The Transcriptional Regulator Id2 Is Critical for Adipose-Resident Regulatory T Cell Differentiation, Survival, and Function

Abstract

Adipose regulatory T cells (aTregs) have emerged as critical cells for the control of local and systemic inflammation. In this study, we show a distinctive role for the transcriptional regulator Id2 in the differentiation, survival, and function of aTregs in mice. Id2 was highly expressed in aTregs compared with high Id3 expression in lymphoid regulatory T cells (Tregs). Treg-specific deletion of Id2 resulted in a substantial decrease in aTregs, whereas Tregs in the spleen and lymph nodes were unaffected. Additionally, loss of Id2 resulted in decreased expression of aTreg-associated markers, including ST2, CCR2, KLRG1, and GATA3. Gene expression analysis revealed that Id2 expression was essential for the survival of aTregs, and loss of Id2 increased cell death in aTregs due to increased Fas expression. Id2-mediated aTreg depletion resulted in increased systemic inflammation, increased inflammatory macrophages and CD8⁺ effector T cells, and loss of glucose tolerance under standard diet conditions. Thus, we reveal an unexpected and novel function for Id2 in mediating differentiation, survival, and function of aTregs that when lost result in increased metabolic perturbation.

Figure 1.. Id2 and Id3 expression in aTregs.

(A) qPCR showing Id2 and Id3 mRNA expression in sorted CD4⁺ CD25⁺ Tregs isolated from the spleen or visceral adipose tissue. Id2 or Id3 expression was normalized to HPRT. Each dot indicates cells isolated from one animal. (B) Flow cytometry histograms showing Id2-YFP or ID3-GFP expression in gated CD4⁺ CD25⁺ Tregs isolated from the spleen or visceral adipose tissue. B6 mice were used as a ‘no reporter’ control. Numbers in histograms indicate the median fluorescence intensity (MFI). Bar graphs indicate the average MFI in Tregs isolated from the indicated tissue. (C) Histograms indicating the frequency of Id2⁺ or Id3⁺ Tregs identified in the spleen (left) and their expression by flow cytometry of ST2 and KLRG1 (right). Yellow peaks indicate Id2-YFP⁺ cells and green peaks indicate Id3-GFP⁺ cells. Grey peaks indicate Id2-YFP⁻ or Id3-GFP⁻ cells, respectively. Data are representative of two independent experiments with 1-3 mice per group. P values were calculated using the student’s t-test.

Figure 2.. Id2-deficiency results in reduced frequency of aTregs.

(A) Flow cytometry plots showing the frequency of Foxp3⁺ CD25⁺ gated CD4⁺ T cells from the indicated tissue in Control (Ctrl) and Id2-deficient (Id2 CKO) mice. Bar graphs indicate the frequency of Foxp3⁺ CD25⁺ Tregs from the adipose, lymph nodes or spleen. Each dot indicates one animal. (B) Flow cytometry plots showing CCR2, ST2 and KLRG1 expression on gated Foxp3⁺ CD25⁺ CD4⁺ T cells in the adipose tissue in Ctrl or Id2 CKO mice. Bar graphs indicate the frequency of CCR2⁺, ST2⁺ and KLRG1⁺ aTregs from wildtype (WT) and Id2 CKO (CKO) mice. (C) Histogram, flow cytometry plots and bar graphs indicating the median fluorescence intensity (MFI) and frequency of GATA3 expression in gated aTregs from WT and Id2 CKO mice. Data are representative of at least four independent experiments with 1-5 mice per group. P values were calculated using the student’s t-test.

Figure 3.. Loss of Id2 decreases survival of aTregs.

WT and Id2 CKO aTregs were sorted from the spleen or adipose tissue of 15-20-week-old male mice for RNA-sequencing. (A) Bar graphs indicating expression of transcripts involved in various pathways (indicated) in Id2 CKO aTregs relative to WT aTregs. (B) Flow cytometry plots and bar graph from Ctrl and Id2 CKO mice showing the frequency of viability dye (zombie)⁺ gated CD4⁺ Foxp3⁺ aTregs. (C) Flow cytometry plots and bar graph indicating the Annexin V frequency and MFI on WT vs CKO aTregs. (D) Histogram and bar graph indicating Fas MFI in WT vs CKO aTregs. Data are representative of two independent experiments with 1-3 mice per group. P values were calculate using the student’s t test.

Figure 4.. Cytokine production by Id2-deficient aTregs.

The total stromal vascular fraction (SVF) isolated from the adipose tissue of WT or Id2 CKO mice was cultured with PMA and ionomycin for 4 hours and cytokine production by gated CD4⁺ Foxp3⁺ aTregs analyzed by flow cytometry. (A) Flow cytometry plots and bar graph indicating IL-10 expression by gated CD4⁺ Foxp3⁺ WT vs CKO aTregs. (B) Flow cytometry plots and bar graph indicating IL-13 expression by gated CD4⁺ Foxp3⁺ WT vs CKO aTregs. (C) Flow cytometry plots and bar graph indicating IFN-γ expression by gated CD4⁺ Foxp3⁺ WT vs CKO aTregs. Data are representative of two independent experiments with 2-3 mice per group. P values were calculate using the student’s t test.

Figure 5.. Physiological function of Id2⁺ aTregs.

(A) CD45.1 mice were irradiated and injected with bone marrow cells from WT or Id2 CKO mice. Four weeks after reconstitution, mice were placed on CD or HFD for 12 weeks. Bar graphs indicating the frequency of CD4⁺ Foxp3⁺ aTregs, ST2⁺ KLRG1⁺, ST2⁺ CCR2⁺ and GATA3⁺ gated aTregs in the indicated BMC under CD or HFD conditions. (B) Flow cytometry and bar graphs indicating the frequency of total macrophages (F4/80⁺), M1 (CD11c⁺ F4/80⁺), M2 (CD206⁺ F4/80⁺), IL-10⁺ F4/80⁺ macrophages and CD8⁺ T cells isolated from the adipose tissue of WT or Id2 CKO mice on CD. (C) Graphs indicating weight, fasting glucose, fasting insulin and blood glucose and insulin over time following GTT. Calculated area under the curve (AUC) from all mice tested by GTT. (D) Graph indicating the expression of TNFα, SAA3, RANTES, IL-6 and A20 mRNA fold change in total adipose tissue SVF versus spleen. Data are representative of two independent experiments with 2-3 mice per group (A, B and D) and one experiment with 6 mice per group (C). P values were calculate using the student’s t test or one-way ANOVA.