Volume 19, Issue 2, Pages 225-234 (April 2017) IGF1 Shapes Macrophage Activation in Response to Immunometabolic Challenge  Olga Spadaro, Christina D. Camell, Lidia Bosurgi, Kim Y. Nguyen, Yun-Hee Youm, Carla V. Rothlin, Vishwa Deep Dixit  Cell Reports  Volume 19, Issue 2, Pages 225-234 (April 2017) DOI: 10.1016/j.celrep.2017.03.046 Copyright © 2017 The Author(s) Terms and Conditions

Cell Reports 2017 19, 225-234DOI: (10.1016/j.celrep.2017.03.046) Copyright © 2017 The Author(s) Terms and Conditions

Figure 1 Myeloid-Specific Ablation of Igf1r Worsens HFD-Associated Obesity (A) Real-time PCR analysis of Igf1 in M1-M2 polarized cultured BMDMs and liver. (B) ELISA assay depicting IGF1 secretion in the supernatant of M1-M2 polarized cultured BMDMs. (C) Igf1r expression in the LysMCre+/− Igf1rfl/fl (myeloid Igf1r −/−, mentioned in the figures as Cre+ Igf1rfl/fl) strain in comparison to the control LysMCre+/− strain (control, mentioned in the figures as Cre+), depicting the amount of myeloid Igf1r allele deletion in in vitro cultured BMDMs. (D) E. coli-phagocytized fluorescent bioparticles in control versus myeloid Igf1r −/− mice, expressed as percentage out of the total protein content in BMDMs per mouse. (E–G) (E) Body weight, (F) fat mass, and (G) lean mass in myeloid Igf1r −/− and controls fed a HFD or normal chow diet. (H) Adjusted energy expenditure (EE) in relation to body weight showed as individual 30’ measurements over 24 hr in control and myeloid Igf1r −/− mice fed a HFD. (I) Resting energy expenditure (REE) averaged-24 values normalized on body weight in control and myeloid Igf1r −/− mice fed a HFD. (J–L) (J) Analysis of covariance (ANCOVA) of EE in relation to both genotype and body weight as covariates. Glucose tolerance test (GTT) and insulin tolerance test (ITT) in myeloid Igf1r −/− and controls fed on HFD (K and L) showed as mg/dl over time or as area under the curve (AUC). All data are presented as mean ± SEM; ∗p < 0.05 (n = 8–10/group/diet). Statistical differences were calculated either by two-tailed Student’s t test or by multiple t test with Holm-Sidak for multiple comparisons corrections (as for ITT-GTT); both males and females were used for the in vitro data; only male mice were used in the HFD cohort. Cell Reports 2017 19, 225-234DOI: (10.1016/j.celrep.2017.03.046) Copyright © 2017 The Author(s) Terms and Conditions

Figure 2 Myeloid IGF1R signaling controls VAT macrophage infiltration and the M2 signature in adipose tissue macrophages (ATM) upon HFD (A and B) (A) Visceral adipose tissue (VAT) weight and (B) stromal vascular fraction (SVF) cells per gram of VAT in male control and myeloid Igf1r −/− mice fed on HFD. (C) The SVF separated from VAT was analyzed for F4/80+ CD11b+ cells by FACS staining in male control and myeloid Igf1r −/− mice fed HFD. (D) F4/80+ CD11b+ cells are shown as both percentages and total cells/g of VAT. (E) Quantitative gene expression analysis of M2-related markers in isolated F4/80+ adipose tissue macrophages from male control and myeloid Igf1r −/− mice fed HFD. All data are presented as mean ± SEM; ∗p < 0.05 (n = 7-9/group); statistical differences were calculated using two-tailed unpaired Student’s t test. Cell Reports 2017 19, 225-234DOI: (10.1016/j.celrep.2017.03.046) Copyright © 2017 The Author(s) Terms and Conditions

Figure 3 The Myeloid IGF1R Signaling Is Not Involved in the Development of Functional Beige Fat and Thermogenic Homeostasis (A) Delta body temperature in 3-month-old myeloid Igf1r −/− and control female mice left at 4°C and 22°C for 24 hr, expressed as percentage of variation over baseline 22°C body temperature for each mouse/genotype (n = 10 per genotype and temperature). (B and C) (B) Brown adipose tissue (BAT) Ucp1 gene expression and (C) UCP1 protein by immunoblot in myeloid Igf1r −/− and controls housed at 22°C or 4°C (n = 3-9 per genotype and temperature). Quantification of the bands (right panel) normalized to tubulin. (D–F) (D) Retroperitoneal fat depot (RPT) Ucp1, (E) Cidea gene expression, and (F) UCP1 immunoblot analysis in myeloid Igf1r −/− and controls housed at 22°C or 4°C (n = 4-7 per genotype and temperature). Each blot depicts three mice per genotype per temperature. Quantification of the bands (right panel) normalized to tubulin. (G) Gating strategy in VAT showing the detection of CD45+GFP+ myeloid cells and within it the F4/80+CD11b+ macrophages and Ly6G+CD11b+ neutrophils. (H) SVF from subcutaneous adipose tissue (SAT) was analyzed for F4/80+ CD11b+ cells in control and myeloid Igf1r −/− female and male mice housed at 4°C and 22°C. (I and J) qPCR analysis of M2-related markers in isolated F4/80+ adipose tissue macrophages from female control and myeloid Igf1r −/− housed at 4°C and 22°C in both VAT (I) and SAT (J) (n = 5 per genotype and temperature; tissue pooled from n = 10 per genotype and temperature). (K) Ucp1 gene expression in VAT of control mice housed at RT or 22°C or 4°C. (L) qPCR analysis of Th gene expression in hippocampus (3-month-old female WT, n = 8), in VAT and SAT isolated F4/80+ macrophages from 4°C and 22°C mice (3-month-old female control and myeloid Igf1r −/−, n = 5) and M2-polarized BMDMs in the presence or absence of dexamethasone (3-month-old female WT, n = 4). (M) Immunoblot analysis for TH in untreated or polarized (M1-M2) BMDMs, in liver and heart (negative control) and hippocampus (positive control) in 4-month-old WT mice (n = 2/tissue or treatment). Data are presented as mean ± SEM; ∗p < 0.05. Statistical differences were calculated by two-way ANOVA with Tukey’s test and by two-tailed paired Student-t test. Cell Reports 2017 19, 225-234DOI: (10.1016/j.celrep.2017.03.046) Copyright © 2017 The Author(s) Terms and Conditions

Figure 4 Myeloid IGF1R Signaling Protects against Nippostrongylus Brasiliensis-Induced Lung Inflammation (A–E)(A-B) Neutrophils (CD11b+ Ly6g+) and (D-E) interstitial macrophages (CD11bHi CD11cInt) were evaluated by FACS in either uninfected or 3- and 5-day post-infection lungs, in 2-month-old control and myeloid Igf1r −/− male mice; percentages and total cell count is shown (B and E). (C) Worms were counted from intestines, at day 3 and 5 after N. Brasiliensis infection, in 2-month-old control and myeloid Igf1r −/− male mice. (F) Insulin tolerance test was performed in infected and uninfected control and in infected myeloid Igf1r −/− male mice (n = 6/genotype/treatment) at day 3 post N. Brasiliensis infection. (G) FACS plot depicting F4/80+ CD11b+ macrophages within the SVF isolated from the VAT of uninfected and infected control and myeloid Igf1r −/− male mice at day 7 post-infection. (H and I) (H) F4/80+ CD11b+ macrophages are represented both as percentages and (I) number of cells. All data are presented as mean ± SEM; ∗p < 0.05. Statistical differences were calculated either by two-tailed paired Student’s t test or by multiple t test with Holm-Sidak for multiple-comparisons corrections (as for ITT). Cell Reports 2017 19, 225-234DOI: (10.1016/j.celrep.2017.03.046) Copyright © 2017 The Author(s) Terms and Conditions