Transcriptomic analysis of hypothalamic microglia and infiltrating monocytes in high-fat diet-induced inflammation

RESUMO

INTRODUÇÃO: Microglia plays a pivotal role in the early stages of diet-induced hypothalamic inflammation. Under a chronic period of high-fat feeding, peripheral monocytes and other immune cells are chemoattracted to the hypothalamic parenchyma, however, their involvement in the inflammatory process has been still poorly explored.

OBJETIVOS: In this study, we aimed to identify the main transcriptomic signature differences and sex dimorphism of hypothalamic microglia and infiltrated monocytes in chow- and high-fat diet (HFD)-fed mice.

MÉTODOS: We performed differential expression gene (DEG) analysis using RNA-Seq data obtained from CX3CR1+ hypothalamic microglia and CCR2+ infiltrating monocytes from CX3CR1GFP/+CCR2RFP/+ mice. Eight-week-old male and female mice from this dual-reporter strain were fed on chow or HFD for four weeks. At the end of the experimental period, whole hypothalami and white adipose tissue were harvested for flow cytometry analysis. Coronal brain slices were also obtained for microscopic analysis. For RNA sequencing, in addition to the dual-reporter strain, we also employed eight-week-old male and female homozygous CX3CR1GFP mice fed exclusively on a chow diet. After the end of the experimental period, we pooled together 15-20 hypothalami per sample (n=5) of each group: (1) CX3CR1GFP/+CCR2RFP/+ HFD-fed male mice; (2) CX3CR1GFP/+CCR2RFP/+ HFD-fed female mice; (3) CX3CR1GFP chow-fed male mice; and (4) CX3CR1GFP chow-fed female mice. CX3CR1+ and CCR2+ cells were isolated by fluorescent-activated cell sorting. Total RNA was extracted from each sample and sequenced throughout low input Takara SMART-Seq v4 library preparation followed by Illumina NovaSeq S2 PE150 sequencing. . All experiments in this study were approved by the ethics committees (CEUA/UNICAMP 5497-1/2019 and CIBIO/FCM 07/2019).

RESULTADOS: Flow cytometry and microscopy analysis reveal that CCR2+ cells infiltrate hypothalamic parenchyma after four weeks of HFD. Bioinformatic analysis shows just 16 DEGs upregulated and 16 DEGs downregulated when comparing CX3CR1+ transcriptomic signature between males and females, independent of the diet. Conversely, we found 1598 DEGs upregulated and 1676 DEGs downregulated in CCR2+ transcriptomic signature when comparing males and females, suggesting an important sex dimorphism in these infiltrating cells. We also found an enormous difference when comparing the transcriptomic signature of CX3CR1+ and CCR2+ cells, both in males (3838 DEGs upregulated; 3350 DEGs downregulated) and females (4036 DEGs upregulated; 3569 DEGs downregulated). KEGG enrichment analysis showed significant DEGs in several metabolic pathways modulated in experimental models of obesity, such as IL-17, chemokine, neurotrophin, and NFkB signaling. Gene ontology (GO) analysis also revealed a massive difference when comparing CX3CR1+ with CCR2+ cells, with hundreds of immune system-related GOs modulated.

CONCLUSÃO: Through a state-of-art approach, this pioneering study defined the main transcriptomic signature differences in CX3CR1+ microglia and infiltrating CCR2+ monocytes from the hypothalamus of chow- and HFD-fed mice. We revealed a paradigm shift regarding sex dimorphism in hypothalamic microglia and unveiled enormous sex dimorphism in these bone-marrow-derived monocytes. This data suggests the existence of a sex-related distinct regulation of chemotaxis by peripheral immune cells. Ongoing experiments using a specific chemokine receptor antagonist will further deeply clarify these findings.

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PALAVRA-CHAVE: microglia; monócitos; quimiotaxia; transcriptoma; obesidade; inflamação