Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
, 18 (6), 665-674

Brown-adipose-tissue Macrophages Control Tissue Innervation and Homeostatic Energy Expenditure

Affiliations

Brown-adipose-tissue Macrophages Control Tissue Innervation and Homeostatic Energy Expenditure

Yochai Wolf et al. Nat Immunol.

Abstract

Tissue macrophages provide immunological defense and contribute to the establishment and maintenance of tissue homeostasis. Here we used constitutive and inducible mutagenesis to delete the nuclear transcription regulator Mecp2 in macrophages. Mice that lacked the gene encoding Mecp2, which is associated with Rett syndrome, in macrophages did not show signs of neurodevelopmental disorder but displayed spontaneous obesity, which was linked to impaired function of brown adipose tissue (BAT). Specifically, mutagenesis of a BAT-resident Cx3Cr1+ macrophage subpopulation compromised homeostatic thermogenesis but not acute, cold-induced thermogenesis. Mechanistically, malfunction of BAT in pre-obese mice with mutant macrophages was associated with diminished sympathetic innervation and local titers of norepinephrine, which resulted in lower expression of thermogenic factors by adipocytes. Mutant macrophages overexpressed the signaling receptor and ligand PlexinA4, which might contribute to the phenotype by repulsion of sympathetic axons expressing the transmembrane semaphorin Sema6A. Collectively, we report a previously unappreciated homeostatic role for macrophages in the control of tissue innervation. Disruption of this circuit in BAT resulted in metabolic imbalance.

Conflict of interest statement

Competing Financial Interests

The authors declare no competing financial interests.

Figures

Fig. 1
Fig. 1. Macrophage-restricted Mecp2 deletion causes spontaneous obesity in adulthood.
(a) Weight of Cx3cr1cre (n=9) and Cx3cr1cre:Mecp2fl/y mice (n=14) over 6 months. *** p<0.001, F20,360=22.24, two- way ANOVA, interaction effect. (b) Weight of Cx3cr1cre (n=4) and Cx3cr1cre:Mecp2fl/y mice (n=7) kept on high-fat diet (HFD) for 8 weeks starting from 6 weeks of age. *** p<0.001, F8,72=5.871, two- way ANOVA, interaction effect. (c) Weight of Cx3cr1creER:Mecp2fl/y (n=13) and Cx3cr1creER (n=14) mice over 6 months, all administered TAM at the age of 6 weeks. *** p<0.001, F26, 624=15.57, two- way ANOVA, interaction effect. (d) Body composition of pre-obese Cx3cr1creER:Mecp2fl/y mice (n=13) and littermate controls (n=4) 6 weeks after TAM treatment (age 12 weeks), and Cx3cr1creER:Mecp2fl/y mice (n=12) and littermate controls (n=5) 12 weeks after TAM treatment (age 18 weeks). *p<0.05, **p<0.01, Mann-Whitney’s U-test.
Fig. 2
Fig. 2. Macrophage-restricted Mecp2 deletion does not impair central functions
(a) Serum leptin levels in pre-obese starved Cx3cr1cre:Mecp2fl/y (n=5) and littermate controls (n=6), age 10 weeks. *p<0.05, Mann-Whitney’s U-Test. (b) Western blot of phosphorylated and total Stat3 in hypothalami isolated from pre-obese starved Cx3cr1cre:Mecp2fl/y mice and littermate controls 45 minutes after leptin or saline administration. (c) qRT-PCR analysis of hypothalami obtained from TAM-treated Cx3cr1creER and Cx3cr1creER:Mecp2fl/y mice (all n=6) for central feeding regulators. *p<0.05,Student's T test, for Npy T9=1.865, p=0.0476, for Lpr T11=2.111, p=0.0292. (d) qRT-PCR analysis of hypothalami obtained from TAM-treated Cx3cr1creER and Cx3cr1creER:Mecp2fl/y mice for inflammation-associated genes (same mice as in Fig 2C). (e) Body temperature differences (Body temperature(t)-body temperature(0)) of TAM-treated Cx3cr1creER (n=10) and Cx3cr1creER:Mecp2fl/y mice mice (n=8) after LPS treatment. Data are pooled from two independent experiments. (f) Daily food consumption of TAM-treated Cx3cr1creER (n=8) and Cx3cr1creER:Mecp2fl/y (n=7) mice divided by dark and light phases. (g) Accumulated food intake of 96 hours of (f)
Fig. 3
Fig. 3. Macrophage-restricted Mecp2 deletion impairs BAT steady state function.
(a) Graphical summary of heat production of Cx3cr1creER mice (n=8) and Cx3cr1creER:Mecp2fl/y littermates (n=7) at 3 and 4 months after TAM, calculated by indirect calorimetry measurements. *p<0.05, 2-way ANNOVA followed by multiple comparisons, Taverage light after 3 months= 3.295, Taverage dark after 3 months= 3.194, Taverage dark after 4 months= 3.154, all df=22. (b) H & E histology of iBAT and visceral WAT (vWAT) of Cx3cr1cre:Mecp2fl/y mice and control littermates at the age of 9 weeks. Pictures are representative of 3 independent mice with similar results. Scale bars, 40 μm. (c) qRT-PCR analysis of BAT obtained from Cx3cr1creER:Mecp2fl/y mice (n=7) and littermates (n=8), 4 months after TAM treatment. Threshold Cycle (CT) for Ucp1- average for Cre mice 15.5 *p<0.05; for Ucp1 T11=2.419, p=0.017, for Dio2 T11=2.082, p=0.0297, Student’s T-test. (d) Representative western blot analysis for Ucp1 and Gapdh in tissues obtained from 10 week old pre-obese Cx3cr1cre:Mecp2fl/y mice and littermates controls (left). Bar graph of densitometry analysis of Ucp1 normalized to Gapdh bands from three independent experiments Cx3cr1cre:Mecp2fl/y (n=10) mice and littermates controls (n=11) (right) **p=0.0028, T18=3.451, Student’s T-test. (e) Weight gain of Cx3cr1cre:Mecp2fl/y (n=5, 5) mice and littermate controls (n=5, 6) under either 22°C or 30°C (thermoneutrality). Mice were examined starting from the age of 5 weeks onwards. *p<0.05, *** p<0.001, two-way ANOVA followed by Tuckey’s post-hoc test, Genotype effect F3,255<0.001.
Fig. 4
Fig. 4. Normal acute cold acclimation and scWAT hyper activation in absence of macrophage Mecp2
(a) Body temperature of pre-obese Cx3cr1cre:Mecp2fl/y (n=7) mice and control littermates (n=4) at 4°C for a period of 8h. (b) Body temperature of pre-obese Cx3cr1creER:Mecp2fl/y mice and control littermates (all n=4) at 4°C for a period of 8h. (c) qRT-PCR of iBAT taken from TAM-treated Cx3cr1creER:Mecp2fl/y and controls in (b) after 8 hours of cold challenge compared to animals housed at room temperature. *p<0.05,Mann-Whitney’s U test. (d) Representative H&E stain of the iBAT of mice in (a). Scale bar, 50 μm. (e) qRT-PCR of scWAT taken from Cx3cr1cre:Mecp2fl/y (n=7) mice and control littermates (n=5) at 4°C for a period of 8h. Threshold Cycle (CT) for ucp1 average for Cx3cr1cre:Mecp2fl/y mice 19.5 *p<0.05, Mann-Whitney’s U test. (f) Western blot analysis for total and phosphorylated Hsl in scWAT of mice in (e). Bar graph of densitometry analysis of pHsl normalized to total Hsl ** p<0.01, Mann-Whitney’s U test. Data are representative of two independent experiments with similar results.
Fig. 5
Fig. 5. Impact of experimental BAT denervation and reduced BAT innervation in the absence of macrophage Mecp2
(a) Western blot analysis of Th, Ucp1 and Vinculin in iBAT obtained from WT mice 10 days after unilateral iBAT denervation. From each mouse, both denervated BAT (designated D) and sham treated control BAT (designated S) are presented. n=4. (b) Individual fat mass measurements of sham lesioned (n=3) and bilaterally denervated C57BL/6 mice (n=5), 0-8 weeks after surgery. *p<0.05, **p<0.01, 2 way ANNOVA followed by Bonferoni’s multiple comparison test, surgery effect F1,6=14.28. (c) qRT-PCR for Th mRNA expression in whole BAT of Cx3cr1creER:Mecp2fl/y mice (n=7) and control littermates (n=8) 4 months after TAM administration. T11=1.904, p=0.0411. (d) Western blot analysis for Th and Vinculin in tissues obtained from 10 week old pre-obese Cx3cr1creER:Mecp2fl/y mice (n=7) and control littermates (n=10). Bar graph of densitometry analysis of Th normalized to Vinculin bands. T15=4.175, ***p=0.008, Student’s T-test. (e) Immunofluorescent stain for Th in BAT of Cx3cr1cre:Mecp2fl/ymice and control littermates (Each group n=6; 6 sections/ mouse; 36 sections/ genotype). Bar graph of iBAT Th+ fibers per mm2.*p=0.0259, T10=2.613, Student’s T test. Scale bar, 25μm. (f) Body weight (left panel, control mean 28gr) and total BAT norepinephrine (right panel, control mean 19.5 ng) in age-matched, Cx3cr1creER (n=12), lean Cx3cr1creER:Mecp2fl/y (n=9) and obese Cx3cr1creER:Mecp2fl/y mice (n=10) 3 months after TAM., *p<0.05, ***p<0.001, one-way ANNOVA followed by Tukey’s multiple comparison test, for body weight F2,28=20.58, for BAT norepinephrine F2,28=11.99.
Fig. 6
Fig. 6. Characterization of brown adipose tissue macrophages.
(a) Two-photon microscopy of iBAT obtained from Th-Cre:Rosa26-tdTomato:Cx3cr1gfp mice. Original image size was: 424.27 µm, y: 424.27 µm, z: 23.69 µm; Z-Stack of 24 images was acquired; Shadow Projection mode (left and middle figures) and Maximum Intensity Projection (MIP) mode (right Fig.) of Zen Imaging Software were used in order to render a represented images of both tdTomato and GFP channels. Data are representative of 6 mice. Scale bars from left to right: 50 μm, 50 μm and 25 μm. (b) FACS analysis of BAT macrophages obtained from Cx3cr1gfp mice at different ages for Cx3cr1/GFP and MHC II expression. n=4 for each age. Bar diagram summarizing data. (c) FACS analysis of iBAT macrophages of Cx3cr1Cre:Rosa26-RFP mouse; cells are gated as in (b). Data are representative of three mice. (d) FACS analysis of iBAT macrophages of TAM-treated Cx3cr1creER:Rosa26-tdTomato mouse, 6 months post TAM treatment. Data are representative of three mice. (e) Fate mapping of indicated macrophage populations in TAM-treated Cx3cr1creER:Rosa26-tdTomato mice. n=3.
Fig. 7
Fig. 7. Mecp2-mutant macrophages overexpress PlexinA4 and might interact with Sema6A-expressing sympathetic axons in BAT
(a) FACS plot indicating sorting gates for tdTomato+ and tdTomato population, isolated from TAM-treated Cx3cr1creER:Rosa26-tdTomato mice. Data are representative of four mice. (b) K-means clustering (K = 8) of 1094 genes differentially expressed by 'CX3CR1' and 'CX3CR1+' BAT macrophages. (c) Bar diagrams illustrating RNA expression of selected genes across macrophage populations presented in (B). ntotal=3, n’cx3cr1-‘=4, n’cx3cr1+’=4. (d) Bar graph showing enrichment of macrophage genes and de-enrichment of adipocyte genes as determined by RNA-seq of ribosome-attached-mRNAs retrieved by IP of BAT from Cx3cr1CreER:Rpl22HA mice, representing fold change over input (mRNA of total tissue) (n=2) (e) Volcano plot of statistical significance (-log10(p value)) against log 2 ratio between translatomes of 'CX3CR1' macrophages of Cx3cr1creER:Rpl22HA (n=2) and Cx3cr1creER:Mecp2fl/y:Rpl22HA mice (n=4), based on the RNA-seq data. Significantly changed genes (at least 2 fold change (1 log2 ratio), P value <0.05)) are indicated by red box (left). Bar diagrams illustrating raw normalized reads of Mecp2 and Plexna4 mRNA expression in Cx3cr1creER:Rpl22HA animals (black bars) and Cx3cr1creER:Mecp2fl/y:Rpl22HA animals (red bars) as determined by RNA-seq (right). (f) RT-PCR analysis of stellate ganglia for Tyrosine hydroxylase and Semaphorin expression. n=3. (g) Micrograph of main sympathetic neuronal fibers innervating the BAT, as visualized in Thy1-YFP mouse. Scale bar, 450 μm. (h) Micrograph of BAT of Sema6a-PLAP (human placental alkaline phosphatase) mouse, illustrating Sema6a expression by sympathetic nerves according to PLAP reporter expression. Scale bar, 100 μm.

Comment in

Similar articles

See all similar articles

Cited by 35 PubMed Central articles

See all "Cited by" articles

References

    1. Wynn TA, Chawla A, Pollard JW. Macrophage biology in development, homeostasis and disease. Nature. 2013;496:445–455. - PMC - PubMed
    1. Ginhoux F, Jung S. Monocytes and macrophages: developmental pathways and tissue homeostasis. Nature Publishing Group. 2014;14:392–404. - PubMed
    1. Okabe Y, Medzhitov R. Tissue biology perspective on macrophages. Nat Immunol. 2016;17:9–17. - PubMed
    1. Chahrour M, Zoghbi HY. The story of Rett syndrome: from clinic to neurobiology. Neuron. 2007;56:422–437. - PubMed
    1. Yona S, et al. Fate mapping reveals origins and dynamics of monocytes and tissue macrophages under homeostasis. Immunity. 2013;38:79–91. - PMC - PubMed

Publication types

MeSH terms

LinkOut - more resources

Feedback