M2 macrophage-secreted Slit3 intensifies sympathetic nerve 1 function in adipose tissue and enhances thermogenesis: A 2 long-term cold adaption way

19 Beiging of white adipose tissue (WAT) is capable of adaptive thermogenesis and 20 dissipating energy. The beiging processes have been associated with the increase of 21 anti-inflammatory M2 macrophages, however the function of M2 macrophage on 22 beiging and the underlying mechanism are not fully understood. Here we identified a 23 macrophage cytokine Slit3 by analyzing the transcriptome of M2 macrophages 24 collected with FACS in inguinal WAT (iWAT) of mice after cold exposure. Once 25 released from macrophages, Slit3 bound to the ROBO1 receptor on sympathetic 26 neuron and activated tyrosine hydrolase (TH) through PKA and CaMKⅡ signaling, 27 and thus stimulated norepinephrine (NE) synthesis and release. NE acts on adipocytes 28 and stimulate thermogenesis. Adoptive transfer of Slit3-overexpressing M2 29 macrophages to iWAT depot acquired local adipocytes with beiging phenotype and 30 enhanced thermogenesis. In addition, mice bearing the myeloid inactivation of Slit3 31 were cold intolerant and gained more weight due to the lowered metabolic rate. 32 Collectively, we demonstrate Slit3 is a macrophage cytokine and promotes beiging 33 and thermogenesis through intensifying the sympathetic nerve function. As the 34 expanded M2 macrophages are integral cell population in adipose tissue, the 35 macrophage-Slit3-sympathetic neuron-adipocyte axis assures the long-term cold 36 adaption. 37 42


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Adipose tissues are important organs for maintaining energy homeostasis. For 44 energy-dealt purpose, adipose tissues have developed into different types. White 45 adipose tissue stores energy in forms of triglycerides after nutrients load, and readily 46 mobilizes to release fatty acids upon energy demands [1] . In contrast, brown adipose 47 tissue dissipates energy by non-shivering thermogenesis [2] . The thermogenic function 48 is fulfilled by mitochondrion-associated uncoupling protein 1 (UCP1), which 49 uncouples oxidative phosphorylation to generate heat instead of ATP [3] . Classical 50 BAT in human beings concentrates at interscapular area in infants and disappears in 51 adulthood [4] . Intriguingly, for the past two decades, studies have revealed a third type 52 of adipose tissue which is aroused in white fat depots [5][6] . Such type of adipose tissue 53 is termed as beige adipose tissue due to its brown-like activity and phenotype [7][8] . As 54 beige adipocytes contain abundant mitochondria and are active in dissipating energy 55 by thermogenesis, they have drawn much attention as a therapeutic target for obesity.

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In adipose tissue, sympathetic nerves are thought to be the main source of NE. It is 69 very interesting that one study reported adipose tissue macrophages also released NE 70 and promoted beiging [16] . However, the conclusion was then questioned by another 71 study which demonstrated that deletion of tyrosine hydroxylase (TH) expression and 72 inhibiting NE synthesis in macrophages failed to impair thermogenesis of adipose 73 tissue [17] . Notwithstanding whether macrophages release NE is controversial, the 74 association between the expansion of anti-inflammatory macrophage (M2) and 75 beiging has been shown in several studies [18][19][20] . So far, most studies focus on 76 revealing the way M2 macrophage is activated, however how M2 macrophages 77 promote beiging is less studied.

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Adipose tissue is composed of heterogeneous cellular populations [21][22] , so the 80 macrophage regulation on beiging may involve communication between macrophages 81 and adipocytes, macrophages and sympathetic nerves, or even other cell types. Here, 82 we found that M2 macrophages in subcutaneous adipose tissue of mouse were 83 activated by cold exposure and were able to synthesize and release Slit3, a secretive

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Slit3 is a cold induced protein secreted by M2 macrophages in iWAT 94 To identify the change in the proportion of M2 macrophages in adipose tissue after 95 cold exposure, we isolated the stromal vascular fraction (SVF) of iWAT from 96 wildtype (WT) mice housed at 22℃ or exposed to 4℃ for 3d, followed by flow 97 cytometry (FCM) analyses. The percentage of M2 macrophages increased 98 significantly after cold exposure (Fig. 1A). Then, we sorted M2 macrophages of the 99 two groups by fluorescence-activated cell sorting (FACS), and the RNA of each group 100 was extracted for transcriptome sequencing. Total 999 genes were identified to change 101 by more than >1.3 fold, of which 527 were elevated in 4℃ cold exposure group 102 versus the 22℃ room temperature (RT) group (Table 1). We performed enrichment 103 analysis of KEGG pathway for prioritizing these candidates, which yielded the most 104 significantly changed pathway of axon guidance including 19 genes (Fig. 1B). Three 105 of the genes belong to the secretory factors, among which the expression abundance 106 of Slit3 is the highest (Fig. 1C). Thus, we proposed Slit3 is a macrophage cytokine. To verify the derivation of the Slit members in adipose tissue, we first analyzed their 109 expression in adipocytes and SVF from three adipose depots. Slit3 is highly expressed 110 in SVF of adipose tissues, especially in iWAT (Fig. S1A). Moreover, the Slit3 mRNA 111 levels were inducible in SVF of iWAT after cold exposure (Fig. S1B). To determine 112 the expression of Slit3 in macrophages, M2 as well as M1 groups of macrophages in 113 iWAT of mice housed at 22℃ or exposed to 4℃ for 3d were sorted by FACS. The 114 mRNA expression of Slit3 was inducible in M2 macrophages (Fig. 1C), but not in M1   The thermal image showed that the iWAT local temperature on the side injected with 137 Slit3-overexpressed M2 macrophages was significantly higher than that on the 138 contralateral side of the GFP control cells (Fig. 2C). At the same time, the volume of 139 adipocytes in the side injected with Slit3-overexpressed M2 macrophages was smaller, 140 as shown in histological sections (Fig. 2D). As anticipated, the protein levels of UCP1, 141 adipose triacylglyceride lipase (ATGL) and phosphorylation of HSL was induced by 142 Slit3 with changes in total HSL (Fig. 2E). Under the same conditions, Slit3 also 143 increased the mRNA levels of thermogenic genes-PGC1α, PRDM16, PPARγ and 144 Cycs; lipolysis genes-ATGL and HSL; and β-Oxidation genes-CPT1β, LCAD, 145 VLCAD and CAD (Fig. 2F). Meanwhile, Slit3 induced a significant increase in iWAT 146 oxygen consumption rates (Fig. 2G). To study the metabolic effects of increased Slit3, 147 M2 macrophges overexpressing Slit3 or GFP were injected into the iWAT of WT mice 148 bilaterally, and whole body energy expenditure was analyzed over the following 60 149 hours using a comprehensive laboratory animal monitoring system (CLAMS). Slit3    s.c. injection of M2 macrophages. We found that after injection of SR59230A, the 189 Slit3-inducing increase in body temperature was dramatically blocked (Fig. 4B).

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Histologically, we found that blockade of β3-AR by SR59230A enlarged the 191 adipocyte size in mice with Slit3 overexpression in M2 macrophages (Fig. 4C).

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We next turned to PKA signaling, a pathway known to be involved in the canonical 194 downstream event of stimulated sympathetic tone. We found that the levels of   found that Slit3 f/f /Lyz2 cre mice were more easily to gain weight than control mice ( Fig.   224 5D), suggesting a lower metabolic rate in Slit3 knockout mice. When exposed to 4 ℃ 225 cold challenge, the ability to sustain core temperature in Slit3 f/f /Lyz2 cre mice was sources in Slit3 f/f /Lyz2 cre mice. Consequently, glycerol production was dramatically 246 reduced in the iWAT of Slit3 f/f /Lyz2 cre mice when exposed to 4℃, although there's no 247 significant difference at 22℃ (Fig. 5J). However, the iWAT oxygen consumption rate 248 was significantly reduced in Slit3 f/f /Lyz2 cre mice at both 22℃ and 4℃ (Fig. 5K).   Given that the Roundabouts (ROBOs) are canonical receptors for Slit3 [27] , we thus 294 determined whether ROBOs are receptors in PC12 sympathetic nerve cells for Slit3.

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The qPCR data indicated that there was only one Robo receptor-Robo1 expressed in 296 PC12 cells (Fig. 6A). Next, we knocked down ROBO1 in PC12 cells by siRNA 297 interference, the phosphorylation of TH (Fig. 7B) and NE production (Fig. 7C) 298 induced by Slit3 were both dramatically suppressed by siROBO1s treatment, as well 299 as phosphorylated PKA substrates and phosphorylated CaMPKII (Fig. 7B). To verify 300 cellular localization of ROBO1 in vivo, we performed IHC of ROBO1 and TH on the 301 two sequential sections respectively, and found positive staining of ROBO1 showed 302 up at TH positive sites (Fig. 7C), which indicate ROBO1 are expressed in sympathetic 303 nerves. Collectively, our results suggest that the Slit3-ROBO1 signaling pathway is 304 necessary for NE production in sympathetic nerve cells.

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Since there are some controversies about whether macrophages express TH [16][17] , we 307 examined TH expression in macrophages. The result showed that both phospho-and 308 total TH were not expressed in both M1 and M2 types of macrophages (Fig. S5A), 309 suggesting that sympathetic neurons are a vital source of NE in adipose tissue.  313 We then explore the factors and signaling for induction of Slit3 in M2 macrophage. 314 We found cold increased Slit3 expression, which was correlated with the increased 315 level of phosphorylated nuclear factor of activated T Cells 2 (NFATC2 or NFAT1) 316 shown by western blot (Fig. 8A). Since only non-phosphorylated NFAT1 can enter  Adipose tissues are highly dynamic and readily change in order to adapt to the 344 environmental alteration such as nutrients load and cold exposure [28][29][30] . Since adipose 345 tissues are heterogeneous and composed of different types of cells [21][22] , the function  In the KEGG analysis of the differential expressed genes, Slit3 is included in axon 362 guidance pathway. It was reported that Slit3 secreted from macrophages interacts with 363 ROBO1 on schwann cells and fibroblasts in the nerve bridge to control axon 364 guidance [37] . Its identity and action as a macrophage derived cytokine are also 365 indicated by another report that Slit3 from osteoclast stimulated osteoblast migration 366 and proliferation [38] . The Slit family in mammalian consists of three members-Slit1,         Supplemental Table S1.  housed at 22℃ or exposed to 4℃ for 3d (n=6/5). in M2 macrophages in iWAT from mice housed at 22℃ or exposed to 4℃ for 3d (n=1 per group). 767 The heat map based on fragments per kilobase of exon model per million mapped fragments 768 (FPKM).

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D. Gene expression of Slit3 in M2 macrophages in iWAT from mice housed at 22℃ or exposed to 770 4℃ for 3d (n=3 per group        Data are presented as mean ± SEM. Data in (B) are analyzed using two-way ANOVA using time 854 and injectedred cell type as covariate and using multiple comparison to test for differences in 855 individual time points. *represent M2 Ad-GFP+vehicle vs. M2 Ad-Slit3+vehicle, # represent M2 856 Ad-Slit3+vehicle vs. M2 Ad-Slit3+SR59230A. 857 Data in (A and E-I) are analyzed using student's t test for comparisons. * p < 0.05, ** p < 0.01, 858 *** p < 0.001, **** p < 0.0001. 859 B. Slit3 levels in iWAT of Slit3 f/f and Slit3 f/f Lyz2 Cre mice that were housed at 22℃ or exposed to 4℃ 864 for 24h was determined by ELISA analyze (n=4-6 per genotype). Results were normalized to the 865 total protein levels.