Journal: Journal of leukocyte biology

Article Title: Rapid externalization of 27-kDa heat shock protein (HSP27) and atypical cell death in neutrophils treated with the sphingolipid analog drug FTY720.

doi: 10.1189/jlb.3VMA1114-522RR

Figure Lengend Snippet: Figure 5. RIP1K and HSP90 are involved in neutrophil death induced by FTY720. (A and B) Flow cytometric quantification of Annexin V+PI+, Annexin V+PI2, and HSP27+ neutrophils after preincubation or not with necrostatin-1 (20 mM) or Z-IETD-FMK (20 mM) for 30 min and treatment or not with FTY720 (10 mM) for 3 h. Data are from 7 independent experiments. *P , 0.05; **P , 0.01; ***P , 0.001. (C and D) Neutrophil lysates analyzed by Western blotting for caspase-8 cleavage or MLKL phosphorylation after cell death induc- tion by FTY720. Where indicated, Q-VD-OPh (20 mM), necrostatin-1 (20 mM), DPI (20 mM), or NSA (5 mM) was added for 30 min before stimulation. GAPDH analysis was performed as loading control. The results of 1 representative experiment out of 4 are presented. (E) Flow cytometric quantification of HSP27+ neutrophils incubated for 30 min in the presence of geldanamycin (10 mM), DMAG (20 mM), or radicicol (10 mM) before treatment with FTY720 (10 mM) for 3 h. Data are from 7 independent experiments. *P , 0.05; **P , 0.01.

Article Snippet: Selective antagonists of S1PRs [JTE013, VPC23019, W146, or FTY720 (S)-phosphate; all from Santa Cruz Biotechnology] were added to neutrophils for 1 h and washed out before FTY720 treatment.

Techniques: Western Blot, Phospho-proteomics, Control, Incubation

Journal: Frontiers in Immunology

Article Title: FTY720 Regulates Mitochondria Biogenesis in Dendritic Cells to Prevent Kidney Ischemic Reperfusion Injury

doi: 10.3389/fimmu.2020.01278

Figure Lengend Snippet: FTY720 treatment induces less immunogenic DCs that have higher mitochondria numbers. (A) 8 day old Veh-DC and FTY-DC were labeled with MitoTracker CMXRos (50 nM). Scale bar, 20 μm. (B) Veh-DC and FTY-DC treated with 100 ng/ml LPS for 24 h or left unstimulated (unstim). Levels of MitoSox (5 μM) and MitoTracker green (100 nM) were measured after 24 h of LPS treatment. (D–E) DCs were seeded in a Seahorse XF-24e analyzer, stimulated with and without LPS for 24 h, and oxygen consumption rate (OCR) was determined during sequential treatments with Oligomycin , FCCP and antimycin A plus rotenone . Quantification of basal OCR and ATP production. (F) Flow cytometry analysis of iNOS expression was determined with and without LPS stimulation. Quantification of percent of iNOS in Veh-DC and FTY-DC after LPS. (G) Nitrite levels were determined in culture supernatants with and without LPS stimulation. mRNA levels of Pgc1a (C) , Il1b (H) , Il10 (I) , and Tnfa (J) in Veh-DC and FTY-DC treated with 100 ng/ml LPS or left unstimulated for 24 h. (K) ELISA of TNFα from the Veh-DC and FTY-DC treated with 100 ng/ml LPS for 24 h. * p ≤ 0.05, ** p ≤ 0.01, and *** p ≤ 0.001, one-way ANOVA followed by Tukey's post-test. Data represent means ± SEM of triplicates. One of three experiments is shown.

Article Snippet: BMDC were treated with 1 μM FTY720 (total of 4 treatments) that was purchased from Cayman Chemicals (Ann Arbor, Michigan).

Techniques: Labeling, Flow Cytometry, Expressing, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Immunology

Article Title: FTY720 Regulates Mitochondria Biogenesis in Dendritic Cells to Prevent Kidney Ischemic Reperfusion Injury

doi: 10.3389/fimmu.2020.01278

Figure Lengend Snippet: FTY-DC require S1pr1 on BMDC to protect kidneys from IRI. (A) Protocol for experimental setup. FTY720 is a ligand for four out of five S1P receptors. Plasma creatinine (PCr, mg/dL) was measured 24 h after IRI. We tested if S1pr1 or S1pr3 were requited for FTY720 dependent regulatory DC phenotype. BMDCs were propagated from either C57BL/6 WT, CD11cCreS1pr1 fl / fl ( S1pr1 −/− DC), or S1pr3 −/− and treated with FTY720. FTY- S1pr1 −/− DC do not protect kidneys from IRI. As demonstrated in our earlier published studies and again confirmed, transfer of S1pr3 −/− DC with or without FTY720 significantly protect kidney from IRI. These studies suggest only S1pr1 are necessary for FTY720 dependent regulatory DC phenotype. Next, we tested if the route of delivery was important for FTY-DC induced protection from kidney IRI. (B) Protocol for experimental setup. Plasma creatinine was measured 24 h after IRI. FTY-DC were injected either intravenous (i.v.), intraperitoneal (i.p.), subcutaneous (s.c.) or as i.v. using Veh-DC that were acutely treated with FTY720 (overnight along with LPS). These studies suggest that FTY-DC only protect kidneys from IRI if injected via i.v. and the FTY-DCs must be propagated in presence of FTY720 at start of the BMDC culture. ** p ≤ 0.01. (C) Protocol for experimental setup. Plasma creatinine was measured 24 h after IRI. In all studies presented we injected FTY-DC 1 day before kidney IRI. We also tested if FTY-DC could be injected after injury. Mice were treated with either NC, Veh-DC or FTY-DC 4 h after bilateral IRI. Mice injected with Veh-DC had higher PCr compared to NC mice and FTY-DC treated mice were significantly protected. (D) Protocol for experimental setup. Plasma creatinine (mg/dL) was measured 24 h after IRI. C57BL/6 FTY-DC induce protection in BALB/c mice and protect kidneys from ischemic injury. Data represent means ± SEM, Unpaired t -test [D], *** p ≤ 0.001; ** p ≤ 0.01 and *** p ≤ 0.001, one-way ANOVA followed by Tukey's post-test.

Article Snippet: BMDC were treated with 1 μM FTY720 (total of 4 treatments) that was purchased from Cayman Chemicals (Ann Arbor, Michigan).

Techniques: Clinical Proteomics, Injection

Journal: Cancer Gene Therapy

Article Title: β3-adrenergic receptor on tumor-infiltrating lymphocytes sustains IFN-γ-dependent PD-L1 expression and impairs anti-tumor immunity in neuroblastoma

doi: 10.1038/s41417-023-00599-x

Figure Lengend Snippet: A Representative cytofluorimetric plots and relative quantification of PD-L1 expression on N2A tumor cells treated in vitro with IFN-γ (100 ng/mL), TNF-α (100 ng/mL) in presence or not of SR59230A (1 µM) for 48 h. B Immunofluorescence and relative quantification of PD-L1 staining on N2A tumor cells co-cultured with lymphocytes obtained from TDLNs of mice treated for 2 days with vehicle or SR59230A. C IFN-γ quantification in co-culture medium after 48 h of tumor cells-lymphocytes co-culture. D Representative cytofluorimetric plots and relative quantification of PD-L1 expression on N2A tumor cells co-cultured with lymphocytes obtained from TDLNs of mice treated for 2 days with vehicle or SR59230A. E Representative cytofluorimetric plots and relative quantification of PD-L1 expression on N2A tumor cells co-cultured with lymphocytes silenced for different β-AR subtypes. F Representative images of tumor mass and relative G tumor growth and H tumor weight of NB obtained from A/J mice inoculated with N2A tumor cells, and treated with SR59230A in presence or not of FTY720. Data are expressed as mean ± SEM. A – E ( n = 3 per group); F – H ( n = 6 per group). Significance was calculated by one-way ( A – E , H ) or two-way ( G ) ANOVA analysis followed by Bonferroni’s post hoc test. ** P < 0.01, *** P < 0.001, **** P < 0.0001 vs Vehicle; ## P < 0.01, ### P < 0.001, #### P < 0.0001 N2A + Lymph (SR) vs N2A + Lymph (Veh); $$ P < 0.01, $$$ P < 0.001 SR59230A vs SR59230A + FTY720; ns not significant.

Article Snippet: Treatment with FTY720 (#6176, Tocris Biotechne) was administered at 2 mg/kg per os (p.o.) from day 6.

Techniques: Quantitative Proteomics, Expressing, In Vitro, Immunofluorescence, Staining, Cell Culture, Co-Culture Assay