DOI: https://doi.org/10.21203/rs.3.rs-1932936/v1
Interleukin-2 was originally thought to be a proinflammatory factor, but recent studies have revealed that low-dose interleukin-2 might have an anti-inflammatory property. The aim of the study was to reveal whether the cytokine inhibited neuroinflammation in a high-fat diet mouse model and to further reveal the mechanism involved.
Mice were treated with a single administration of an AAV-interleukin-2 or AAV-LUC vector. Then, the mice were fed a normal or high-fat diet for 12 weeks, followed by a 4-week intervention period. During the intervention period, some of the mice were treated with CREB inhibitor 666 − 15. Then, cognitive function and depression-like behavior were assessed using the Morris water maze, sucrose preference test and tail suspension test. The expression of p-CREB, several microglial polarizations and inflammasome markers were measured using western blotting. The rate of pyroptosis and expansion and activation of Tregs were assessed using flow cytometry.
A high-fat diet caused cognitive impairment and depression-like behavior in the mice. Meanwhile, the high-fat diet also inhibited the expansion and activation of Tregs, promoted microglial M1 polarization, activated the NLRP3 inflammasome and pyroptosis in the hippocampus, and eventually induced significant neuroinflammation in the hippocampus. Low-dose IL-2 using an AAV vector reversed these cognitive, behavioral and pathophysiological abnormalities. However, 666 − 15 treatment weakened the protective effect of IL-2 and aggravated cognitive impairment, neuroinflammation and all other abnormalities in the mice.
Low-dose interleukin-2 alleviated neuroinflammation and cognitive impairment by activating CREB signaling in high-fat diet mice.
A growing body of evidence suggests that obesity or a long-term high-fat diet (HFD) contributes to the development of cognitive impairment (Wang et al. 2017; Wang et al. 2000). In this process, inflammation is regarded as a main mechanism for the disease (Miller and Spencer 2014; Guillemot-Legris and Muccioli 2017). Briefly, excessive lipids in the body lead to a systematic inflammatory response, which in turn causes a variety of pathophysiological abnormalities in the brain, especially neuroinflammation. Together, these abnormalities induce neuronal dysfunction and death, and constitute the pathophysiological basis of cognitive impairment in obesity.
Currently, there are several potential mechanisms involved in the regulation of neuroinflammation in the brain. First, regulatory T cells (Tregs) are one type of T cell that controls autoimmune response in the body. They can inhibit the activation of autoreactive T cells and prevent the occurrence of autoimmune diseases (Kleinewietfeld et al. 2014). Although the role of Tregs in neuroinflammation has not been fully determined, it is widely believed that these cells have an anti-inflammatory effect in the brain (Baruch et al. 2015). Second, microglia are generated and distributed in the brain and play a central role in the regulation of neuroinflammation (Kwon and Koh 2020). Depending on external stimulation, microglia can exhibit a proinflammatory response (M1 phenotype) or anti-inflammatory response (M2 phenotype) (Subhramanyam et al. 2019). Promoting its polarization to the M2 phenotype is an important therapeutic target against neuroinflammation. Third, the inflammasome is a complex of proteins that can identify endogenous and endogenous threats (Sutterwala et al. 2014). It can activate caspase-1, promote the maturation and release of multiple cytokines, lead to neuronal pyroptosis and trigger a strong inflammatory response (Sutterwala et al. 2014). Several types of inflammasomes have been discovered up to now, and the NLRP3 inflammasome has been confirmed to be related to the regulation of neuroinflammation in obese and HFD models (Sobesky et al. 2016; Mirzaei et al. 2018). In the body, these and other unproven mechanisms independently or interrelatedly regulate neuroinflammation, clear potential threats and maintain a stable internal environment. Dysfunction of these mechanisms may lead to the occurrence of neurological disease.
Interleukin-2 (IL-2) is a type of cytokine that is closely associated with the central nervous system. Previous studies have revealed that peripheral IL-2 can pass through the blood–brain barrier, and hippocampal neurons express a large amount of IL-2 receptors (Waguespack et al. 1994; Dansokho et al. 2016). For a long period of time, IL-2 has been regarded as a neurotoxic or proinflammatory factor. In fact, its biological function is much more complex than expected.
For instance, one study suggests that IL-2 provides trophic support for neurons and glia and enhances neurite branching and dendritic development (Awatsuji et al. 1993). Another study reported that knockout of IL-2 causes abnormal changes in the hippocampal structure of mice (Beck et al. 2005). These findings partly explain the protective effect of IL-2 on cognitive function.
IL-2 can also exert its biological effect by regulating the immune system (Petitto et al. 2015). On the one hand, one study suggests that IL-2 supports the survival and function of Tregs, thus inhibiting inflammation and autoimmunity (Klatzmann and Abbas 2015). In this study, IL-2 was proven to selectively activate Tregs without activating effector T cells, which explains why cytokines play an anti-inflammatory role rather than promoting inflammation (Klatzmann and Abbas 2015). On the other hand, microglia are a source and target of IL-2, and there is a close relationship between microglia and the NLPR3 inflammasome (Schneider et al. 2012; Hanslik and Ulland 2020). Therefore, it is necessary to explore whether microglia and the NLPR3 inflammasome are involved in the anti-inflammatory effect of IL-2, especially in a HFD model.
It is worth mentioning that all of the above neuroprotective effects and anti-inflammatory effects are achieved by low-dose IL-2. The “low-dose IL-2” refers to an appropriate dose of IL-2 that can expand and activate Tregs, but can not affect effector T cells (Churlaud et al. 2014).
Based on the above, we conducted an in vivo experiment to determine whether low-dose IL-2 exerts a protective effect on neuroinflammation and cognitive function in a mouse model of HFD and to further reveal the potential pathophysiological and molecular mechanism involved.
2.1 Ethical standards and flow chart
The study was approved by the Ethics Committees of Tianjin Haihe Hospital and Tianjin Medical University General Hospital. Flow charts of the whole experiment was shown in supplemental Fig. 1.
A total of 70 C57BL/6J mice were purchased from a laboratory animal center at the Academy of Military Medical Sciences (Beijing, China). All mice were male and approximately 4 weeks old. At the beginning of the experiments, there was a seven-day adaptation period. During this period, they were placed in several cages at 25°C, subjected to a 24-hour light cycle (i.e., 12-hour light/12-hour dark) and fed a balanced diet and adequate water.
Then, the mice were randomly divided into seven groups: the ND group, ND + LUC group, ND + IL2 group, HFD group, HFD + LUC group, HFD + IL2 group and HFD + IL2 + CREBI group. Each group contained 10 mice. ND indicates “normal diet”, LUC indicates “luciferase”, IL2 indicates “interleukin-2”, HFD indicates “high-fat diet”, and CREBI indicates “CREB signaling inhibitor”.
After grouping, the mice in the ND + IL2 group, HFD + IL2 group and HFD + IL2 + CREBI group were treated with a single administration of an AAV-IL-2 vector. Meanwhile, the mice in the ND + LUC group and HFD + LUC group were challenged with a single administration of an AAV-LUC vector. Recombinant rAAV8 vectors were obtained through triple transfection of HEK-293T cells (Churlaud et al. 2014). The transgenes were luciferase (LUC) and murine IL-2, and the promoters were hybrid cytomegalovirus enhancer/chicken beta-actin constitutive promoter (CAG). The vectors were intraperitoneally injected only once with 1010 viral genomes of AAV8-CAG-IL2 or AAV8-CAG-LUC.
Based on the previous study, 1010 viral genomes of AAV8-CAG-IL2 may allow stable expression and release of IL-2 for at least 20 weeks after the transfection. During this period, the average peripheral circulating of IL-2 did not exceed 200 pg/ml, and can be regarded as “low-dose” (Churlaud et al. 2014).
After treatment, there was a 12-week modeling process. During this period, mice in the HFD group, HFD + LUC group, HFD + IL2 group and HFD + IL2 + CREBI group were fed a HFD (D12492, Research Diets). In this type of food, 60% of the energy comes from fats, 20% from proteins, and the remaining 20% from carbohydrates. During the same period, the mice in the other three groups were provided a normal diet (D12450B, Research Diets). In this type of food, only 10% of the energy comes from fats, and 20% and 70% separately from proteins and carbohydrates.
The body weights of the mice were routinely monitored. At the end of the 12-week modeling period, fasting blood specimens were collected from their caudal veins. Blood glucose and lipids were measured using a Hitachi 7170 automatic biochemical analyzer (Japan).
Then, the mice in the HFD + IL2 + CREBI group were intravenously injected with CREB signaling inhibitor 666 − 15 at a dose of 15 mg/kg three times a week for 4 weeks (Wang et al. 2020). The mice in the other groups were intravenously injected with 0.9% saline three times a week for 4 weeks. During this period, the dietary types in these groups were the same as what they were fed in the modeling process.
After the intervention, cognitive function was assessed using the Morris water maze (Mulder and Pritchett 2003). Briefly, the research equipment was mainly a circular pool filled with opaque water. The pool was randomly divided into four quadrants, one of which had a platform underwater that could hold one mouse standing. A video-tracking system (TSE Systems, Germany) was mounted above the pool to record the movement of the mice. On the first day, each mouse was placed in the pool for 2 minutes to familiarize themselves with the experimental environment. From the second day to the fifth day, the test was carried out. Each mouse was randomly placed anywhere in the pool. “Escape latency” from entering the water to boarding the platform was recorded. If the escape latency exceeded 60 seconds, the mouse was artificially placed on the platform for 30 seconds, and its escape latency was recorded as 60 seconds. Then, the underwater platform was withdrawn. Each mouse was put back into the pool for 120 seconds, and “the percentage of time the mouse spent in the target quadrant” and “the number of times it passed the target quadrant” were reported.
Depression-like behavior was evaluated using a sucrose preference test (Moore et al. 2018). Briefly, each mouse was fed in a separate cage. In the first two days, each mouse was provided with two bottles. One bottle was filled with normal water, and the other bottle was filled with 1% sucrose water. The mouse could freely choose which kind of water to drink. On the third day, the mice were not fed any diet. On the fourth day, each mouse was again provided with the two bottles and was allowed to drink freely for 6 hours. Then, the consumption of both types of water was recorded. Total consumption indicated the sum of sucrose water consumption and normal water consumption. The sucrose preference rate (%) indicated the ratio of sucrose water consumption and total consumption.
Depression-like behavior was also evaluated using the tail suspension test (Moore et al. 2018). Briefly, each mouse was hung upside down on a crossbar with its head 15 cm above the ground. A camera system was adopted to record the movement of the mouse for 6 minutes. The length of the time during which its limbs were completely immobile or moved slightly was reported.
After the above cognitive and behavioral tests, the mice were euthanized. Subsequently, the skull was cut from the dorsal side and the entire brain was obtained with sterile forceps. Separate the brain into two hemispheres from the midline using a sterile scalpel. In each hemisphere, the olfactory bulb, cerebellum, medulla and pons were first removed. Then, peel off the meninges and move aside the striatum. A curved kidney-like structure can be visualized in the distal region of the hemisphere, which was the hippocampus. Carefully isolate the hippocampus for the following experiment.
Tissue from the left hippocampus was mixed with the correct amount of homogenization buffer and smashed using an electrical homogenizer. The homogenized tissue was centrifuged at 600 g for 8 minutes, and the obtained supernatants were centrifuged at 12,000 g for 15 minutes. Then, the pellets were mixed with the homogenization buffer and centrifuged at 12,000 g for 10 minutes. The obtained pellets were mixed with homogenization buffer to form tissue homogenates. The latter were refrigerated for the following western blotting and enzyme-linked immunosorption assay (ELISA).
Tissue from the right hippocampus was soaked and cleaned using double distilled water. Then, the tissue was cut into small pieces and digested with 0.2% trypsin for more than 30 minutes. When the tissue was dispersed, the tissue fragments were removed and filtered with a 100-mesh nylon filter. The obtained cell suspensions were centrifuged at 500 rpm for 12 minutes. The cells were mixed with phosphate-buffered saline to form hippocampal cell suspensions for further flow cytometry.
The expression of IL-2, CD206, Arg-1, CD86, iNOS, NLRP3, Caspase-1 and ASC in hippocampal tissue homogenates was measured using western blotting. Briefly, the amount of total protein was measured using a PierceTM modified Lowry protein assay kit (Thermo Fisher Scientific). Fifty micrograms of the protein was separated using 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a nitrocellulose membrane (Bio-Rad). The latter was blocked with 5% skimmed milk at room temperature for 120 minutes. After that, the membrane was incubated with a variety of primary antibodies, including anti-IL-2 (#PA5-94995, Thermo Fisher Scientific, 1:1000), anti-CD206 (#24595, CST, 1:1000), anti-Arg-1 (#93668, CST, 1:1000), anti-CD86 (#19589, CST, 1:1000), anti-iNOS (#13120, CST, 1:1000), anti-NLRP3 (#15101, CST, 1:1000), anti-Caspase-1 (#24232, CST, 1:1000), anti-IL-1β (#31202, CST, 1:1000) and anti-GAPDH (#5174, CST, 1:1000 dilution), at 4°C for 16 hours. Then, the membrane was incubated with an anti-rabbit IgG horseradish peroxidase-linked secondary antibody (#7074, CST). Visualization of the obtained immunoreactive bands was performed using chemiluminescence detection (Immolilon Western). The intensity of the bands was assessed using ImageJ software.
The expression of tumor necrosis factor-α (TNF-α), IL-1β, IL-4, IL-10, brain-derived neurotrophic factor (BDNF), p-ERK1/2 and p-CREB in hippocampal tissue homogenates was measured using ELISA. ELISA kits for TNF-α, IL-1β, IL-4, IL-10 and BDNF were purchased from Thermo Fisher Scientific (USA). ELISA kits for p-ERK1/2 and p-CREB were separately obtained from Enzo Life Sciences (USA) and MyBioSource (China). The level of IL-2 in peripheral circulation was also measured using ELISA. ELISA kits for IL-2 was obtained from eBioscience (USA). All experiments were performed according to the manufacturers’ instructions.
2.11 Flow cytometry
The pyroptosis rate in the hippocampal cell suspensions was measured using flow cytometry. For this process, a commercial FAM-FLICA caspase-1 detection kit was adopted according to the manufacturer’s instructions. Briefly, the prepared cells were stained with FAM-FLICA and PI at room temperature for 20 minutes. Fluorescence intensity was determined using a Coulter Epics XL flow cytometer. The percentage of double-positive cells out of the total cells indicated the rate of pyroptosis.
The expansion and activation of Tregs were also assessed using flow cytometry. Briefly, the prepared cells were stained with several monoclonal antibodies at 4°C for 20 minutes. The antibodies included CD3-PE, CD8-Alexa700, CD4-HorizonV500, CD25-PeCy7, NKp46-APC and B220-FITC (eBioscience). To detect intracellular FOXP3, the cells were fixed and permeabilized using a fixation buffer/permeabilization buffer (eBioscience) and stained with Foxp3-E450 (eBioscience). The cells were measured using a Coulter Epics XL flow cytometer. Tregs indicated CD4+CD25+Foxp3+ cells. The mean fluorescence intensity of CD25 represented the activity of Tregs.
2.12 Statistical analysis
Continuous variables in the study are expressed as the mean ± standard deviation. Differences in several continuous variables were measured using one-way ANOVA with the LSD test. A P value < 0.05 suggested that the difference was statistically significant. All analyses were conducted using SPSS 23.0 (USA).
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.
It is well known that obesity induces a systemic inflammatory response in the body, which in turn leads to neuroinflammation in the brain (Karczewski et al. 2018). In this study, we fed mice a HFD for 12 weeks and successfully caused neuroinflammation in the hippocampus. Meanwhile, the mice showed cognitive impairment and depression-like behavior. Therefore, the modeling strategy almost completely simulated the true course of disease development and helped to ensure the reliability of the conclusions obtained from the following experiments.
As a cytokine, IL-2 is mainly generated by activated T cells and is widely distributed throughout the body. It contributes to the expansion and activation of T cells, B cells, NK cells and macrophages, thus playing an important role in promoting an inflammatory response against various endogenous and exogenous threats. Therefore, IL-2 has long been thought to be a proinflammatory factor (Malek 2008). In fact, the effect of IL-2 on regulating the inflammatory response is complex. Churlaud et al. suggested that IL-2 was a key cytokine supporting the survival and function of Tregs, and low-dose IL2 treatment promoted the expansion of Tregs without inducing effector T cell activation and prevented autoimmune diseases in a mouse model (Churlaud et al. 2014). Alves et al. suggested that IL-2 improved amyloid pathology, synaptic failure and memory in Alzheimer’s disease mice, which was related to controlling inflammation caused by the cytokine by suppressing Tregs (Alves et al. 2017). Therefore, the study adopted low-dose IL-2 to treat HFD mice and revealed that IL-2 can inhibit neuroinflammation through a variety of mechanisms or pathways.
In addition to regulating Tregs, a study found that low-dose IL-2 can regulate microglial polarization and NLRP3 inflammasome activity. Ma et al. reported that Tregs protected against brain damage by alleviating the inflammatory response in neuromyelitis optica spectrum disorder. Briefly, the absence of Tregs induced microglial M1 activation and proinflammatory cytokine release. In contrast, Treg transfer skewed microglia toward an alternative activation phenotype, thereby inhibiting the inflammatory response (Ma et al. 2021). Park et al. suggested that NLRP3 deficiency increased the amount and suppressive activity of Tregs, whereas NLRP3 overexpression reduced Foxp3 expression and Treg abundance (Park et al. 2019). All these findings confirmed the association of IL-2 with microglial function and NLRP3 signaling and supported the conclusion of the present study.
In this study, a recombinant rAAV8 vector containing the IL-2 transgene was constructed, and then the vectors were injected into the abdominal cavity of the mice. This method of administration has two advantages over traditional abdominal injection. First, unlike with traditional injection, the recombinant adenovirus vectors only need to be injected once. Tissue infected with adenoviruses can continuously express endogenous IL-2 for approximately 20 weeks (Alves et al. 2017). Second, after vector injection, the peripheral IL-2 concentration in the study never exceed 100 pg/ml in the IL-2-treated mice. According to previous studies, these concentrations should be defined as “low-dose” and were able to expand and activate Tregs without affecting the effector T cells (Churlaud et al. 2014; Alves et al. 2017).
After IL-2 intervention, the present study confirmed the reactivation of BDNF-ERK-CREB signaling, which was originally inhibited by the HFD. BDNF is an important factor for neuronal survival and synaptic plasticity. It regulates several intracellular pathways, such as MAPK/ERK signaling. Through its downstream signaling molecules, p-ERK1/2 eventually regulates the phosphorylation and activation of CREB. BDNF-ERK-CREB signaling plays a crucial role in neuronal survival, synaptic plasticity and cognitive function. In this study, we provided some findings to prove that the signaling pathway was also involved in the regulation of neuroinflammation.
As mentioned above, IL-2 is mainly generated by activated T cells. Previous studies further revealed that IL-2 was able to regulate and response to the CREB signaling in this type of cells (Guyot et al. 1998; Bostik et al. 2007). In the present study, AAV-IL-2 transfection increased the expression of p-CREB in the hippocampus of the HFD mice. Meanwhile, the CREB inhibitor can inhibit the hippocampal expression of IL-2 in the same model. So, the relationship between IL-2 and CREB signaling was complex. These finding gave us a hint that IL-2 played an important role in neuroinflammation and cognitive function, which should be fully explored in the future.
There were several limitations that merited further research. First, the study found that IL-2 can alleviate neuroinflammation by regulating a variety of mechanisms, such as Tregs, microglia and NLRP3 signaling. However, these mechanisms were likely to have mutual influence, and the study failed to explore their relationship. Second, a previous study revealed that most of the adenovirus vectors injected into the body were distributed in the peripheral tissue; that is, the increased IL-2 in the brain mainly came from the peripheral tissue (Alves et al. 2017). This finding suggested that peripheral cytokines might significantly affect neuroinflammation and cognitive function, which should be fully investigated in the future. Third, the study failed to explore the potential effect of the CREB inhibitor on the conclusion. However, our previous study had excluded the potential effect of the reagent on cell activity in a HT-22 cell line. So, we did not believe that this limitation was sufficient to interfere with the conclusion of the study.
In conclusion, the study suggested that low-dose IL-2 can promote the expansion and activation of Tregs, induce microglial M2 polarization, inhibit NLRP3 inflammasome activity and pyroptosis in the hippocampus, and ultimately improve cognitive impairment and depression-like behavior in HFD mice. The study also revealed that the protective effect of IL-2 mentioned above might be associated with the activation of BDNF-ERK-CREB signaling in the hippocampus.
Funding
The authors declare that no funds, grants, or other support were received during the preparation of this manuscript.
Competing Interests
The authors have no relevant financial or non-financial interests to disclose.
Author Contributions
Zheng Chen and Feng Wang contributed to the study concepts and design. All authors performed the experimental operation and data analysis. Zheng Chen and Feng Wang prepared the manuscript.
Data Availability
The data can not be shared at present, because this is an ongoing study.
Ethics approval
The study was approved by the Ethics Committees of Tianjin Haihe Hospital and Tianjin Medical University General Hospital.
Consent to participate
This was an animal study, and no human was included.
Consent to publish
The authors agreed to submit and publish this article in your journal.
Acknowledgments
We want to thank Prof. JQ from TIANJIN UNIVERSITY (China) for the funding and laboratory support for this project.