Background
Parkinson’s disease (PD) is a common age-related neurodegenerative disease that is induced by multitudinous environmental and inherited factors [
1]. PD is characterized by the progress degeneration of dopaminergic (DA) neurons in the substantia nigra pars compacta (SNc), as well as clinical motor dysfunction [
2]. The nigrostriatal DA neurons are vulnerable to multiple insults, including inflammatory attacks. Neuroinflammation and systemic inflammation are considered important contributors to the pathogenesis of PD [
3,
4], where results of epidemiological studies show a reduced risk of PD with the use of anti-inflammatory medications, specifically non-steroidal anti-inflammatory drugs [
5,
6]. The efficacy of anti-inflammatory drugs within the PD clinical treatment and compatibility with motor symptom controlling drugs requires further examination.
Most researches have focused on the neuroinflammation process of PD, while systemic inflammatory responses, especially peripheral inflammation acts as an inescapable risk factor during the pathological process of PD [
7,
8]. The neurovascular function becomes altered in aging and neurodegenerative disorders, leading to abnormal states, such as increased blood brain barrier (BBB) permeability and failure of enzymatic function [
9]. When inflammatory reactions occur, the infiltration of peripheral inflammatory cytokines and immune cells permeate through the BBB to induce the degeneration of dopaminergic neurons in the SNc [
10]. The peripheral inflammation amplifies the inflammatory cascade in the central nervous system (CNS) and further exacerbates neurodegeneration [
11]. The ability to reduce or control the systemic inflammatory response has important significance in developing novel anti-inflammatory medicines for PD therapy.
Previous reports show that the inflammatory cytokines, such as tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interferon-γ (IFN-γ), have higher levels in the serum of PD patients [
12]. IL-1β is a key pro-inflammatory cytokine throughout the pathological damage of PD [
13], and our previous study had shown that the increased IL-1β levels were primarily produced by nod-like receptor protein 3 (NLRP3) inflammasome activation in the midbrain and the microglia of PD model [
14]. The application of IL-1β receptor blockers to postpone the process of PD remains controversial in experimental animal studies, due to uncontrollable factors in regulating the production and maturation of IL-1β [
15,
16]. The activation of inflammasome promotes the maturation and the release of several pro-inflammatory cytokines, such as IL-1β and IL-18. The activation of inflammasomes requires tight control to prevent excessive inflammation [
17,
18]. Further research is needed to determine if the inflammasome could be a linking bridge in regulating systemic inflammation from the peripheral tissues to CNS, which will allow us to understand the exact events that lead to inflammation in PD.
The current study established acute 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP) and chronic MPTP/probenecid (MPTP/p)-induced PD model with C57BL/6J mice in order to determine the role that NLRP3 inflammasomes play in the liver and the brain involved during the pathogenesis of PD. We used siRNA wrapped with lentivirus (LV3-siNlrp3) to reduce the liver NLRP3 expression via tail vein injection. The siNlrp3 inhibited the MPTP-induced activation of NLRP3 inflammasome in the liver and pro-inflammatory cytokine release particularly in IL-1β. The LV3-siNlrp3 suppressed the activation and the proliferation of microglia and alleviated the loss of DA neurons in the SNc in MPTP-injected mice. Our findings suggested that hepatic NLRP3 could act as a potential target for the linkage of inflammatory response from the liver to the brain during the pathogenesis of PD.
Methods
Animals and treatments
Three to 4-month-old male C57BL/6J mice were purchased from Nanjing Medical University (Nanjing, Jiangsu, China) and maintained in light/dark (12-h light/12-h dark), temperature (22–24 °C), and humidity-controlled rooms. The mice were fed standard food with free access to drinking water. All experiments were performed in strict accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.
The mice were injected with the negative control siRNA wrapped with lentivirus (LV3-NC) or the
Nlrp3 siRNA wrapped with lentivirus (LV3-GFP-siNlrp3 or LV3-siNlrp3) (20 μl per mice, 10
9 TU/ml) via the tail vein [
19]. The siRNA wrapped with lentivirus was synthesized by Genepharma (Shanghai, China) for gene silencing of mouse
Nlrp3. After 1 week, the mice were injected with MPTP (20-mg/kg body weight) administered four times at 2-h intervals via intraperitoneal and euthanized after either 90 min, 120 min, 240 min, or 7 days post injection (the total dose per mouse was 80-mg/kg body weight) [
20] for acute MPTP PD model. For chronic MPTP/p PD model, the mice were injected subcutaneously with 20 mg/kg MPTP (Sigma, St. Louis, MO, USA) in saline and 1 h later intraperitoneally with 250 mg/kg probenecid in DMSO every 3.5 days over a period of 5 weeks. The mice were euthanized 7 days after the last injection. The control mice were treated with saline only [
21]. The midbrains, livers, and serum samples were collected during these time periods. The siRNA sequences were (5′ to 3′): LV3-NC TTCTCCGAACGTGTCACGT and LV3-siNlrp3 GGTTCTGAGCTCCAACCATTC.
Liver histology
The livers were prefixed by perfusion with 4% paraformaldehyde in 0.01 M phosphate buffer (PBS; PH 7.4, 4 °C) and dehydrated using graded 20% sucrose for 3 days and then 30% sucrose for 3 days. The liver tissues were then sectioned on Leica freezing microtomes at 10 μm using a freezing microtome. Liver biopsy sections (10 μm) were stained with hematoxylin and eosin (HE staining) and then evaluated by a double-blinded hepatopathologist or by directly observed green fluorescent protein (GFP) with a fluorescence microscope (Additional file
1).
Analysis of monoamine oxidase B (MAO-B) activity
All enzymatic assays were performed in a phosphate buffer (pH 7.5) supplemented with 0.1% Triton X-100 at 37 °C. The activities of the recombinant MAO-B proteins were normalized with the ELISA kit (Senbeijia Biomart, Nanjing, Jiangsu, China), where p-tyramine hydrochloride was used as a substrate. The activity was assayed by monitoring the rate of resorufin formation at 560 nm, in accordance with the manufacturer’s guidelines. All measurements were performed in triplicate. Lysates from tissues were prepared from RIPA lysis buffer. The protein concentration was detected with a BCA assay kit (Beyotime Biotechnology, Nanjing, Jiangsu, China). All plates were read on a microplate reader (Thermo Fisher Scientific, USA) at 562 nm. The activity of MAO-B was analyzed in unit per milligram pre protein.
Immunohistochemistry
The protocol for immunohistochemical staining was described in previous literature [
21]. The brain was prefixed via perfusion with 4% paraformaldehyde in 0.01 M PBS (PH 7.4, 4 °C). The brain tissues were sectioned on a Leica freezing microtome at 30-μm sections through the midbrain (from approximately − 2.5 to − 3.88 mm from bregma, according to the whole mouse brain atlas) using a freezing microtome. Midbrain sections were washed three times with 0.01 M PBS, 3% H
2O
2 was added for 30 min to eliminate endogenous peroxidase, the sections were washed with PBS three times, and then the sections were incubated for 1 h in blocking a solution (0.3% Triton X-100 and 5% bovine serum albumin (BSA) in PBS), followed by incubation overnight with either primary antibody (anti-Tyrosine hydroxylase (TH) antibody (1:4000, Sigma) or anti-ionized calcium binding adaptor molecule 1 (IBA-1) antibody (1:1000, wako)) in order to detect DA neurons or microglia. The samples were then for 1 h with secondary antibodies. Immunoreactivity was visualized via incubation in DAB. Control staining was performed without the primary antibodies. The specimens were observed under Microbrightfield Stereo Investigator software (Microbrightfield, Williston, VT, USA) for visualization and photography.
Enzyme-linked immunosorbent assay (ELISA)
Following euthanasia, the blood from mice was laid to rest 4 h at room temperature. The blood samples were centrifuged at 3000g for 10 min, and the serum was transferred for the ELISA test. The concentrations of IL-1β, caspase-1, TNF-α, IL-12, and IL-18 in serum were measured by the mouse ELISA Kits (R&D, USA), in accordance to the manufacturer’s instructions. The plates were read on a microplate reader (Thermo Fisher Scientific, USA) at 450 nm.
The mice were euthanized 90, 120, or 240 min post MPTP injection. The striata were dissected, immediately frozen, and stored at − 80 °C until ready for analysis. On the day of the assay, the tissue samples were sonicated in 10 vol of 5% tricholoracetic acid containing 5 μg/ml of 4-phenylpyridine (Sigma, USA) as the internal standard. The samples were centrifuged at 3000g for 10 min and then 20 μl of the supernatant was injected onto a cation-exchange Ultracyl-CS column (Waters chromatographic system, Japan). The mobile phase consisted of 90% a solution with 0.1 M acetic acid and 75 mM triethylamine-HCl (pH 2.35 adjusted with formic acid), and 7% acetonitrile. The flow rate was 1.5 ml/min. An external calibration curve was used to express the final amount in the tissue sample as microgram per gram (μg/mg) wet tissue for MPP+.
Cell culture for BMDMs
Bone marrow-derived macrophages (BMDMs) were derived from tibia and the femoral bone marrow cells and cultured for 7 days in Dulbecco’s modified essential media complemented with 10% fetal bovine serum, 1% penicillin/streptomycin (vol/vol), and 50 nM granulocyte-macrophage colony stimulating factor (GM-CSF). The purity of BMDM culture was > 95%, which was as determined with immunocytochemistry.
Western blotting analysis
The midbrain, the liver, and the BMDM cells protein lysates were fractionated with a RIPA lysis buffer. The protein was electrophoresed through a 10–15% SDS-polyacrylamide gel and blotted through the PVDF-membrane. The membranes were probed with the following primary antibodies: rabbit anti-Caspase-1/pro-caspase-1 (1:500, Millipore, USA), mouse anti-IL-1β/pro-IL-1β (1:1000, Sigma, USA), mouse anti-NLRP3 (1:1000, Adipogen, USA), rabbit anti-NLRP1 (1:5000, Cell Signaling, USA), rabbit anti-NLRP2 (1:1000, abcam, USA), goat anti-NLRC4 (1:2000, Santa Cruze, USA), rabbit anti-AIM2 (1:5000, Santa Cruze, USA), and mouse-β-actin (1:1000, Sigma, USA). The blots were incubated secondary antibodies and the signals were detected by the enhanced chemiluminescence (ECL) (Pierce, Rockford, IL, USA). The membranes were analyzed using an Image Quant LAS 4000 Chemiluminescence Imaging System (GE Healthcare, USA).
Statistical analysis
Data was initially examined for equal variance and then subjected to two-way repeated-measures ANOVA using time and treatments as variables, with Turkey’s post hoc tests at the treatment. Student’s t tests were used for single variant analyses. In all studies, n indicated the number of animals used in each group and a critical value of p < 0.05 was used. All values were reported as mean ± SEM.
Discussion
Elevated levels of inflammatory cytokines in the brain, peripheral organs, cerebral spinal fluid (CSF), and serum of PD patients support the existence of functional interconnections between the immune and nervous systems [
28]. Several reports showed that DA neuronal loss in PD originates from neuroinflammation and is triggered by systemic circulating inflammatory molecules [
10,
28]. Furthermore, innate immunity is the first line of defense in infection, which plays a vital role in tissue repair, clearance of apoptotic cells, and cellular debris [
29]. Within innate immunity, inflammasomes act as an important immune defense in central and peripheral tissues [
30]. The production and the maturation of IL-1β/IL-18 were controlled by the activation of NLRP inflammasomes [
31,
32]. Our previous study showed that the microglial NLRP3 inflammasome activation is responsible for the DA neuronal degeneration in a MPTP-induced PD mouse model [
14]. The roles that the liver inflammasome play in neurological damage of PD remain unknown.
It has been considered that the liver is the first line of defense against MPTP. Damage to other tissues may be secondary to the liver in response to MPTP challenge. MPTP toxicity is mainly due to inhibition of complex I in the mitochondrial electron transport chain, and the generation of ROS. ROS can induce a series of inflammatory events, which subsequently aggravates ROS generation. Excessive ROS in the liver can exacerbate the inflammatory responses by activating several pro-inflammatory signaling, such as NF-κB, MAPK, and JAK-STAT pathways. Consequently, nuclear NF-κB p65 subunit will activate signal 1 of NLRP3 inflammasome and upregulate the expression of NLRP3. Therefore, ROS accumulation contributes to MPTP-induced NLRP3 upregulation in the liver. In the present study, we attempt to confirm that further changes in the brain are the consequence of the levels of hepatic inflammation, without impact on the levels of MPP+ and MAO-B activity in the brain.
Growing evidence indicates that systemic inflammation aggravates the progress of PD, and inhibiting inflammatory cytokine production or blocking cytokine receptor can alleviate DA neuron damage in multiple animal models of PD [
33]. However, the ability to alleviate the inflammatory molecules from peripheral tissues to CNS remains unknown. The liver is the main metabolic organ in the periphery and a large number of inflammatory cytokines originate from the mammal liver, so we analyzed the MPTP-induced inflammatory response in the liver for a pointcut to mimic systemic inflammation. An amount of inflammasomes (e.g., NLRP1, NLRP2, NLRP3, NLRC4, AIM2) was present in both the CNS and the peripheral tissues [
34]. In the present study, we clarified that the amplitude of change in NLRP3 is the most remarkable whether in the midbrain, the liver, or the BMDM in the acute and the chronic MPTP model. Therefore, we speculated that NLRP3 inflammasome may be the most sensitive to MPTP challenge among NLRP family members.
We then used the LV3-siNlrp3 to downregulate the expression of hepatic NLRP3 protein via tail vein injection. The MPTP acute model of PD was established 7 days later. The siNlrp3 could significantly decrease the NLRP3 expression in the liver, although it had almost no impact in other organs, such as the brain. The results found that the reduction of NLRP3 in the liver could decrease the release of pro-inflammatory cytokines from the liver into serum and brain, even inhibiting MPTP-induced microglia activation and DA neuron loss in the mouse SNc, without affecting the metabolism of MPTP. These findings suggested that the NLRP3 in the liver mediated the immune signaling and played an unexpected role in the central nerve injury of PD. The downregulation of the NLRP3 expression in the liver attenuated MPTP-triggered systemic inflammation and inhibited the activation of the microglia and the loss of the DA neurons in the midbrain. This study demonstrated that the inhibition of the NLRP3 inflammasome activation in the liver could alleviate the MPTP-induced neural injury, which could provide novel target for modulating systemic inflammation in the pathogenesis of PD. Compared with traditional anti-inflammatory drugs, targeting NLRP3 can selectively inhibit IL-1β/IL-18 production without impact on some other beneficial cytokines, such as IL-4 and IL-10. Therefore, NLRP3 inhibitors may have the characteristics of the higher selectivity and the fewer side effects.
The predominant innate immune cells in the brain are microglia, although macrophages and astrocytes also contribute to the innate immune reposes in the CNS [
35]. BMDM is a common macrophage that assumes the regulation of immune and inflammation in the periphery [
36]. The BMDM-released pro-inflammatory cytokines (e.g., TNF-α, IL-1β) and even the BMDMs themselves could transfer from the BBB into the brain, which in turn transformed into microglia, where it played a crucial role of immunoregulation [
37]. The downregulation of NLRP3 in the liver did not change the expression of NLRP3 protein in the BMDM but did decrease the release of pro-inflammatory factors from the BMDM, such as IL-1β and caspase-1. We speculate that NLRP3-targeted siRNA does not reach the bone marrow, and the changes in other inflammatory markers in BMDM might be due to general levels of liver inflammation. Therefore, the siNlrp3 injection may secondarily suppress the MPTP-induced NLRP3 inflammasome activation, rather than downregulating the NLRP3 protein expression in the BMDMs. The results suggested that the tail vein injection of the siNlrp3 reduced the pro-inflammatory cytokines permeating the BBB into the brain. This meant that inhibition of the NLRP3 inflammasome in the liver contributed to alleviating inflammatory molecules spreading into the brain and delayed the progress of MPTP-induced neuroinflammation and DA neuron damage.
At last, MPTP-induced animal models of PD merely show an acute, severe, and pure dopaminergic deficiency and display a homogeneous behavioral disturbance. Meanwhile, cognitive, emotional, and other nondopaminergic signs are difficult to evaluate in MPTP-induced animal model. Furthermore, there are significant differences in species between humans and animals; thus, pharmacological effective drugs in MPTP animal models always have no curative effect in clinical application. Therefore, we need to further develop clinical trials to get patient data support and clarify the exact key molecules and mechanisms involved in inflammation from the periphery to the brain in the pathogenesis of PD.