection.

Avances en inmunosupresión en trasplante hepático / Advances in immunossuppression in liver transplantation

The history of Immunosuppresslon is a long one. From the utilization of steroids and azathloptlne In the 50's to the design of humanized molecules that specifically block cell surface receptors. Liver transplantation is one of the procedures that benefit the most with the development of new immunosuppressors and is also one of the reasons to create a new branch in research and clinical practice: transplant medicine. It also set the standards for research in the "immunologic tolerance" field. The cornerstone in the post-liver transplant stage is the utilization of calcineurin inhibitors combined with new anti-metabolites and monoclonal antibodies. All these settings conforms a promising field in the research of new and better immunosuppressing agents.

Se ha recorrido mucho camino desde el diseño de la inmunosupresión en la década de los 50's. Desde la utilización de los esteroides y la azatioprina hasta el desarrollo de moléculas humanizadas, que bloquean específicamente receptores de superficie celular para inducir tolerancia del injerto, ha transcurrido medio siglo. El trasplante hepático ha sido uno de los procedimientos más beneficiados con el desarrollo de las nuevas drogas inmunosupresoras y ha dado origen a una nueva rama de la medicina: la medicina de trasplantes. También ha sentado las bases de investigación tendiente a lograr la "tolerancia inmunológica" del órgano trasplantado. La piedra angular en la inmunosupresión postrasplante hepático es la utilización de los inhibidores de calcineurina que, en combinación con nuevos antimetabolitos y anticuerpos monoclonales, dibujan un futuro promisorio en la búsqueda de mejores agentes.