CCR2+ cells infiltrate the hypothalamus of mice fed a high-fat diet.

a) CX3CR1GFP/+CCR2RFP/+ dual-reporter mutant mice generation. b) Schematic representation of the experimental protocol for analysis of HFD-induced CCR2+ peripheral-cell chemotaxis towards the hypothalamus. c) Flow cytometry analysis of CX3CR1GFP+ and CCR2RFP+ cells in the white adipose tissue and in the hypothalamus of HFD-fed mice. d) Measured event rate detected by flow cytometer of CX3CR1GFP+ and CCR2RFP+ cells isolated from the hypothalamus of HFD-fed male and female mice. e) Coronal brain sections of mediobasal hypothalamus (MBH) from chow- and 4-week HFD-fed mice CX3CR1GFP/+CCR2RFP/+. f) Coronal brain sections of MBH from 4 weeks HFD-fed mice CX3CR1GFP/+CCR2RFP/+ immunostained for CD169 (Sialoadhesin). White arrows indicate overlap between CD169+ cells with CX3CR1+ cells or with CCR2+ cells. 3V = Third ventricle, Scale Bar = 50 μm.

CX3CR1+ resident and CCR2+ recruited cells sorted from the hypothalamus of HFD-fed mice express classical markers of microglia and other immune cells.

a) Schematic representation of the experimental protocol for sorting and sequencing CX3CR1+ and CCR2+ cells from the hypothalamus of chow- and HFD-fed mice. b) Cx3cr1 gene expression and c) Ccr2 gene expression of CX3CR1+ and CCR2+ cells sorted from the hypothalamus of HFD-fed mice. Analysis of d-m) classical microglial markers, and n-x) bone marrow-derived immune cell markers in the transcriptome of CX3CR1+ and CCR2+ cells sorted from the hypothalamus of HFD-fed mice. To perform RNA-sequencing we have employed a total of 200 mice of each sex fed on HFD and 100 mice of each sex fed on chow diet. They were divided into 5 independent experiments. To get the total RNA amount from CCR2+ cells in the hypothalamus of HFD- fed mice that was enough for library construction and RNA-seq, CCR2+ samples were pooled together in 3 samples, but only 2 samples could be sequenced due to the final RNA integrity and amount.

Differential gene expression (DGE) analysis of CX3CR1+ resident microglia and CCR2+ infiltrating cells sorted from the hypothalamus of HFD-fed mice show an enormous difference in their transcriptomic signature.

A-b) Venn diagram showing the number of DGEs for chow and HFD diet comparison and sex comparison, respectively. c) Heatmap of up and downregulated DEGs when comparing CX3CR1+ resident and CCR2+ infiltrating cells from HFD-fed female mice. d) Heatmap of up and downregulated DEGs when comparing CX3CR1+ resident and CCR2+ infiltrating cells from HFD-fed male mice. e) Heatmap of up and downregulated DEGs when comparing CCR2+ infiltrating cells from HFD-fed male and female mice. hfd_cx_f = Cx3cr1_hfd_female vs. Cx3cr1_chow_female; hfd_cx_m = Cx3cr1_hfd_male vs. Cx3cr1_chow_male; chow_cx = Cx3cr1_chow_male vs. Cx3cr1_chow_female; hfd_cx = Cx3cr1_hfd_male vs. Cx3cr1_hfd_female; hfd_cc_f = Cx3cr1_hfd_female vs. Ccr2_hfd_female; hfd_cc_m = Cx3cr1_hfd_male vs. Ccr2_hfd_male; hfd_cc = Ccr2_hfd_male vs. Ccr2_hfd_female.

Various differential gene expression (DGE) found in CCR2+ infiltrating cells from the hypothalamus of HFD-fed mice belong to chemotaxis pathways.

a) KEGG enrichment analysis shows the distribution of DGEs in distinct metabolic pathways. b-c) Ontology analysis for DGEs related to chemotaxis from CCR2+ cells sorted from the hypothalamus of HFD-fed female and male mice, respectively. d) Heatmap of up and downregulated DEGs related to chemotaxis when comparing CX3CR1+ resident and CCR2+ infiltrating cells from HFD-fed male and female mice.

Number of DEGs for each comparison

CCR2+ infiltrating cells from the hypothalamus of HFD-fed mice express a broad of chemokine receptors.

a-i) Chemokine receptors gene expression in the transcriptome of CX3CR1+ and CCR2+ cells sorted from the hypothalamus of HFD-fed mice.

CXCL10/interferon γ-induced protein 10 kDa (IP-10) is highly expressed in CCR2+ infiltrating cells from the hypothalamus of HFD-fed mice.

a-c) Cxcl9, Cxcl10 and Ifng gene expression in the transcriptome of CX3CR1+ and CCR2+ cells sorted from the hypothalamus of HFD-fed mice. d-e) Ontology analysis for DGEs related to interferon signaling pathways from CCR2+ cells sorted from the hypothalamus of HFD-fed female and male mice, respectively.

CXCL10 neutralization has a mild impact on reducing CCR2+ and CXCR3+ cell chemotaxis towards the hypothalamus of HFD-fed mice.

a) Schematic representation of the experimental protocol for CXCL10 central neutralization. B) Coronal brain sections from 4 weeks HFD-fed CCR2RFP mice showing the CCR2+ cells distribution in the hypothalamic parenchyma upon CXCL10 central neutralization. 3V = Third ventricle, Scale Bars = 50 μm. c) Coronal brain sections from 4 weeks HFD-fed female CCR2RFP/+ mice immunostained for CXCR3 upon CXCL10 central neutralization. D) Coronal brain sections from 4 weeks HFD-fed male CCR2RFP/+ mice immunostained for CXCR3 upon CXCL10 central neutralization. 3V = Third ventricle, Scale Bars = 25 μm. e-f) Hypothalamic Mrna levels of several chemokine receptors and chemokines in HFD-fed female (light gray and purple bars) and male (light gray and green bars) CCR2RFP/+ mice upon CXCL10 central neutralization. For qualitative confocal image analysis, we have used 3 samples per group. For RT-Qpcr of the hypothalamus, we have used 5-6 samples per group. Two-tailed Mann-Whitney tests were used for statistical analyses. *p<0.05 and **p<0.01 in comparison with respective Anti-IgG treated groups.

CXCL10 central neutralization in HFD-fed female mice.

a) Percentual of body weight gain from Day 0 to Day 28 of the experimental protocol. b) Delta body weight during the experimental period. c) Brown adipose tissue weight and d) White adipose tissue (retroperitoneal depot) weight at Day 28. e) Leptin, f) Triglycerides, and g) Total cholesterol plasma levels at Day 28. h) Weekly food intake measurement during the experimental period. i) Cumulative food intake during the experimental period. j) 90-minute food intake measurement after 16 hours of fasting. k) Hypothalamic mRNA levels of neuropeptides involved in food intake control. l) Intraperitoneal glucose tolerance test on Day 24. m) 6h-fasting blood glucose levels. n) Insulin plasma levels at Day 28. o) Hypothalamic mRNA levels of inflammatory genes. p) O2 consumption; q) CO2 production; r) Energy Expenditure and s) Respiratory Quotient at Day 24. Data were expressed as mean ± SEM of 8-10 mice per group (in two independent experiments). To perform qRT-PCR we have used 6 mice/group. To perform biochemical analysis in plasma we have used 8-10 mice/group. To perform ipGTT we have used 4 mice/group. To perform indirect calorimetry, we have used 4 mice/group. Two-way ANOVA followed by Sidak’s post-hoc test and Mann-Whitney test were used for statistical analyses. *p<0.05, **p<001, ***p<0.001, ****p<0.0001 in comparison with IgG treated group.

CXCL10 central neutralization in HFD-fed male mice.

a) Percentual of body weight gain from Day 0 to Day 28 of the experimental protocol. b) Delta body weight during the experimental period. c) Brown adipose tissue weight and d) White adipose tissue (retroperitoneal depot) weight at Day 28. e) Leptin, f) Triglycerides and g) Total cholesterol plasma levels at Day 28. h) Weekly food intake measurement during the experimental period. i) Cumulative food intake during the experimental period. j) 90-minute food intake measurement after 16 hours of fasting. k) Hypothalamic mRNA levels of neuropeptides involved in food intake control. l) Intraperitoneal glucose tolerance test at Day 24. m) 6h- fasting blood glucose levels. n) Insulin plasma levels at Day 28. o) Hypothalamic mRNA levels of inflammatory genes. p) O2 consumption; q) CO2 production; r) Energy Expenditure and s) Respiratory Quotient at Day 24. Data were expressed as mean ± SEM of 8-10 mice per group (in two independent experiments). To perform qRT-PCR we have used 6 mice/group. To perform biochemical analysis in plasma we have used 8-10 mice/group. To perform ipGTT we have used 4 mice/group. To perform indirect calorimetry, we have used 4 mice/group. Two-way ANOVA followed by Sidak’s post-hoc test and Mann-Whitney test were used for statistical analyses. *p<0.05, **p<001, ***p<0.001, ****p<0.0001 in comparison with IgG treated group.

AMG487 treatment attenuates CCR2+ and CXCR3+ cell chemotaxis towards the hypothalamus of HFD-fed mice.

a) Schematic representation of the experimental protocol for CXCR3 systemic blockage. b) Coronal brain sections from 4 weeks HFD-fed CCR2RFP/+ mice showing the CCR2+ cells distribution in the hypothalamic parenchyma upon AMG487 treatment. 3V = Third ventricle, Scale Bars = 50 μm. c) Coronal brain sections from 4 weeks HFD-fed female CCR2RFP/+ mice immunostained for CXCR3 upon AMG487 treatment. d) Coronal brain sections from 4 weeks HFD-fed male CCR2RFP/+ mice immunostained for CXCR3 upon AMG487 treatment. 3V = Third ventricle, Scale Bars = 25 μm. e-f) Hypothalamic mRNA levels of several chemokine receptors and chemokines in HFD-fed female (light gray and pink bars) and male (light gray and blue bars) CCR2RFP/+ mice upon AMG487 treatment. For qualitative confocal image analysis, we have used 3 samples per group. For RT-qPCR of the hypothalamus, we have used 7-8 samples per group. Two-tailed Mann-Whitney tests were used for statistical analyses. *p<0.05 and **p<0.01 in comparison with respective VEH-treated groups.

CXCR3 systemic blockage in HFD-fed female mice.

a) Percentual of body weight gain from Day 0 to Day 28 of the experimental protocol. B) Delta body weight during the experimental period. C) Brown adipose tissue weight and d) White adipose tissue (retroperitoneal depot) weight at Day 28. E) Leptin, f) Triglycerides and g) Total cholesterol plasma levels at Day 28. H) Weekly food intake measurement during experimental period. I) Cumulative food intake during the experimental period. J) 90 min food intake measurement after 16h-fasting. K) Hypothalamic mRNA levels of neuropeptides involved in food intake control. L) Intraperitoneal glucose tolerance test at Day 24. M) 6h-fasting blood glucose levels. N) Insulin plasma levels at Day 28. O) Hypothalamic mRNA levels of inflammatory genes. P) O2 consumption; q) CO2 production; r) Energy Expenditure and s) Respiratory Quotient at Day 24. Data were expressed as mean ± SEM of 14-16 mice per group (in four independent experiments). To perform qRT-PCR we have used 8 mice/group. To perform biochemical analysis in plasma we have used 8-10 mice/group. To perform ipGTT we have used 5 mice/group. To perform indirect calorimetry, we have used 4-5 mice/group. Two-way ANOVA followed by Sidak’s post-hoc test and Mann-Whitney test were used for statistical analyses. *p<0.05, **p<001, ***p<0.001, ****p<0.0001 in comparison with VEH treated group.

CXCR3 systemic blockage in HFD-fed male mice.

a) Percentual of body weight gain from Day 0 to Day 28 of the experimental protocol. b) Delta body weight during the experimental period. c) Brown adipose tissue weight and d) White adipose tissue (retroperitoneal depot) weight at Day 28. e) Leptin, f) Triglycerides, and g) Total cholesterol plasma levels at Day 28. h) Weekly food intake measurement during the experimental period. i) Cumulative food intake during the experimental period. j) 90-minute food intake measurement after 16 hours of fasting. k) Hypothalamic mRNA levels of neuropeptides involved in food intake control. l) Intraperitoneal glucose tolerance test on Day 24. m) 6h-fasting blood glucose levels. n) Insulin plasma levels at Day 28. o) Hypothalamic mRNA levels of inflammatory genes. p) O2 consumption; q) CO2 production; r) Energy Expenditure and s) Respiratory Quotient at Day 24. Data were expressed as mean ± SEM of 14-16 mice per group (in four independent experiments). To perform qRT-PCR we have used 8 mice/group. To perform biochemical analysis in plasma we have used 8-10 mice/group. To perform ipGTT we have used 5 mice/group. To perform indirect calorimetry, we have used 4-5 mice/group. Two-way ANOVA followed by Sidak’s post-hoc test and Mann-Whitney test were used for statistical analyses. *p<0.05, **p<001, ***p<0.001, ****p<0.0001 in comparison with VEH treated group.

Ovariectomy and ovariectomy with estradiol replacement modulate hypothalamic chemokine and neuropeptides.

a) Experimental design of the ovariectomy and estradiol replacement protocol performed in C57BL/6J female mice; b) Body mass gain from Day 7 to Day 35 of the experimental protocol. c) Total food intake during the experimental period. d) Fasting blood glucose levels, and d) Retroperitoneal white adipose tissue depot weight at Day 35. Hypothalamic mRNA levels of f) chemokines; g) chemokine receptors; h) neuropeptides; i) inflammatory genes. Data are expressed as mean ± SEM of 4-6 mice per group. One-way and Two-way ANOVA followed by Sidak’s post-hoc test and Mann-Whitney test were used for statistical analyses. *p<0.05 and **p<001, in comparison with the Sham group.

CXCR3 systemic blockage in HFD-fed female mice.

a) Total 24h-Energy expenditure and b) Locomotor activity at Day 24 of the experimental protocol. c) Delta body weight during the experimental period. c) Hypothalamic mRNA levels of ER stress genes; d) Hypothalamic mRNA levels of mitochondrial function; e) Hepatic mRNA levels of genes of lipid and glucose metabolism; f) White adipose tissue mRNA levels of genes of lipid metabolism and thermogenesis. Data were expressed as mean ± SEM of 4-6 mice per group. To perform indirect calorimetry and locomotor activity, we have used 5-6 mice/group. To perform qRT-PCR we have used 4-6 mice/group. Two-way ANOVA followed by Sidak’s post-hoc test and Mann-Whitney test were used for statistical analyses.

CXCR3 systemic blockage in HFD-fed male mice.

a) Total 24h-Energy expenditure and b) Locomotor activity at Day 24 of the experimental protocol. c) Delta body weight during the experimental period. c) Hypothalamic mRNA levels of ER stress genes; d) Hypothalamic mRNA levels of mitochondrial function; e) Hepatic mRNA levels of genes of lipid and glucose metabolism; f) White adipose tissue mRNA levels of genes of lipid metabolism and thermogenesis. Data were expressed as mean ± SEM of 4-6 mice per group. To perform indirect calorimetry and locomotor activity, we have used 5-6 mice/group. To perform qRT-PCR we have used 4-6 mice/group. Two-way ANOVA followed by Sidak’s post-hoc test and Mann-Whitney test were used for statistical analyses. *p<0.05 and **p<001, in comparison with VEH treated group.