Neuroscience

CXCR3-expressing myeloid cells recruited to the hypothalamus protect against diet-induced body mass gain and metabolic dysfunction

Natalia F. Mendes author has email address
Ariane M. Zanesco
Cristhiane F. Aguiar
Gabriela F. Rodrigues-Luiz
Dayana C. da Silva
Jonathan F. Campos
Niels O. S. Câmara
Pedro M. M. de Moraes-Vieira
Eliana P. de Araújo
Licio A. Velloso author has email address

School of Medical Sciences, Department of Translational Medicine (Section of Pharmacology), University of Campinas, Brazil
Laboratory of Cell Signaling, Obesity and Comorbidities Research Center, University of Campinas, Brazil
Laboratory of Immunometabolism, Institute of Biology - University of Campinas, Brazil
Department of Microbiology, Immunology and Parasitology, Federal University of Santa Catarina, Brazil
Laboratory for Transplantation Immunobiology, Institute of Biomedical Sciences, University of Sao Paulo, Brazil
Faculty of Nursing, University of Campinas, Brazil
National Institute of Science and Technology on Neuroimmunomodulation, Rio de Janeiro, Brazil

https://doi.org/10.7554/eLife.95044.1

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Figures and data

CCR2+ cells infiltrate the hypothalamus of mice fed a high-fat diet.
a) CX3CR1^GFP/+CCR2^RFP/+ 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 CX3CR1^GFP+ and CCR2^RFP+ cells in the white adipose tissue and in the hypothalamus of HFD-fed mice. d) Measured event rate detected by flow cytometer of CX3CR1^GFP+ and CCR2^RFP+ cells isolated from the hypothalamus of HFD-fed male and female mice. e) Coronal brain sections of mediobasal hypothalamus (MBH) from chow- and 4 weeks HFD-fed mice CX3CR1^GFP/+CCR2^RFP/+. f) Coronal brain sections of MBH from 4 weeks HFD-fed mice CX3CR1^GFP/+CCR2^RFP/+ 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 in 5 independent experiments. In order to get 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 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 hypothalamus of HFD-fed mice belongs 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.

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 CCR2^RFP 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 CCR2^RFP mice immunostained for CXCR3 upon CXCL10 central neutralization. d) Coronal brain sections from 4 weeks HFD-fed male CCR2^RFP 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) CCR2^RFP mice upon CXCL10 central neutralization. For qualitative confocal image analysis, we have used 3 samples per group. For RT-qPCR of 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 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 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 following 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 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 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 following 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 CCR2^RFP 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 CCR2^RFP mice immunostained for CXCR3 upon AMG487 treatment. d) Coronal brain sections from 4 weeks HFD-fed male CCR2^RFP 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) CCR2^RFP mice upon AMG487 treatment. For qualitative confocal image analysis, we have used 3 samples per group. For RT-qPCR of 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 following 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 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 following 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

Number of DEGs for each comparison

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