Journal: Rheumatology (Oxford, England)

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

doi: 10.1093/rheumatology/keae320

Figure Lengend Snippet: Figure 1. FTY720 alleviated renal injury in EAV rats. (A, B) Representative images of glomeruli (A) and tubulointerstitium (B) of PAS staining for kidneys of rats (scale bar: 100 mm). (C, D) Representative images of glomerular (C) and tubulointerstitial (D) neutrophil infiltration in EAV rats (scale bar: 50 mm). (E, F) Amelioration of haematuria (E) and proteinuria (F) of EAV rats following FTY720 treatment. (G, H) Assessment of crescent formation (G) and TIN score (H) in EAV rats following FTY720 treatment. (I, J) Quantification of infiltrating neutrophils in glomeruli (I) and tubulointerstitium (J). Data are presented as means (S.D.) from five rats per group. P < 0.05, P < 0.01, P < 0.001; ns: not significant. DAPI: 40,6-diamidino-2-phenylindole; EAV: experimental autoimmune vasculitis; HSA: human serum albumin; MPO: myeloperoxidase; PAS: periodic acid–Schiff; TIN: tubulointerstitial nephritis

Article Snippet: To explore the pathway in FTY720-induced inhibition of ANCA-medicated neutrophil activation, cells were preincubated with 10 mM PPARα antagonist GW6471 (S2798, Selleck Chemicals, Houston, TX, USA), or 100 nM PPARα agonist GW7647 (HY-13861, MedChem Express, Monmouth Junction, NJ, USA) for 12 h and 3 mM CPT1 inhibitor etomoxir (HY-50202, MedChemExpress) for 30 min before treatment with FTY720 [25–28].

Techniques: Staining

Journal: Rheumatology (Oxford, England)

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

doi: 10.1093/rheumatology/keae320

Figure Lengend Snippet: Figure 2. RNA sequencing analysis and gene enrichment analysis of renal cortex from rats. (A, B) Heat map (A) and scatter plot (B) presenting the distribution of DEGs between vehicle-treated and FTY720-treated groups. Fold change >2.0 or <0.5, P < 0.05. (C) A cluster profiler of the 10 most prominently enriched pathways identified in RNA sequencing and gene enrichment analysis between vehicle-treated and FTY720-treated groups. (D, F) Gene set enrichment analysis of FAO (D), PPAR signalling pathway (E) and respiratory burst pathway (F). FTY represents EAV rats treated with FTY720; AAV represents EAV rats treated with vehicle. AAV: ANCA-associated vasculitis; DEG: differentially expressed gene; FAO: fatty acid oxidation; FC: fold change; FDR: false discovery rate; NES: normalized enrichment score; PPAR: peroxisome proliferator activated receptor

Article Snippet: To explore the pathway in FTY720-induced inhibition of ANCA-medicated neutrophil activation, cells were preincubated with 10 mM PPARα antagonist GW6471 (S2798, Selleck Chemicals, Houston, TX, USA), or 100 nM PPARα agonist GW7647 (HY-13861, MedChem Express, Monmouth Junction, NJ, USA) for 12 h and 3 mM CPT1 inhibitor etomoxir (HY-50202, MedChemExpress) for 30 min before treatment with FTY720 [25–28].

Techniques: RNA Sequencing

Journal: Rheumatology (Oxford, England)

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

doi: 10.1093/rheumatology/keae320

Figure Lengend Snippet: Figure 4. FTY720 inhibited ANCA-induced neutrophil activation while upregulating FAO. (A) Identification of increased neutrophil marker CD11b of differentiated HL-60 cells. (B) Inhibitory effect of FTY720 on respiratory burst of differentiated HL-60 cells stimulated with serum from AAV patients. (C) Inhibitory effect of FTY720 on respiratory burst of isolated human neutrophils activated by MPO-ANCA positive IgG. (D) Gene expression of the top six upregulated genes in FAO in RNA sequencing was measured in differentiated HL-60 cells treated with the 10% serum from AAV patients and FTY720 or vehicle. (E) mRNA level of PPARα and protein expression of CPT1a and PPARα in differentiated HL-60 cells treated with the 10% serum from AAV patients and FTY720 or vehicle. Data are shown as means (S.D.). P < 0.05, P < 0.01, P < 0.001; ns: not significant. AAV: ANCA-associated vasculitis; ABCD3: ATP binding cassette subfamily D member 3; CPT1a: carnitine palmitoyltransferase 1A; ECHDC2: enoyl-CoA hydratase domain containing 2; FAO: fatty acid oxidation; GAPDH: glyceraldehyde 3-phosphate dehydrogenase; HSD17b4: 17β-hydroxysteroid dehydrogenase 4; MFI: mean fluorescence intensity; PPARα: peroxisome proliferator activated receptor α; ROS: reactive oxygen species; SESN2: sestrin 2; SLC27A2: solute carrier family 27 member 2

Article Snippet: To explore the pathway in FTY720-induced inhibition of ANCA-medicated neutrophil activation, cells were preincubated with 10 mM PPARα antagonist GW6471 (S2798, Selleck Chemicals, Houston, TX, USA), or 100 nM PPARα agonist GW7647 (HY-13861, MedChem Express, Monmouth Junction, NJ, USA) for 12 h and 3 mM CPT1 inhibitor etomoxir (HY-50202, MedChemExpress) for 30 min before treatment with FTY720 [25–28].

Techniques: Activation Assay, Marker, Isolation, Gene Expression, RNA Sequencing, Expressing, Binding Assay, Fluorescence

Journal: Rheumatology (Oxford, England)

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

doi: 10.1093/rheumatology/keae320

Figure Lengend Snippet: Figure 5. FTY720 exerted an inhibitory effect on ANCA-induced neutrophil activation via the PPARα–CPT1a pathway. (A) Determination of CPT1a- knockdown efficiency by qPCR and western blot. (B, C) The inhibitory effect of FTY720 on respiratory burst in differentiated HL-60 cells upon stimulation with 10% serum from AAV patients was reversed by CPT1 antagonist etomoxir (B) and CPT1a knockdown (C). (D, E) The effect of GW6471 (a PPARα antagonist) on the inhibition of FTY720 on respiratory burst (D) and NET formation (E). (F, G) The effect of combination of GW7647 (a PPARα agonist) and etomoxir (a CPT1 inhibitor) on the inhibition of FTY720 on respiratory burst (F) and NET formation (G). (H) Representative images of NET formation with or without FTY720, etomoxir, GW6471 (scale bar: 50 mm). Data are shown as means (S.D.). P < 0.05, P < 0.01, P < 0.001; ns: not significant. AAV: ANCA-associated vasculitis; CPT1a: carnitine palmitoyltransferase 1A; Eto: etomoxir; GAPDH: glyceraldehyde 3-phosphate dehydrogenase; MFI: mean fluorescence intensity; PPARα: peroxisome proliferator activated receptor α; ROS: reactive oxygen species

Article Snippet: To explore the pathway in FTY720-induced inhibition of ANCA-medicated neutrophil activation, cells were preincubated with 10 mM PPARα antagonist GW6471 (S2798, Selleck Chemicals, Houston, TX, USA), or 100 nM PPARα agonist GW7647 (HY-13861, MedChem Express, Monmouth Junction, NJ, USA) for 12 h and 3 mM CPT1 inhibitor etomoxir (HY-50202, MedChemExpress) for 30 min before treatment with FTY720 [25–28].

Techniques: Activation Assay, Knockdown, Western Blot, Inhibition, Fluorescence

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