Hypothalamic glial cells isolated by MACS reveal that microglia and astrocytes induce hypothalamic inflammation via different processes under high-fat diet conditions

Glial cells can mediate hypothalamic inflammatory processes induced in response to a high-fat diet (HFD). We used magnetic-activated cell sorting (MACS) to isolate microglia and astrocytes from hypothalamus of mice fed HFD and examined changes in expression of inflammation-related cytokines and markers related to glial cell activation status. Hypothalamus from male C57BL6 mice fed a chow diet (chow) or HFD for 1, 3, or 28 days were collected and microglia and astrocytes were isolated by MACS. After confirming cell viability by fluorescence activated cell sorting, mRNA expression levels of inflammation-related cytokines and markers of glial cell activation status were examined by qRT-PCR, which revealed that both glial cell types isolated by MACS retained specificity. On day 3 of HFD, both CD86 and TNF alpha mRNA expression was significantly increased in microglia relative to the chow group. In astrocytes, TNF alpha mRNA expression levels were similar between the chow and HFD groups on day 3, but anti-inflammatory cytokine IL-10 levels were significantly increased. On day 7 of HFD, TNF alpha expression in microglia decreased to levels comparable to the chow group while that in astrocytes remained unchanged. On day 28 of HFD, TNF alpha levels were significantly increased in both microglia and astrocytes, which had increased mRNA expression of CD86 and MAO-B, respectively. For both glial cell types, results for TNF alpha expression assessed by RT-PCR and immunohistochemical analysis were similar. These results indicate that the role of microglia and astrocytes in hypothalamic inflammation under HFD conditions changed with time and these changes were accompanied by changes in the activation status of glial cells. Our data suggest that early after initiating HFD, hypothalamic astrocytes suppress diet-induced inflammation at least in part by secreting IL-10, whereas continued HFD feeding impairs this suppressive function such that both microglia and astrocytes promote hypothalamic inflammation.