3.1 Metabolic disorders in the mice
In supplemental Figure 2, the levels of body weight, fasting glucose, total cholesterol and triglycerides were significantly higher in the four HFD groups than that in the three ND groups (P<0.05, P<0.05, P<0.05, P<0.05, respectively). In addition, there was no difference in these four metabolic markers among the three ND groups or among the four HFD groups (P>0.05). These findings indicated that a HFD induced significant metabolic disorders in the mice, and the AAV transfection and CREB inhibitor did not affect this process.
3.2 Cognitive function in the mice
In Figure 1, the “escape latency” was longer in the HFD group than in the ND group (P<0.05), and the “percentage of time spent in the target quadrant” and “number of times crossing the platform area” were lower in the HFD group than in the ND group (P<0.05, P<0.05, respectively). Compared with the HFD group, IL-2 intervention in the HFD+IL2 group decreased the “escape latency” and increased the “percentage of time spent in the target quadrant” and “number of times crossing the platform area” (P<0.05, P<0.05, P<0.05, respectively). In the HFD+IL2+CREBI group, CREB inhibitor 666-15 reversed the effect of IL-2 on these three cognitive markers (P<0.05, P<0.05, P<0.05, respectively). These findings indicate that IL-2 intervention alleviated the cognitive impairment induced by HFD through the activation of CREB signaling.
3.3 Depression-like behavior in the mice
In Figure 2, there was no difference in “total consumption” among all the experimental groups (P = 0.994). The “sucrose preference rate” was lower in the HFD group than in the ND group (P<0.05), and “immobility time” was higher in the HFD group than in the ND group (P<0.05). IL-2 treatment in the HFD+IL2 group increased the “sucrose preference rate” and decreased the “immobility time” (P<0.05, P<0.05, respectively). In the HFD+IL2+CREBI group, 666-15 redecreased the “sucrose preference rate” and reincreased the “immobility time” (P<0.05, P<0.05, respectively). These findings indicated that IL-2 improved the depression-like behavior caused by HFD through the activation of CREB signaling.
3.4 IL-2 levels in the mice
In supplemental Figure 3, the peripheral level of IL-2 was significantly higher in the groups with AVV-IL2 than that in the other groups without AVV-IL2 from 4 weeks to 16 weeks after transfection (P<0.05), which indirectly confirmed the AAV infection. More importantly, the peripheral level of IL-2 never exceed 100 pg/ml, which can be considered as “low-dose”.
In the hippocampus, the relative level of IL-2 was lower in the HFD group than in the ND group (P<0.05). In the HFD+IL2 group, IL-2 treatment increased the level of IL-2 (P<0.05). In the HFD+IL2+CERBI group, 666-15 inhibited the level of IL-2 compared with the HFD+IL2 group (P<0.05)
3.5 Inflammatory response in the hippocampus
In Figure 3, the levels of TNF-α and IL-1β were higher in the HFD group than in the ND group (P<0.05, P<0.05, respectively), and the levels of IL-4 and IL-10 were lower in the HFD group than in the ND group (P<0.05, P<0.05, respectively). In the HFD+IL2 group, IL-2 intervention inhibited the levels of TNF-α and IL-1β (P<0.05, P<0.05, respectively). Meanwhile, IL-2 intervention in the same group increased the levels of IL-4 and IL-10 (P<0.05, P<0.05, respectively). In the HFD+IL2+CREBI group, the CREB inhibitor reversed the effect of IL-2 on these inflammatory factors (P<0.05, P<0.05, P<0.05, P<0.05, respectively). These findings indicate that IL-2 intervention inhibited the inflammatory response induced by HFD through the upregulation of CREB signaling.
3.6 Expansion and activation of Tregs in the hippocampus
In Figure 4, the “percentage of Tregs in CD4+ in hippocampus” and “mean fluorescence intensity of CD25 in Tregs” were lower in the HFD group than in the ND group (P<0.05, P<0.05, respectively). In the HFD+IL2 group, IL-2 intervention increased the levels of these two markers (P<0.05, P<0.05, respectively). In the HFD+IL2+CERBI group, the CREB inhibitor redecreased the “percentage of Tregs in CD4+ in hippocampus” and “mean fluorescence intensity of CD25 in Tregs” (P<0.05, P<0.05, respectively). These findings indicated that a HFD inhibited the expansion and activation of Tregs and that IL-2 alleviated the effect of a HFD through the activation of CREB signaling.
3.7 Microglial polarization in the hippocampus
In Figure 5, the expression of CD206 and Arg-1 was decreased in the HFD group compared with the ND group (P<0.05, P<0.05, respectively). Meanwhile, the expression of CD86 and iNOS was increased in the HFD group compared with the ND group (P<0.05, P<0.05, respectively). In the HFD+IL2 group, IL-2 upregulated the expression of CD206 and Arg-1 and downregulated the expression of CD86 and iNOS compared with the HFD group (P<0.05, P<0.05, P<0.05, P<0.05, P<0.05, respectively). In the HFD+IL2+CREBI group, the CREB inhibitor reversed the effect of IL-2 on these four markers (P<0.05, P<0.05, P<0.05, P<0.05, respectively). These findings indicated that IL-2 promoted microglial M2 polarization through the activation of CREB signaling.
3.8 NLRP3 inflammasome and pyroptosis in the hippocampus
In Figure 6, the expression of NLRP3, caspase-1 and ASC was higher in the HFD group than in the ND group (P<0.05, P<0.05, P<0.05, respectively). The rate of pyroptosis was also higher in the HFD group than in the ND group (P<0.05). In the HFD+IL2 group, the expression of the three proteins and the rate of pyroptosis were lower than those in the HFD group (P<0.05, P<0.05, P<0.05, P<0.05, respectively). In the HFD+IL2+CREBI group, expression of the three proteins and rate of pyroptosis significantly reincreased (P<0.05, P<0.05, P<0.05, P<0.05, respectively). The findings indicated that IL-2 inhibited NLRP3 inflammasome activity and pyroptosis through the activation of CREB signaling.
3.9 BDNF-ERK-CREB signaling in the hippocampus
In Figure 7, the expression of BDNF, p-ERK1/2 and p-CREB was lower in the HFD group than in the ND group (P<0.05, P<0.05, P<0.05, respectively). In the HFD+IL2 group, IL-2 intervention significantly upregulated the expression of the three proteins (P<0.05, P<0.05, P<0.05, respectively). In the HFD+IL2+CREBI group, the CREB inhibitor downregulated the expression of p-CREB but did not affect the expression of the other two proteins (P<0.05, P>0.05, and P>0.05, respectively). These findings indicated that a HFD inhibited the activity of BDNF-ERK-CREB signaling and that IL-2 may upregulate this signaling again.