 in brown adipose tissue.

Obesity has reached pandemic proportions in the Western world and represents a major risk factor for the development of type 2 diabetes, cardiovascular diseases and cancer 1,2. It results from an imbalance between energy intake and energy expenditure causing an increment of fat mass in adipose tissue or liver. Adipose tissue can be classified as white adipose tissue (WAT), brown adipose tissue (BAT) or beige adipose tissue (BgAT) [3][4][5][6]. WAT participates mainly in energy storage and release 3 whereas BAT and BgAT can metabolize stored lipids to produce heat by a process known as nonshivering thermogenesis or adaptive thermogenesis 4,5. This type of thermogenesis occurs in the mitochondria and is mediated by uncoupling protein 1 (UCP1), which facilitates the uncoupling of electrons from the synthesis of adenosine triphosphate (ATP) towards heat production 4. Obesity is correlated with an increase in WAT mass and impaired BAT activity 7,8. It has been proposed that potentiating thermogenesis in BAT could influence weight gain in humans 9,10.
Adipose tissues are rich in effectors of both innate and adaptive immunity 11,12 whose numbers are altered with obesity 13,14. The contribution of the innate immune system and specifically adipose tissue macrophages to low-grade inflammation and homeostasis in adipose tissue has been well documented [15][16][17]. Macrophage accretion in adipose tissue can increase inflammatory cytokines and lead to chronic low-grade inflammation [17][18][19]. With regards to BAT, alternatively activated macrophages have been suggested to influence BAT homeostasis and thermogenesis 20. However, these findings were recently challenged 21 raising questions about the involvement of macrophages in BAT activation.
While investigating the role of innate immunity in obesity, we previously identified myeloid-resident neuropilin-1 (NRP1) as necessary for healthy weight gain and maintaining glucose tolerance in obesity 22. NRP1 is a single pass transmembrane receptor that binds multiple ligands (e.g. semaphorins, VEGFs) and receptors (e.g. VEGFR2, integrins, plexins), influences intracellular signaling 23, axonal guidance 24 and neuronal and vascular development 25. Adipose tissue macrophages (ATMs) devoid of NRP1 are less efficient at internalizing lipids and shift their metabolism towards a more pro-inflammatory carbohydrate-based glycolytic metabolism 22. Here we investigated the role of NRP1 expressing myeloid cells on BAT biology and the control of thermogenesis.

Results
Interscapular brown adipose tissue macrophages express high levels of neuropilin-1. In order to investigate the transcriptomic signatures of interscapular brown adipose tissue (iBAT) macrophages compared to resident macrophage populations of other tissues, we analyzed the transcriptomes of fluorescence-activated cell sorted (FACS) native macrophages across various organs 26. We ran Gene Set Variation Analysis (GSVA) to identify enriched gene sets in iBAT-resident macrophages (iBAT-MPs) compared to macrophages from other tissues. We found enrichment in distinct gene sets across all macrophage populations (Fig. 1A, Supplemental Table S1). Six differential gene expression analyses by DESeq2 were performed between iBAT-MPs and all the other tissue macrophage populations; we identified 624 significantly upregulated and 375 significantly downregulated genes in a minimum of 5 out of 6 comparisons that were associated with iBAT-MPs (Fig. 1B, Supplemental Table S2-S3). Interestingly, by analyzing Gene Ontology (GO) biological processes, we found transcript enrichment in iBAT-MPs for cell migration involved in vasculogenesis, semaphorin-plexin signalling pathways and positive regulation of cell migration involved in sprouting angiogenesis (Fig. 1C, Supplemental Table S4). In order to identify specific genes of interest associated with the iBAT-MP function, we determined the most recurrent genes within the top 15 Gene Ontology (GO) biological processes. Of interest, the second-most frequently associated gene with the iBAT-MP enrichment signature was neuropilin-1 (Nrp1) (Fig. 1C-inset, Supplemental Table S5), a gene which we had previously demonstrated to play a critical role in white adipose tissue macrophage function 22. Therefore, we assessed Nrp1 expression across screened macrophages and found iBAT-MP to express the highest levels of Nrp1 (Fig. 1D). These data raise the possibility that NRP1 may play a role in the function of iBAT-resident macrophages.
Neuropilin-1-expressing myeloid cells influence interscapular brown adipose tissue composition in diet-induced obesity. BAT mass and activity are reported to significantly decrease with obesity, age and in diabetic patients [27][28][29]. Given that loss of BAT function can impact the accumulation of body fat, we investigated the effect of a myeloid-specific knockdown of Nrp1 in an experimental model of obesity. Eightweek-old LysM-Cre:Nrp1 fl/fl mice or LysM-Cre:Nrp1 wt/wt control littermates were fed either a high-fat diet (HFD; 60% fat calories) or a matched regular diet (RD; 10% fat calories) for 12 weeks ( Fig. 2A). Consistent with our previous work 22, HFD-fed LysM-Cre:Nrp1 fl/fl mice gained significantly more weight when compared to control LysM-Cre:Nrp1 wt/wt mice (Fig. 2B, C) despite not increasing food intake (Supplemental Figure 1A-B). Following diet-induced obesity, we analyzed distinct adipose tissues: (1) eWAT, which consists of a bilateral intraabdominal visceral depot attached to the epididymis, (2) interscapular brown adipose tissue (iBAT), which is localized between the scapulae and (3) subcutaneous adipose tissue (iWAT), located between the skin and the muscle fascia anterior to the lower segment of the hind limbs (Supplemental Figure 1C). To determine the degree of adiposity, we measured the total weight of eWAT, iBAT and iWAT fat pads ( Fig. 2D-F), and in order to determine if weight gain has an effect on adiposity, we normalized these values to whole-body weight (Supplemental Fig. 1D-G). At the beginning of the diet paradigms (0 weeks), LysM-Cre:Nrp1 fl/fl and control mice showed similar weights of total iBAT (Fig. 2D) while a slight increase in baseline eWAT and iWAT weight was noted in LysM-Cre:Nrp1 fl/fl mice (Fig. 2E,F). When the data were normalized to whole-body weight, only eWAT weight was increased (Supplemental Figure 1D-F). Surprisingly, after 12 weeks of diet, LysM-Cre:Nrp1 fl/fl mice on HFD showed significantly higher total or normalized weights of both iBAT and iWAT when compared to LysM-Cre:Nrp1 wt/wt mice, whereas eWAT showed a trend, but was not significant, despite eWAT weight being significantly higher after 4 weeks of diet in LysM-Cre:Nrp1 fl/fl mice ( Fig. 2D-F, Supplemental Figure 1D-M). Fat pad weights did not vary between genotypes on RD at this later time point (Fig. 2D-F). Consistent with this, histological assessment of iBAT with H&E staining revealed that HFD-fed LysM-Cre:Nrp1 fl/fl mice showed signs of hypertrophy in iBAT when compared to LysM-Cre:Nrp1 wt/wt mice (Fig. 2G).
Adipose tissue vasculature plays an essential role in nutrient and oxygen supply to iBAT as well as in heat dissipation [30][31][32]. We therefore assessed iBAT vascularization by CD31 immunofluorescence staining on sections of iBAT from HFD-fed mice. We observed a decrease in vascularized area per analyzed section ( Fig. 2H-J). Importantly, when we accounted for iBAT hypertrophy (iBAT weight), we observed a significant increase in total vessel area and total vessel length in iBAT from LysM-Cre:Nrp1 fl/fl mice as would be expected from an increased organ size (Supplemental Figure 1N-O). We also assessed blood vessel lacunarity, a morphological measure pertaining to gaps and heterogeneity, and detected a significant increase in vascular lacunarity per section, in iBAT from LysM-Cre:Nrp1 fl/fl mice (Fig. 2K). Brown adipose tissue is also highly innervated by sympathetic nerve fibers, which regulate thermogenesis, and changes in innervation can influence lipid storage in iBAT 26,[33][34][35]. We therefore used the iDISCO method adapted for adipose tissue 36 to clear iBAT from HFD-fed mice and stained for tyrosine hydroxylase (TH) to visualize sympathetic axons and evaluate the impact of myeloid specific Nrp1knockdown on sympathetic innervation. 3D reconstructions of 4-5 randomly selected regions in iBAT were performed to evaluate sympathetic fiber length (Fig. 2L). While we detected a decrease in fiber length in LysM-Cre:Nrp1 fl/fl mice (Fig. 2M), when we normalized fiber length to total iBAT weight to account for the effect of iBAT expansion on innervation, we observed a significant increase in the amount of fibers as expected for a larger tissue (Supplemental Figure 1P). We also evaluated fiber density and we detected a significant decreased fiber density in LysM-Cre:Nrp1 fl/fl mice (Fig. 2N), due to adipocyte