IGF1 Shapes Macrophage Activation in Response to Immunometabolic Challenge

Report

IGF1 Shapes Macrophage Activation in Response to Immunometabolic Challenge Graphical Abstract

Authors Olga Spadaro, Christina D. Camell, Lidia Bosurgi, Kim Y. Nguyen, Yun-Hee Youm, Carla V. Rothlin, Vishwa Deep Dixit

Correspondence [email protected]

In Brief Endocrine IGF1 plays pleiotropic functions and provides signals to macrophages to sustain tissue development and homeostasis. In this work, Spadaro et al. show that M2-like macrophages are an important source of IGF1 itself and that the myeloid-derived IGF1R signaling regulates immune metabolism. Host adaptation to high-fatdiet-induced obesity and helminth clearance requires myeloid IGF1R, but not the response to cold-stress.

Highlights d

Ablation of myeloid IGF1R worsens diet-induced obesity

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M2-like macrophage activation is sustained by the IGF1R signaling

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Myeloid IGF1R promotes resolution of inflammation upon helminth infection

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Cold stress-induced metabolic adaptation does not involve myeloid IGF1R signaling

Spadaro et al., 2017, Cell Reports 19, 225–234 April 11, 2017 ª 2017 The Author(s). http://dx.doi.org/10.1016/j.celrep.2017.03.046

Cell Reports

Report IGF1 Shapes Macrophage Activation in Response to Immunometabolic Challenge Olga Spadaro,1,2 Christina D. Camell,1,2 Lidia Bosurgi,2 Kim Y. Nguyen,1,2 Yun-Hee Youm,1,2 Carla V. Rothlin,2 and Vishwa Deep Dixit1,2,3,4,* 1Section

of Comparative Medicine and Program on Integrative Cell Signaling and Neurobiology of Metabolism of Immunobiology 3Yale Center for Research on Aging Yale School of Medicine, New Haven, CT 06520, USA 4Lead Contact *Correspondence: [email protected] http://dx.doi.org/10.1016/j.celrep.2017.03.046 2Department

SUMMARY

In concert with their phagocytic activity, macrophages are thought to regulate the host immunometabolic responses primarily via their ability to produce specific cytokines and metabolites. Here, we show that IL-4-differentiated, M2-like macrophages secrete IGF1, a hormone previously thought to be exclusively produced from liver. Ablation of IGF1 receptors from myeloid cells reduced phagocytosis, increased macrophages in adipose tissue, elevated adiposity, lowered energy expenditure, and led to insulin resistance in mice fed a high-fat diet. The investigation of adipose macrophage phenotype in obese myeloid IGF1R knockout (MIKO) mice revealed a reduction in transcripts associated with M2-like macrophage activation. Furthermore, the MIKO mice infected with helminth Nippostrongylus brasiliensis displayed delayed resolution from infection with normal insulin sensitivity. Surprisingly, cold challenge did not trigger an overt M2-like state and failed to induce tyrosine hydroxylase expression in adipose tissue macrophages of control or MIKO mice. These results show that IGF1 signaling shapes the macrophage-activation phenotype. INTRODUCTION Somatotrophic endocrine hormones such as GH, ghrelin, and IGF1 are known to influence the innate immune system (Dixit et al., 2004; Qin et al., 2014). Macrophages play indispensable roles in development (Pollard, 2009), homeostasis, and host defense (Okabe and Medzhitov, 2016; Wynn et al., 2013). The macrophage activation paradigm was initially thought to follow a binary division between a pro-inflammatory (M1) or an anti-inflammatory (M2) state. Indeed, in vitro, inductive signals of IFNg (Th1) or IL-4 (Th2) produce a distinct classical or alternative activated macrophage phenotype, respectively (Sica and Mantovani, 2012). However, it has now become increasingly clear

that, within a tissue microenvironment, macrophages possess a wide spectrum of activation states that is influenced by an array of tissue-derived growth factors, cytokines, chemokines, lipokines, and hormones (Murray et al., 2014; Xue et al., 2014). Interestingly, the liver-derived endocrine hormone IGF1, which is an established regulator of somatotropic axis, provides signals to macrophages that are implicated in muscle regeneration (Lu et al., 2011; Saclier et al., 2013), neuronal survival, and adipose tissue function (Chang et al., 2016; Ueno et al., 2013). Intriguingly, however, reduction in IGF1 signaling extends healthspan and lifespan in model organisms (Holzenberger et al., 2003). Furthermore, mutations that lead to reduced activity of IGF1R in humans are also associated with increased lifespan (Erikson et al., 2016; Kenyon 2010; Suh et al., 2008). Our prior studies showed that IGF1R deletion in macrophages regulates inflammation and reduction in GH receptor is associated with reduced adipose tissue inflammasome activation (Spadaro et al., 2016). These studies suggested that macrophages may produce IGF1. Indeed, bioinformatics analyses of existing databases (Immgen) and additional studies showed that macrophages highly express IGF1 (Spadaro et al., 2016; Tonkin et al., 2015). It has been hypothesized that local production of hormones by immune cells is an additional means of homeostatic cellular control (Besedovsky and del Rey 1996), especially as autocrine and paracrine levels of macrophage-derived IGF1 could be substantially higher instead of being diluted from a distal endocrine source such as liver. This led us to ask the question of whether macrophage’s ability to secrete and respond to IGF1 is part of the immune-metabolic cross-talk that controls macrophage function in metabolic stress and host defense. IGF1is involved in nutrient-sensing response to insulin, as liver-specific IGF1R-deficient mice develop muscle insulin resistance (Ferna´ndez et al., 2001) and IGF1 treatment has been shown to improve insulin action in small clinical studies (Moses et al., 1996; Thrailkill et al., 1999). Recent studies identified that in addition to macrophages, adipocytes also express IGF1 and that local, adipose-derived IGF1 influences adaptation to metabolic stress (Chang et al., 2016). We showed that macrophages activated by IL-4 into a M2-like polarized state secrete higher levels of IGF1 and express IGF1R, suggesting auto/paracrine role of this classical hormone in macrophage function. Here, we report that myeloid-cell IGF1 signaling integrates

Cell Reports 19, 225–234, April 11, 2017 ª 2017 The Author(s). 225 This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

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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.

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immune-metabolic interactions to facilitate macrophage activation status. RESULTS AND DISCUSSION Myeloid-Cell-Specific Deletion of IGF1R Predisposes to HFD-Induced Obesity We recently showed that ablation of IGF1R in macrophages led to reduced caspase-1 cleavage in response to canonical Nlrp3 activator such as ATP (Spadaro et al., 2016). This suggested that macrophages have a functional somatotropic axis that impacts immune-metabolic function. Interestingly, we found that Igf1 mRNA shows the highest expression in bone marrow-derived macrophages (BMDMs) that are skewed in vitro into an IL-4driven M2 phenotype, consistent with previous reports on IGF1 expression under M2-polarizing stimuli (Arkins et al., 1993; Martinez et al., 2006) (Figure 1A). Furthermore, compared to M0 or M1 macrophages, the M2-like cells secrete significantly higher levels of IGF1 (Figure 1B). In order to describe the role of IGF1-IGF1R in vivo, we generated mice with myeloid-cell-specific ablation of IGF1R using LysM-Cre, which caused efficient deletion of IGF1R in macrophages (LysMcre+/ , Igf1rfl/fl, or MIKO) (Figure 1C and Figure S1B). The functional analysis of IGF1R-deficient macrophages revealed impaired E.coli (K12) bioparticles elicited phagocytic activity (Figure 1D) concordant with increased IGF1 secretion from M2-like macrophages (Figure S1A). Further analyses found that ablation of IGF1 axis did not, however, impair the potential of BMDMs to change the expression of either Nos2, Il18, and Il6 or Arg1, Ccl22, and Chil3, respectively associated with M1- or M2-like signatures (Figure S1C andS1D). Given that microphage activation is linked to adiposity (Donath and Shoelson 2011; Gregor and Hotamisligil 2011; Han et al., 2013; Vandanmagsar et al., 2011), we sought to determine the effects of myeloid-cell-specific ablation of IGF1R in the inflammatory-driven environment of high-fat-diet-induced obesity (DIO). Upon high-fat diet feeding, compared to control mice, the male MIKO mice gain significantly more fat mass, and they show less lean mass compared to their littermate controls (Figures 1E-G). The MIKO-HFD (high-fat diet) mice show a slight increase in total body weight that was not statistically significant (Figure 1E). To understand the cause of increased adiposity in MIKO mice, we analyzed several metabolic outcomes and found no significant difference for respiratory exchange rate (RER or RQ, Figures S1E-S1G), locomotor activity (Figures S1H and S1I), or food or water intake (Figures S1L–O). However, MIKO mice show an altered energy balance with decreased energy

expenditure (EE) (Figure 1H) ascribed to the resting expenditure (REE) (Figures 1I, S1F). Notably, when these values are normalized by body weight (Tscho¨p et al., 2011) using the analysis of covariance (ANCOVA), change in EE between the two genotypes (Figure 1J) does not reach statistical significance. Also, the ablation of IGF1R in macrophages did not affect glucose levels in response to ipGTT (Figure 1K), but MIKO displayed increased insulin resistance (Figure 1L) during an intraperitoneal insulin tolerance test (ipITT), with no difference in the mice fed a chow diet (Figures S1P-S1Q). Together, these data suggest that the myeloid IGF1-IGF1R interaction influences whole body metabolism in response to a high-fat diet. Myeloid IGF1R Signaling Controls Adipose Tissue Mass and M2-Like Macrophage Activation under High-Fat Diet-Induced Metabolic Stress Consistent with our prior metabolic data, MIKO mice show increased visceral (VAT) (Figure 2A) and subcutaneous (SAT) (Figure S2C) mass with increased number of stromal-vascular cells (SVF) in VAT (Figure 2B, S2C). This higher cellularity in SVF fraction was mainly attributed to an increase in F4/80+ CD11b+ adipose tissue macrophages (ATM) (Figures 2C and 2D) in MIKO VAT in HFD-fed conditions. No difference was observed for B220+ MHCII+ B lymphocytes in VAT or SAT (Figure S2A,S2B,S2I, and S2N) or F4/80+CD11b+ ATMs in SAT (Figure S2H and S2M) between the two genotypes upon HFD. Also, myeloid IGF1R deletion did not alter the systemic CD4+ and CD8+ T cell subsets or cause any alterations in the thymic T cell development in mice (Figure S2Land O), suggesting that macrophage IGF1 signaling is dispensable for maintenance of T cell homeostasis. To examine the activation status of tissue macrophages, the F4/80+ cells were positively selected from VAT of control and MIKO mice fed an HFD and compared to macrophages isolated from spleen. Interestingly, consistent with exacerbated obesity, myeloid deletion of IGF1R is associated with a reduction in mRNA expression of Ccl17, Ccl22, Ccl24, Retnla, Tgfb1, and Irf4 (Figure 2E) that associate with M2-like alternative macrophage activation. Importantly, ablation of IGF1 signaling in splenic macrophages did not affect the genes regulated in VAT suggesting microenvironment-specific responses influence macrophage function. In addition, no difference in pro-inflammatory Il1b, Tnf, and Il6 genes were detected, and other M2-associated transcription factors (Pparg and Stat6) or anti-inflammatory Il-10 gene expression was not significantly different (Figure S2D).

(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.

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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.

To investigate the dependence of the observed phenotype exclusively on the macrophage population, we gated on the F4/80low CD11blow myeloid population and found no differences between MIKO and littermate controls (Figure S2E). Also, the higher expression of CD206+ cells within the F4/80hi CD11bhi in comparison to the F4/80low CD11blow myeloid population further confirms the purity of the gated macrophage population (Figure S2F andS2G) in these mice (Lumeng et al., 2008; Wentworth et al., 2010). Taken together, these data show that the macrophage IGF1R signaling influences adipose tissue homeostasis by sustaining a partial M2-like signature in the complex spectrum of macrophage responses in obesity. Macrophage IGF1R Signaling Is Dispensable for Adaptation to Cold-Induced Metabolic Stress Unlike obesity, which is a state of positive energy balance, survival during cold stress requires the host to mobilize energy reserves with induction of adipose lipolysis to regulate core body temperature. Several studies have demonstrated that in response to cold, white adipocyte precursors differentiate into a population of brown-like adipocytes that express UCP1 for heat production (Chabowska-Kita and Kozak, 2016; Harms and Seale, 2013). Furthermore, exposure of mice to cold stress of 4 C was found to induce M2-like macrophage polarization within brown and white adipose tissue (Nguyen et al., 2011; Qiu et al., 2014; Rao et al., 2014) that aids adaptive thermogenesis and

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maintenance of homeostasis. Given a reduction in M2-like signatures in MIKO mice exposed to HFD-induced caloric excess, we next investigated the role of macrophage IGF1 signaling in a mouse model of cold-induced adaptive thermogenesis that induces M2-like responses. We found that ablation of IGF1R had no effect on core body temperature upon 24 hr of cold challenge at 4 C (Figure 3A). As established before (Guerra et al., 1998) cold stress induced mRNA, as well as protein, expression of UCP1 in brown adipose tissue (BAT) (Figures 3B and 3C). MIKO mice showed elevation of UCP1 expression in BAT of cold-stressed mice (Figures 3B and C), but no difference in body temperature (Figure 3A), body weight (Figure S3A) or blood glucose (Figure S3B) was detectable between the control and MIKO mice. Analysis of white retro peritoneal fat (RPT) in mice exposed to 4 C cold stress shows 10x increase of Ucp1 and Cidea as ‘‘browning/beiging’’ markers upon 24 hr cold challenge, while ablation of macrophage IGF1R had no effect on these genes and UCP1 protein levels (Figures 3D-3F). Genes regulating lipolysis were also induced evenly in response to cold in both genotypes (Figures S3D and E); interestingly, at 22 C, MIKO show increased expression of the same genes, consistent with their reduced subcutaneous fat mass (Figure S3C-E). These data suggested that macrophage IGF1-IGF1R may not be relevant to whole-body metabolism during cold stress. To definitively asses the myeloid contribution to cold-induced thermogenic responses, we utilized the double reporter mT/mG

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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).

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(Muzumdar et al., 2007) transgenic mice, in which, after LysMCre-mediated intrachromosomal recombination, the membrane tomato (mT) sequence is excised, allowing the pCA promoter to drive expression of membrane targeted eGFP protein (mG). This can be visualized as change of color from red (in non-recombined cells) to green (in myeloid Cre+ recombined cells) (Figure S3F). Additionally, we created triple-transgenic mice using this approach and deleted IGF1R such that all GFP+ (Cre recombined) expressing cells lack Igf1r allele (Figure S3G). The gating strategy for quantifying GFP+ adipose tissue myeloid cells is shown in Figure 3G. No change in macrophage frequency is observed upon cold challenge in SAT from females in both genotypes (Figures 3H and S3I), while male MIKO mice show increase in ATM frequency at both 22 C and 4 C (Figures 3H and S3J). In WT female mice, cold did not alter LysMlin+F4/80 CD11b+Ly6G+ neutrophils, while in males 24 hr cold exposure reduced the frequency of these cells in SAT (Figure S3K and S3L). Instead, deletion of myeloid IGF1R is associated with cold-induced increase in neutrophil frequency within the SAT of female mice only (Figure S3K). To examine gene expression of ATMs, we selected F4/80+ cells from VAT and SAT depots. Cold challenge of 4 C for 24 hr did not affect the limited number of analyzed genes thought to reflect an M2-like alternative macrophage phenotype in VAT or SAT (Figures 3I and 3J), but this condition did upregulate Ucp1 expression in total VAT (Figure 3K). Furthermore, ablation of IGF1R in myeloid cells had no impact on genes involved in M2-like responses in purified ATMs in VAT and SAT of coldstressed mice (Figures 3I and 3J). Given that alternatively activated macrophages are reported to produce catecholamines in cold-stress conditions to regulate adipose biology (Nguyen et al., 2011), we also tested whether macrophage IGF1/IGF1R signaling impacts this alternate mechanism. Surprisingly, realtime PCR did not detect tyrosine hydroxylase (Th) mRNA in F4/ 80+ ATMs of mice exposed to 22 C or 4 C, and neither immunoblot analysis detected the TH protein expression in BMDMs skewed to M2-like macrophages (Figures 3L and 3M). These data are consistent with a recent report in which macrophages isolated from adipose tissue of cold challenged mice did not express Th (Chang et al., 2016), thus suggesting that, in the sub-

population of F4/80+ cells that were analyzed in our study, the macrophage M2-like state or potential catecholamine production from these cells did not account for adipose homeostasis and adaptation to cold. Taken together, these data demonstrate that the macrophage-IGF1 axis does not impact immunometabolic homeostasis in a cold-stressed state. Myeloid IGF1 Signaling Delays Resolution of Helminth Infection without Impairing Insulin Action Prior studies have demonstrated that helminth infection induces type 2 immune response to control adipose tissue homeostasis and improves glucose tolerance in mice (Wu et al., 2011). Furthermore, in response to tissue or cellular injury, IGF1 secretion by connective tissue stimulates reparative cell types to replicate in different contexts (Clemmons, 2007). Mechanisms of tissue repair are also elicited by protective type 2 immune responses during helminth parasites infection and involve M2 macrophage-secreted growth factor and cytokines (Chan et al., 2016; Gause et al., 2013). In order to assess the contribution of myeloid IGF1-IGF1R signaling in model of helminth-type 2 elicited immunity, we subcutaneously inoculated Nippostrongylus Brasiliensis (Nippo) larvae in young male MIKO and control mice. At days 3 and 5 after infection, we collected lung, bronchial alveolar lavage (BAL) fluid, and intestine, as the larvae first enter the lung between 24 hr and 48 hr post-infection, then migrate to the small intestine, where they fully mature and lay eggs (Chen et al., 2012). Infiltration of Ly6G+ CD11b+ lung neutrophils and CD11bHiCD11cInt lung interstitial macrophages increases at day 3 after infection and declines at day 5 when animals recover (Chen et al., 2012). Ablation of IGF1R in myeloid lineage led to increase in frequency and number of lung neutrophils at day 3 of infection (Figures 4A and 4B), which was reflected by increased worm counts at day 5 (Figure 4C) and increased retention of interstitial lung macrophages at day 3 (Figures 4D and 4E), suggesting a sustained inflammatory response in MIKO compared to control. Analysis of BAL fluid revealed no difference in RBCs and WBCs in infected mice of either genotype (Figure S4A and S4B). Consistent with this result, no changes in Ly6G+ CD11b+ BAL neutrophils (Figure S4C) or in CD3+ BAL lymphocytes

(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.

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were observed (Figure S4D). CD11bHiCD11cInt Arg1+ and CD11bHiCD11cInt RELMa+ interstitial M2 responses increased progressively through day 3 and 5 post-infection with no difference between control and MIKO mice (Figure S4E). These data suggest that ablation of IGF1 signaling in myeloid cells increases the interstitial lung macrophages and neutrophils, but analysis of hemorrhaging in BAL did not reveal overt lung damage. Helminth parasite-dependent host responses induce alternative macrophage activation and impact whole-body metabolism (Guigas and Molofsky, 2015). Therefore, we next performed an ipITT on uninfected control, infected control, and MIKO mice. In line with previous reports (Wu et al., 2011), compared to uninfected animals, the WT mice infected with Nippo had improved insulin sensitivity. Surprisingly, compared to WT infected animals, the MIKO mice did not show any significant difference in insulin sensitivity (Figure 4F). Consistent with these data, ablation of IGF1R in myeloid cells did not affect the frequency or number of F4/80+ CD11b+ in VAT (Figures 4G-4I) or SAT (Figure S4F) of mice exposed to helminths. Collectively, these data suggest that lack of IGF1R signaling in myeloid cells delays resolution of Nippo infection without compromising insulin action. Prior studies show that macrophages are required in helminthelicited responses, as their deletion worsens the disease (Chen et al., 2012). Thus, in MIKO mice, in which neutrophil responses are affected, it is likely that the delayed resolution from infection is predominantly due to macrophage IGF1R signaling. Strikingly, conditional inactivation of insulin receptor (IR) in myeloid lineage cells protects against insulin resistance and adipose tissue macrophage accumulation (Mauer et al., 2010). As IR and IGF1R can form heterodimers (Cubbon et al., 2016), further studies are required to delineate the contribution of IR signaling and whether this mechanism regulates an overall modest host response observed in the MIKO mice. Interestingly, recent studies have shown that adipocyte-derived IGF1, but not myeloid-derived IGF1, regulates perigonadal adipocyte number in HFD feeding, implying a role for adipose-secreted IGF1 (Chang et al., 2016). Although it is now evident that macrophages do not display binary M1-M2 polarization, macrophages secrete IGF1, and an auto/paracrine IGF-IGF1R signaling in myeloid compartment facilitates a reparative M2like macrophage activation program. Our studies show that ablation of IGF1R in macrophages, which disrupts local as well as endocrine IGF1-mediated signaling, impacts adipose tissue homeostasis, and influences macrophage activation status.

EXPERIMENTAL PROCEDURES Experimental Animals The LysMCre (B6.129P2-Lyz2tm1(cre)Ifo), the Igf1R-flox (B6.129-Igf1rtm2Arge), and the mT/mG (B6.129(Cg)-Gt(ROSA)26Sortm4(ACTB-tdTomato,-EGFP)Luo/J) strains were purchased from Jackson Laboratories. The mice were multihoused and fed ad libitum either with normal chow diet (5002; LabDiet) or with a high-fat diet (60% HFD, Research Diets D12492). Igf1Rfl/fl were crossed to LysMCre/Cre to generate the littermate Igf1Rfl/ LysMCre/ . Heterozygous mice were inter-crossed to generate experimental LysMCre/ Igf1Rfl/fl and control LysMCre/ . For the triple-transgenic model, LysMCre/CreIgf1Rfl/fl heterozygous mice were first inter-crossed with the mT/mGfl/fl in order to generate the triple-heterozygous-transgenic Igf1Rfl/ LysMCre/ mT/mGfl/ ;triple-heterozygous mice were then inter-crossed to generate the experimental LysMCre/ Igf1Rfl/fl mT/mGfl/ and the control LysMCre/ mT/mGfl/ mouse model. All animals were kept according to guidelines issued by Yale University’s Institutional Animal Care and Use Committee (IACUC). Cell Culture Differentiation of BMDMs has been previously described (Spadaro et al., 2016). IGF1 secretion in supernatants was measured by ELISA (ab100695-IGF1 Abcam) according to the manufacturer’s protocols. The phagocytosis assay is based on the Vybrant Phagocytosis Assay Kit, (Life Technologies, V6694) according to the manufacturer’s protocol. Measurements of EE and Body Composition The TSE PhenoMaster System (V3.0.3) Indirect Calorimetry System was used to monitor energy expenditure, activity, and food and water consumption of mice in individual chambers at 30 min intervals. Oxygen consumption (O2) and carbon dioxide production (CO2) are the parameters sampled in each cage to determine energy expenditure (EE). EE was then analyzed either after normalization by body weight or using the ANCOVA analysis (SPSS software). Activity measurements were taken via infrared sensors, while food and water intake were measured through weight sensors located on food and water dispensers hanging within the cage. Body composition was measured in vivo by magnetic resonance imaging (EchoMRI; Echo Medical Systems). Glucose and Insulin Tolerance Test For the Insulin Tolerance Test (ITT), mice were fasted for 4 hr and injected with human insulin solution (Sigma Aldrich, I9278) at an amount of 0.4 U/kg for chow-fed mice and 0.6 U/Kg for HFD-fed mice. For the Glucose Tolerance Test (GTT), both chow and HFD-fed mice were fasted for 12 hr and intraperitoneally injected with 1.5 g/Kg of 10% glucose solution (Sigma Aldrich, G8270). For both the ITT and the GTT, blood glucose was measured from the tail vein using a glucometer (Breeze, Bayer Health Care) over 15’, 30’ and 60’ time points up to 90’ for the ITT and 180’ for the GTT. Cold Challenge Mice were acutely shifted from 22 C to 4 C for either 24 hr or 36 hr in prechilled cages w/o nestlet, in groups of two per cage with food ad libitum.

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 postinfection 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 HolmSidak for multiple-comparisons corrections (as for ITT).

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Rectal temperatures of mice were measured using a Physitemp thermometer. Parasite Infection and Downstream Analysis 650 viable third-stage larvae of N. brasiliensis were injected subcutaneously in WT and MIKO mice as described previously (Chan et al., 2016). Animals were sacrificed at day 3 and day 5 post-infection. Broncho-alveolar (BAL) fluid, lung, and small intestine were collected. Total cell numbers from BAL and lung tissue were counted, and leukocytes were analyzed by flow cytometry. Worm burdens were determined as described previously(Chan et al., 2016). Flow Cytometry Antibodies used were CD45-BV711, CD45 PE-Cy7, F4/80-eF450, CD11bPerCPCy5.5, CD11b-APC-Cy7, B220-APC, MHC II-PE, CD11c-eF450, Ly6G-APC, Ly6G-PECy7, Biotinylated Anti-Murine RELMa (Peprotech), AF647 Streptavidin, and Arg1-APC. Data were acquired on a BD-LSRII and analyzed using FlowJo vX. Statistical Analysis Statistical significance was calculated by two-tailed Student’s t test, by analysis of variance (ANOVA), and by Multiple t test with either Holm-Sidak or Tukey’s test for multiple comparisons corrections when appropriate (GraphPad Prism 6 software). p < 0.05 was considered significant for all the analyses. SUPPLEMENTAL INFORMATION Supplemental Information includes Supplemental Experimental Procedures, four figures, and one table and can be found with this article online at http:// dx.doi.org/10.1016/j.celrep.2017.03.046. AUTHOR CONTRIBUTIONS O.S. designed and conducted all the experiments, analyzed and interpreted the data, and participated in writing the manuscript. C.D.C. participated in performing FACS analysis and in the interpretation of the data. L.B. performed the infection experiments. K.Y.N. assisted in generation and validation of IGF1R mice. Y.H.Y. conducted the study on the thymus. C.V.R. conducted the infection study design and data analyses. V.D.D. conceived and supervised the project, interpreted the data, and wrote the manuscript. ACKNOWLEDGMENTS Research in the Dixit lab is supported in part by US National Institutes of Health (NIH) grants P01AG051459, AG051459, and AI105097. C.C. is supported through an NIA-AG043608 career development supplement award. C.V.R. is an HHMI Faculty Scholar and is supported by NIH grant AI089824, and L.B is supported by an American Italian Cancer Foundation Post-Doctoral Fellowship. Received: August 23, 2016 Revised: January 28, 2017 Accepted: March 14, 2017 Published: April 11, 2017 REFERENCES Arkins, S., Rebeiz, N., Biragyn, A., Reese, D.L., and Kelley, K.W. (1993). Murine macrophages express abundant insulin-like growth factor-I class I Ea and Eb transcripts. Endocrinology 133, 2334–2343. Besedovsky, H.O., and del Rey, A. (1996). Immune-neuro-endocrine interactions: facts and hypotheses. Endocr. Rev. 17, 64–102. Chabowska-Kita, A., and Kozak, L.P. (2016). The critical period for brown adipocyte development: Genetic and environmental influences. Obesity (Silver Spring) 24, 283–290.

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