FTY720 Treatment Research Articles

Behavioral Analyses in Dark Agouti Rats Following Repeated Systemic Treatment With Fingolimod (FTY720).

Studies in experimental animals revealed that acute and chronic treatment with small-molecule immunosuppressive drugs lead to neurobehavioral alterations in rodents. Against this background, this study investigated behavioral alterations in rats after repeated administration of FTY720, an immunosuppressive drug used for the treatment of multiple sclerosis, employing the open field, elevated plus maze, and dark/light tests. Compared to controls, repeated FTY720 treatment affected behavior in rats, reflected by a reduction in distance traveled as well as increased time engaged in freezing in the open field and elevated plus maze. Furthermore, the time spent freezing in the elevated plus maze test positively correlated with FTY720 concentrations in the amygdala and insular cortex, two brain regions involved in regulation of emotionality. Since no changes in plasma corticosterone levels were observed, stress effects due to treatment, behavioral testing, or handling can be ruled out. The present findings indicate that treatment with FTY720 did not induce typical anxiety-like behavioral patterns in otherwise healthy rats as seen following treatment with other immunosuppressive drugs. Nevertheless, it remains of great importance to evaluate behavioral effects in clinical practice to shed more light onto possible detrimental side effects emerging during treatment with small-molecule immunosuppressive drugs.

Intrapulmonary T Cells Are Sufficient for Schistosoma-Induced Pulmonary Hypertension.

Schistosomiasis is a parasitic infection that can cause pulmonary hypertension (PH). Th2 CD4 T cells are necessary for experimental Schistosoma-PH. However, if T cells migrate to the lung to initiate, the localized inflammation that drives vascular remodeling and PH is unknown. Mice were sensitized to Schistosoma mansoni eggs intraperitoneally and then challenged using tail vein injection. FTY720 was administered, which blocks lymphocyte egress from lymph nodes. T cells were quantified using flow cytometry, PH severity via heart catheterization, and cytokine concentration through ELISA. FTY720 decreased T cells in the peripheral blood, and increased T cells in the mediastinal lymph nodes. However, FTY720 treatment resulted in no change in PH or type 2 inflammation severity in mice sensitized and challenged with S. mansoni eggs, and the number of memory and effector CD4 T cells in the lung parenchyma was also unchanged. Notably, intraperitoneal Schistosoma egg sensitization alone resulted in a significant increase in intravascular lymphocytes and T cells, including memory T cells, although there was no significant change in parenchymal cell density, IL-4 or IL-13 expression, or PH. Blocking T cell migration did not suppress PH following Schistosoma egg challenge. Memory CD4 T cells, located in the lung intravascular space following egg sensitization, appear sufficient to cause type 2 inflammation and PH.

FTY720/Fingolimod mitigates paclitaxel-induced Sparcl1-driven neuropathic pain and breast cancer progression.

Paclitaxel is among the most active chemotherapy drugs for the aggressive triple negative breast cancer (TNBC). Unfortunately, it often induces painful peripheral neuropathy (CIPN), a major debilitating side effect. Here we demonstrate that in naive and breast tumor-bearing immunocompetent mice, a clinically relevant dose of FTY720/Fingolimod that targets sphingosine-1-phosphate receptor 1 (S1PR1), alleviated paclitaxel-induced neuropathic pain. FTY720 also significantly attenuated paclitaxel-stimulated glial fibrillary acidic protein (GFAP), a marker for activated astrocytes, and expression of the astrocyte-secreted synaptogenic protein Sparcl1/Hevin, a key regulator of synapse formation. Notably, the formation of excitatory synapses containing VGluT2 in the spinal cord dorsal horn induced by paclitaxel was also inhibited by FTY720 treatment, supporting the involvement of astrocytes and Sparcl1 in CIPN. Furthermore, in this TNBC mouse model that mimics human breast cancer, FTY720 administration also enhanced the anti-tumor effects of paclitaxel, leading to reduced tumor progression and lung metastasis. Taken together, our findings suggest that targeting the S1P/S1PR1 axis with FTY720 is a multipronged approach that holds promise as a therapeutic strategy for alleviating both CIPN and enhancing the efficacy of chemotherapy in TNBC treatment.

FTY720 ameliorates experimental MPO-ANCA-associated vasculitis by regulating fatty acid oxidation via the neutrophil PPARα-CPT1a pathway.

Increasing studies demonstrated the importance of C5a and anti-neutrophil cytoplasmic antibody (ANCA)-induced neutrophil activation in the pathogenesis of ANCA-associated vasculitis (AAV). Sphingosine-1-phosphate (S1P) acts as a downstream effector molecule of C5a and enhances neutrophil activation induced by C5a and ANCA. The current study investigated the role of a S1P receptor modulator, FTY720, in experimental autoimmune vasculitis (EAV) and explored the immunometabolism-related mechanisms of FTY720 in modulating ANCA-induced neutrophil activation. The effects of FTY720 in EAV were evaluated by quantifying haematuria, proteinuria, crescent formation, tubulointerstitial injury and pulmonary haemorrhage. RNA sequencing of renal cortex and gene enrichment analysis were performed. The proteins of key identified pathways were analysed in neutrophils isolated from peripheral blood of patients with active AAV and normal controls. We assessed the effects of FTY720 on ANCA-induced neutrophil respiratory burst and neutrophil extracellular traps formation (NETosis). FTY720 treatment significantly attenuated renal injury and pulmonary haemorrhage in EAV. RNA sequencing analyses of renal cortex demonstrated enhanced fatty acid oxidation (FAO) and peroxisome proliferator-activated receptor (PPAR) signalling in FTY720-treated rats. Compared with normal controls, patients with active AAV showed decreased FAO in neutrophils. FTY720-treated differentiated HL-60 cells showed increased expression of carnitine palmitoyltransferase 1a (CPT1a) and PPARα. Blocking or knockdown of CPT1a or PPARα in isolated human neutrophils and HL-60 cells reversed the inhibitory effects of FTY720 on ANCA-induced neutrophil respiratory burst and NETosis. FTY720 attenuated renal injury in EAV through upregulating FAO via the PPARα-CPT1a pathway in neutrophils, offering potential immunometabolic targets in AAV treatment.

FTY720 Suppresses Pathogenic Retinal Müller Cell Activation and Chronic Progression by Inhibiting the mTOR/NF-κB Signaling Pathway and Regulating Autophagy

Purpose FTY720 is an agonist of the Sphingosine-1-phosphate (S1P) receptor 1, 3, 4, and 5 and a functional antagonist of the S1P1 receptor; it can inhibit the activation of mTOR/NF-κB and has therapeutic potential in inflammatory disease. This study was designed to determine the role of the inflammatory process in diabetic retinopathy and investigate the effect of FTY720 on high glucose (HG)-induced rat retinal Müller cells (rMC-1 cells). Methods In the present study, the role of FTY720 in inhibiting inflammation and its underlying mechanism were investigated. rMC-1 cells were treated without or with HG, FTY720, CQ, or RAP. Cell viability was examined by CCK-8 assay; cell activation was assessed by western blot analysis and IF staining; and cell migration was evaluated by a scratch wound healing assay. The expression of inflammation-associated proteins and autophagy-related proteins was evaluated by transmission electron microscopy, AO staining, MDC-labeled autophagic vacuoles, western blot analysis and ELISA. Results Western blot analysis and IF staining showed that the level of the rMC-1 cell marker GFAP was decreased, while GS was increased in FTY720 groups compared to that in the HG group. The healing assay results showed that compared with HG treatment, FTY720 treatment significantly reduced cell migration. Western blot analysis, ELISA and IF staining showed that compared with HG, FTY720 reduced proinflammatory proteins by inhibiting the mechanistic target of the mTOR/NF-κB signaling pathway and regulating autophagy. Conclusions This study suggests that in an HG-induced rMC-1 cell model, FTY720 significantly inhibited the production of inflammatory cytokines by inhibiting mTOR/NF-κB signaling and regulating autophagy. These findings were associated with a decrease in rMC-1 cell injury, suggesting that FTY720 or related compounds may be valuable modulators of HG-induced retinal injury.

Abstract 6860: Fluorescent tracking reveals that radiation therapy influences the migration and plasticity of Tregs within the tumor microenvironment

Abstract Radiation therapy (RT) has long been a cornerstone in cancer treatment, yet the mechanisms underlying its impact on the immune system remain incompletely understood. RT is recognized for its ability to induce DNA damage within cancer cells, leading to cancer cell death. For those cancer cells that do not die as a result of radiation, tumor infiltration by immune cells is closely associated with treatment outcomes. T cell infiltration is a dynamic process, with subsets taking up residence in the tumor while the majority recirculate through lymph nodes into the peripheral blood. T regulatory cells (Tregs) have been recognized as potent suppressors of anti-tumor immune responses, dampening immune-mediated clearance of cancer cells. While RT can initially contribute to an improved anti-tumor immune response, this beneficial effect is often counterbalanced by the presence of suppressive Tregs. Tregs can infiltrate the tumor microenvironment, ultimately impeding and overwhelming the immune response generated by RT. Our objective was to gain insights into the migratory patterns of Tregs to and from tumor sites and to elucidate how RT influences the dynamics of Treg recirculation. Utilizing the established photoconvertible Kaede mouse model we can track photoconverted cells following RT. Following photoconversion, photoconverted T cells were observable within the tumor and subsequently moved to the TdLN and on to other NdLN at later time points. In the TdLN of mice bearing MC38, Moc1, and Moc2 tumors, Tregs represented a significantly higher proportion of photoconverted cells compared to non-Treg CD4 or CD8 T cells. To validate their active departure from the tumor site, mice underwent FTY720 treatment, resulting in a reduction in both photoconverted Treg and non-Treg migration to the TdLN. When RT was administered to the tumor, there was a decrease in photoconverted Treg and non-Treg CD4 T cells within the TdLN, consistent with the expected cytotoxic effects on the irradiated tumor upstream. To focus on recruitment back from the TdLN, mice were analyzed after photoconversion of the TdLN. This revealed that a majority of TdLN Tregs exhibited a preference for relocating to the tumor, with only a minority migrating to the NdLN. The newly entered Tregs were analyzed in the tumor, at D1 and D3 post RT, and we found that these Tregs exhibited increased expression of CCR8, CD69, KLRG1 and Ly6C as compared to those in non-irradiated tumors, suggesting that these are recent entrants with limited suppressive capacity. These findings illuminate the intricate dynamics between RT, Tregs, and the use of site-specific photoconversion to track immune cell movement and changes over time to understand treatment responses. Our ongoing studies focus on the interplay between the Treg and the tumor environment that dictates the success of CD8 T cell mediated control of residual cancer cells following RT. Citation Format: David J. Friedman, Tiffany Blair, Gwen Kramer, Aanchal Preet Kaur, Jason Baird, Alejandro Alice, Megan Potuznik, Marka R. Crittenden, Michael J. Gough. Fluorescent tracking reveals that radiation therapy influences the migration and plasticity of Tregs within the tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6860.

The sphingosine 1-phosphate analogue, FTY720, modulates the lipidomic signature of the mouse hippocampus.

The small-molecule drug, FTY720 (fingolimod), is a synthetic sphingosine 1-phosphate (S1P) analogue currently used to treat relapsing-remitting multiple sclerosis in both adults and children. FTY720 can cross the blood-brain barrier (BBB) and, over time, accumulate in lipid-rich areas of the central nervous system (CNS) by incorporating into phospholipid membranes. FTY720 has been shown to enhance cell membrane fluidity, which can modulate the functions of glial cells and neuronal populations involved in regulating behaviour. Moreover, direct modulation of S1P receptor-mediated lipid signalling by FTY720 can impact homeostatic CNS physiology, including neurotransmitter release probability, the biophysical properties of synaptic membranes, ion channel and transmembrane receptor kinetics, and synaptic plasticity mechanisms. The aim of this study was to investigate how chronic FTY720 treatment alters the lipid composition of CNS tissue in adolescent mice at a key stage of brain maturation. We focused on the hippocampus, a brain region known to be important for learning, memory, and the processing of sensory and emotional stimuli. Using mass spectrometry-based lipidomics, we discovered that FTY720 increases the fatty acid chain length of hydroxy-phosphatidylcholine (PCOH) lipids in the mouse hippocampus. It also decreases PCOH monounsaturated fatty acids (MUFAs) and increases PCOH polyunsaturated fatty acids (PUFAs). A total of 99 lipid species were up-regulated in the mouse hippocampus following 3 weeks of oral FTY720 exposure, whereas only 3 lipid species were down-regulated. FTY720 also modulated anxiety-like behaviours in young mice but did not affect spatial learning or memory formation. Our study presents a comprehensive overview of the lipid classes and lipid species that are altered in the hippocampus following chronic FTY720 exposure and provides novel insight into cellular and molecular mechanisms that may underlie the therapeutic or adverse effects of FTY720 in the central nervous system.

The functional antagonist of sphingosine-1-phosphate, FTY720, impairs gut barrier function.

FTY720 or fingolimod is a known functional antagonist of sphingosine-1-phosphate (S1P), and it is effective in treating multiple sclerosis and preventing inflammatory bowel disease (IBD). Evidence shows that its use in mice can increase the susceptibility to mucosal infections. Despite the significant contribution of S1P to barrier function, the effect of the administration of FTY720 on the mucosal barrier has never been investigated. In this study, we looked into how FTY720 therapy affected the function of the gut barrier susceptibility. Administration of FTY720 to C57BL/6 mice enhances the claudin-2 expression and reduces the expression of claudin-4 and occludin, as studied by qPCR, Western blot, and immunofluorescence. FTY720 inhibits the Akt-mTOR pathway to decrease occludin and claudin-4 expression and increase claudin-2 expression. FTY720 treatment induced increased colonic inflammation, with notably greater immune cell infiltration, colon histopathology, and increased production of TNF-α, IFN-γ, CXCL-1, and CXCL-2 than that in control mice. Taking into account the close association of "the leaky gut" and gut dysbiosis among the major diseases, we therefore can infer that the vigilance of gut pathology should be maintained, where FTY720 is used as a treatment option.

Fingolimod (FTY720), an FDA-approved sphingosine 1-phosphate (S1P) receptor agonist, restores endothelial hyperpermeability in cellular and animal models of dengue virus serotype 2 infection.

Extensive vascular leakage and shock is a major cause of dengue-associated mortality. At present, there are no specific treatments available. Sphingolipid pathway is a key player in the endothelial barrier integrity; and is mediated through the five sphingosine-1-phosphate receptors (S1PR1-S1PR5). Signaling through S1PR2 promotes barrier disruption; and in Dengue virus (DENV)-infection, there is overexpression of this receptor. Fingolimod (FTY720) is a specific agonist that targets the remaining barrier-protective S1P receptors, without targeting S1PR2. In the present study, we explored whether FTY720 treatment can alleviate DENV-induced endothelial hyperpermeability. In functional assays, in both in vitro systems and in AG129 animal models, FTY720 treatment was found effective. Upon treatment, there was complete restoration of the monolayer integrity in DENV serotype 2-infected human microvascular endothelial cells (HMEC-1). At the molecular level, the treatment reversed activation of the S1P pathway. It significantly reduced the phosphorylation of the key molecules such as PTEN, RhoA, and VE-Cadherin; and also, the expression levels of S1PR2. In DENV2-infected AG129 mice treated with FTY720, there was significant improvement in weight gain, in overall clinical symptoms, and in survival. Whereas 100% of the DENV2-infected, untreated animals died by day-10 post-infection, 70% of the FTY720-treated animals were alive; and at the end of the 15-day post-infection observation period, 30% of them were still surviving. There was a significant reduction in the Evan's-blue dye permeability in the organs of FTY720-treated, DENV-2 infected animals; and also improvement in the hemogram, with complete restoration of thrombocytopenia and hepatic function. Our results show that the FDA-approved molecule Fingolimod (FTY720) is a promising therapeutic intervention in severe dengue.

Fingolimod (FTY720) Hinders Interferon-γ-Mediated Fibrotic Scar Formation and Facilitates Neurological Recovery After Spinal Cord Injury.

Following spinal cord injury (SCI), fibrotic scar inhibits axon regeneration and impairs neurological function recovery. It has been reported that T cell-derived interferon (IFN)-γ plays a pivotal role in promoting fibrotic scarring in neurodegenerative disease. However, the role of IFN-γ in fibrotic scar formation after SCI has not been declared. In this study, a spinal cord crush injury mouse was established. Western blot and immunofluorescence showed that IFN-γ was surrounded by fibroblasts at 3, 7, 14, and 28 days post-injury. Moreover, IFN-γ is mainly secreted by T cells after SCI. Further, in situ injection of IFN-γ into the normal spinal cord resulted in fibrotic scar formation and inflammation response at 7 days post-injection. After SCI, the intraperitoneal injection of fingolimod (FTY720), a sphingosine-1-phosphate receptor 1 (S1PR1) modulator and W146, an S1PR1 antagonist, significantly reduced T cell infiltration, attenuating fibrotic scarring via inhibiting IFN-γ/IFN-γR pathway, while in situ injection of IFN-γ diminished the effect of FTY720 on reducing fibrotic scarring. FTY720 treatment inhibited inflammation, decreased lesion size, and promoted neuroprotection and neurological recovery after SCI. These findings demonstrate that the inhibition of T cell-derived IFN-γ by FTY720 suppressed fibrotic scarring and contributed to neurological recovery after SCI.

Antigen-specific reactivation of brain tissue resident memory CD8 T cells directly induces severe neuroinflammation and blood-brain-barrier disruption

Abstract Intracranial infection with neurotropic Theiler’s Murine Encephalomyelitis Virus induces robust generation of brain TRMs against the immunodominant antigen, VP2 121–130, following viral clearance in C57BL/6 mice. While the presence of TRMs in the brain is documented, neurological consequences of TRM reactivation and their causal role in neuropathology remain understudied. We disabled peripheral CD8 T cells via anti CD8 depleting strategies and FTY720 treatment to determine the causal link between brain TRM reactivation and neuropathology. We depleted peripheral T cells and then administrated VP2 peptide IV to reactivate TRMs 60–200 days post infection as this elegant technique isolates responses of brain TRMs away from other branches of memory. We used state-of-the-art flow cytometry and MRI analysis to evaluate neurological consequences of TRM reactivation. TRM reactivation induced severe neuroinflammation in the brain as evident by infiltration of myeloid cells and blasting of T cells. MRI detected gadolinium leakage marking severe blood-brain-barrier (BBB) disruption when brain TRMs were reactivated. Depletion of peripheral CD8 T cells with low-dose anti CD8 antibodies or their sequestration via FTY720 did not alter neuroinflammation indicating brain TRM reactivation causally initiates neuropathology. Confirming this, tissue depletion of all TRMs in the brain using high-dose anti CD8 antibodies prevented both neuroinflammation and neuropathology. Hence, TRM reactivation causes severe neuroinflammation and BBB disruption from within the brain independently of peripheral T cells. Targeting brain TRM reactivation is a novel potential therapeutic for patients with many acute and chronic neurological diseases. Supported by grants from NIH (K99NS117799-01A1 (KA), R01NS103212 (AJJ), and RF1NS122174(AJJ)

Effect of FTY-720 on Pulmonary Fibrosis in Mice via the TGF-β1 Signaling Pathway and Autophagy.

We investigated whether FTY-720 might have an effect on bleomycin-induced pulmonary fibrosis through inhibiting TGF-β1 pathway, and up-regulating autophagy. The pulmonary fibrosis was induced by bleomycin. FTY-720 (1 mg/kg) drug was intraperitoneally injected into mice. Histological changes and inflammatory factors were observed, and EMT and autophagy protein markers were studied by immunohistochemistry and immunofluorescence. The effects of bleomycin on MLE-12 cells were detected by MTT assay and flow cytometry, and the related molecular mechanisms were studied by Western Blot. FTY-720 considerably attenuated bleomycin-induced disorganization of alveolar tissue, extracellular collagen deposition, and α-SMA and E-cadherin levels in mice. The levels of IL-1β, TNF-α, and IL-6 cytokines were attenuated in bronchoalveolar lavage fluid, as well as protein content and leukocyte count. COL1A1 and MMP9 protein expressions in lung tissue were significantly reduced. Additionally, FTY-720 treatment effectively inhibited the expressions of key proteins in TGF-β1/TAK1/P38MAPK pathway and regulated autophagy proteins. Similar results were additionally found in cellular assays with mouse alveolar epithelial cells. Our study provides proof for a new mechanism for FTY-720 to suppress pulmonary fibrosis. FTY-720 is also a target for treating pulmonary fibrosis.

Repurposing drugs against Alzheimer's disease: can the anti-multiple sclerosis drug fingolimod (FTY720) effectively tackle inflammation processes in AD?

Therapeutic approaches providing effective medication for Alzheimer's disease (AD) patients after disease onset are urgently needed. Previous studies in AD mouse models and in humans suggested that physical exercise or changed lifestyle can delay AD-related synaptic and memory dysfunctions when treatment started in juvenile animals or in elderly humans before onset of disease symptoms. However, a pharmacological treatment that can reverse memory deficits in AD patients was thus far not identified. Importantly, AD disease-related dysfunctions have increasingly been associated with neuro-inflammatory mechanisms and searching for anti-inflammatory medication to treat AD seems promising. Like for other diseases, repurposing of FDA-approved drugs for treatment of AD is an ideally suited strategy to reduce the time to bring such medication into clinical practice. Of note, the sphingosine-1-phosphate analogue fingolimod (FTY720) was FDA-approved in 2010 for treatment of multiple sclerosis patients. It binds to the five different isoforms of Sphingosine-1-phosphate receptors (S1PRs) that are widely distributed across human organs. Interestingly, recent studies in five different mouse models of AD suggest that FTY720 treatment, even when starting after onset of AD symptoms, can reverse synaptic deficits and memory dysfunction in these AD mouse models. Furthermore, a very recent multi-omics study identified mutations in the sphingosine/ceramide pathway as a risk factor for sporadic AD, suggesting S1PRs as promising drug target in AD patients. Therefore, progressing with FDA-approved S1PR modulators into human clinical trials might pave the way for these potential disease modifying anti-AD drugs.

Tissue-resident, memory CD8+ T cells are effective in clearing intestinal Eimeria falciformis reinfection in mice.

Eimeria, a cousin of malarial parasites, causes coccidiosis that results in huge losses in the poultry industry. Although live coccidiosis vaccines have been developed and used widely for the successful control of the disease, the mechanism underlying protective immunity remains largely unknown. Using Eimeria falciformis as a model parasite, we observed that tissue-resident memory CD8+ T (Trm) cells accumulated in cecal lamina propria following E. falciformis infection in mice, especially after reinfection. In convalescent mice challenged with a second infection, E. falciformis burden diminished within 48-72h. Deep-sequencing revealed that CD8+ Trm cells were characterized by rapid up-regulation of effector genes encoding pro-inflammatory cytokines and cytotoxic effector molecules. While FTY720 (Fingolimod) treatment prevented the trafficking of CD8+ T cells in peripheral circulation and exacerbated primary E. falciformis infection, such treatment had no impact on the expansion of CD8+ Trm cells in convalescent mice receiving secondary infection. Adoptive transfer of cecal CD8+ Trm cells conferred immune protection in naïve mice, indicating that these cells provide direct and effective protection against infection. Overall, our findings not only explain a protective mechanism of live oocyst-based anti-Eimeria vaccines but also provide a valuable correlate for assessing vaccines against other protozoan diseases.

Transplanted human iPSC-derived vascular endothelial cells promote functional recovery by recruitment of regulatory T cells to ischemic white matter in the brain

BackgroundIschemic stroke in white matter of the brain induces not only demyelination, but also neuroinflammation. Peripheral T lymphocytes, especially regulatory T cells (Tregs), are known to infiltrate into ischemic brain and play a crucial role in modulation of inflammatory response there. We previously reported that transplantation of vascular endothelial cells generated from human induced pluripotent stem cells (iVECs) ameliorated white matter infarct. The aim of this study is to investigate contribution of the immune system, especially Tregs, to the mechanism whereby iVEC transplantation ameliorates white matter infarct.MethodsiVECs and human Tregs were transplanted into the site of white matter lesion seven days after induction of ischemia. The egress of T lymphocytes from lymph nodes was sequestered by treating the animals with fingolimod (FTY720). The infarct size was evaluated by magnetic resonance imaging. Immunohistochemistry was performed to detect the activated microglia and macrophages, T cells, Tregs, and oligodendrocyte lineage cells. Remyelination was examined by Luxol fast blue staining.ResultsiVEC transplantation reduced ED-1+ inflammatory cells and CD4+ T cells, while increased Tregs in the white matter infarct. Treatment of the animals with FTY720 suppressed neuroinflammation and reduced the number of both CD4+ T cells and Tregs in the lesion, suggesting the importance of infiltration of these peripheral immune cells into the lesion in aggravation of neuroinflammation. Suppression of neuroinflammation by FTY720 per se, however, did not promote remyelination in the infarct. FTY720 treatment negated the increase in the number of Tregs by iVEC transplantation in the infarct, and attenuated remyelination promoted by transplanted iVECs, while it did not affect the number of oligodendrocyte lineage cells increased by iVEC transplantation. Transplantation of Tregs together with iVECs into FTY720-treated ischemic white matter did not affect the number of oligodendrocyte lineage cells, while it remarkably promoted myelin regeneration.ConclusionsiVEC transplantation suppresses neuroinflammation, but suppression of neuroinflammation per se does not promote remyelination. Recruitment of Tregs by transplanted iVECs contributes significantly to promotion of remyelination in the injured white matter.

FTY720 Attenuates LPS-Induced Inflammatory Bone Loss by Inhibiting Osteoclastogenesis via the NF-κB and HDAC4/ATF Pathways.

Osteoclast (OC) abnormalities lead to many osteolytic diseases, such as osteoporosis, inflammatory bone erosion, and tumor-induced osteolysis. Exploring effective strategies to remediate OCs dysregulation is essential. FTY720, also known as fingolimod, has been approved for the treatment of multiple sclerosis and has anti-inflammatory and immunosuppressive effects. Here, we found that FTY720 inhibited osteoclastogenesis and OC function by inhibiting nuclear factor kappa-B (NF-κB) signaling. Interestingly, we also found that FTY720 inhibited osteoclastogenesis by upregulating histone deacetylase 4 (HDAC4) expression levels and downregulating activating transcription factor 4 (ATF4) expression levels. In vivo, FTY720 treatment prevented lipopolysaccharide- (LPS-) induced calvarial osteolysis and significantly reduced the number of tartrate-resistant acid phosphatase- (TRAP-) positive OCs. Taken together, these results demonstrate that FTY720 can inhibit osteoclastogenesis and ameliorate inflammation-induced bone loss. Which may provide evidence of a new therapeutic target for skeletal diseases caused by OC abnormalities.

Intranasal Vaccination with rePcrV Protects against Pseudomonas aeruginosa and Generates Lung Tissue-Resident Memory T Cells.

Tissue-resident memory T (TRM) cells are immune sentinels that bear a key role in the local immune system and rapidly respond to infection. Our previous studies showed that mucosal immunization via intranasal pathways was more effective than intramuscular route. However, the mechanism of enhanced protective immunity remains unclear. Here, we formulated a Pseudomonas aeruginosa vaccine composed of type III secretion protein PcrV from P. aeruginosa and curdlan adjuvant and then administered by the intranasal route. Flow cytometry and immunofluorescence staining showed that the ratio of CD44+CD62L-CD69+CD4+ TRM cells induced by this vaccine was significantly increased, and IL-17A production was notably enhanced. Further analysis revealed that vaccinated mice can protect against the P. aeruginosa challenge even after administration with FTY720 treatment. What is more, our results showed that CD4+ TRM might be involved in the recruitment of neutrophils and provided partial protection against Pseudomonas aeruginosa. Taken together, these data demonstrated that CD4+ TRM cells were elicited in lung tissues after immunization with rePcrV and contributed to protective immunity. Furthermore, it provided novel strategies for the development of vaccines for P. aeruginosa and other respiratory-targeted vaccines.

FTY720 Reduces Lipid Accumulation by Upregulating ABCA1 through Liver X Receptor and Sphingosine Kinase 2 Signaling in Macrophages

Formation of foam cells as a result of excess lipid accumulation by macrophages is a pathological hallmark of atherosclerosis. Fingolimod (FTY720) is an immunosuppressive agent used in clinical settings for the treatment of multiple sclerosis and has been reported to inhibit atherosclerotic plaque development. However, little is known about the effect of FTY720 on lipid accumulation leading to foam cell formation. In this study, we investigated the effects of FTY720 on lipid accumulation in murine macrophages. FTY720 treatment reduced lipid droplet formation and increased the expression of ATP-binding cassette transporter A1 (ABCA1) in J774 mouse macrophages. FTY720 also enhanced the expression of liver X receptor (LXR) target genes such as FASN, APOE, and ABCG1. In addition, FTY720-induced upregulation of ABCA1 was abolished by knockdown of sphingosine kinase 2 (SphK2) expression. Furthermore, we found that FTY720 treatment induced histone H3 lysine 9 (H3K9) acetylation, which was lost in SphK2-knockdown cells. Taken together, FTY720 induces ABCA1 expression through SphK2-mediated acetylation of H3K9 and suppresses lipid accumulation in macrophages, which provides novel insights into the mechanisms of action of FTY720 on atherosclerosis.

Targeting Extracellular Vesicle Secretion in Combination with Venetoclax Synergistically Induces Apoptosis in FLT3-ITD+ AML

Introduction Intrinsic or acquired resistance to apoptosis is a hallmark of human cancers, including different subtypes of acute myeloid leukemia (AML). Among different mechanisms of achieving resistance, disposal of pro-apoptotic factors via extracellular vesicle (EV) release has been described for several cancer models. In AML, RNA and protein trafficking by leukemia-derived EVs has emerged as a mediator of cell-cell communication and a crucial factor in reprogramming the bone marrow (BM) niche into a leukemia permissive microenvironment. In our study, we aimed to characterize the content of primary AML-derived EVs with a special focus on apoptotic factors, and to evaluate the effect of EV release inhibition with FTY720, a FDA approved oral drug for treatment of multiple sclerosis, on apoptosis in combination with venetoclax (VEN) treatment, an inhibitor of the anti-apoptotic protein Bcl-2. Methods BM-derived EVs were purified from healthy donors (n=3), or patients with newly diagnosed AML (FLT3-WT and -ITD, n=10) after written informed consent and approval by the ethics committee in accordance with the Declaration of Helsinki. EVs were characterized by transmission electron microscopy (TEM), western blotting and nano tracking analysis (NTA). Protein and RNA from EVs were isolated and used for proteomic and RNA analyses. The apoptotic response of FLT3-WT (OCI-AML2, OCI-AML3) or FLT3-ITD (MOLM-13, MV4-11) AML cell lines and primary samples to EV release inhibitor FTY720 was assessed by flow cytometry with AnnexinV/PI. FTY720-induced EV release inhibition and intracellular EV accumulation was determined by Western Blot analysis of EV markers and visualized by immunostaining. Finally, the combination treatment of FTY720 with VEN was tested. Results TEM and immunoblotting confirmed a pure vesicle fraction (30-150 nm) with expression of characteristic markers enriched (CD63, CD81, HSP70) or absent (GAPDH, ß-actin) in EVs. NTA showed no differences in EV numbers released by AML compared to healthy cells. However, AML EVs were significantly larger in size. EV content was significantly different comparing healthy to AML-derived EVs and bioinformatic analysis from RNA sequencing revealed enrichment in pathways involved in regulation of apoptosis specifically in AML FLT3-ITD EVs (Figure 1). Of note, hsa-miR-196b-5p target interaction analysis identified BCL-2 as an exemplary anti-apoptotic target gene (Figure 2). Concordantly, AML cell lines harboring FLT3-ITD showed higher sensitivity to the EV release inhibitor FTY720 compared to FLT3-WT (IC50 = 6.2µM, 6.6µM, 13.7µM and 10.2µM respectively for MOLM-13, MV4-11, OCI-AML2 and OCI-AML3). FTY720 treatment induced intracellular EV accumulation and modulated EV marker expression confirming its effect on the EV machinery. Interestingly, FTY720 co-treatment potentiated apoptosis induced by VEN alone in both FLT3-ITD cell lines while FTL3-WT cells were still resistant to VEN and healthy hematopoiesis was unaffected. Conclusions Taken together, these results suggest that FLT3-ITD AML cells establish a protective mechanism against apoptosis by disposing of positive regulators of apoptosis via EV release. Preventing the disposal of the pro-apoptotic factors in combination with BCL-2 inhibition sensitizes AML FLT3-ITD cells to apoptosis. Additional evaluation in primary patient material and further studies to identify the factors responsible for sensitization to apoptosis upon EV release inhibition are ongoing. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Limited impact of fingolimod treatment during the initial weeks of ART in SIV-infected rhesus macaques

Antiretroviral therapy (ART) is not curative due to the persistence of a reservoir of HIV-infected cells, particularly in tissues such as lymph nodes, with the potential to cause viral rebound after treatment cessation. In this study, fingolimod (FTY720), a lysophospholipid sphingosine-1-phosphate receptor modulator is administered to SIV-infected rhesus macaques at initiation of ART to block the egress from lymphoid tissues of natural killer and T-cells, thereby promoting proximity between cytolytic cells and infected CD4+ T-cells. When compared with the ART-only controls, FTY720 treatment during the initial weeks of ART induces a profound lymphopenia and increases frequencies of CD8+ T-cells expressing perforin in lymph nodes, but not their killing capacity; FTY720 also increases frequencies of cytolytic NK cells in lymph nodes. This increase of cytolytic cells, however, does not limit measures of viral persistence during ART, including intact proviral genomes. After ART interruption, a subset of animals that initially receives FTY720 displays a modest delay in viral rebound, with reduced plasma viremia and frequencies of infected T follicular helper cells. Further research is needed to optimize the potential utility of FTY720 when coupled with strategies that boost the antiviral function of T-cells in lymphoid tissues.