chemical treatments fty720  (Tocris)

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: Mucosal Immunology

Article Title: IL-1α is required for T cell-driven weight loss after respiratory viral infection

doi: 10.1016/j.mucimm.2024.02.005

Figure Lengend Snippet: Timing of T cell recruitment to lungs affects disease timing in RSV. 6-7 weeks old BALB/c SPF mice were intranasally infected 7.7 x 10 6 PFU/ml RSV A2 subtype and culled on day 1, 3, 5, and 7 post infection. Body weight (A) and food consumed (B) were monitored daily. Immune cell populations were measured using flow cytometry (C) at each time course, and live viral plaques (D) and RSV L gene (E) were quantified. 6-7 weeks old BALB/c SPF mice were injected with 25µg of FTY720 or PBS only daily from day -2 to day 6. Mice were intranasally infected with 7.7 x 10 6 PFU/ml RSV infection on day 0. Body weight (F) and food (G) were monitored daily from day -2 to day 14. At day 7, viral load was determined using RSV L gene qPCR on RNA extracted from the left lung lobe (H) and flow cytometry was used to analyze the number of CD8 T cell (I), CD4 T cells (J) and antigen-specific CD8 + T cells (K). At day 14, viral load was determined using RSV L gene qPCR (L) and flow cytometry was used to analyze the number of CD8 T cell (M), CD4 T cells (N) and antigen-specific CD8 T cells (O). N = 5, each dot represents an individual mouse (H-O); or mean +/-SEM –A - E, F-G). Significance calculated by ordinary one-way ANOVA and post test. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001. Experiment was repeated twice.

Article Snippet: For FTY720 treatment, FTY720 (6176, BioTechne,Minneapolis, MN, USA) was diluted in PBS to a final concentration of 25μg in 250 μl PBS.

Techniques: Infection, Flow Cytometry, Injection

Journal: Neuron

Article Title: Astrocyte growth is driven by the Tre1/S1pr1 phospholipid-binding G protein-coupled receptor

doi: 10.1016/j.neuron.2023.11.008

Figure Lengend Snippet: (A) Images of spinal cord astrocyte membrane labeled with myrGFP (green) and nuclei labeled with H2AmCherry (magenta) in 6 dpf Tg(slc1a3b:myrGFP-P2A-H2AmCherry) transgenic control (N=15, dorsal view; N=3 lateral view) and s1pr1vo88/vo88 mutants (N=17, dorsal view; N=5, lateral view). N, number of animals. Scale bar, 20 µm. (B) Images of sparsely labeled individual astrocytes by slc1a3b:myrGFP-P2A-H2Amcherry DNA constructs and the representative IMARIS 3D-rendering surface (grey) at 6 dpf in the spinal cord of control and s1pr1vo88/vo88 mutant zebrafish. Scale bar, 20 µm. (C) Quantification of individual astrocyte volumes in control (N=18) and s1pr1vo88/vo88 mutants (N=23) at 6 dpf, related to (B). N, number of animals. Data points represent single astrocytes. ****, p<0.0001; unpaired t test. Error bars, mean values ± S.D. (D) Images of 6 dpf Tg(slc1a3b:myrGFP-P2A-H2AmCherry) larval brain in control (N=5) and s1pr1vo88/vo88 mutants (N=6). Dashed lines mark astrocyte processes densely infiltrated neuropil in the forebrain, midbrain, and hindbrain. N, number of animals. Scale bar, 50 µm. (E) Time-lapse still images of astrocyte process dynamics labeled with myrGFP (green) in control and s1pr1vo88/vo88 mutants at 3 dpf. Dashed boxes mark the regions shown to the right. Scale bar, 20 µm. (F) Quantification of astrocyte individual process extension and retraction displacement speed in control (N=10) and s1pr1vo88/vo88 mutants (N=8) at 3 dpf. N, number of animals. Data points represent single astrocyte processes tracked. *, p<0.05; ***, p<0.001; unpaired t test. Error bars, mean values ± S.D. (G and I) Images of 6 dpf Tg(slc1a3b:myrGFP-P2A-H2AmCherry) transgenic larval spinal cord astrocytes after treatment with DMSO, 1 µM FTY720, or 1 µM Ex26 at 2–4 dpf (G) or 4–6 dpf (I). Dashed boxes represent 4 independent 10 µm x 10 µm areas in the astrocyte process-enriched regions were used to quantify the GFP coverage area percentage. Scale bar, 20 µm. (H) Quantification of relative GFP coverage area percentage at 6 dpf in the astrocyte process-enriched regions in DMSO (N=12), FTY720 (N=12), and Ex26 (N=12) after 2–4 dpf treatment. N, number of animals. Data points represent average GFP coverage area percentage of the 4 independent areas in a single fish. ****, p<0.0001; one-way ANOVA with multiple comparisons. (J) Quantification of relative GFP coverage area percentage at 6 dpf in the astrocyte process-enriched regions in DMSO (N=10), FTY720 (N=12), and Ex26 (N=11) after 4–6 dpf treatment. N, number of animals. Data points represent average GFP coverage area percentage of the 4 independent areas in a single fish. ****, p<0.0001; one-way ANOVA with multiple comparisons. See also Figure S7 and Video S2.

Article Snippet: Acquired time-lapse images were Airyscan processed, and followed by Bleach correction and 3D drift correction using Fiji (ImageJ) software before analysis. . Chemical treatments FTY720 and Ex26 (Tocris Bioscience) were dissolved in DMSO to a stock concentration of 100 mM.

Techniques: Membrane, Labeling, Transgenic Assay, Control, Construct, Mutagenesis