Journal: Molecular Metabolism

Article Title: Elevating adipose eosinophils in obese mice to physiologically normal levels does not rescue metabolic impairments

doi: 10.1016/j.molmet.2017.12.004

Figure Lengend Snippet: Eosinophil levels in eAT, blood, bone marrow, liver, and intestine of rIL5 injected mice . (A ) Male C57BL/6J mice were fed either chow or HFD for 8 weeks and simultaneously IP injected twice weekly with rIL5 protein. (B) Flow cytometry gating strategy for eosinophils (CD45 + , live, F4/80 lo , CD11b lo , SiglecF + ) and macrophages (CD45 + , live, F4/80 hi , CD11b hi , SiglecF − ). (C) Percent eAT eosinophils negatively correlate with body weight (slope = −0.96, R 2 = 0.75). (D) Treatment with rIL5 elevates eAT eosinophils in HFD-fed mice, compared to saline and vehicle controls, back to chow-fed levels [n = 4–11]. (E) No difference in percent eosinophils in blood, bone marrow, or liver of rIL5 treated mice, but intestine does show an increase [n = 2–8]. Data are shown as means ± SEM. eAT = epididymal adipose tissue; HFD = high fat diet. Key: chow = hashed bars; HFD = open bars; saline = white; vehicle = grey; rIL5 = black. * P < 0.05, compared to saline; *** P < 0.0005, compared to saline; ˆˆ P < 0.005, compared to vehicle.

Article Snippet: During the entire 8-week period, mice also received 50 ng recombinant IL5 (rIL5; R&D Systems, Minneapolis, MN) protein (in 0.001% BSA as carrier protein) by intraperitoneal (IP) injection twice per week to elevate eosinophils.

Techniques: Injection, Flow Cytometry, Saline

Journal: Molecular Metabolism

Article Title: Elevating adipose eosinophils in obese mice to physiologically normal levels does not rescue metabolic impairments

doi: 10.1016/j.molmet.2017.12.004

Figure Lengend Snippet: Inflammatory profile of obese eAT with elevated eosinophils . (A ) Representative 40× images of eAT from HFD-fed obese mice treated with saline, vehicle, or rIL5. Macrophages (red, F4/80) are seen in abundance in all groups, whereas eosinophils (green, SiglecF) are increased in rIL5 treated mice. Magnified view shows the donut-shaped nucleus ( * ) of eosinophils juxtaposed to the spherical nucleus (#) of macrophages [n = 3]. (B) Treatment with rIL5 does not alter percent eAT macrophages or (C) protein expression of pro-inflammatory marker MHCII [n = 6–11]. (D) Gene expression analysis of macrophage marker, Adgre1 (F4/80), and an array of macrophage polarization genes were not statistically different between groups in eAT [n = 6–8]. Data are shown as means ± SEM. eAT = epididymal adipose tissue, HFD = high fat diet. Key: saline = white; vehicle = grey; rIL5 = black.

Article Snippet: During the entire 8-week period, mice also received 50 ng recombinant IL5 (rIL5; R&D Systems, Minneapolis, MN) protein (in 0.001% BSA as carrier protein) by intraperitoneal (IP) injection twice per week to elevate eosinophils.

Techniques: Saline, Expressing, Marker, Gene Expression

Journal: Molecular Metabolism

Article Title: Elevating adipose eosinophils in obese mice to physiologically normal levels does not rescue metabolic impairments

doi: 10.1016/j.molmet.2017.12.004

Figure Lengend Snippet: Weight gain, body composition, and glucose tolerance in mice with elevated AT eosinophils . (A) HFD-fed mice gained more weight over 8 weeks compared to chow, with no difference in (B) total weight gained between vehicle or rIL5 treated mice [n = 4]. HFD-fed vehicle and rIL5 treated mice have equally altered total fat and lean mass by (C) percent or (D) grams at study completion [n = 4–12]. (E) HFD-fed mice have increased body, eAT, and sAT weight, but no further difference with rIL5 [n = 4]. (F) Glucose tolerance is impaired by HFD-feeding, but not rescued by rIL5 treatment [n = 4]. Data are shown as means ± SEM. AT = adipose tissue; HFD = high fat diet; eAT = epididymal AT; sAT = subcutaneous AT. Key: chow = hashed bars, dotted lines; HFD = open bars, solid lines; vehicle = grey; rIL5 = black. * P < 0.05, compared to chow; ** P < 0.005, compared to chow; *** P < 0.0005, compared to chow; **** P < 0.0001, compared to chow; ˆ P < 0.005, compared to vehicle.

Article Snippet: During the entire 8-week period, mice also received 50 ng recombinant IL5 (rIL5; R&D Systems, Minneapolis, MN) protein (in 0.001% BSA as carrier protein) by intraperitoneal (IP) injection twice per week to elevate eosinophils.

Techniques:

Journal: Molecular Metabolism

Article Title: Elevating adipose eosinophils in obese mice to physiologically normal levels does not rescue metabolic impairments

doi: 10.1016/j.molmet.2017.12.004

Figure Lengend Snippet: Triglyceride tolerance in mice with elevated AT eosinophils . Fasted HFD-fed mice treated with vehicle or rIL5 for 8 weeks were given a bolus of TGs (i.e. olive oil) and showed no difference between groups in (A) plasma TG [n = 8] (B) plasma FFA [n = 4] (C) blood glucose [n = 8] (D) or plasma cholesterol [n = 8] over the course of 5 h. Data are shown as means ± SEM. HFD = high fat diet; TG = triglycerides; FFA = free fatty acids. Key: vehicle = grey; rIL5 = black.

Article Snippet: During the entire 8-week period, mice also received 50 ng recombinant IL5 (rIL5; R&D Systems, Minneapolis, MN) protein (in 0.001% BSA as carrier protein) by intraperitoneal (IP) injection twice per week to elevate eosinophils.

Techniques: Clinical Proteomics

Journal: Molecular Metabolism

Article Title: Elevating adipose eosinophils in obese mice to physiologically normal levels does not rescue metabolic impairments

doi: 10.1016/j.molmet.2017.12.004

Figure Lengend Snippet: Metabolic parameters of rIL5 treated mice during a mixed-meal test . HFD-fed mice treated with vehicle or rIL5 for 8 weeks were fasted and then allowed access to HFD ad libitum for 3 h. (A) Both groups consumed the same amount of food [n = 6–7]. (B) A spike in blood glucose occurred at 0.5 h following refeeding with no difference between groups [n = 6–7]. (C) Insulin also rose at 0.5 h and then held relatively steady in both groups [n = 6–7]. Plasma (D) TG [n = 6–7], (E) FFA [n = 4], and (F) cholesterol [n = 5–7] did not vary between groups upon refeeding. Data are shown as means ± SEM. HFD = high fat diet; TG = triglycerides; FFA = free fatty acids. Key: vehicle = grey; rIL5 = black.

Article Snippet: During the entire 8-week period, mice also received 50 ng recombinant IL5 (rIL5; R&D Systems, Minneapolis, MN) protein (in 0.001% BSA as carrier protein) by intraperitoneal (IP) injection twice per week to elevate eosinophils.

Techniques: Clinical Proteomics

Journal: Molecular Metabolism

Article Title: Elevating adipose eosinophils in obese mice to physiologically normal levels does not rescue metabolic impairments

doi: 10.1016/j.molmet.2017.12.004

Figure Lengend Snippet: Insulin signaling in eAT with elevated eosinophils . (A) Representative western blots of eAT showed an increase in the ratio pAKT/AKT upon insulin stimulation in HFD-fed mice that received vehicle or rIL5 for 8 weeks [n = 3–4]. β-Actin is shown as a protein loading control. (B) Quantification of western blots revealed no difference in the eAT insulin signaling response via pAKT/AKT between vehicle and rIL5 treated mice. Data are shown as means ± SEM. eAT = epididymal adipose tissue; HFD = high fat diet. Key: vehicle = grey; rIL5 = black; insulin injected = dotted bars; no-insulin saline control injection = open bars. * P < 0.05, compared to no insulin stimulation.

Article Snippet: During the entire 8-week period, mice also received 50 ng recombinant IL5 (rIL5; R&D Systems, Minneapolis, MN) protein (in 0.001% BSA as carrier protein) by intraperitoneal (IP) injection twice per week to elevate eosinophils.

Techniques: Western Blot, Control, Injection, Saline

Journal: Molecular Metabolism

Article Title: Elevating adipose eosinophils in obese mice to physiologically normal levels does not rescue metabolic impairments

doi: 10.1016/j.molmet.2017.12.004

Figure Lengend Snippet: Cold challenge: Energy balance in mice with elevated AT eosinophils . HFD-fed mice that previously received vehicle or rIL5 for 8 weeks were individually housed in metabolic cages to measure an array of physiological parameters for 2 days at RT and 2 days at 4 °C [n = 8]. (A) Average daily body weight did not vary between vehicle and rIL5 treated mice. (B) Average daily food intake and (C) average daily movement were increased in the dark cycle compared to light cycle, but with no difference between vehicle and rIL5. (D) The respiratory quotient oscillated between light and dark cycles to equal degrees in vehicle and rIL5 treated mice, both during RT and the 4 °C cold challenge; indicating both groups had the same energy substrate utilization. (E) Energy expenditure increased upon 4 °C cold challenge to maintain body heat, but did not vary between vehicle and rIL5 treated mice. Both treatment groups lost the same amount of (F) body mass, (G) fat mass, and (H) lean mass. Data are shown as means ± SEM. eAT = epididymal adipose tissue; sAT = subcutaneous AT. RT = room temperature, set at 21 °C; HFD = high fat diet. Key: vehicle = grey bars/lines; rIL5 = black bars/lines.

Article Snippet: During the entire 8-week period, mice also received 50 ng recombinant IL5 (rIL5; R&D Systems, Minneapolis, MN) protein (in 0.001% BSA as carrier protein) by intraperitoneal (IP) injection twice per week to elevate eosinophils.

Techniques:

Journal: Molecular Metabolism

Article Title: Elevating adipose eosinophils in obese mice to physiologically normal levels does not rescue metabolic impairments

doi: 10.1016/j.molmet.2017.12.004

Figure Lengend Snippet: Cold challenge: Beiging capacity of white AT with elevated eosinophils . AT of HFD-fed mice that received vehicle or rIL5 for 8 weeks was examined after 2 days of RT or 4 °C cold exposure. After cold exposure, the presence of increased eosinophils was verified in (A) sAT and (C) eAT of rIL5 treated mice compared to vehicle controls [n = 5–7]. Cold exposure alone was able to increase eosinophils in sAT but not eAT. (B) Ucp1 gene expression in sAT was increased ∼307-fold in the cold compared to RT, with no further increase in rIL5 treated mice [n = 4–5]. (D) Ucp1 gene expression in eAT trended towards an increase under cold conditions, but no difference was observed between vehicle and rIL5 treated mice [n = 5–7]. Data are shown as means ± SEM. eAT = epididymal adipose tissue; sAT = subcutaneous AT; RT = room temperature, set at 21 °C; HFD = high fat diet; Ucp1 = uncoupling protein 1. Key: vehicle = grey bars; rIL5 = black bars.

Article Snippet: During the entire 8-week period, mice also received 50 ng recombinant IL5 (rIL5; R&D Systems, Minneapolis, MN) protein (in 0.001% BSA as carrier protein) by intraperitoneal (IP) injection twice per week to elevate eosinophils.

Techniques: Gene Expression

Journal: Biochemical pharmacology

Article Title: The JAK1/2 inhibitor baricitinib suppresses eosinophil effector function and restricts allergen-induced airway eosinophilia.

doi: 10.1016/j.bcp.2021.114690

Figure Lengend Snippet: Fig. 1. Differential expression and phosphoryla tion of STAT1 in eosinophils from allergic and healthy non-allergic donors. (A) Purified eosino phils from allergic (n = 10) and healthy donors (n = 12) were fixed and permeabilized, stained with the respective isotype control or specific antibodies against total-STAT1 and phospho-STAT1 and measured by flow cytometry. Data were analyzed as fold increase in fluorescence intensity with respect to the isotype control and reported as phospho-STAT1/ total-STAT1 (mean ± SEM). ** P < 0.01; Student’s t-test. (B-D) Purified eosinophils from healthy donors were pretreated with (B) vehicle (n = 7), (C) bar icitinib (BARI, 200 nM, n = 6) or (D) tofacitinib (TOFA, 200 nM, n = 6) for 30 min at 37 ◦C, stimu lated with vehicle or IL-5 (300 pM) for 10 min at 37 ◦C, fixed and permeabilized, stained with isotype control or a phospho-STAT1 antibody and analyzed by flow cytometry. Data are expressed as fold in crease in fluorescence intensity with respect to the isotype control (mean ± SEM). * P < 0.05; Student’s t-test.

Article Snippet: In some experiments eosinophils from healthy donors were pretreated with vehicle, baricitinib (200 nM; TargetMol) or tofacitinib (200 nM; TargetMol) for 30 min at 37 ◦C and stimulated with vehicle or IL-5 (100 pM) for 10 min at 37 ◦C prior to the staining.

Techniques: Quantitative Proteomics, Purification, Staining, Control, Flow Cytometry, Fluorescence

Journal: Biochemical pharmacology

Article Title: The JAK1/2 inhibitor baricitinib suppresses eosinophil effector function and restricts allergen-induced airway eosinophilia.

doi: 10.1016/j.bcp.2021.114690

Figure Lengend Snippet: Fig. 2. Baricitinib and tofacitinib similarly inhibit eosinophil differentiation and JAK1/2 activation. Bone marrow cells were collected from the femurs and tibiae of three female BALB/c mice and cultured at 106/mL in media supplemented with 100 ng/mL SCF and 100 ng/mL FLT3L from day 0 to day 4. On day 4, media were replaced with media containing 10 ng/mL IL-5 supplemented with or without baricitinib (BARI, 200 nM) or tofacitinib (TOFA, 200 nM). (A) Total cells, (B) Siglec F positive cells and (C) CCR3 positive cells were enumerated by flow cytometry on the indicated days. On day 12 (D) JAK1 and (E) JAK2 phosphorylation was measured by flow cytometric intracellular staining. Data are shown as mean ± SEM from 3 independent experiments, ** P < 0.01, *** P < 0.005; (A-C) two-way ANOVA and (D and E) one- way ANOVA.

Article Snippet: In some experiments eosinophils from healthy donors were pretreated with vehicle, baricitinib (200 nM; TargetMol) or tofacitinib (200 nM; TargetMol) for 30 min at 37 ◦C and stimulated with vehicle or IL-5 (100 pM) for 10 min at 37 ◦C prior to the staining.

Techniques: Activation Assay, Cell Culture, Flow Cytometry, Phospho-proteomics, Staining

Journal: Biochemical pharmacology

Article Title: The JAK1/2 inhibitor baricitinib suppresses eosinophil effector function and restricts allergen-induced airway eosinophilia.

doi: 10.1016/j.bcp.2021.114690

Figure Lengend Snippet: Fig. 3. Effects of baricitinib and tofacitinib on eosinophil migratory responsiveness. (A) Purified eosinophils were seeded on L-lysine coated chamber slides and pretreated with baricitinib (200 nM), tofacitinib (200 nM) or vehicle for 30 min at room temperature. F-actin polymerization was induced by stimulation with IL- 5 (1 nM) at 37 ◦C for 15 min. Adherent cells were fixed, permeabilized and blocked prior to F-actin staining with Phalloidin-Texas Red. Slides were mounted with mounting media with DAPI and fluorescence images were taken by an Olympus IX70 microscope connected with a Hamamatsu ORCA-ER digital camera. Olympus xcellence® imaging analysis software 1.1 was used for acquiring images. Representative pictures of 4 independent experiments are shown. (B-E) Purified eosinophils (n = 4–16) were pretreated with vehicle, baricitinib (BARI, 200 nM or 500 nM) (B and D) or tofacitinib (TOFA, 200 nM or 500 nM) (C and E) for 30 min at 37 ◦C and were allowed to migrate towards eosinophil chemoattractants: IL-5 300 pM, complement 5a 10 nM, CCL11 10 nM and PGD2 30 nM for 60 min in a 48-well micro- Boyden chamber. Migrated cells were enumerated by flow cytometry (BD FACSCanto II, high flow-rate for 30 s). Data are shown as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.005; Student’s t-test.

Article Snippet: In some experiments eosinophils from healthy donors were pretreated with vehicle, baricitinib (200 nM; TargetMol) or tofacitinib (200 nM; TargetMol) for 30 min at 37 ◦C and stimulated with vehicle or IL-5 (100 pM) for 10 min at 37 ◦C prior to the staining.

Techniques: Purification, Staining, Fluorescence, Microscopy, Imaging, Software, Flow Cytometry

Journal: Biochemical pharmacology

Article Title: The JAK1/2 inhibitor baricitinib suppresses eosinophil effector function and restricts allergen-induced airway eosinophilia.

doi: 10.1016/j.bcp.2021.114690

Figure Lengend Snippet: Fig. 4. Effects of baricitinib and tofacitinib on neutrophil chemotaxis. PMNL (n = 14) were pre treated with vehicle, baricitinib (BARI, 500 nM) or tofacitinib (TOFA, 500 nM) for 30 min at 37 ◦C and were allowed to migrate towards neutrophil chemo attractants: IL-8 (10 nM) and complement 5a (10 nM) for 60 min in a 48-well micro-Boyden chamber. Migrated cells were enumerated by flow cytometry (BD FACSCanto II, high flow-rate for 30 min). Data are shown as mean ± SEM. * P < 0.05; Student’s t- test.

Article Snippet: In some experiments eosinophils from healthy donors were pretreated with vehicle, baricitinib (200 nM; TargetMol) or tofacitinib (200 nM; TargetMol) for 30 min at 37 ◦C and stimulated with vehicle or IL-5 (100 pM) for 10 min at 37 ◦C prior to the staining.

Techniques: Chemotaxis Assay, Flow Cytometry

Journal: Biochemical pharmacology

Article Title: The JAK1/2 inhibitor baricitinib suppresses eosinophil effector function and restricts allergen-induced airway eosinophilia.

doi: 10.1016/j.bcp.2021.114690

Figure Lengend Snippet: Fig. 5. Effects of baricitinib and tofacitinib on eosinophil ROS production. Purified eosinophils were pretreated with vehicle, baricitinib (BARI, 500 nM) or tofacitinib (TOFA, 500 nM) for 30 min at 37 ◦C and ROS production was induced by IL-5 (A, n = 4–8), complement 5a (B, n = 6), CCL11 (C, n = 5–8) and PGD2 (D, n = 7–9). ROS production was detected by flow cytometry using a dihydrorhodamin-123 based assay. Data are expressed as geometric mean fluorescence intensity (MFI) and shown as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.005; two-way ANOVA.

Article Snippet: In some experiments eosinophils from healthy donors were pretreated with vehicle, baricitinib (200 nM; TargetMol) or tofacitinib (200 nM; TargetMol) for 30 min at 37 ◦C and stimulated with vehicle or IL-5 (100 pM) for 10 min at 37 ◦C prior to the staining.

Techniques: Purification, Flow Cytometry, Fluorescence

Journal: Biochemical pharmacology

Article Title: The JAK1/2 inhibitor baricitinib suppresses eosinophil effector function and restricts allergen-induced airway eosinophilia.

doi: 10.1016/j.bcp.2021.114690

Figure Lengend Snippet: Fig. 6. Effects of baricitinib and tofacitinib on eosinophil phagocytosis and degranulation. Purified eosinophils were pretreated with vehicle, baricitinib (500 nM and 2.5 µM) (A, n = 4) or tofacitinib (500 nM and 2.5 µM) (B, n = 4) for 30 min at 37 ◦C, incubated with opsonized Escherichia coli-FITC and analyzed by flow cytometry. Phagocytosis is presented as the percentage of E. coli-FITC-positive eosinophils. Data are shown as mean ± SEM. * P < 0.05, ** P < 0.01; One-way ANOVA. (C) Purified eosinophils were pretreated with vehicle, baricitinib or tofacitinib (500 nM) for 30 min at 37 ◦C and then stimulated with complement 5a for 30 min at 37 ◦C. The release of EPO activity into the supernatant was determined by photometry. Data were expressed as a percentage of the vehicle response (unstimulated) and are shown as the mean ± SEM; n = 14. Two-way ANOVA.

Article Snippet: In some experiments eosinophils from healthy donors were pretreated with vehicle, baricitinib (200 nM; TargetMol) or tofacitinib (200 nM; TargetMol) for 30 min at 37 ◦C and stimulated with vehicle or IL-5 (100 pM) for 10 min at 37 ◦C prior to the staining.

Techniques: Purification, Incubation, Flow Cytometry, Activity Assay

Journal: Biochemical pharmacology

Article Title: The JAK1/2 inhibitor baricitinib suppresses eosinophil effector function and restricts allergen-induced airway eosinophilia.

doi: 10.1016/j.bcp.2021.114690

Figure Lengend Snippet: Fig. 7. Effects of baricitinib and tofacitinib on eosinophil apoptosis. Purified eosinophils were cultured in vehicle or IL-5 100 pM-treated medium, supplemented with or without baricitinib (BARI, 500 nM) or tofacitinib (TOFA, 500 nM) for 18 h. Apoptosis and necrosis were detected by flow cytometry using FITC-annexin-V/ propidium iodide (PI). Annexin/PI double-negative cells were identified as live cells (A), annexin positive/PI negative cells as early apoptotic cells (B), annexin/PI double-positive cells as late apoptotic cells and annexin negative/PI positive cells as necrotic cells (D). Data are expressed as % of total cells and shown as mean ± SEM of 11 independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.005; two-way ANOVA.

Article Snippet: In some experiments eosinophils from healthy donors were pretreated with vehicle, baricitinib (200 nM; TargetMol) or tofacitinib (200 nM; TargetMol) for 30 min at 37 ◦C and stimulated with vehicle or IL-5 (100 pM) for 10 min at 37 ◦C prior to the staining.

Techniques: Purification, Cell Culture, Flow Cytometry

Journal: Biochemical pharmacology

Article Title: The JAK1/2 inhibitor baricitinib suppresses eosinophil effector function and restricts allergen-induced airway eosinophilia.

doi: 10.1016/j.bcp.2021.114690

Figure Lengend Snippet: Fig. 8. Effects of baricitinib and tofacitinib on HDM-induced lung inflammation and airway hyperresponsiveness. (A) On day 1, eight-week-old female BALB/c mice were sensitized intranasally with 1 µg HDM and challenged by intranasal application of 10 µg HDM per day from day 7 to 12. From day 8 to 14 mice received intragastric applications of tofacitinib (TOFA, 50 µmol, twice a day) and baricitinib (BARI, 50 µmol, once a day). (B) Gating strategy for lung single cell suspensions. After single cell and CD45pos gating, eosinophils were identified as CD11cneg/Siglec Fpos and alveolar macrophages as CD11cpos/Siglec Fpos cells. CD11c/ Siglec F double negative cells were characterized as CD11bpos/Ly6Gpos neutrophils, CD11bpos/Ly6Gneg monocytes and FSC/SSClow CD11bneg/Ly6Gneg lymphocytes. On day 15, (C) lungs, (D) spleens, and (E) bone marrow were taken from six mice per group and inflammatory cells were analyzed by flow cytometry: Total CD45pos

Article Snippet: In some experiments eosinophils from healthy donors were pretreated with vehicle, baricitinib (200 nM; TargetMol) or tofacitinib (200 nM; TargetMol) for 30 min at 37 ◦C and stimulated with vehicle or IL-5 (100 pM) for 10 min at 37 ◦C prior to the staining.

Techniques: Single Cell, Flow Cytometry

Journal: Microorganisms

Article Title: Heat-Inactivated Selenium Nanoparticle-Enriched Lactobacillus Enhance Mucosal IgA Responses and Systemic Responses of Clostridium perfringens Multi-Epitope Vaccine Correlated with TGF-β and NF-κB Pathways in Mice

doi: 10.3390/microorganisms14010180

Figure Lengend Snippet: The cytokines induced by HiSeL in mice. The levels of TGF-β and IL-5 were measured at 28 dpi in the serum using ELISA, and the mRNA expression levels of IFN-γ, IL-12, TNF-α, IL-10, IL-1β, and IL-4 in the spleen were measured by RT-qPCR. The data are presented as mean ± SD (* p < 0.05, ** p < 0.01, **** p < 0.0001).

Article Snippet: TGF-β1 and IL-5 in serum were tested by ELISA kits (Boster Bioengineering, Wuhan, China) following the manufacturer’s protocol.

Techniques: Enzyme-linked Immunosorbent Assay, Expressing, Quantitative RT-PCR