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. 2024 May 15:15:1375138.
doi: 10.3389/fimmu.2024.1375138. eCollection 2024.

Dynamic changes of immunocyte subpopulations in thermogenic activation of adipose tissues

Yuqing Ye  1 , Huiying Wang  1 , Wei Chen  1 , Zhinan Chen  1 , Dan Wu  1 , Feng Zhang  1 , Fang Hu  1
Affiliations

Dynamic changes of immunocyte subpopulations in thermogenic activation of adipose tissues

Yuqing Ye et al. Front Immunol. .

Abstract

Objectives: The effects of cold exposure on whole-body metabolism in humans have gained increasing attention. Brown or beige adipose tissues are crucial in cold-induced thermogenesis to dissipate energy and thus have the potential to combat metabolic disorders. Despite the immune regulation of thermogenic adipose tissues, the overall changes in vital immune cells during distinct cold periods remain elusive. This study aimed to discuss the overall changes in immune cells under different cold exposure periods and to screen several potential immune cell subpopulations on thermogenic regulation.

Methods: Cibersort and mMCP-counter algorithms were employed to analyze immune infiltration in two (brown and beige) thermogenic adipose tissues under distinct cold periods. Changes in some crucial immune cell populations were validated by reanalyzing the single-cell sequencing dataset (GSE207706). Flow cytometry, immunofluorescence, and quantitative real-time PCR assays were performed to detect the proportion or expression changes in mouse immune cells of thermogenic adipose tissues under cold challenge.

Results: The proportion of monocytes, naïve, and memory T cells increased, while the proportion of NK cells decreased under cold exposure in brown adipose tissues.

Conclusion: Our study revealed dynamic changes in immune cell profiles in thermogenic adipose tissues and identified several novel immune cell subpopulations, which may contribute to thermogenic activation of adipose tissues under cold exposure.

Keywords: adipose tissues; bioinformatics; cold exposure; immune cells; thermogenesis.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Schematic plot of the workflow of the analysis. RT, Room Temperature; TN, Thermal Neutrality.
Figure 2
Figure 2
Cibersort analysis of immune cell changes during different cold periods in BAT and SAT. Dynamic expression of naïve CD4+T cells (A), naïve CD8+T cells (B), memory CD4+T cells (C), memory CD8+T cells (D), M1 macrophages (E), M2 macrophages (F), Treg cells (G), γδT cells (H), and NK resting cells (I) in different cold periods in BAT and SAT. (J) Correlation analysis of these immune cells in BAT. (K) Correlation analysis of these immune cells in SAT. Statistical data were assessed by unpaired two-tailed Student’s t test. */#P<0.05, **/##P<0.01, ***/###P<0.001, ****/####P<0.0001. BAT, Brown Adipose Tissues; SAT, Subcutaneous Adipose Tissues.
Figure 3
Figure 3
mMCP-counter analysis of immune cell changes during different cold periods in BAT and SAT. Dynamic expression of T cells (A), CD8 T cells (B), B-derived cells (C), NK cells (D), Monocytes (E), Monocytes/macrophages (F), Fibroblasts (G), Endothelial cells (H), and Vessels (I) in different cold periods in BAT and SAT. (J) Correlation analysis of these immune cells in BAT. (K) Correlation analysis of these immune cells in SAT. Statistical data were assessed by unpaired two-tailed Student’s t test. */#P<0.05, **/##P<0.01, ***/###P <0.001, ****/####P<0.0001. BAT, Brown Adipose Tissues; SAT, Subcutaneous Adipose Tissues.
Figure 4
Figure 4
Reanalysis of Single-cell sequencing data (GSE207706) in BAT upon 4d-cold exposure. (A) Unsupervised clustering of Lineage+ stromal vascular fraction (SVF), shown as t-distributed stochastic neighbor embedding (tSNE). (B) The proportion of each cell type in SVF under cold adaptation. (C) Unsupervised clustering of T cells shown as t-distributed stochastic neighbor embedding (tSNE). (D) The proportion of each cell type in cold adaptation of T cells.
Figure 5
Figure 5
Validation of the changes in monocytes and NK cells under cold exposure. (A) Flow chart of the experiment. 8-10w male mice were divided into four groups, and brown and white adipose tissues were collected separately at RT and upon 1d, 3d, and 7d cold exposure for RT-qPCR, immunofluorescence, and flow cytometry. (B) UCP1 mRNA expression in BAT (left) and SAT (right) of mice at RT and upon 1d-3d-7d cold exposure. (C) Representative flow cytometry plots and quantification of the proportion of monocytes in CD45+ cells in BAT of mice at RT and upon 1d-3d-7d cold exposure. (D) Representative flow cytometry plots and quantification of the proportion of NK cells in CD45+ cells in BAT of mice at RT and upon 1d-3d-7d cold exposure. (E) NK1.1 mRNA expression in BAT SVF of mice at RT and upon 1d-3d-7d cold exposure. (F, G) Representative images of the localization of NK1.1+ NK cells with CD31+ endothelial cells (F) or TH+ sympathetic neurons (G) in BAT of mice at RT. Scale bar, 10μm. (H, I) Representative images of the localization of CD11B+ CD115+ monocytes with CD31+ endothelial cells (H) or TH+ sympathetic neurons (I) in BAT of mice at RT. Scale bar, 10μm. (J) Pearson correlation coefficient of the fluorescence intensity between these cells (F–I) in BAT of mice at RT. (K, L) Representative images of cold-induced changes in CD11B+CD115+ monocytes (K) and NK1.1+ NK cells (L) in BAT of mice at RT and upon 1d-3d-7d cold exposure. Scale bar, 50μm. All samples were biologically independent replicates (n=4-6 in each group). Data were presented as mean ± SEM. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. RT, Room Temperature; BAT, Brown Adipose Tissues; SAT, Subcutaneous Adipose Tissues; SVF, Stromal Vascular Fraction; Sac, Sacrifice.
Figure 6
Figure 6
Changes of T cell subpopulations under cold exposure. (A) Representative flow cytometry plots and quantification of the proportion of CD4+ T cell subpopulations and CD8+ T cell subpopulations in CD45+ cells in BAT of mice at RT and upon 1d-3d-7d cold exposure. (B) Representative images of the localization of CD44-CD62L+ naïve cells (yellow arrows), CD44+CD62L+ memory cells (grey arrows), and CD31+ endothelial cells in BAT of mice at RT. Scale bar, 10μm. (C) Representative images of the localization of CD44-CD62L+naïve cells (yellow arrows), CD44+CD62L+ memory cells (grey arrows), and TH+ sympathetic neurons in BAT of mice at RT. Scale bar, 10μm. (D) Pearson correlation coefficient of the fluorescence intensity between these cells (B, C) in BAT of mice at RT. (E) Representative images of cold-induced changes in CD44-CD62L+naïve cells and CD44+CD62L+ memory cells in BAT of mice at RT and upon 1d-3d-7d cold exposure. Scale bar, 50μm. All samples were biologically independent replicates (n=4-6 in each group). Data were presented as mean ± SEM. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. RT, Room Temperature; BAT, Brown Adipose Tissues.
Figure 7
Figure 7
Changes of macrophage subpopulations and endothelial cells under cold exposure. (A, B) Representative flow cytometry plots and quantification of the proportion of M1 and M2 macrophages in CD45+ cells in BAT (A) and SAT (B) of mice at RT and upon 1d-3d-7d cold exposure. (C) Representative flow cytometry plots and quantification of the proportion of endothelial cells in CD45- cells in BAT of mice at RT and upon 1d-3d-7d cold exposure. (D) CD31 mRNA expression in BAT SVF of mice at RT and upon 1d-3d-7d cold exposure. (E) Representative flow cytometry plots and quantification of the proportion of endothelial cells in CD45- cells in SAT of mice at RT and upon 1d-3d-7d cold exposure. (F) CD31 mRNA expression in SAT SVF of mice at RT and upon 1d-3d-7d cold exposure. All samples were biologically independent replicates (n=4-6 in each group). Data we represented as mean ± SEM. *P<0.05, **P<0.01, ***P<0.001, ****P<0. 0001. RT, Room Temperature; BAT, Brown Adipose Tissues; SAT, Subcutaneous Adipose Tissues; SVF, Stromal Vascular Fraction.
Figure 8
Figure 8
Summary of immune cell changes under cold exposure in BAT. (A) Summary of immune cell changes in BAT of mice at RT and upon 1d-3d-7d cold exposure shown as a heatmap. (B) Upon cold stimulation, the proportions of monocytes, naïve and memory T cells are increased, while the proportion of NK cells is decreased.
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Grants and funding

The author(s) declare financial support was received for the research, authorship, and/or publication of this article. This work was supported by grants from National Key R&D Program, China (2022YFA0806102; 2020YFA0803604), National Natural Science Foundation of China (91957113, 31871180), and Health Commission of Hunan Province key project (W20242006) to FH. Natural Science Foundation of Hunan Province (2023JJ40794) to FZ, and the Fundamental Research Funds for the Central Universities of Central South University (2021zzts0382) to YY.

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