Regulatory T cells in the mouse hypothalamus control immune activation and ameliorate metabolic impairments in high-calorie environments

Abstract

The hypothalamus in the central nervous system (CNS) has important functions in controlling systemic metabolism. A calorie-rich diet triggers CNS immune activation, impairing metabolic control and promoting obesity and Type 2 Diabetes (T2D), but the mechanisms driving hypothalamic immune activation remain unclear. Here we identify regulatory T cells (Tregs) as key modulators of hypothalamic immune responses. In mice, calorie-rich environments activate hypothalamic CD4⁺ T cells, infiltrating macrophages and microglia while reducing hypothalamic Tregs. mRNA profiling of hypothalamic CD4⁺ T cells reveals a Th1-like activation state, with increased Tbx21, Cxcr3 and Cd226 but decreased Ccr7 and S1pr1. Importantly, results from Treg loss-of function and gain-of-function experiments show that Tregs limit hypothalamic immune activation and reverse metabolic impairments induced by hyper-caloric feeding. Our findings thus help refine the current model of Treg-centered immune-metabolic crosstalk in the brain and may contribute to the development of precision immune modulation for obesity and diabetes.

Fig. 1. Identification of CD4⁺T cells and CD4⁺CD25⁺Foxp3GFP⁺Tregs in brains of healthy lean mice.

a, b Representative immune fluorescence staining of brain cryosections (12 µm) of Balb/c mice (a) stained for glial fibrillary acidic protein (GFAP, astrocyte marker, green), CD4 (T-cell marker, red) and DAPI (nuclei, blue) and b stained for CD3 (T-cell marker, green), Foxp3 (Treg marker, red) and DAPI (nuclei, blue). Stainings were obtained from >3 independent experiments. The scale bar is 20 µm. c Anti-GFP DAB staining of 30 µm vibratome sections of brains from Foxp3GFP Balb/c reporter mice to detect Tregs in the brain. The scale bar is 50 µm or 25 µm as indicated. Stainings were obtained from >3 independent experiments. d, e Representative immune fluorescence staining of brain cryosections (12 µm) stained for lectin (vessel, green), CD4 (CD4 T-cell marker, red) and DAPI (nuclei, blue) for the quantification of vessel-associated (d) vs. vessel distinct (e). The scale bar is 50 µm or 20 µm as indicated in the figure. f Summary graph for the quantification of vessel-associated (depicted as circles) vs. distinct from vessel (depicted as squares) CD4⁺T cells. Dots represent counts per high power field (HPF) as in (d, e), obtained from multiple sections. Mean ± SEM. Two-tailed unpaired Student’s t test, P < 0.0001. g, h Flow cytometric analysis of g CD4⁺T cells from mouse brain without hypothalamus (referred to as rest brain) and hypothalamus. CD4⁺T cells are analyzed as being positive for CD4 and negative for a panel of exclusion markers (live dead stain, B220, CD8a, CD11b, CD11c, CD14, F4/80). h Flow cytometric analysis of CD25⁺Foxp3GFP⁺ Tregs (% of live CD4⁺) from mouse rest brain and hypothalamus. Tregs were pre-gated on CD4⁺ and negative for exclusion markers as in (g). Source data are provided as a Source Data file. ****P < 0.0001.

Fig. 2. A high-calorie diet promotes hypothalamic immune activation in local CD4⁺T cells.

a, b Representative immune fluorescence staining of Balb/c brain cryosections (12 µm) stained for lectin (vessel, green), CD4 (CD4 T-cell marker, red) and DAPI (nuclei, blue) for the quantification of vessel-associated (a) vs. vessel distinct (b) after 16 weeks HFHS diet. The scale bar is 50 µm or 20 µm as indicated. c Summary graph for the quantification of vessel-associated vs. not vessel-associated CD4⁺T cells upon exposure to high-fat high-sugar (HFHS) diet for 16 weeks. Vessel-associated CD4⁺ T cells are depicted as circles, T cells distinct from vessels as triangles and were quantified as counts per high power field (HPF) across multiple sections. Mean ± SEM. Two-tailed Mann–Whitney U test, P < 0.0001. d, e Summary graph for the quantification of d vessel-associated CD4⁺T cells and e T cells distinct from vessels per high power field (HPF) upon exposure to a standard diet (SD) or high-fat high-sugar (HFHS) diet for 16 weeks. Mean ± SEM. Two-tailed Mann–Whitney U test d P = 0.1531; e P = 0.2198. (f) Representative anti-GFP DAB immunostainings in brains of CD4GFP mice fed a standard diet or HFHS diet for 6 months. Vibratome sections were cut with 30 µm and the scale bar is 20 µm. Stainings were obtained from >3 experiments. g Quantification of CD25^hiFoxp3⁺Treg frequencies (% of CD4⁺) in rest brain vs. hypothalamus of Foxp3GFP Balb/c mice exposed to HFHS diet for 8–48 weeks. n = 4–16 biological replicates from ≥4 independent experiments. Depicted are box-and-whisker plots (min to max with all data points). One-way ANOVA with Šidák post hoc test, multiple comparisons vs. lean values. Adjusted P values for hypothalamus: P(8 weeks) = 0.0660; P(16 weeks) <0.0001; P(21 weeks) <0.0001; P(48 weeks) <0.0001. Adjusted P values for rest brain: P(8 weeks) > 0.9999; P(16 weeks) = 0.9882; P(21 weeks) = 0.6420; P(48 weeks) = 0.9131. h Representative FACS plots for the analysis of activated CD62L^low CD4⁺ T cells in mouse Foxp3GFP Balb/c brains with or without exposure to HFHS diet (SD gray, 16 weeks HFHSD red). i Quantification of (H) in % of CD4⁺ T cells. Depicted are box-and-whisker plots (min-to-max values with all data points); n = 8 or n = 15 biological replicates from >4 independent experiments. One-way ANOVA with Šidák post hoc test. Adjusted P values: P(hypothalamus) <0.0001; P(rest brain) = 0.6028). j Box-and-whisker plots (min-to-max values with all data points) for activated CD44^hiCD4⁺ T cells from mouse Foxp3GFP Balb/c brains exposed to SD or HFHS diet; n = 8 biological replicates from four independent experiments. One-way ANOVA with Šidák post hoc test, adjusted P values: p(hypothalamus) = 0.0011; P(rest brain) = 0.0004). k–m Representative FACS plots for hypothalamic CD44^hiCD62L^low effector memory T cells (as % of CD4⁺) from the hypothalamus (k) or rest brain (l) of Foxp3GFP Balb/c mice. m summary plot depicted as box-and-whisker plots (min to max with all data points). N = 5–19 biological replicates. One-way ANOVA with Šidák post hoc test, adjusted P values: p(hypothalamus) = 0.0323; P(rest brain) = 0.0381). n, o Lymph node-residing CD4⁺Foxp3GFP⁺Tregs and CD4⁺CD62L^low T cells of Foxp3GFP Balb/c mice fed the standard or the HFHS diet. n = 8 biological replicates from 2 to 4 independent experiments. Data are depicted as box and whisker plot (min-to-max values with all data points). n One-way ANOVA with Šidák post hoc test, adjusted P values: P(SD vs. 16 weeks) = 0.0699; P(SD vs. 48 weeks) = 0.0056; P(16 weeks vs. 48 weeks) = 0.6880). o One-way ANOVA with Šidák post hoc test, adjusted P values: P(SD vs. 16 weeks) = 0.1038; P(SD vs. 48 weeks) = 0.5281; P(16 weeks vs. 48 weeks) = 0.8587). p Quantification of Treg frequencies from rest brain vs. hypothalamus of Foxp3GFP C57Bl/6J mice exposed to standard diet or HFHS diet for 8 or 16 weeks. Depicted are box-and-whisker plots (min to max with all data points). n = 4−8 biological replicates from 2 to 4 independent experiments. One-way ANOVA with Šidák post hoc test, adjusted P values: hypothalamus: P(SD vs. 8 weeks) <0.0001; P(SD vs 16 weeks) <0.0001; P(8 vs. 16 weeks) >0.9999; rest brain: P(SD vs. 8 weeks) = 0.2470; P(SD vs. 16 weeks) = 0.6984; P(8 vs. 16 weeks) = 0.9663). q Treg frequencies in the hypothalamus and rest brain of genetically obese ob/ob mice on SD. Depicted are box-and-whisker plots (min to max with all data points). n = 8 biological replicates from two independent experiments. Two-tailed unpaired t test, P < 0.0001. Source data are provided as a Source Data file. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Fig. 3. Transcriptome of hypothalamus-residing CD4⁺T cells reflects diet-induced immune activation.

a–d DESeq2-normalized read counts regulated more than 2.5-fold (HFHS diet vs. SD) were functionally annotated to Gene Ontology Biological Processes (GOBP) level 5 using DAVID Bioinformatics Resources 6.7. Terms are depicted as percentage GO-term gene coverage for a hypothalamic CD4⁺CD25⁻CD44^low+int T cells and b hypothalamic CD4⁺CD25^highFoxp3GFP⁺T cells from Foxp3GFP Balb/c mice exposed to 16 weeks SD or HFHS diet (pooled from n = 9 animals). Regulated genes were annotated to selected immunological GOBP terms and color-coded by log₁₀-fold change; c hypothalamic CD4⁺CD25⁻CD44^low+int T cells and d CD4⁺CD25^highFoxp3GFP⁺T cells from Foxp3GFP Balb/c mice exposed to HFHS diet. e, f Heatmap of the fold change of selected differentially regulated genes in response to HFHS diet vs. SD in hypothalamic CD4⁺CD25⁻CD44^low+int T cells (e) or CD4⁺CD25^highFoxp3GFP⁺T cells (f) of Foxp3GFP Balb/c mice. g Gene expression analysis of FACS-sorted CD4⁺CD25⁻CD44^low+int T cells from Foxp3GFP Balb/c mice exposed to 16 weeks SD or HFHS diet by qPCR. n = 4 biological replicates per group from two independent experiments. Mean ± SEM. Unpaired two-tailed t test, P(Ccr7) = 0.0801; P(Cd226) = 0.0670; P(Cxcr3) = 0.0188; P(Ifng) = 0.0169. h, i VENN diagram of regulated genes within selected immunological GO terms of hypothalamic (h) CD4⁺CD25⁻ T cells and i CD4⁺CD25^high T cells from ob/ob mice or C57Bl/6J wild-type mice fed the HFHS diet. Source data are provided as a Source Data file. *P < 0.05.

Fig. 4. A high-calorie diet promotes immune activation of microglia and infiltrating macrophages.

a Representative FACS plots for CD45^intCD11b⁺ microglia and CD45^hiCD11b⁺ infiltrating macrophages in brains from Foxp3GFP Balb/c reporter mice fed the standard or the HFHS diet. b Frequencies of CD45^hiCD11b⁺ macrophages after 1 week of HFHS diet. Depicted are Box-and-whisker plots (min-to-max values with single data points), n = 8 from two independent experiments. Two-tailed unpaired t test; P = 0.0064. c–f Analyses of MHCII expression and costimulatory markers (CD80, CD86 and CD40) on CD45^intCD11b⁺ microglia of Foxp3GFP Balb/c mice fed a standard or HFHS diet for 16 weeks. Depicted are Box-and-whisker plots (min-to- max values with single data points), n = 8 biological replicates from two independent experiments. Two-tailed unpaired t test; P(CD80) < 0.0001; P(CD86) = 0.0028; P(MHCII) < 0.0001; P(CD45) < 0.0001. g Representative FACS plots for MHCII expression on hypothalamic microglia of Cx3cr1^GFP reporter mice exposed to standard or HFHS diet for 12 weeks. h–j Volcano plot (h) of the pairwise comparison between proteomes of CD45^intCD11b⁺Cx3cr1GFP⁺ microglia fed a standard diet or exposed to the HFHS diet for 36 weeks. Expression fold changes (t-test difference, log₂) were calculated and plotted against the t test P value (–log₁₀). 1D annotation enrichment for the most enriched or depleted annotation term (Corum, GOBP, GOCC, GOMF, KEGG, Pfam) is depicted on top of the volcano plot including the Benjamini–Hochberg-corrected false-discovery rate. i All proteins quantified in the total proteome analysis were grouped using unsupervised hierarchical clustering of the z-scored MaxLFQ-intensities across the indicated experimental groups. j Most significantly regulated proteins of i were grouped using unsupervised hierarchical clustering of the z-scored MaxLFQ-intensities across the indicated experimental groups. Source data are provided as a Source Data file. **P < 0.01; ****P < 0.0001.

Fig. 5. Treg loss-of-function experiments reflect their impact in lowering diet-induced hypothalamic immune activation.

a–f Analyses of frequencies of CD80⁺, CD86⁺, and MHCII⁺ cells in CD45^intCD11b⁺ microglia of a–c C57Bl/6J or d–f Balb/c mice on standard diet examined 2 weeks after Treg depletion using mCD25 antibodies. Depicted are Box-and-whisker plots (min-to-max values with single data points). n = 6 biological replicates from two independent experiments. Two-tailed unpaired t test, P(a) = 0.1054; P(b) = 0.0499; P(c) = 0.0491; P(d) = 0.0292; P(e) = 0.3581; P(f)= 0.3513. g–i Identification of frequencies of CD80⁺, CD86⁺, and MHCII⁺ cells in CD45^hiCD11b⁺ macrophages of C57Bl/6J mice on standard diet examined 2 weeks after Treg depletion using mCD25 antibodies. Depicted are Box-and-whisker plots (min-to-max values with single data points). Two-tailed unpaired t test, P(g) = 0.4956; P(h) = 0.0013; P(i) = 0.0326. Source data are provided as a Source Data file. *P < 0.05. **P < 0.01.

Fig. 6. Loss of Foxp3⁺Tregs enhances diet-induced hypothalamic immune activation.

Tregs were depleted using i.p. administration of diphtheria toxin (DTX) in Foxp3 DTR mice. a Representative FACS plots for macrophages and microglia after maintenance on the HFHS diet for 1 week with or without Treg depletion using Foxp3 DTR mice and ablation with DTX. b Quantification of CD45^hiCD11b⁺ infiltrating macrophages. Data are depicted as box-and-whisker plots (min-to-max values with single data points). N = 6 biological replicates. Two-tailed unpaired Student’s t test with P = 0.0040. c–e Immune activation of CD45^intCD11b⁺ microglia as seen from CD80⁺, CD86⁺ and MHCII⁺ expressing cells. Data are depicted as box-and-whisker plots (min-to-max values with single data points). N = 6−8 biological replicates. f–j Representative FACS plots and quantification of immune activation in infiltrating macrophages as seen from CD80⁺, CD86⁺ and MHCII⁺ expressing cells. Data are depicted as box-and-whisker plots (min-to-max values with single data points). N = 6 biological replicates. k Volcano plot of proteome analysis of FACS-sorted CD45^intCD11b⁺ microglia from Foxp3 DTR mice without (n = 4) or with (n = 6) DTX-mediated Treg depletion exposed to 2 weeks HFHS diet. l, m Activation status and proliferative capacity of brain-residing CD4⁺T cells (1 week HFHS diet) with or without DTX-mediated Treg depletion. n = 4–6 biological replicates per group from two independent experiments. Depicted are Box-and-whisker plots (min-to-max values with single data points). Two-tailed unpaired t test, P(b) = 0.0040; P(c) = 0.0014; P(d) = 0.0440; P(e) = 0.3135; P(h) = 0.0124; P(i) = 0.065; P(j) = 0.0272; P(l, hypothalamus) = 0.0008; P(l, rest brain) = 0.0001; P(m, hypothalamus) = 0.0346; P(m, rest brain) <0.0001; Source data are provided as a Source Data file. *P < 0.05; **P < 0.01; ****P < 0.0001.

Fig. 7. In vivo Treg expansion by anti-IL2/IL2 antibody complexes improves metabolic indices in mice fed a HFHS diet.

a, b Treg frequencies (a) and activation of non-Tregs (b) analyzed by flow cytometry in peripheral blood upon Treg expansion using anti-IL2/IL2 antibody complexes. N = 9 biological replicates per group. Mean ± SD. Two-way RM ANOVA with Šidák post hoc test, exact P values are provided in the Source Data file. c Corresponding body weight curves from (a). n = 9 per group. Mean ± SD. Two-way RM ANOVA with Šidák post hoc test. Exact P values are provided in the Source Data file. d Ccl5 gene expression analysis of hypothalami from (a). Mean ± SEM. Two-tailed unpaired t test, P = 0.0193. e Subcutaneous adipose tissue (SAT) and visceral adipose tissue (VAT) weights at the end of the study represented in grams and % of body weight. Each dot represents a biological replication. Mean ± SEM. Two-tailed unpaired t test, P(SAT, mg) = 0.0053; P(SAT, %BW) = 0.0064; P(VAT, mg) = 0.0027; P(VAT, %BW) = 0.0026. f, g ipGTT after 4 weeks (f) and 8 weeks (g) of Treg expansion. Each dot represents a biological replication. Mean ± SD. Two-tailed unpaired t test, P(4 weeks) <0.0001; P(8 weeks) <0.0001. Source data are provided as a Source Data file. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Fig. 8. Transferred Foxp3⁺Tregs reduce hypothalamic immune activation induced by the HFHS diet and improve metabolic indices.

a Representative FACS plots for CD45^intCD11b⁺ microglia and CD45^hiCD11b⁺ infiltrating macrophages in brains from C57Bl/6J mice fed the HFHS diet for 2 weeks with or without peripheral Foxp3GFP⁺Treg transfer (i.v.). b Box-and-whisker plots (min-to-max values with single data points), for the frequencies of infiltrating macrophages of mice. n = 8 biological replicates from two independent experiments. Two-tailed unpaired t test, P = 0.0053. c–e Identification of frequencies of CD80⁺ (P = 0.0256), CD86⁺ (P = 0.1207) and MHCII⁺ (P = 0.8314) cells in CD45^intCD11b⁺ microglia. Box-and-whisker plots (min-to-max values with single data points). n = 6 biological replicates from two independent experiments. Two-tailed unpaired t test. f Frequencies of brain-residing CD4⁺CD62L^lowT cells. n = 6 biological replicates per group from two independent experiments. Box-and-whisker plots (min-to-max values with single data points). Two-tailed unpaired t test, p(hypothalamus) = 0.0004; P(rest brain) <0.0001. g, h Representative FACS plots (g) for the identification of activated CD4⁺CD44^hiT cells in mice with or without i.v. Treg transfer. h Quantification of (g). n = 6 biological replicates per group from two independent experiments. Box-and-whisker plots (min-to-max values with single data points). Two-tailed unpaired t test, P(hypothalamus) = 0.0024; P(rest brain) <0.0001. i, j ipGTT of mice fed a HFHSD for 4 weeks (wild-type C57Bl6J, i) or 8 weeks (wild-type Balb/c, j) that received vehicle or i.v. Treg transfers every 2 weeks. N = 4–6 biological replicates per group. Mean ± SD. Two-tailed unpaired t test, P(4 weeks) = 0.0531; P(8 weeks) <0.0001. Source data are provided as a Source Data file. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Fig. 9. Transferred Foxp3⁺Tregs reduce hypothalamic immune activation induced by the HFHS diet and improve metabolic indices.

a Localization of transferred CD4⁺CD25^highFoxp3GFP⁺ Tregs after i.c. injection analyzed by fluorescence microscopy. Cryosections were stained for astrocytes (GFAP, red), nuclei (DAPI, blue) and GFP (transferred GFP+ Tregs, green). Localization of the i.c. transfer was shown in three independent experiments. Scale bar is 50 µm or 20 µm as indicated. b Representative FACS plots for CD45^intCD11b⁺ microglia and CD45^hiCD11b⁺ macrophages in brains from Balb/c mice fed the HFHS diet for 2 weeks. Prior to exposure to the HFHS diet, animals received i.c. Treg or cellular control transfers. c–f Frequencies of CD45^hiCD11b⁺ macrophages (c, P = 0.0307) and expression CD80⁺ (P = 0.0037), CD86⁺ (P < 0.0001), MHCII⁺ (P = 0.0040) cells in CD45^intCD11b⁺ microglia (d–f). n = 6 biological replicates from two independent experiments. Box-and-whisker plots (min-to-max values with single data points). Two-tailed unpaired t test. g ipGTT of mice that received either i.c. control T cells or Tregs and were fed a HFHS diet for 2 weeks. Mean ± SD. n = 3 biological replicates per group. Two-tailed unpaired t test. Source data are provided as a Source Data file. P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.