Journal: Nature immunology

Article Title: A self-sustained loop of inflammation-driven inhibition of beige adipogenesis in obesity

doi: 10.1038/ni.3728

Figure Lengend Snippet: a-c) Adoptive transfer experiments; a 1:1 mixture of PKH26-red labeled Cre - α 4 f/f and PKH67-green labeled Cre + α 4 f/f cells (monocytes or splenic T cells) was simultaneously transferred into obese wild-type mice. The numbers of Cre - α 4 f/f and Cre + α 4 f/f macrophages ( a ) or T cells ( c ) accumulated in inguinal subcutaneous adipose tissue (SAT) or visceral adipose tissue (VAT) and the numbers of Cre - α 4 f/f and Cre + α 4 f/f monocytes/macrophages accumulated in the lymph nodes draining the SAT (SAT dLN) and lymph nodes draining the VAT (VAT dLN) ( b ) were analyzed by flow cytometry. Data (percentage of labeled cells in defined cells) are presented as relative to control. The percentage of labeled Cre - α 4 f/f cells in defined cells was set as 100% in each tissue; n=5 recipient mice in a,b and n=4 recipient mice in c ; data in ( b ) are from one experiment, data in ( a ), ( c ) are representative of two experiments. d) Vcam1 mRNA expression was determined in VAT and SAT of lean mice, fed a normal diet (ND, n=8 mice) or obese mice (fed a HFD, n=10 mice), in mature adipocytes from VAT and SAT of lean (ND, n=6 mice) or obese mice (HFD, n=6 mice), and in CD31 + endothelial cells from SAT of lean (ND, n=5 mice) or obese mice (HFD, n=6 mice). 18S expression was used for normalization and Vcam1 expression of ND-fed mice was set as 1 in each case. Data are from one experiment (SAT endothelial cells) or representative of two experiments (VAT, SAT, VAT adipocytes, SAT adipocytes). ( e ) Comparison of Vcam1 mRNA expression between CD45 - CD31 + endothelial cells and adipocytes from SAT of obese mice is shown. Vcam1 expression of endothelial cells was set as 1; n=6 mice; data are from one experiment. f) Primary mouse adipocytes were treated without (control; Con) or with TNF or palmitate. The surface expression of VCAM-1, expressed as Median Fluorescence Intensity (MFI) units, analyzed by flow cytometry, is shown (n=6 separate cell isolations; data are representative of two experiments). In the right panel, a representative flow cytometry plot depicting VCAM-1 expression of Control-, TNF-, or palmitate-treated adipocytes, as well as isotype control (Iso), is shown. g) Adhesion assay of bone marrow mononuclear cells (BMM) to TNF- or palmitate-pretreated 3T3-L1 adipocytes was performed in the presence of ICAM-1- or VCAM-1-blocking antibodies or respective isotype controls (ICAM-1 Iso or VCAM-1 Iso). The percentage of adherent cells is shown. Shown is one experiment performed in triplicate; representative of 3 experiments. h) Adhesion of BMM from Cre + α 4 f/f or Cre - α 4 f/f mice to TNF- or palmitate-pretreated 3T3-L1 adipocytes (data from separate BMM isolations from n=6 Cre + α 4 f/f mice and n=8 Cre - α 4 f/f mice; pooled from 3 experiments). The percentage of adherent cells is shown. Data are presented as mean ± SEM. *P < 0.05. Mann-Whitney U-test in ( a ), ( c ), ( d ), ( e ), ( f ), Student's t-test in ( b ), ( g ), ( h ).

Article Snippet: Human VCAM1 and UCP1 expression were measured by quantitative real-time RT-PCR using the following probes: human VCAM1 (Hs01003370_m1, ThermoFisher Scientific), UCP1 (Hs00222453_m1, ThermoFisher Scientific).

Techniques: Adoptive Transfer Assay, Labeling, Flow Cytometry, Control, Expressing, Comparison, Fluorescence, Cell Adhesion Assay, Blocking Assay, MANN-WHITNEY

Journal: Nature immunology

Article Title: A self-sustained loop of inflammation-driven inhibition of beige adipogenesis in obesity

doi: 10.1038/ni.3728

Figure Lengend Snippet: a) Energy expenditure (EE, expressed as kcal/h) of obese Cre + α 4 f/f (n=4 mice) and Cre - α 4 f/f (n=5 mice) mice was assessed by a metabolic cage monitoring system for 3 days (left). Average EE in light and dark period is also displayed (right). b) Gene expression in SAT of obese Cre + α 4 f/f or Cre - α 4 f/f mice was evaluated by qPCR (n=8 mice for Cre + α 4 f/f and 10-11 mice for Cre - α 4 f/f ). 18S expression was used for normalization of mRNA expression and the respective gene expression of obese Cre - α 4 f/f mice was set as 1. c) Representative cropped blot images showing immunoblotting for UCP1 (and actin) in SAT from 2 obese Cre + α 4 f/f and 2 Cre - α 4 f/f mice. Actin was used as loading control. d-f) Obese Cre + α 4 f/f or Cre - α 4 f/f mice were challenged with a temperature of 4°C for 12 h. d) Core temperature of obese Cre + α 4 f/f (n=4 mice) or Cre - α 4 f/f (n=6 mice) mice during cold exposure. e) Representative cropped blot images showing immunoblotting (left) for UCP1 (and actin) in SAT of 2 obese Cre + α 4 f/f and 2 obese Cre - α 4 f/f mice exposed to cold. Densitometric analysis (right) of UCP1 immunobloting from a total of 4 Cre + α 4 f/f mice and 5 Cre - α 4 f/f mice is shown. The protein amounts of UCP1 were normalized against actin; the UCP1 amounts (normalized over actin) in SAT from Cre - α 4 f/f mice were set as 1. f) UCP1 staining in SAT from Cre + α 4 f/f or Cre - α 4 f/f mice exposed to cold. g) Mature white and brown adipocytes were isolated from SAT and BAT of obese mice (n=6 mice) after tissue digestion and Vcam1 mRNA expression was determined. 18S expression was used for normalization and Vcam1 expression of SAT was set as 1. Data are presented as mean ± SEM. Data in ( a ), ( d ) and ( e ) are from one experiment; data in ( b ) are pooled from 3 experiments; data in ( c ) and ( g ) are representative of 2 experiments. In ( f ) representative images are from analysis performed on 5 Cre - α 4 f/f and 4 Cre + α 4 f/f mice. *P < 0.05. ANCOVA in ( a ) and Mann-Whitney U-test in ( b ) – ( g ). Abbreviations: Ucp1 , uncoupling protein 1; Cidea , cell death-inducing DNA fragmentation factor-like effector A; Cox8b , cytochrome c oxidase subunit 8b; Cox7a1 , Cytochrome c oxidase subunit 7A1; Acsl1 , acyl-CoA synthetase long-chain family member 1

Article Snippet: Human VCAM1 and UCP1 expression were measured by quantitative real-time RT-PCR using the following probes: human VCAM1 (Hs01003370_m1, ThermoFisher Scientific), UCP1 (Hs00222453_m1, ThermoFisher Scientific).

Techniques: Gene Expression, Expressing, Western Blot, Control, Staining, Isolation, MANN-WHITNEY

Journal: Nature immunology

Article Title: A self-sustained loop of inflammation-driven inhibition of beige adipogenesis in obesity

doi: 10.1038/ni.3728

Figure Lengend Snippet: a-b) Primary adipocytes were cultured with T3 and norepinephrine to induce a thermogenic response, prior to co-culture in the absence or presence of ( a ) M1-like polarized pro-inflammatory BMDMs or ( b ) M1-like polarized primary SAT-derived macrophages [M1MΦ in the figure are M1-like polarized BMDMs in ( a ) or SAT macrophages in ( b )] for 1 h. Co-culture was performed in direct contact (in the same well). Experiments were performed in the absence (control; Con) or presence of α 4 -inhibitor (α 4 -inh.). a ) Ucp1 mRNA expression was analyzed in adipocytes after separation from the BMDMs. 18S expression was used for normalization and Ucp1 expression in adipocytes cultured in the absence of macrophages and in the absence of α 4 -inhibitor was set as 1 (n=5 separate primary adipocyte isolations). b ) The mRNA expression of Ucp1 was analyzed in adipocytes after separation from SAT macrophages. 18S expression was used for normalization and Ucp1 expression in adipocytes cultured in the absence of macrophages and in the absence of α 4 -inhibitor was set as 1 (n=5 separate primary cell isolations from SAT). c-d) Primary adipocytes (pre-stimulated with T3 and norepinephrine) were co-cultured in the absence or presence of M2-like polarized primary SAT-derived macrophages (M2MΦ) in direct contact ( c , in the same well) or in an indirect way ( d , in transwell co-culture system, wherein adipocytes and macrophages were in the lower and upper compartment, respectively). Experiments were performed in the absence (control; Con) or presence of α 4 -inhibitor (α 4 -inh.). The mRNA expression of Ucp1 was analyzed in adipocytes after separation from the macrophages. 18S expression was used for normalization and Ucp1 expression in adipocytes cultured in the absence of macrophages was set as 1 (n=5 separate primary cell isolations from SAT). e-f) Primary adipocytes (pre-stimulated with T3 and norepinephrine) were co-cultured in the absence (control; Con) or presence of M1-like polarized BMDMs (direct contact) for 30 min. The amounts of phospho-p38 ( e ) and phospho-Erk ( f ) in adipocytes were detected by flow cytometry. Phospho-p38 or phospho-Erk protein amounts (MFI) in adipocytes cultured in the absence of M1MΦ were set as 1 (n=4 separate primary adipocyte isolations). g) Primary adipocytes were pre-treated with T3 and norepinephrine for 3 h, washed and then incubated in the absence (control; Con) or presence of an Erk inhibitor for 30 min. After washing, direct co-cultures between adipocytes and M1-like BMDMs were performed for 1 h and the mRNA expression of Ucp1 was studied in adipocytes after separation from the macrophages. The mRNA expression of Ucp1 in the absence of BMDMs and without Erk-inhibitor pre-treatment was set as 1 (n=4 separate primary adipocyte isolations). h, i) T3- and norepinephrine-pre-treated adipocytes were co-cultured with M1-like BMDMs (M1MΦ) in direct contact for 30 min in the absence (control; Con) or presence of α 4 -inhibitor (α 4 -inh.). Phospho-p38 ( h ) and phospho-Erk ( i ) in adipocytes were detected by flow cytometry and their respective amount (MFI) in the presence of M1MΦ but in the absence of α 4 -inhibitor was set as 1 (n=4 separate primary adipocyte isolations). j) Primary adipocyte progenitor cells (AP) were isolated from SAT of wild-type C57BL/6 lean (ND, n=4 mice) and obese (HFD, n=6 mice) mice by FACS-sorting (AP cells were defined as CD31 - CD45 - Sca1 + PDGFRα + cells) and Vcam1 mRNA expression was determined. 18S expression was used for normalization and Vcam1 expression of ND-fed mice was set as 1. k) AP were isolated from SVF of SAT of wild-type mice with FACS-sorting (CD31 - CD45 - Sca1 + PDGFRα + cells). Cells were treated without (control; Con) or with TNF. Vcam1 mRNA expression was analyzed. 18S expression was used for normalization and Vcam1 expression of control-treated cells (Con) was set as 1 (n=4 separate cell isolations). l) AP were isolated as in ( k ) and treated with T3 and norepinephrine. Thereafter, AP were co-cultured in the absence or presence of M1-like polarized pro-inflammatory BMDMs (M1MΦ) in direct contact with each other. Experiments were performed in the absence (control; Con) or presence of α 4 -inhibitor (α 4 -inh.). The mRNA expression of Ucp1 in AP was detected after macrophage separation. 18S expression was used for normalization and Ucp1 expression in the absence of macrophages and in the absence of α 4 -inh. was set as 1 (n=5 separate primary adipocyte progenitor isolations). Data are presented as mean ± SEM. Data in ( b ), ( c ), ( d ) are pooled from 3 experiments; data in ( a ), ( e ), ( f ), ( g ), ( h ), ( i ), ( k ) are representative of 2 experiments; data in ( j ) and ( l ) are from one experiment. *P < 0.05. Mann-Whitney U-test in ( a )-( l ).

Article Snippet: Human VCAM1 and UCP1 expression were measured by quantitative real-time RT-PCR using the following probes: human VCAM1 (Hs01003370_m1, ThermoFisher Scientific), UCP1 (Hs00222453_m1, ThermoFisher Scientific).

Techniques: Cell Culture, Co-Culture Assay, Derivative Assay, Control, Expressing, Flow Cytometry, Incubation, Isolation, MANN-WHITNEY

Journal: Nature immunology

Article Title: A self-sustained loop of inflammation-driven inhibition of beige adipogenesis in obesity

doi: 10.1038/ni.3728

Figure Lengend Snippet: a-d ) Peritoneal macrophages were FACS-sorted from wild-type C57BL/6 mice or Cre + α 4 f/f and Cre - α 4 f/f mice. a-b) Macrophages were cultured on plates pre-coated with Fc or VCAM-1-Fc. a) Wild-type macrophages (n=4 separate cell isolations) were incubated on pre-coated plates for 12 h in the absence (control; Con) or presence of the α 4 -inhibitor (α 4 -inh.) and macrophage TNF expression was analyzed by flow cytometry. Data are expressed as percentage of TNF-positive macrophages (F4/80 + CD11b + ). b) Isolated Cre + α 4 f/f (n=4 separate cell isolations) or Cre - α 4 f/f (n=5 separate cell isolations) macrophages were incubated on plates pre-coated with Fc or VCAM-1-Fc for 6 h and TNF expression was analyzed by flow cytometry. Data are presented as percentage of TNF-positive macrophages (F4/80 + CD11b + ). c-d) Macrophages were co-cultured in a direct cell-to-cell contact mode with 3T3-L1 adipocytes for 12 h. c) Wild-type macrophages were co-cultured with adipocytes in the absence (control, Con) or presence of α 4 -inhibitor (α 4 -inh.) and macrophage TNF expression was analyzed by flow cytometry. Data are presented as percentage of TNF-positive macrophages (defined as CD45 + F4/80 + CD11b + ) (n=4 separate macrophage isolations). d) Cre + α 4 f/f or Cre - α 4 f/f macrophages were co-cultured with adipocytes in a direct cell-to-cell contact mode and macrophage TNF expression was analyzed by flow cytometry. Data are expressed as percentage of TNF-positive macrophages (CD45 + F4/80 + CD11b + ) (n=4 separate macrophage isolations). Data in ( a )-( d ) are representative of 2 experiments. e-f) Primary adipocytes were cultured in the absence or presence of M1-like or M2-like polarized SAT-derived macrophages in a transwell co-culture system (adipocytes, lower compartment; macrophages, upper compartment). The experiments were performed in the presence or absence of the α 4 -inh. or TNF-blocking antibody, as indicated. After 24 h of incubation, adipocyte VCAM-1 expression was analyzed by flow cytometry and is expressed as MFI units (n=4 separate primary cell isolations from SAT in e and n=5 separate primary cell isolations from SAT in f ). Data in ( e )-( f ) are pooled from 2 experiments. g) Correlation analysis between mRNA expression of UCP1 and VCAM1 in human SAT (n=61 human samples). h) Human subcutaneous pre-adipocytes were differentiated to adipocytes and 10 days after starting differentiation were treated without (control; Con) or with TNF or palmitate for 12 h. VCAM1 expression was analyzed and ACTB expression was used for normalization. VCAM1 expression of control-treated adipocytes was set as 1. Data are pooled from 4 experiments. i) Human inflammatory macrophages were allowed to adhere to TNF-pre-treated differentiated human adipocytes in the absence (control; Con) or presence of α 4 -inhibitor (α 4 -inh.). The percentage of adherent cells is shown (n=4 separate macrophage isolations). Data are representative of 2 experiments. j) Differentiated human adipocytes were treated with T3 and norepinephrine to induce a thermogenic response, washed and then co-cultured without or with human M1-like inflammatory macrophages (M1MΦ) in the same well (direct contact) for 1 h. Experiments were performed in the absence (control; Con) or presence of α 4 -inhibitor (α 4 -inh.). The expression of UCP1 in adipocytes (after exclusion of CD45-expressing cells) was detected; ACTB expression was used for normalization. UCP1 expression in adipocytes cultured in the absence of M1MΦ and in the absence of α 4 -inhibitor was set as 1. Shown is one representative experiment (n=4-5 cell culture treatments); similar results were observed in 2 experiments. Data are mean ± SEM.; *P < 0.05. Mann-Whitney U-test was used in ( a )-( f ) and ( h )-( j ) and one-tailed Pearson Correlation analysis in ( g ).

Article Snippet: Human VCAM1 and UCP1 expression were measured by quantitative real-time RT-PCR using the following probes: human VCAM1 (Hs01003370_m1, ThermoFisher Scientific), UCP1 (Hs00222453_m1, ThermoFisher Scientific).

Techniques: Cell Culture, Incubation, Control, Expressing, Flow Cytometry, Isolation, Derivative Assay, Co-Culture Assay, Blocking Assay, MANN-WHITNEY, One-tailed Test