Hypothalamic inflammation after LPS administration
The declined expression of proinflammatory cytokines (TNF-a and IL-1b) in the hypothalamus at 1 week and 2 week post HFD feeding has been reported [14, 16]. Previously, we have shown that IL-1b was increased in the hypothalamus at 2 month (8 week) after HFD feeding . Thus, to determine whether intermittent systemic inflammation intensified hypothalamic inflammation in HFD-fed mice, we peripherally administered a high dose of LPS (1 mg/kg) at three time points (1 week, 2 weeks, and 8 weeks) during the early HFD administration period (Fig. 1A). The expression of proinflammatory cytokines (TNF-a and IL-1b) in the hypothalamus was examined 24 h after each LPS injection using QPCR analysis (Fig. 1A). As shown in Fig. 2A, the protein levels of TNF-a and IL-1b in the plasma were increased 24 h after the first LPS injection in chow- and HFD-fed mice. Similarly, compared to that detected in the chow-saline and HFD-saline groups, the mRNA expression of TNF-a and IL-1b in the hypothalamus of chow- and HFD-fed mice was significantly upregulated by the first LPS injection (Fig. 3A). However, the plasma level of TNF-a in HFD-fed mice was not affected, and its level in chow-fed mice showed a decreasing trend 24 h after the second LPS injection (Fig. 2B). Note that an increase in plasma IL-1b protein was detected in mice after HFD feeding for two weeks. Interestingly, the second LPS injection resulted in a reduction in the plasma level of IL-1b in chow- and HFD-fed mice when compared to the relative control group (Fig. 2A). Nevertheless, a non-significant difference in the expression of the two genes in the hypothalamus of animals from the four groups was observed after the second LPS injection (Fig. 3A). The plasma levels and gene expression of TNF-a and IL-1b in the hypothalamus were not significantly different from those detected in the chow-saline and HFD-saline groups (Fig. 2C and Fig. 3A). Despite that microglia accumulation in the hypothalamic ARC region was evident at 24 h after each LPS injection into chow or HFD-fed mice (Fig. S1), we noticed that the three intermittent injections of LPS caused no change in the body weight of mice continuously fed either chow or a HFD for 5 months (Fig. 3B). Moreover, LPS administrations had no effect on food intake in HFD-fed mice, although it did reduce food intake in chow-fed group (Fig. S2). Overall, the results demonstrate that the three intermittent peripheral injections of LPS administered at early time points during HFD feeding may have primed the response of immune cells and hypothalamic cells to chronic HFD feeding.
Chronic HFD feeding attenuates LPS administration-induced exploratory behavior in mice.
Given that systemic inflammation induced by a bolus injection of LPS can evoke mice to develop anxious and depressive behavior later [24, 25], the behaviors of animals from the four groups were analyzed using the EPM and OFT after approximately 5 months of HFD feeding (Fig. 1A). The animals in the Chow-saline and HFD-saline groups spent similar amounts of time in the open arms and showed no difference in the number of entries into the open arm (Fig. 4A). Surprisingly, intermittent LPS administration increased the time spent in and number of entries into the open arms of the chow-fed group (Chow-LPS), whereas this behavior was significantly suppressed in HFD-LPS mice. No differences in the time spent in the closed arms or the number of closed-arm entries were observed between the four animal groups (Fig. S3). The results of the other behavioral assay, the OFT, also indicated that compared to animals from the other three groups, animals from the chow-LPS group spent more time in the center and exhibited an increased number of entries into the center (Fig. 4B). These observations reveal that the three intermittent injections of LPS at early time points induced chow-fed animals to develop intensive exploratory-like behavior at a later time point (i.e., 5 months in this study), whereas chronic HFD feeding suppressed LPS-induced exploratory behavior in mice.
Intermittent LPS injections mediate ARC microglia in response to chronic HFD feeding
We previously reported that HFD feeding for 2, 3, and 4 months prolongs the accumulation of Iba1+ microglia with activated shapes in the ARC . Here, the results showed that hypertrophic microglia were continuously observed in the ARC when mice were fed a HFD (the HFD-saline group) for up to 5 months (Fig. 5A, arrowheads). LPS administration at early time points caused an increase in the number and cell body size of ARC microglia in chow-fed mice compared to mice in the Chow-saline group (Fig. 5A, arrowheads; Fig. 5B). However, the number and cell size of microglia in the chow-LPS group tended to be decreased compared with those observed in the HFD-saline group (Fig. 5B). Moreover, intermittent LPS injections significantly reduced the number of ARC microglia in HFD-fed mice compared to mice in the HFD-saline and chow-LPS groups (Fig. 5B). Moreover, ARC microglia in the HFD-LPS group displayed reduced cell body sizes (Fig. 5A, arrows). These findings indicate that intermittent LPS administration at early time points might modulate the sensitivity of ARC microglia to persistent HFD feeding.
Suppression of microglial activation in different brain regions by intermittent LPS injections combined with chronic HFD feeding
Since LPS administration can induce microglial activation in emotion-associated brain regions, such as the basolateral amygdala (BLA) and nucleus accumbens (NAc) , microglia in the two brain regions collected from animals from the four groups (i.e., the chow-saline, HFD-saline, chow-LPS, and HFD-LPS groups) at 5 months after HFD feeding were examined(Fig. 1). As shown in Fig. 6A (arrowheads), early LPS administration in chow-fed mice increased cell size in the BLA, although the number of BLA microglia was not increased in chow-fed mice (Fig. 6B). However, the cell body size of BLA microglia was significantly reduced in the HFD-LPS group (Fig. 6A, arrows) compared to the Chow-LPS and HFD-Saline groups (Fig. 6B). As in the BLA, early LPS administration and chronic HFD feeding did not change the level of microglial activation in the NAc (Fig. 6A). However, NAc microglia were found to exhibit a larger cell size in HFD-fed mice than in mice from the chow-saline, chow-LPS, and HFD-LPS groups (Fig. 6B). The results reveal that the interplay of peripherally injected of LPS and HFD feeding suppressed the activity of microglia in the BLA and NAc.
Furthermore, we investigated whether early LPS administration affected microglial activation in emotion-associated cortical areas, specifically the ACC and the insula, in chow- and HFD-fed mice (Fig. 1). Although no change in the number of ACC microglia was induced in chow- or HFD-fed mice by early LPS administration, the body size of ACC microglia was increased in chow-fed mice that received early LPS administration (Fig. 7A, arrowheads; Fig. 7B). Although most ACC microglia in the HFD-fed mice that received early LPS administration were small (Fig. 7A, arrows), there was no significant difference compared to the chow-LPS group (Fig. 7B). Microglia in the insula were examined, and it was found that the microglial cell size was increased in the insula of the chow-LPS and HFD-saline groups (Fig. 7C, arrowheads; Fig. 7D); however, the number of microglia in the insula was not changed in either of the two animal groups. However, a reduction in the cell size of microglia in the insula was observed in the HFD-LPS group compared to the chow-LPS and HFD-saline groups (Fig. 7C, arrows; Fig. 7D). The data suggest that although microglial activation in emotion-associated brain regions was induced by either chronic HFD feeding or early LPS administration, the combination of early LPS administration and chronic HFD feeding did not excite microglia, suggesting that LPS administration might mediate microglia at early time points to regulate the influence of chronic HFD feeding.
Enhancement of the elaboration of astrocytic processes in the ARC and BLA by early LPS administration and chronic HFD feeding
Given that the complex elaboration of astrocytic processes reflects the multiple functions of astrocytes in the CNS [26, 27], the morphology of astrocytes in distinct brain regions of animals from the four groups was examined at 5 months using immunofluorescence for the astrocytic cytoskeleton protein GFAP (Fig. 1). The results showed that early LPS administration combined with chronic HFD feeding enhanced the complexity of astrocytic processes and GFAP intensity in the ARC when compared to those observed in both the chow-saline and chow-LPS groups (Fig. 8A, arrowheads; Fig. 8B). Chronic HFD feeding for 5 months also increased GFAP expression in ARC astrocytes compared to that in the chow-saline group (Fig. 8B). Note that an insignificant difference in GFAP intensity was observed between the chow-saline and chow-LPS groups.
Interestingly, early LPS administration induced the elaboration of astrocytic processes and caused an increase in GFAP expression in the BLA of chow-fed mice (Fig. 8A, arrowheads; Fig. 8B) while chronic HFD feeding did not affect the expression of GFAP in the BLA at 5 months (Fig. 8B and 8D). The expression of GFAP in the BLA, like the elaboration of astrocytic processes (Fig. 8A, arrowheads), was much higher in the HFD-LPS group than in both the chow-saline and HFD-saline groups (Fig. 8B). However, differences astrocytic morphology in the NAc, ACC, and insula were insignificant between the four animal groups (Fig. S2). However, astrocytes in the insula displayed elaborate cell processes after chronic HFD feeding for 5 months (Fig. S4, arrowheads). The finding that the elaboration of astrocytic processes in the ARC and BLA was enhanced by early LPS administration and chronic HFD feeding indicates that early LPS exposure resulted in the susceptibility of astrocytes in the two brain regions to continuous exposure to HFD feeding.