Journal: The Journal of Experimental Medicine

Article Title: Tissue-specific pathways extrude activated ILC2s to disseminate type 2 immunity

doi: 10.1084/jem.20191172

Figure Lengend Snippet: Circulating ILC2s phenotypically differ during the course of N. brasiliensis infection. (A – D) Arg1 Yarg Il5 Red5 Il13 Sm13 (YRS) triple-reporter mice were infected with N. brasiliensis and analyzed at the indicated time points. Uninfected mice (Naive) were examined as controls. (A) Expression of KLRG1 and IL-5 by cells gated on CD45 + Lin − cells isolated from peripheral blood (left) or small intestine lamina propria and lung (right). Expression of ST2 and IL-17RB or Arg1 by ILC2s gated as indicated in the top panels. (B and C) Frequencies of KLRG1 + IL-5 (Red5) + ILC2s (B) and percentages of IL-13 reporter–positive ILC2s (C) in peripheral blood. DL, detection limit. (D) Flow cytometry plots of blood (left) or lung (right) ILC2s gated on CD45 + Lin − IL-5 + cells from d5 or d12, highlighting activation as assessed by IL-13 (Sm13) expression and the expression of ST2, Arg1 (Yarg), and IL-17RB. (E) Serum IL-13 levels from WT C57BL/6J mice infected with N. brasiliensis treated with or without FTY720 (FTY) and analyzed at the indicated time points. The x axis represents days after infection. Data displayed as mean ± SEM. u.i., uninfected. Data are from one experiment representative of at least two independent experiments (A, D, and E) or pooled from multiple independent experiments (B and C). *, P < 0.05.

Article Snippet: FTY720 (Tocris) was dissolved in normal saline.

Techniques: Infection, Expressing, Isolation, Flow Cytometry, Activation Assay

Journal: The Journal of Experimental Medicine

Article Title: Tissue-specific pathways extrude activated ILC2s to disseminate type 2 immunity

doi: 10.1084/jem.20191172

Figure Lengend Snippet: Circulating ILC2s phenotypically differ during the course of N. brasiliensis infection. (A) Arg1 Yarg Il5 Red5 Il13 Sm13 (YRS) triple-reporter mice were infected with N. brasiliensis, and blood (left panels) and lungs (right panels) were analyzed on d5 after infection for IL-5 (Red5) and IL-13 (Sm13) expression in ILCs (gated on CD45 + Lin − ) and CD4 T cells (gated on CD45 + CD4 + ). (B) Mice were infected with N. brasiliensis in the presence or absence of FTY720 (1 mg/kg), and blood (top panels) and lung (bottom panels) ILC2s (gated on CD45 + Lin − GATA3 + ) were examined on d5 or d12 for IL-13 (Sm13) expression and Ki-67 labeling. (C and D) Mice were infected with N. brasiliensis and were i.v. injected with 3 µg of APC-Cy7–labeled CD45.2 antibody on d5 and euthanized 3 min later. (C) Top panels indicate the intravascular (IV) or tissue-resident (Tissue) ILC2s gated on CD45 + Lin − IL5 (Red5) + from the blood, lung, or mLNs (mesLN). Middle and bottom panels show the expression of ST2 and IL-17RB (middle panels) or ST2 and CD69 (bottom panels) from the intravascular or tissue-resident fraction from tissues as indicated. (D) Percentage of tissue and blood (IV) ILC2s in the lung on d5. Data are from one experiment representative of at least two independent experiments. *, P < 0.05; **, P < 0.005. ns, no significant difference.

Article Snippet: FTY720 (Tocris) was dissolved in normal saline.

Techniques: Infection, Expressing, Labeling, Injection

Journal: The Journal of Experimental Medicine

Article Title: Tissue-specific pathways extrude activated ILC2s to disseminate type 2 immunity

doi: 10.1084/jem.20191172

Figure Lengend Snippet: Circulating ILC2s are dependent on tissue extrusion. (A–H) C57BL/6J mice were infected with N. brasiliensis , injected i.p. with saline or FTY720 (1 mg/kg daily from day of infection), and analyzed at the indicated time points. (A) Gating of ILC2s (pregated on live CD45 + Lin − ) from blood or lung. (B) Total number of ILC2s in the blood (left) or lung (right) at d5. (C–E) Flow cytometry plots showing the expression of ST2 and IL-17RB on blood and lung ILC2s from d5 (C) and d12 (E) and quantification of the percentage of IL-17RB + ST2 − ILC2s in the blood and lung on d5 (D). (F) Total number of ILC2s in the blood (left) or lung (right) at d12. Data are representative of two independent experiments with n ≥ 3 individual mice per group. *, P < 0.05; **, P < 0.005; ***, P < 0.0005; ****, P < 0.0001. N.b. , N. brasiliensis ; ns, no significant difference.

Article Snippet: FTY720 (Tocris) was dissolved in normal saline.

Techniques: Infection, Injection, Saline, Flow Cytometry, Expressing

Journal: Journal of Clinical Investigation

Article Title: FTY720 reactivates cryptococcal granulomas in mice through S1P receptor 3 on macrophages

doi: 10.1172/jci136068

Figure Lengend Snippet: Figure 2. BAF212 increases survival in mice after primary infection with WT C.neoformans H99. (A) Mice received compound daily via gavage (FTY720, n = 8; BAF312, n = 8; or vehicle control [H2O], n = 8) starting 2 days before intranasal infection with C. neoformans H99 and survival of mice was monitored. Survival curves were compared using the log-rank (Mantel- Cox). **P = 0.0083. (B) After 40 days of daily compound administration, BAF312 mice that survived were euthanized and analyzed for CFUs in the lung and the brain, n = 4. All error bars represent SEM. (C) NBD-FTY720 was added to the media of either mammalian cells (J774A.1) or C. neoformans cells (Δgcs1 and H99) at a concentration of 2 μg/mL. NBD-FTY720 was extracted from the media and evaluated via thin layer chromatography, n = 3 independent experiments.

Article Snippet: FTY720 and BAF312 were provided by Novartis Pharma AG whereas NBDFTY720 (Cayman Chemicals), FTY720P (Echelon or Cayman Chemicals), CYM5541 (Cayman Chemicals), TY52156 (Tocris), CAY10444 (Cayman Chemicals), U73122 (Tocris), Manoalide (Santa Cruz Biotechnology), and LY294002 (Cayman Chemicals) were purchased.

Techniques: Infection, Control, Concentration Assay, Thin Layer Chromatography

Journal: Journal of Clinical Investigation

Article Title: FTY720 reactivates cryptococcal granulomas in mice through S1P receptor 3 on macrophages

doi: 10.1172/jci136068

Figure Lengend Snippet: Figure 3. FTY720 and BAF312 both cause a decrease in T cells and innate immune cells in the lung and blood. Mice were infected for 30 days with C. neoformans Δgcs1 before FTY720, BAF312, or vehicle control (H2O) administration. For the blood analysis, n = 4 mice per group at each time point and for lungs, n = 4 mice per group at each time point (1 day before compound administration [day –1], day 5, and day 20 after compound administration). Experi- ment was conducted 2 times. For lung samples, leukocytes were labeled intravascularly to distinguish between cells in the lung parenchyma and vascula- ture before staining to identify immune cell populations. Statistical significance was determined using 2-way repeated measures ANOVA with Bonferroni’s multiple comparisons post hoc test. P values were corrected for multiplicity using the Bonferroni’s adjustment. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 compared with the control. Adjusted P values available in Supplemental Table 1.

Article Snippet: FTY720 and BAF312 were provided by Novartis Pharma AG whereas NBDFTY720 (Cayman Chemicals), FTY720P (Echelon or Cayman Chemicals), CYM5541 (Cayman Chemicals), TY52156 (Tocris), CAY10444 (Cayman Chemicals), U73122 (Tocris), Manoalide (Santa Cruz Biotechnology), and LY294002 (Cayman Chemicals) were purchased.

Techniques: Infection, Control, Labeling, Staining

Journal: Journal of Clinical Investigation

Article Title: FTY720 reactivates cryptococcal granulomas in mice through S1P receptor 3 on macrophages

doi: 10.1172/jci136068

Figure Lengend Snippet: Figure 4. FTY720 does not induce a major change in lung cytokine profiles compared with BAF312. Mice were infected for 30 days with C. neoformans Δgcs1 before FTY720, BAF312, or vehicle control (H2O) administration. For the lung analysis, n = 4 mice per group at each time point (before compound administration [day –1], day 5, and day 20 after compound administration) were used. Cytokine levels (pg/mL) were normalized to protein concentration (mg/mL) determined by the Bradford assay. All error bars represent SEM.

Article Snippet: FTY720 and BAF312 were provided by Novartis Pharma AG whereas NBDFTY720 (Cayman Chemicals), FTY720P (Echelon or Cayman Chemicals), CYM5541 (Cayman Chemicals), TY52156 (Tocris), CAY10444 (Cayman Chemicals), U73122 (Tocris), Manoalide (Santa Cruz Biotechnology), and LY294002 (Cayman Chemicals) were purchased.

Techniques: Infection, Control, Protein Concentration, Bradford Assay

Journal: Journal of Clinical Investigation

Article Title: FTY720 reactivates cryptococcal granulomas in mice through S1P receptor 3 on macrophages

doi: 10.1172/jci136068

Figure Lengend Snippet: Figure 5. Treatment with FTY720 but not BAF312 affects macrophage organization in the granuloma structure. (A) Mice were infected for 30 days with C. neoformans Δgcs1 before FTY720, BAF312, or vehicle control (H2O) administration. At day 60 after compound administration lungs were processed for F4/80 immunohistochemistry and Verhoeff–Van Gieson (VVG) staining. Scale bars: 200 μm (top), 50 μm (middle and bottom). Black box indicates enlarged area. The dashed line indicates the fibrotic granuloma layer identified by the red collagen and black elastin staining in VVG. (B) The mean intensity of F4/80 staining inside and outside the bounds of the granuloma, as delineated by the collagen deposition seen in VVG, was quantified using Image J soft- ware, n = 4. All error bars represent SEM. Comparisons were done with 1-way ANOVA with Bonferroni’s multiple comparisons post hoc test. P value was corrected for multiplicity using the Bonferroni’s adjustment. **P = 0.0021 compared with control.

Article Snippet: FTY720 and BAF312 were provided by Novartis Pharma AG whereas NBDFTY720 (Cayman Chemicals), FTY720P (Echelon or Cayman Chemicals), CYM5541 (Cayman Chemicals), TY52156 (Tocris), CAY10444 (Cayman Chemicals), U73122 (Tocris), Manoalide (Santa Cruz Biotechnology), and LY294002 (Cayman Chemicals) were purchased.

Techniques: Infection, Control, Immunohistochemistry, Staining

Journal: Journal of Clinical Investigation

Article Title: FTY720 reactivates cryptococcal granulomas in mice through S1P receptor 3 on macrophages

doi: 10.1172/jci136068

Figure Lengend Snippet: Figure 6. Treatment with FTY720 but not BAF312 affects M2 macrophage polar- ization in the granuloma structure. (A) Mice were infected for 30 days with C. neoformans Δgcs1 before FTY720, BAF312, or vehicle control (H2O) administration. At day 60 after compound administration, lungs were processed for CD38 (gray) and EGR2 (magenta) immunohistochemistry to assess for M1 and M2 macrophage polariza- tion, respectively. Scale bars: 200 μm (top), 50 μm (bottom). (B) The relative intensity of CD38 and EGR2 staining inside versus outside the bounds of the granuloma, as delineated by the collagen deposition seen in VVG of Figure 5, was quantified using Image J software, n = 3. All error bars rep- resent SEM. Comparisons were done with 2-way ANOVA with Bonferroni’s multiple comparisons post hoc test. P value was corrected for multiplicity using Bonferroni’s adjustment. *P = 0.0393, **P = 0.0084, ****P < 0.0001 compared with the control.

Article Snippet: FTY720 and BAF312 were provided by Novartis Pharma AG whereas NBDFTY720 (Cayman Chemicals), FTY720P (Echelon or Cayman Chemicals), CYM5541 (Cayman Chemicals), TY52156 (Tocris), CAY10444 (Cayman Chemicals), U73122 (Tocris), Manoalide (Santa Cruz Biotechnology), and LY294002 (Cayman Chemicals) were purchased.

Techniques: Infection, Control, Immunohistochemistry, Staining, Software

Journal: Journal of Clinical Investigation

Article Title: FTY720 reactivates cryptococcal granulomas in mice through S1P receptor 3 on macrophages

doi: 10.1172/jci136068

Figure Lengend Snippet: Figure 7. FTY720 impairs phagocytosis and ROS in macrophages. (A) A schematic of pathways downstream of S1PR3 in macrophages as well as the reported activity of the compounds used in C and D is shown. (B) Primary alveolar macrophages isolated from WT mice were treated overnight with 1 nM of indicated compound, n = 3. Cells were subsequently coincubated with opsonized C. neoformans Δgcs1 and phagocytic index was calculated by microscopic observation. Experiment was conducted 3 times. (C) Primary alveolar macrophages isolated from WT mice were treated with indicated compound for 1 hour, n = 3 each. Phagocytic index was performed as in B. Experiment was conducted 3 times. (D) Primary alveolar macrophages from WT mice were treated over- night with the respective compound and analysis of ROS production was performed, n = 3. Experiment was conducted 3 times. All error bars represent SEM and statistical comparisons were done using the 2-sided Student’s t test (B: *P = 0.0313) or 1-way ANOVA with Bonferroni’s multiple comparisons post hoc test. P values were corrected for multiplicity using the Bonferroni’s adjustment (C: ***P = 0.0098, ****P < 0.001, *****P < 0.0001; D: **P = 0.0218).

Article Snippet: FTY720 and BAF312 were provided by Novartis Pharma AG whereas NBDFTY720 (Cayman Chemicals), FTY720P (Echelon or Cayman Chemicals), CYM5541 (Cayman Chemicals), TY52156 (Tocris), CAY10444 (Cayman Chemicals), U73122 (Tocris), Manoalide (Santa Cruz Biotechnology), and LY294002 (Cayman Chemicals) were purchased.

Techniques: Activity Assay, Isolation

Journal: Traffic (Copenhagen, Denmark)

Article Title: ENDOCYTOSIS OF LIGAND ACTIVATED SPHINGOSINE 1-PHOSPHATE RECEPTOR 1 MEDIATED BY THE CLATHRIN-PATHWAY

doi: 10.1111/tra.12343

Figure Lengend Snippet: (A-D) HeLa cells not expressing (A) or transiently expressing 3xHA-S1PR1-eGFP (B-D) were incubated with anti-HA antibody to label receptor at the cell surface and then follow their internalization upon incubation with DMSO only (Carrier) or the S1PR1 ligands (S1P or FTY720P). Fluorescent transferrin-Alexa Fluor 647 was used to monitor the clathrin-based receptor mediated endocytosis of transferrin. HA antibody was used to follow the uptake of S1P1R. Images for each condition correspond to a center plane obtained using spinning disc confocal microscopy from which quantification data was obtained. An acid wash step at the end of the experiment was used to remove the surface bound transferrin or HA antibodies. Scale bar 10 μm.

Article Snippet: Reagents Sphingosine-1-phosphate (S1P) and FTY720-phosphate (FTY720P) were from Echelon Bioscience (Salt Lake City, UT), poly-d-lysine (Sigma) and collagen (BD Biosciences).

Techniques: Expressing, Incubation, Confocal Microscopy

Journal: Traffic (Copenhagen, Denmark)

Article Title: ENDOCYTOSIS OF LIGAND ACTIVATED SPHINGOSINE 1-PHOSPHATE RECEPTOR 1 MEDIATED BY THE CLATHRIN-PATHWAY

doi: 10.1111/tra.12343

Figure Lengend Snippet: (A) Effect of clathrin or AP2 depletion in the uptake of S1PR1 in Hela cells transiently expressing 3xHA-S1P1-eGFP. Cells were depleted of clathrin or of its endocytic adaptor AP2 by transduction with lentiviruses encoding shRNA specific for clathrin heavy chain (CLC) or the μ2 subunit of AP2 (AP2); scrambled shRNA was used as a negative control. The cells were incubated with anti-HA antibody to follow the uptake of S1PR1 upon addition of DMSO only (Carrier) or the ligands (S1P or FTY720P). Transferrin-Alexa Fluor 647 was used to monitor its clathrin-based receptor mediated endocytosis. The representative images correspond to a center plane from which quantification data was obtained. An acid wash step at the end of the experiment was used to remove surface bound transferrin and anti-HA antibodies. Scale bar 10 μm.

Article Snippet: Reagents Sphingosine-1-phosphate (S1P) and FTY720-phosphate (FTY720P) were from Echelon Bioscience (Salt Lake City, UT), poly-d-lysine (Sigma) and collagen (BD Biosciences).

Techniques: Expressing, Transduction, shRNA, Negative Control, Incubation

Journal: Traffic (Copenhagen, Denmark)

Article Title: ENDOCYTOSIS OF LIGAND ACTIVATED SPHINGOSINE 1-PHOSPHATE RECEPTOR 1 MEDIATED BY THE CLATHRIN-PATHWAY

doi: 10.1111/tra.12343

Figure Lengend Snippet: (A) MEF cells from mice expressing (β-Arrestin1/2+/+) or not (β-Arrestin1/2−/−) β-arrestin1 and β-arrestin2 and stably expressing 3xHA-S1PR1-eGFP were incubated with anti-HA antibody in the presence of DMSO only (Carrier) or the ligands S1P or FTY720P. Images for each condition correspond to a center plane obtained using spinning disc confocal microscopy. An acid wash step at the end of the experiment was used to remove most of the surface bound anti-HA antibodies. Scale bar 10 μm.

Article Snippet: Reagents Sphingosine-1-phosphate (S1P) and FTY720-phosphate (FTY720P) were from Echelon Bioscience (Salt Lake City, UT), poly-d-lysine (Sigma) and collagen (BD Biosciences).

Techniques: Expressing, Stable Transfection, Incubation, Confocal Microscopy

Journal: Traffic (Copenhagen, Denmark)

Article Title: ENDOCYTOSIS OF LIGAND ACTIVATED SPHINGOSINE 1-PHOSPHATE RECEPTOR 1 MEDIATED BY THE CLATHRIN-PATHWAY

doi: 10.1111/tra.12343

Figure Lengend Snippet: HEK 293A cells stably expressing 3xHA-S1PR1-mCherry were incubated or not with anti-HA antibody to label receptor at the cell surface and then follow their internalization upon incubation with DMSO only (carrier) or the S1PR1 ligands (S1P or FTY720P). Fluorescent transferrin-Alexa Fluor 647 was used to monitor the clathrin-based receptor mediated endocytosis of transferrin. Data obtained using flow cytometry was from more than 10,000 cells analyzed per condition.

Article Snippet: Reagents Sphingosine-1-phosphate (S1P) and FTY720-phosphate (FTY720P) were from Echelon Bioscience (Salt Lake City, UT), poly-d-lysine (Sigma) and collagen (BD Biosciences).

Techniques: Stable Transfection, Expressing, Incubation, Flow Cytometry

Journal: Traffic (Copenhagen, Denmark)

Article Title: ENDOCYTOSIS OF LIGAND ACTIVATED SPHINGOSINE 1-PHOSPHATE RECEPTOR 1 MEDIATED BY THE CLATHRIN-PATHWAY

doi: 10.1111/tra.12343

Figure Lengend Snippet: (A) HeLa cells transiently expressing 3xHA-S1PR1-eGFP were incubated with anti-HA antibody in the absence (− dynasore-OH) or presence (+ dynasore-OH) of the modified more potent cell permeable inhibitor of dynamin, dynasore-OH. Following brief 15 min incubation with dynasore-OH, the media was replaced for 30 min with a new solution only containing DMSO, S1P or FTY720P. Fluorescent transferrin-Alexa Fluor 647 was used to follow the efficiency of the clathrin-based endocytic pathway. Images obtained using spinning disc confocal microscopy correspond to a center plane from which quantification data was obtained. An acid wash step at the end of the experiment was used to remove the surface bound transferrin and anti-HA antibodies. Scale bar, 10 μm.

Article Snippet: Reagents Sphingosine-1-phosphate (S1P) and FTY720-phosphate (FTY720P) were from Echelon Bioscience (Salt Lake City, UT), poly-d-lysine (Sigma) and collagen (BD Biosciences).

Techniques: Expressing, Incubation, Modification, Confocal Microscopy

Journal: ACS Nano

Article Title: Enhancing Lung Recovery: Inhaled Poly(lactic- co -glycolic) Acid Encapsulating FTY720 and Nobiletin for Lipopolysaccharide-Induced Lung Injury, with Advanced Inhalation Tower Technology

doi: 10.1021/acsnano.3c12532

Figure Lengend Snippet: (A) The number distribution of coencapsulated FTY720 and NOB NPs was prepared by the emulsification method using PLGA. (B) The morphology of the FTY720-NOB-PLGA NPs with a field of view of 30K.

Article Snippet: FTY720 was purchased from TargetMol Chemicals Inc. (USA).

Techniques: Emulsification

Journal: ACS Nano

Article Title: Enhancing Lung Recovery: Inhaled Poly(lactic- co -glycolic) Acid Encapsulating FTY720 and Nobiletin for Lipopolysaccharide-Induced Lung Injury, with Advanced Inhalation Tower Technology

doi: 10.1021/acsnano.3c12532

Figure Lengend Snippet: (A) FTIR spectra of the (a) FTY720, (b) NOB, (c) PLGA, and (d) FTY720-NOB-PLGA NPs with wave numbers from 500 to 4000 cm –1 . The FTY720-NOB-PLGA NPs underwent drug release at 1× PBST and 37 °C. Concentrations of FTY720 and NOB were determined at wavelengths of 200 and 330 nm and expressed as (B) grams of cumulative drug and (C) percent of total cumulative drug.

Article Snippet: FTY720 was purchased from TargetMol Chemicals Inc. (USA).

Techniques:

Journal: ACS Nano

Article Title: Enhancing Lung Recovery: Inhaled Poly(lactic- co -glycolic) Acid Encapsulating FTY720 and Nobiletin for Lipopolysaccharide-Induced Lung Injury, with Advanced Inhalation Tower Technology

doi: 10.1021/acsnano.3c12532

Figure Lengend Snippet: (A) Schematic diagram illustrating the design of the animal model. (B) Evaluation of IL-6 and TNF-α secretion in bronchoalveolar lavage fluid (BALF) from LPS-induced lung injury mice ( n = 3 per group) after 24 h of treatment with PBS, NOB-PLGA NPs, and FTY720-NOB-PLGA NPs using an ELISA kit. (C) Histopathological examination of lung tissues in mice ( n = 3 per group) with LPS-induced lung injury treated with PBS, NOB-PLGA NPs, and FTY720-NOB-PLGA NPs at 24 h, displayed at an original magnification of 100×. (D) Panoramic images of NF-κB obtained through the TissueFAXs platform and quantification of NF-κB expression percentage in the entire lung section (*** p < 0.001, ** p < 0.01, and * p < 0.05).

Article Snippet: FTY720 was purchased from TargetMol Chemicals Inc. (USA).

Techniques: Animal Model, Enzyme-linked Immunosorbent Assay, Expressing

Journal: ACS Nano

Article Title: Enhancing Lung Recovery: Inhaled Poly(lactic- co -glycolic) Acid Encapsulating FTY720 and Nobiletin for Lipopolysaccharide-Induced Lung Injury, with Advanced Inhalation Tower Technology

doi: 10.1021/acsnano.3c12532

Figure Lengend Snippet: (A) Schematic diagram of the animal model examining the endurance of FTY720-NOB-PLGA NPs’ efficacy over a 48 h period. (B) Assessment of IL-6 and TNF-α secretion in bronchoalveolar lavage fluid (BALF) from LPS-induced lung injury mice ( n = 3 per group) 48 h post-treatment with PBS, NOB-PLGA NPs, and FTY720-NOB-PLGA NPs using an ELISA kit. (C) Lung pathology evaluation in mice ( n = 3 per group) with LPS-induced lung injury treated with different drugs at 48 h, displayed at an original magnification of 100×. (D) Panoramic images of NF-κB obtained through the TissueFAXs platform, along with quantification of NF-κB expression percentage in the entire lung section (*** p < 0.001, ** p < 0.01, and * p < 0.05).

Article Snippet: FTY720 was purchased from TargetMol Chemicals Inc. (USA).

Techniques: Animal Model, Enzyme-linked Immunosorbent Assay, Expressing

Journal: ACS Nano

Article Title: Enhancing Lung Recovery: Inhaled Poly(lactic- co -glycolic) Acid Encapsulating FTY720 and Nobiletin for Lipopolysaccharide-Induced Lung Injury, with Advanced Inhalation Tower Technology

doi: 10.1021/acsnano.3c12532

Figure Lengend Snippet: (A) Schematic illustration of FTY720-NOB-PLGA NPs inhalation for 30 min daily over three consecutive days, followed by sacrifice at 96 h. (B) Comparison of the impact of different treatments (PBS and FTY720-NOB-PLGA NPs; 30 min/day for 3 days) on IL-6 and TNF-α secretion in BALF using an ELISA kit. (C) Lung pathology assessment in mice ( n = 3 per group) with LPS-induced lung injury treated with different drugs for three consecutive days. (D) Panoramic NF-κB images obtained through the TissueFAXs platform, and quantification of NF-κB expression percentage in the entire lung section. Mean ± SD values are presented, and p -values were determined using the Student’s t test (*** p < 0.001, ** p < 0.01, and * p < 0.05).

Article Snippet: FTY720 was purchased from TargetMol Chemicals Inc. (USA).

Techniques: Comparison, Enzyme-linked Immunosorbent Assay, Expressing

Journal: ACS Nano

Article Title: Enhancing Lung Recovery: Inhaled Poly(lactic- co -glycolic) Acid Encapsulating FTY720 and Nobiletin for Lipopolysaccharide-Induced Lung Injury, with Advanced Inhalation Tower Technology

doi: 10.1021/acsnano.3c12532

Figure Lengend Snippet: Histological analysis of lung tissue sections from control, LPS-induced acute lung injury, and post-treatment with FTY720-NOB-PLGA NPs. (A) Tissue sections were stained using H&E and immunohistochemical staining for MPO, CD68, and CD3. Positive cells are brown (magnification, 400×). Scale bar, 10 μm. (B) Quantification of neutrophils (MPO), macrophages (CD68), and T cells (CD3) per high-power field (HPF) derived from IHC data ( n = 3 per group). Data are presented as mean ± SD. Statistical significance was assessed using the Student’s t test (*** p < 0.001, ** p < 0.01).

Article Snippet: FTY720 was purchased from TargetMol Chemicals Inc. (USA).

Techniques: Control, Staining, Immunohistochemical staining, Derivative Assay

Journal: ACS Nano

Article Title: Enhancing Lung Recovery: Inhaled Poly(lactic- co -glycolic) Acid Encapsulating FTY720 and Nobiletin for Lipopolysaccharide-Induced Lung Injury, with Advanced Inhalation Tower Technology

doi: 10.1021/acsnano.3c12532

Figure Lengend Snippet: Multiplexed mIF assay of immune cell infiltration. (A) Representative images from the mIF assay on lung tissue from mice post-LPS stimulation with or without FTY720-NOB-PLGA nanoparticle inhalation treatment, showing staining for MPO (neutrophils), CD68 (macrophages), CD3 (T cells), and DAPI (nuclei). Scale bar, 100 μm. (B) The bar graph illustrates the proportion of neutrophils, macrophages, and T cells infiltrating the lungs of mice following LPS stimulation with or without FTY720-NOB-PLGA nanoparticle inhalation treatment. Data are presented as mean ± SD, calculated from four fields per mouse lung. Statistical significance was determined using the Student’s t test (*** p < 0.001).

Article Snippet: FTY720 was purchased from TargetMol Chemicals Inc. (USA).

Techniques: Staining

Journal: ACS Nano

Article Title: Enhancing Lung Recovery: Inhaled Poly(lactic- co -glycolic) Acid Encapsulating FTY720 and Nobiletin for Lipopolysaccharide-Induced Lung Injury, with Advanced Inhalation Tower Technology

doi: 10.1021/acsnano.3c12532

Figure Lengend Snippet: Body weight, organ function, and respiratory rate in control group and treatment group. (A) Body weight percentage of mice in response to LPS and FTY720-NOB-PLGA NPs. The values presented are the mean ± SD. p values were obtained using the Student’s t test (*** p < 0.001). (B) Blood biochemical results of the FTY720-NOB-PLGA NPs following inhalation into mice ( n = 5). The results show mean and standard deviation of alanine aminotransferase (ALT), creatinine (CRE), total bilirubin (TBIL), and blood urea nitrogen (BUN). (C) Respiratory parameters: Mean of breathing frequency was analyzed during day 1 to day 3 through plethysmography associated with the Allay restrainer.

Article Snippet: FTY720 was purchased from TargetMol Chemicals Inc. (USA).

Techniques: Control, Standard Deviation

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: Cells were incubated with FTY720 and FTY720 (S)-phosphate (both from Santa Cruz Biotechnology, Santa Cruz, CA, USA) or cycloheximide (Sigma, St. Louis, MO, USA) at 3 3 10/ml for 3 h. In some cases, neutrophils were preincubated for 30 min with necrostatin-1 or Z-IETD-FMK (both from Sigma); Q-VD-OPh (R&D Systems, Minneapolis, MN, USA); NSA (Tocris, Bristol, United Kingdom); geldanamycin, 17-DMAG, and radicicol (all from InvivoGen, San Diego, CA, USA); OA (Santa Cruz Biotechnology); or DPI (Sigma).

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