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Macrophage IRX3 promotes diet-induced obesity and metabolic inflammation

Abstract

Metabolic inflammation is closely linked to obesity, and is implicated in the pathogenesis of metabolic diseases. FTO harbors the strongest genetic association with polygenic obesity, and IRX3 mediates the effects of FTO on body weight. However, in what cells and how IRX3 carries out this control are poorly understood. Here we report that macrophage IRX3 promotes metabolic inflammation to accelerate the development of obesity and type 2 diabetes. Mice with myeloid-specific deletion of Irx3 were protected against diet-induced obesity and metabolic diseases via increasing adaptive thermogenesis. Mechanistically, macrophage IRX3 promoted proinflammatory cytokine transcription and thus repressed adipocyte adrenergic signaling, thereby inhibiting lipolysis and thermogenesis. JNK1/2 phosphorylated IRX3, leading to its dimerization and nuclear translocation for transcription. Further, lipopolysaccharide stimulation stabilized IRX3 by inhibiting its ubiquitination, which amplified the transcriptional capacity of IRX3. Together, our findings identify a new player, macrophage IRX3, in the control of body weight and metabolic inflammation, implicating IRX3 as a therapeutic target.

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Fig. 1: Irx3 deficiency in macrophages decreases body weight and fat composition.
Fig. 2: Irx3 deficiency in macrophages increases cold-induced thermogenesis by BAT and scWAT.
Fig. 3: IRX3 of macrophages inhibits adipocyte lipolysis and thermogenesis through repression of adrenergic signaling.
Fig. 4: IRX3 promotes proinflammatory cytokine expression in macrophages.
Fig. 5: LPS stimulation inhibits degradation of IRX3 and increases its phosphorylation.
Fig. 6: Phosphorylated IRX3 acts as a transcriptional factor to promote proinflammatory gene expression.
Fig. 7: IRX3 deficiency in macrophages improves glucose homeostasis, insulin sensitivity and liver steatosis.

Data availability

Sequencing data generated for this study have been deposited in the Gene Expression Omnibus database under accession number GSE178574. Source data are provided with this paper.

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Acknowledgements

We thank members of the laboratory of Y.Q. and Y. Liu (Wuhan University) for helpful comments on the manuscript. We thank C.-C. Hui (University of Toronto) for generous sharing of Irx3fl/fl, Irx3/ and Irx5−/− mice and mouse Irx3 cDNA. We thank Z. Dong (Tsinghua University) for generous sharing of CD1d tetramer (loaded with PBS-57) from the National Institutes of Health Tetramer Core Facility. We also thank H. Deng and X. Meng in Proteinomics Facility at Technology Center for Protein Sciences, Tsinghua University, for protein MS analysis. This work was supported by grants from National Key R&D Program of China (2018YFA0800702), National Natural Science Foundation of China (31671227 and 91642113) and the Thousand Young Talents Program of the Chinese government (to Y.Q.).

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Authors

Contributions

J.Y., D.W. and C.Z. designed and performed the main experiments with assistance from T.Y., Y.Z., H.S., K.X., X.H. and Z.W.; J.Y., D.W., C.Z. and Y.Q. discussed and interpreted the results from the study; and J.Y., D.W. and Y.Q. conceived of the study, supervised the work and wrote the paper.

Corresponding author

Correspondence to Yifu Qiu.

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The authors declare no competing interests.

Additional information

Peer review information Nature Immunology thanks Andrew Hogan and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. L. A. Dempsey was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

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Extended data

Extended Data Fig. 1 IRX3 deficiency in macrophages decreases fat accumulation, while does not affect food intake and activity in HFD-fed and ND-fed mice.

a-d, Representative images of H&E-stained sections of different tissues (a), and distribution of adipocyte size of BAT (b), scWAT (c) and eWAT (d) from Irx3f/f and Irx3f/fLyz2Cre mice fed HFD (n = 4). e-h, Representative images of H&E-stained sections of different tissues (e), and distribution of adipocyte size of BAT (f), scWAT (g) and eWAT (h) from Irx3f/f and Irx3f/fLyz2Cre mice fed ND (n = 3). i, j, Locomotor activity and food intake of Irx3f/f and Irx3f/fLyz2Cre mice fed HFD (i) (n = 10 for Irx3f/f, n = 9 for Irx3f/fLyz2Cre mice in locomotor activity and n = 4 in food intake) and ND (j) (n = 8 for Irx3f/f, n = 9 for Irx3f/fLyz2Cre mice in locomotor activity and n = 9 in food intake). All data represent means ± s.e.m. Statistical significance was determined by unpaired two-tailed Student’s t-test (b-d, f-h, i, j).

Source data

Extended Data Fig. 2 IRX3 deficiency in macrophages increases adipose tissues lipolysis.

a, b, Locomotor activity (a) and food intake (b) of Irx3f/f and Irx3f/fLyz2Cre mice housed at room temperature (22 °C) and cold environment (4 °C) (n = 6). c-e,Quantitative RT-PCR analysis of lipogenic gene expression in BAT (c), scWAT (d) and eWAT (e) of Irx3f/f and Irx3f/fLyz2Cre mice housed at 4 °C for 48 h (n = 6 for Irx3f/f and n = 5 for Irx3f/fLyz2Cre mice). f, RER from mice in Fig. 2a (n = 6). g, Quantitative RT-PCR analysis of thermogenic gene expression in eWAT of Irx3f/f and Irx3f/fLyz2Cre mice housed at 4 °C for 48 h (n = 5 or 6 for Irx3f/f and n = 5 or 6 for Irx3f/fLyz2Cre mice). h, i, NE-induced RER from mice in Fig. 3a (n = 5). All data represent means ± s.e.m. Statistical significance was determined by two-way ANOVA with Bonferroni’s multiple-comparisons test (f) or unpaired two-tailed Student’s t-test (a-e and g-i).

Source data

Extended Data Fig. 3 IRX3 promotes proinflammatory cytokine expression in macrophages to inhibit adipocyte lipolysis and thermogenesis.

a, Serum concentrations of adrenaline, noradrenaline and dopamine of Irx3f/f and Irx3f/fLyz2Cre mice housed at room temperature (22 °C) or 4 °C for 48 h (n = 5). b, c, Quantitative RT-PCR analysis of macrophage marker expression in BAT (b) and scWAT (c) of Irx3f/f and Irx3f/fLyz2Cre mice housed at 4 °C for 48 h (n = 6 for Irx3f/f and n = 4 or 5 for Irx3f/fLyz2Cre mice). d, Flow cytometry analysis of macrophage numbers in scWAT of Irx3f/f and Irx3f/fLyz2Cre mice housed at 4 °C for 48 h (n = 5). e, Quantitative RT-PCR analysis of M2 marker expression in scWAT of Irx3f/f and Irx3f/fLyz2Cre mice housed at 4 °C for 48 h (n = 6 for Irx3f/f and n = 5 or 6 for Irx3f/fLyz2Cre mice). f, g, Quantitative RT-PCR analysis of proinflammatory gene expression in BAT (f) and eWAT (g) of Irx3f/f and Irx3f/fLyz2Cre mice housed at 4 °C for 48 h (n = 6 for Irx3f/f and n = 5 for Irx3f/fLyz2Cre mice). h, Quantitative RT-PCR analysis of proinflammatory gene expression in peritoneal macrophage of Irx3f/f and Irx3f/fLyz2Cre mice (n = 3). i, Immunoblot analysis of beige adipocytes pretreated with 5 ng/ml cytokines and followed by a co-treatment of 50 nM NE for 8 hrs. Independent experiments were performed three times with similar results. j, k, Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses of genes significantly upregulated (log2FC > 0.5, Padj<0.05). All data represent means ± s.e.m. Statistical significance was determined by unpaired two-tailed Student’s t-test (a-h).

Source data

Extended Data Fig. 4 LPS stimulation inhibits IRX3’s degradation, while increases its phosphorylation.

a, Immunoblot analysis of RAW 264.7 cells overexpressing IRX3-Flag and then treated with LPS for 12 h followed by CHX treatment for indicated time, and IRX3 protein level was quantified by ImageJ. b, Ubiquitination site prediction by ‘Ubisite’. c, Immunoblot analysis of RAW 264.7 cells overexpressing IRX3 (WT) and its ubiquitination site mutants and then treated with CHX for indicated time, and quantification of IRX3 protein level by ImageJ. d, Immunoblot analysis and phos-tag SDS-PAGE analysis of RAW 264.7 cells overexpressing IRX3-HA plus indicated kinases in RAW 264.7 cells and then treated with or without LPS for 4 h. Independent experiments were performed three times with similar results. e, JNK phosphorylation site prediction by ‘GPS 5.0’. f, Alignment of IRX3 sequences in different species. IP-MS-identified Ser phosphorylation sites (S361 and S389) were highlighted in red. g, h, Quantitative RT-PCR analysis of proinflammatory gene expression in human THP-1 monocytes overexpressing GFP or hIRX3 (g) or in Cas9 stable THP-1 monocytes infected with control or hIRX3-gRNA AAVs (h) (n = 3). (i-l) Quantitative RT-PCR analysis of proinflammatory gene expression in differentiated human THP-1 macrophages overexpressing hIRX3 (i) or hIRX3-shRNA (j), or in hMDMs overexpressing hIRX3 (k) or hIRX3-shRNA (l) (n = 3). Statistical significance was determined by unpaired two-tailed Student’s t-test (g-l).

Source data

Extended Data Fig. 5 Profiling of crown-like structure and different immune cell populations in Irx3f/f and Irx3f/fLyz2Cre mice scWAT.

a, b, Representative images of F4/80 IHC staining (a) and quantification of CLS (b) in scWAT from Irx3f/f and Irx3f/fLyz2Cre mice fed HFD (n = 6). c, d, Gating strategy for analysis of different subpopulations of macrophages (c) and other immune cells (d) in scWAT of Irx3f/f and Irx3f/fLyz2Cre mice fed HFD. e-i, Flow cytometry analysis of different subpopulations of macrophages (e), T cells, B cells (f), NK cells (g), CD4+ T cells, CD8+ T cells, Treg cells (h) and NKT cells (i) (n = 4 in e, n = 5 for Irx3f/f and n = 6 for Irx3f/fLyz2Cre mice in f, h and n = 6 in g, i). All data represent means ± s.e.m. Statistical significance was determined by unpaired two-tailed Student’s t-test (b, e-i).

Source data

Extended Data Fig. 6 IRX3 depletion in neutrophils does not affect the HFD-induced metabolic dysfunctions.

a-d, Body weight (a), tissues weight (b), GTT (c) and ITT (d) of Irx3f/f and Irx3f/fS100a8Cre mice fed HFD (n = 6). e, In situ hybridization of Irx3 RNA (red) in conjunction with staining for the neuronal marker NeuN (green) and DAPI (Blue) in the sagittal brain sections of Irx3f/f and Irx3f/fLyz2Cre mice. f, Quantitative RT-PCR analysis of Irx3 expression in the whole brains and hypothalami of Irx3f/f and Irx3f/fLyz2Cre mice (n = 4). g, Quantitative RT-PCR analysis of proinflammatory gene expression in WT and Irx5−/− BMDM (n = 3). h, Luciferase assay of Il1a, Il1b and Il6 promoter-driven luciferase reporter in HEK 293 T cells co-transfected with GFP or IRX5 plasmid for 24 h (n = 3). i, Model of how macrophage IRX3 controls proinflammatory cytokine expression and thus inhibits adipocyte thermogenesis and lipolysis. All data represent means ± s.e.m. Statistical significance was determined by two-way ANOVA with Bonferroni’s multiple-comparisons test (a, c and d) or unpaired two-tailed Student’s t-test (b, f-h).

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Yao, J., Wu, D., Zhang, C. et al. Macrophage IRX3 promotes diet-induced obesity and metabolic inflammation. Nat Immunol 22, 1268–1279 (2021). https://doi.org/10.1038/s41590-021-01023-y

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