Journal: Nature metabolism

Article Title: Eosinophils regulate adipose tissue inflammation and sustain physical and immunological fitness in old age

doi: 10.1038/s42255-020-0228-3

Figure Lengend Snippet: (a) Experimental protocol. (b) Frequencies of eosinophils, macrophages and calculated eosinophil:macrophage ratios in eWAT of Aged-PBS (n=8) and Aged-yNEU (n=9) mice. (c) mRNA expression levels for Tnfα, Il1β and IL6 in eWAT of Aged-PBS (n=8) and Aged-yNEU mice (n=9). Data are presented as fold induction over Aged-PBS controls. (d) Average time on Rotarod of Aged-PBS controls (n=8) and Aged-yNEU mice (n=9). (e) Total numbers of lin–, Sca-1+, c-kit+ hematopoietic stem cells (LSKs) in Aged-PBS (n=4) and Aged-yNEU (n=4) mice. (f) Numbers of common myeloid progenitors (CMP), common lymphoid progenitors (CLP), and granulocyte/monocyte progenitors (GMP) in the bone marrow of Aged-PBS (n=4) and Aged-yNEU (n=4) mice. (g) Frequencies of neutrophils in eWAT of young (n=5), Aged-PBS (n=4) and Aged-yNeu (n=5) mice. (h) Experimental protocol of bone marrow derived eosinophil (BMDE) transfers. (i) Calculated ATE:ATM ratios in eWAT of Aged-PBS (n=5) and Aged-BMDE (n=4) mice as measured by flow cytometry. (j) IL-6 protein levels in eWAT of Aged-PBS (n=6) and Aged-BMDE (n=5) mice. (k) Pre- and post-treatment IL-6 plasma protein levels in Aged-PBS (n=6) and Aged-BMDE (n=5). (l) Intra-group and (m) inter-group comparison of pre- and post-treatment average time on wheel (Rotarod test) in Aged-PBS (n=6) and Aged-BMDE (n=5) mice. Delta in performances in (l) is calculated relative to baseline (post- minus pre-treatment results). Statistical significance was calculated by Wilcoxon matched pairs signed rank test (k, l), by unpaired two-tailed Student’s t test (b, c, d, e, f, I, j, m) or by one-way ANOVA followed by two-tailed post-hoc Dunnett’s multiple comparison test against the aged-PBS treated group (g). Data are pooled from two independently performed experiments (except for (g-m) only one experiment has been performed) and shown as individual data points with mean ± SEM. Data are shown as mean ± SEM. *p < 0.05, **p < 0.01.

Article Snippet: Briefly, 3.3 × 10 3 Lin − cells were cultured in MethoCult base medium (STEMCELL Technologies) supplemented with 15% FCS, 20% BIT (50 mg/ml BSA in Iscove’s modified Dulbecco’s medium, 1.44 U/ml rh-insulin [Actrapid, Novo Nordisk], and 250 ng/ml human Holo Transferrin [ProSpec]), 100 μM 2-β-mercaptoethanol, 100 U/ml penicillin, 100 μg/ml streptomycin, 2 mM L-glutamine, and a cytokine mix of 50 ng/ml rm-SCF, 10 ng/ml rm-IL-3, 10 ng/ml rh-IL-6, and 50 ng/ml rm-Flt3-ligand (all from ProSpec).

Techniques: Expressing, Derivative Assay, Flow Cytometry, Two Tailed Test

Journal: Nature metabolism

Article Title: Eosinophils regulate adipose tissue inflammation and sustain physical and immunological fitness in old age

doi: 10.1038/s42255-020-0228-3

Figure Lengend Snippet: Aged mice (18 months) were adoptively transferred with sort-purified GFP+ eosinophils on two subsequent days. The following day eosinophil recruitment into different tissues was assessed by flow cytometry. (a) Representative flow plots of transferred GFP+ and resident GFP– eosinophils in indicated tissues. (b) Number of GFP+ and GFP– resident eosinophils in indicated tissues. Data are representative of n=4 per group. One out of two independently performed experiments is shown. (c) Experimental protocol. (d) Frequencies of ATEs and ATMs and the calculated ATE:ATM ratios in eWAT of young (n=19), Aged-PBS (n=23), Aged-yEOS (n=13) and Aged-yEOSIL−4−/− (n=13) mice. (e) Protein levels in eWAT of young (n=24), Aged-PBS (n=26), Aged-yEOS (n=18) and Aged-yEOSIL−4−/− (n=16) mice. (f) Plasma protein levels for IL-6 and IL-1β in young (n=20), Aged-PBS (n=35), Aged-yEOS (n=32) and Aged-yEOSIL−4−/− (n=14) mice and CCL2 plasma protein levels in young (n=26), Aged-PBS (n=37), Aged-yEOS (n=22) and Aged-yEOSIL−4−/− (n=7) mice. Statistical significance was calculated by one-way ANOVA followed by two-tailed post-hoc Dunnett’s multiple comparison test against the Aged-PBS group (d, e, f) and wherever possible, results are shown as individual data points with mean bars ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: Briefly, 3.3 × 10 3 Lin − cells were cultured in MethoCult base medium (STEMCELL Technologies) supplemented with 15% FCS, 20% BIT (50 mg/ml BSA in Iscove’s modified Dulbecco’s medium, 1.44 U/ml rh-insulin [Actrapid, Novo Nordisk], and 250 ng/ml human Holo Transferrin [ProSpec]), 100 μM 2-β-mercaptoethanol, 100 U/ml penicillin, 100 μg/ml streptomycin, 2 mM L-glutamine, and a cytokine mix of 50 ng/ml rm-SCF, 10 ng/ml rm-IL-3, 10 ng/ml rh-IL-6, and 50 ng/ml rm-Flt3-ligand (all from ProSpec).

Techniques: Purification, Flow Cytometry, Two Tailed Test

Journal: Nature metabolism

Article Title: Eosinophils regulate adipose tissue inflammation and sustain physical and immunological fitness in old age

doi: 10.1038/s42255-020-0228-3

Figure Lengend Snippet: (a) Gating strategy for ATMs and ATEs in scWAT of young (3 months) and aged (20 months) mice. (b) Calculated ATE:ATM ratio in scWAT of young (n=10), Aged-PBS (n=10), Aged-yEOS (n=6) and Aged-yEOSIL−4−/− (n=7) mice. (c) Representative photographs of H&E stained histological scWAT sections of indicated treatment groups. (d) Quantification of adipocyte hypertrophy in Young (n=11), Aged-PBS (n=10), Aged-yEOS (n=9) and Aged-yEOSIL−4−/− (n=12) mice by ImageJ. (e) IL-6 and CCL2 protein levels in scWAT of Young (n=15), Aged-PBS (n=14), Aged-yEOS (n=12) and Aged-yEOSIL−4−/− (n=13) mice. Statistical significance was calculated by one-way ANOVA followed by two-tailed post-hoc Dunnett’s multiple comparison test against the aged-PBS treated group. Data (e) are pooled from 2 independently performed experiments or performed once (a-d) and shown as individual data points with mean bars ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: Briefly, 3.3 × 10 3 Lin − cells were cultured in MethoCult base medium (STEMCELL Technologies) supplemented with 15% FCS, 20% BIT (50 mg/ml BSA in Iscove’s modified Dulbecco’s medium, 1.44 U/ml rh-insulin [Actrapid, Novo Nordisk], and 250 ng/ml human Holo Transferrin [ProSpec]), 100 μM 2-β-mercaptoethanol, 100 U/ml penicillin, 100 μg/ml streptomycin, 2 mM L-glutamine, and a cytokine mix of 50 ng/ml rm-SCF, 10 ng/ml rm-IL-3, 10 ng/ml rh-IL-6, and 50 ng/ml rm-Flt3-ligand (all from ProSpec).

Techniques: Staining, Two Tailed Test

Journal: Nature metabolism

Article Title: Eosinophils regulate adipose tissue inflammation and sustain physical and immunological fitness in old age

doi: 10.1038/s42255-020-0228-3

Figure Lengend Snippet: (a) Experimental protocol. (b) Frequencies of eosinophils, macrophages and calculated eosinophil:macrophage ratios in eWAT of Aged-PBS (n=8) and Aged-yNEU (n=9) mice. (c) mRNA expression levels for Tnfα, Il1β and IL6 in eWAT of Aged-PBS (n=8) and Aged-yNEU mice (n=9). Data are presented as fold induction over Aged-PBS controls. (d) Average time on Rotarod of Aged-PBS controls (n=8) and Aged-yNEU mice (n=9). (e) Total numbers of lin–, Sca-1+, c-kit+ hematopoietic stem cells (LSKs) in Aged-PBS (n=4) and Aged-yNEU (n=4) mice. (f) Numbers of common myeloid progenitors (CMP), common lymphoid progenitors (CLP), and granulocyte/monocyte progenitors (GMP) in the bone marrow of Aged-PBS (n=4) and Aged-yNEU (n=4) mice. (g) Frequencies of neutrophils in eWAT of young (n=5), Aged-PBS (n=4) and Aged-yNeu (n=5) mice. (h) Experimental protocol of bone marrow derived eosinophil (BMDE) transfers. (i) Calculated ATE:ATM ratios in eWAT of Aged-PBS (n=5) and Aged-BMDE (n=4) mice as measured by flow cytometry. (j) IL-6 protein levels in eWAT of Aged-PBS (n=6) and Aged-BMDE (n=5) mice. (k) Pre- and post-treatment IL-6 plasma protein levels in Aged-PBS (n=6) and Aged-BMDE (n=5). (l) Intra-group and (m) inter-group comparison of pre- and post-treatment average time on wheel (Rotarod test) in Aged-PBS (n=6) and Aged-BMDE (n=5) mice. Delta in performances in (l) is calculated relative to baseline (post- minus pre-treatment results). Statistical significance was calculated by Wilcoxon matched pairs signed rank test (k, l), by unpaired two-tailed Student’s t test (b, c, d, e, f, I, j, m) or by one-way ANOVA followed by two-tailed post-hoc Dunnett’s multiple comparison test against the aged-PBS treated group (g). Data are pooled from two independently performed experiments (except for (g-m) only one experiment has been performed) and shown as individual data points with mean ± SEM. Data are shown as mean ± SEM. *p < 0.05, **p < 0.01.

Article Snippet: Briefly, 3.3 × 10 3 Lin − cells were cultured in MethoCult base medium (STEMCELL Technologies) supplemented with 15% FCS, 20% BIT (50 mg/ml BSA in Iscove’s modified Dulbecco’s medium, 1.44 U/ml rh-insulin [Actrapid, Novo Nordisk], and 250 ng/ml human Holo Transferrin [ProSpec]), 100 μM 2-β-mercaptoethanol, 100 U/ml penicillin, 100 μg/ml streptomycin, 2 mM L-glutamine, and a cytokine mix of 50 ng/ml rm-SCF, 10 ng/ml rm-IL-3, 10 ng/ml rh-IL-6, and 50 ng/ml rm-Flt3-ligand (all from ProSpec).

Techniques: Expressing, Derivative Assay, Flow Cytometry, Two Tailed Test

Journal: Nature metabolism

Article Title: Eosinophils regulate adipose tissue inflammation and sustain physical and immunological fitness in old age

doi: 10.1038/s42255-020-0228-3

Figure Lengend Snippet: (a) Experimental protocol. (b) Number of CSFE-labeled eosinophils in eWAT of control treated Aged-yEOS (n=4) and PT pre-treated Aged-yEOSPT mice (n=5) as measured by flow cytometry. One out of two independently performed experiments is shown. (c) Experimental protocol. (d) eWAT IL-6 protein levels in Aged-PBS (n=10) and Aged-yEOSPT (n=10) mice. (e) Intra- and inter-group (f) comparison of average time on wheel (Rotarod test) of Aged-yEOS (n=10) and Aged-yEOSPT (n=10) mice. (g) Intra- and inter-group (h) comparison of maximal force (grip strength) of Aged-yEos (n=10) and Aged-yEOSPT (n=10) mice. Delta in performances for (f) and (h) were calculated relative to baseline (post- minus pre-treatment results). The experiment (c-h) was done once. (i) Experimental protocol. (j) IL-6 plasma levels relative to baseline (pre-treatment) in Aged-ISO (n=16) and Aged-aIL6 (n=15) mice. (k) Intra- and inter-group (l) comparison of average time on wheel (Rotarod test) of Aged-PBS (n=16) and Aged-aIL6 (n=15) mice. (m) Intra- and inter-group (n) comparison of maximal force (grip strength) of Aged-PBS (n=16) and Aged-aIL6 (n=15) mice. Delta in performance was calculated relative to baseline (post- minus pre-treatment results). Data (i-n) are pooled from two independently performed experiments. Statistical significance was calculated by Wilcoxon matched pairs signed rank test (e, g, k, m) or by unpaired two-tailed Student’s t test (b, d, f, h, j, l, n) and data are shown as individual data points with mean ± SEM. ns: not significant, *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: Briefly, 3.3 × 10 3 Lin − cells were cultured in MethoCult base medium (STEMCELL Technologies) supplemented with 15% FCS, 20% BIT (50 mg/ml BSA in Iscove’s modified Dulbecco’s medium, 1.44 U/ml rh-insulin [Actrapid, Novo Nordisk], and 250 ng/ml human Holo Transferrin [ProSpec]), 100 μM 2-β-mercaptoethanol, 100 U/ml penicillin, 100 μg/ml streptomycin, 2 mM L-glutamine, and a cytokine mix of 50 ng/ml rm-SCF, 10 ng/ml rm-IL-3, 10 ng/ml rh-IL-6, and 50 ng/ml rm-Flt3-ligand (all from ProSpec).

Techniques: Labeling, Flow Cytometry, Two Tailed Test

Journal: Nature metabolism

Article Title: Eosinophils regulate adipose tissue inflammation and sustain physical and immunological fitness in old age

doi: 10.1038/s42255-020-0228-3

Figure Lengend Snippet: The age-related decrease of eosinophil frequency in epidydimal white adipose tissue (eWAT) is associate with the occurrence of inflamm-aging, frailty and immunosenescence. Our study demonstrates that these aging phenotypes are reversible upon young donor eosinophil transfers which dampen local and systemic inflammation leading to improved physical and immune fitness. The observed rejuvenation of the aged host upon young donor eosinophil transfers is dependent on their migration into eWAT since pertussis toxin treatment of the cells abrogates these effects. Systemic blocking of IL-6 only partially phenocopies physical improvements observed upon transfer of eosinophils.

Article Snippet: Briefly, 3.3 × 10 3 Lin − cells were cultured in MethoCult base medium (STEMCELL Technologies) supplemented with 15% FCS, 20% BIT (50 mg/ml BSA in Iscove’s modified Dulbecco’s medium, 1.44 U/ml rh-insulin [Actrapid, Novo Nordisk], and 250 ng/ml human Holo Transferrin [ProSpec]), 100 μM 2-β-mercaptoethanol, 100 U/ml penicillin, 100 μg/ml streptomycin, 2 mM L-glutamine, and a cytokine mix of 50 ng/ml rm-SCF, 10 ng/ml rm-IL-3, 10 ng/ml rh-IL-6, and 50 ng/ml rm-Flt3-ligand (all from ProSpec).

Techniques: Migration, Blocking Assay

Journal: Nature Communications

Article Title: TREM-1 links dyslipidemia to inflammation and lipid deposition in atherosclerosis

doi: 10.1038/ncomms13151

Figure Lengend Snippet: ( 1 ) TREM-1 skews myeloid differentiation towards increased monocyte output by yet undetermined direct or indirect mechanisms, likely acting together with microbial or dietary PAMPs which become systemically available following HFCD feeding. ( 2 ) These factors may also be involved on the upregulation of TREM-1 on peripheral blood myeloid cell subsets. ( 3 ) TREM-1 is further upregulated on Ly6C hi monocytes following their transmigration to the intima and exposure to oxLDL. ( 4 ) Activation of monocytes via TREM-1 may be mediated by neutrophil-derived PGLYRP1 (not depicted) or by HMGB1 released by activated or necrotic macrophages. ( 5 ) TREM-1-mediated activation promotes the expression of scavenger receptors CD36, MSR1 and the LDLR and thereby increases lipid uptake. TREM-1 further promotes foam cell formation by interfering with intracellular cholesterol transport and efflux mechanisms (not depicted). ( 6 ) CD36 facilitates TLR4/6 assembly and NLRP3 activation (not depicted) leading to increased pro-inflammatory cytokine and ROS production, which may contribute to additional lipid peroxidation. Concurring TREM-1-mediated activation could further amplify these pro-inflammatory processes. ( 7 ) Increased uptake of oxLDL leads to the differentiation of Ly6C hi monocytes to Ly6C − macrophage foam cells through a Ly6C int TREM-1 + MHCII + intermediary stage.

Article Snippet: A total of 5 × 10 3 lin − BM cells were cultured in MethoCult base medium (STEMCELL Technologies, Grenoble, France) supplemented with 15% FCS, 20% BIT (50 mg ml −1 BSA in Iscove's modified Dulbecco's medium, 1.44 U ml −1 rh-insulin (Actrapid, Novo Nordisk, Denmark), and 250 ng ml −1 human Holo Transferrin [ProSpec, USA]), 100 μM 2-β-mercaptoethanol, 100 U ml −1 penicillin, 100 μg ml −1 streptomycin, 2 mM L -glutamine, and a cytokine mix of 50 ng ml −1 rm-SCF, 10 ng ml −1 rm-IL-3, 10 ng ml −1 rh-IL-6, and 50 ng ml −1 rm-Flt3-ligand (all from ProSpec, USA).

Techniques: Transmigration Assay, Activation Assay, Derivative Assay, Expressing