Potential role of BAY11-7082, a NF-κB blocker inhibiting experimental autoimmune encephalomyelitis in C57BL/6J mice via declining NLRP3 inflammasomes

Abstract

Multiple sclerosis (MS) is an inflammatory autoimmune demyelinating disease of the central nervous system. NOD-like receptor pyrin domain-containing 3 (NLRP3) inflammasome is implicated in the pathogenesis of MS and its animal model, experimental autoimmune encephalomyelitis (EAE). However, the exact mechanism by which NLRP3 inflammasome is involved in the development of MS and EAE is not clear. NF-kappaB (NF-κB) is associated with the activity of NLRP3 inflammasomes, but the role of NF-κB is controversial. We sought to demonstrate that both NF-κB and NLRP3 contribute to development of MS and EAE, and NF-κB pathway is positively correlated with NLRP3 activation in EAE. The inhibitor of NF-κB and NLRP3, BAY11-7082, can prevent and treat EAE. BAY11-7082 (5 and 20 mg/kg/i.p.) was intraperitoneally administered to EAE mice at the time of second injection of pertussis toxin (BAY11-7082 prevention group) or at the onset of symptoms (BAY11-7082 treatment group). mRNA expressions of NLRP3 were determined by qPCR. Protein expressions of NLRP3, NF-κB p65, and phosphorylated p65 were determined by western blotting. Serum levels of inflammatory cytokines were measured by cytometric bead array. Mice treated with BAY11-7082 (both prevention and treatment groups) showed lower clinical scores and attenuated pathological changes. NLRP3 inflammasome and activity of NF-κB in spinal cord of EAE mice was higher than that in control group. However, the level of NLRP3 inflammasome decreased in BAY11-7082 prevention and treatment groups. BAY11-7082 is a promising therapeutic agent for MS. NLRP3 activation in EAE maybe related with NF-κB pathway.

BAY11-7082, a NF-?B blocker, inhibits experimental autoimmune encephalomyelitis in C57BL/6J mice via declining NLRP3 inflammasomes.

Graphical Abstract

Graphical Abstract.

Introduction

Multiple sclerosis (MS) is an inflammatory autoimmune disease of the central nervous system (CNS) that typically affects young and middle-aged people. It is the second most common cause of disability due to CNS disease in young adults [1]. Treatment modalities for MS need to be improved. Currently, glucocorticoids, interferon (IFN)-β, and natalizumab are the main drugs used for treatment of MS; however, these drugs are expensive and associated with several side effects. Investigation of the pathological mechanism of MS in order to identify novel treatment modalities is of much clinical relevance. Experimental autoimmune encephalomyelitis (EAE) is the classical animal model of MS that is widely used for studying on the pathological mechanism and treatment methods in MS.

Inflammasomes are multiprotein complexes that can sense pathogen-associated molecular patterns (PAMPs) and damage-associated molecular signals (DAMS). Inflammasomes are involved in the initiation and development of inflammation via activation of interleukin (IL)-1β and IL-18 synthetic pathways [2]. Recent studies have shown a strong correlation between inflammasomes and neurological diseases, including MS. However, the exact mechanism by which NOD-like receptor pyrin domain-containing 3 (NLRP3) inflammasomes is involved in the development of MS and EAE is not clear. NF-kappaB (NF-κB) is a crucial inflammatory molecule and associated with NLRP3 activation. However, the role of NF-κB in this process is controversial. According to some research results, NF-κB has been shown to be one of the activation signals for inflammasomes [3–5], but in Zhong’s report, NF-κB restricts inflammasome activation [6]. The function and relationship of NLRP3 inflammasome and NF-κB in inflammatory process deserve to further study, which helps to investigate the pathogenesis and therapy in the patients with MS.

BAY11-7082 is an inhibitor of NF-κB and NLRP3. Since NLRP3 contributes to the development of EAE, the current study aims to explore the role of NF-κB pathway and NLRP3 inflammasome in the pathogenesis of EAE, and to assess the effect of BAY11-7082 on the development of EAE.

Materials and methods

Ethics statement

The EAE model in mice was approved by the Research Animal Ethics Committee, The First Hospital, Jilin Province, Changchun, China.

Animals

Female C57BL/6J mice aged 4–6 weeks were purchased from the Beijing Vital River Laboratory Animal Technology Co. Ltd. (Beijing, China). Mice were housed in ventilated cages in groups of 4–6 mice per cage. All experimental procedures complied with the regulations for the management of laboratory animals in the Jilin Province.

Antigen

MOG35–55 peptide was synthesized by GenScript (USA). The sequence is MEVGWYRSPFSRVVHLYRNGK. Heat-killed Mycobacterium tuberculosis (H37Ra) was from Difco BD company (USA). BAY11-7082 was purchased from Beyotime (China). Antibodies against NLRP3, NF-κB p65, and NF-κB-phosphorylated p65 were purchased from Cell Signaling Technology (USA).

Induction and evaluation of EAE

EAE was induced by immunizing mice via subcutaneous injection of 200 μg MOG35-55 in 100 µl complete Freund’s adjuvant (CFA, Sigma-Aldrich, St. Louis, MO, USA) containing 8 mg/ml heat-killed M. tuberculosis (strain H37 RA; Difco, Franklin Lakes, NJ, USA) and 100 µl phosphate-buffered saline (PBS) into the back of mice on day 0. Mice were also intraperitoneally injected with pertussis toxin (PTX, Gibco, Grand Island, NY, USA) (300 ng per mouse) on days 0 and 2 post immunization (p.i.), respectively.

Using a blinded protocol, two examiners assessed clinical signs of mice with EAE immediately before immunization (day 0) and thereafter every 2 days until the mice were sacrificed. The clinical symptoms of EAE were scored using a 6-point scale: 0, normal; 1, tail limpness; 2, single hind leg weakness; 3, paralysis of both hind limbs; 4, quadriplegia; 5, death. 0.5, intermediate clinical signs.

Treatment of EAE

The animals were randomly divided into four groups (n = 20 mice in prevention group with BAY11-7082 and the corresponding shame group; n = 15 mice in treatment group with BAY11-7082 and the corresponding shame group) as the below.

Prevention group with BAY11-7082: EAE mice received intraperitoneal injection of BAY11-7082 (Sigma-Aldrich, USA), dissolved in 1% (v/v) dimethyl sulfoxide (DMSO, Gibco, Grand Island, NY, USA) in PBS at a dose of 5 or 20 mg/kg on day 2 p.i. by every 2 days until the mice were sacrificed. Treatment group with BAY11-7082: EAE mice received intraperitoneal injection of BAY11-7082 at a dose of 5 or 20 mg/kg at the onset of symptoms, i.e. day 10 p.i., by every 2 days. Sham group for BAY11-7082 prevention group: EAE mice were injected only vehicle (1% DMSO in PBS) when mice in prevention group with BAY11-7082 received BAY11-7082 injection. Sham group for BAY11-7082 treatment group: EAE mice were injected only vehicle (1% DMSO in PBS) when mice in treatment group with BAY11-7082 were subjected to BAY11-7082 treatment.

For the sham group and BAY11-7082 prevention group, 7–8 mice were scarified at onset, i.e. day 10 p.i., or peak phases, day 21 p.i., while the mice in the treatment group with BAY11-7082 were scarified at peak phase.

To analyze the changes in molecular biology and pathology caused by BAY11-7082, mice in BAY11-7082 prevention group (20 mg/kg/i.p.), treatment group (20 mg/kg/i.p.) and sham group were sacrificed at different phases.

Pathological analysis

Mice were anesthetized with 10% chloral hydrate, and perfused with cold saline and 4% buffered formaldehyde. The brains and spinal cords (lower thoracic–lumbar) of mice were sliced (10 μm), and pathological changes were analyzed by hemoxylin and eosin (H&E) and Luxol Fast Blue (LFB) stainings. Cells infiltrating into the CNS were identified by H&E staining, and intact myelin was identified by LFB staining.

Reverse Transcription and real-time PCR

At onset phase and peak phase, mice in BAY11-7082 prevention group, BAY11-7082 treatment group, and corresponding shame group were anesthetized with 10% chloral hydrate and perfused with cold PBS. The spinal cord (from forth thorax spinal cord to fifth lumbar spinal cord) from EAE mice were removed and washed once with PBS, then were immediately frozen in liquid nitrogen. One half of spinal cord were collected for RNA extraction, and the other half of spinal cord were stored for western blot analysis. Total RNA was extracted from spinal cord using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). RNA was subjected to reverse transcription using kit (Takara, Japan). Real-time PCR was conducted using LightCycler quantitative PCR (Stratagene, Santa Clara, CA, USA) with SYBR Green JumpStartTM Taq ReadyMixTM kit (Takara, Japan). Expression level was normalized to that of β-actin in the same sample and then normalized to the control. The sequences of the primer pairs are as follows:

• β-actin: forward: 5ʹ-CCCACTCCTAAGAGGAGGATG-3;

• β-actin: reverse: 5ʹ-AGGGAGACCAAAGCCTTCAT-3;

• NLRP3: forward: 5ʹ-ATGCTGCTTCGACATCTCCT-3;

• NLRP3: reverse: 5ʹ-AACCAATGCGAGATCCTGAC-3.

Western blot

At onset phase and peak phase, mice were anesthetized and perfused with cold PBS. The spinal cord from mice were collected as described in ‘Reverse transcription and real-time PCR’ section. The tissues were subjected to lysis for 30 s. Protein concentration was determined by the BCA method (BioTeke, China). The samples were loaded into 12% Tris/Gly gel, subjected to SDS-PAGE, and transferred onto a PVDF membrane (Millipore, Billerica, MA, USA). The following antibodies were used: anti-NLRP3 monoclonal antibody (Cell Signaling Technology, Danvers, MA, USA, 15101, 1:1000); rabbit anti-NF-κB p65 and anti-NF-κB p-p65 monoclonal antibody (Cell Signaling Technology, Danvers, MA, USA, 8242 and 3033, 1:1000); β-actin (Bioss, China, bsm-33139M, 1:2000), and the corresponding HRP-conjugated secondary antibodies (Bioss, China, bs-0295G-HRP, 1:1000). The gels were developed with enhanced chemiluminescence kit (Amersham Imager 600, GE, USA). The developed gel of NF-κB p65 was washed with stripping buffer (Beyotime, China, Beijing, P0025N) and then incubated with anti-NF-κB p-p65 monoclonal antibody. Densitometric analysis of western blots was done using Image J software.

Cytometric bead array

Mice were anaesthetized for eyeball removal to facilitate blood sample collection. Blood samples of mice were collected by removing eyeballs. A total of 0.6–1 ml blood was collected from each mouse. Serum was obtained by centrifugation of blood samples at 3000 rpm for 15 min at 4°C (Eppendorf 5801R centrifuge, Germany) and the supernatant collected for further analysis. The levels of IL-1β and IL-6 were measured using the cytometric bead array (CBA) kit (BD Company, USA) as per the manufacturer’s instructions. The process was briefly as follows: diluting the standard sample serially according to the concentration gradient, and preparing one tube containing only Assay Diluent to serve as the 0 pg/ml negative control. Preparing the captured beads as instruction of the product. Mixing the captured beads with samples well and incubating the mixture at room temperature for 1.5 h. Adding 1 ml washing buffer to the mixture and then centrifuging and discarding supernatant. Incubating the mixture of residual liquid and 50 μl PE detection reagent at room temperature for 1.5 h. Washing, centrifuging, discarding supernatant and supplying 300 μl washing buffer and detecting the samples by flow cytometry. Limit of detection of CBA IL-6 kits is 5 pg/ml, and the limit of detection of CBA IL-1β is 274 fg/ml.

Statistical analysis

The results were presented as mean ± standard deviation (SD). The distribution of data was determined by Shapiro–Wilk and Kolmogorov–Smirnov tests. Between-group differences were assessed using Student’s independent t-test. Variations among any two groups were analyzed by univariate ANOVA followed by Dunnett’s test. Abnormal distribution data were analyzed with Kruskal–Wallis test. All statistical analyses were performed using SPSS v21 software (IBM, USA). Statistical significance was defined as P < 0.05.

Results

Preventive application of BAY11-7082 at dose of 5 and 20 mg/kg i.p. reduced the severity of the disease: the low-dose group developed the disease on day 11, and the high-dose group developed the disease on day 13 of immunization (Fig. 1a). Administration of BAY11-7082 at the time that EAE mice presented with symptoms also decreased the average score of clinical symptoms and peak stage score of mice when compared with sham group, either at dose of 5 or 20 mg/kg/i.p. (Fig. 1b). These results suggest that prophylactic administration of BAY11-7082 at dose of 5 and 20 mg/kg/i.p. both can delay the disease progression of EAE mice, but the effect of high-dose group is more significant. No matter at dose of 5 or 20 mg/kg/i.p., the effect of BAY11-7082 is more significant when administration before the symptom appears (Fig. 1c and d). Taking 20 mg/kg/i.p. as an example, the comparison of clinical symptoms score is shown in Fig. 1e.

Fig. 1.

(a) Clinical score of mice in sham group, BAY11-7082 prevention group with different dose (5 and 20 mg/kg/i.p.). (b) Clinical score of mice in sham group, BAY11-7082 treatment group with different dose (5 and 20 mg/kg/i.p.). (c) Clinical symptom scores of mice at peak in the BAY11-7082 prevention group, treatment group (5 mg/kg/i.p.), and solvent groups, respectively. (d) Clinical symptom scores of mice at peak in the BAY11-7082 prevention group, treatment group (20 mg/kg/i.p.), and solvent groups, respectively. (e) Mean clinical score of mice in sham group, BAY11-7082 prevention group, and BAY11-7082 treatment group (20 mg/kg/i.p.). For prevention group and treatment group, BAY11-7082 was administered to EAE mice on day 2 or at the onset of clinical symptoms, respectively. For sham group, the mice received vehicle (1% DMSO in PBS). Mean clinical score ± SD are presented. ∗P: comparisons between EAE mice in BAY11-7082 prevention group or treatment group and sham-treated control EAE mice. P < 0.05. ∗∗P: comparisons between EAE mice in BAY11-7082 treatment group and sham-treated control EAE mice. P < 0.05.

Pathological analysis

Infiltration of inflammatory cells in spinal cord is common pathological feature in EAE mice. In this research, we detect the pathological changes of spinal cord in BAY11-7082 prevention (20 mg/kg/i.p.) and treatment groups (20 mg/kg/i.p.) and sham group mice. In BAY11-7082 prevention and treatment groups, the amount of inflammatory cells was significantly reduced compared with sham group; the inhibiting effect was particularly prominent in the prevention group (Fig. 2a–c). Demyelination in the spinal cord is the main pathological change in EAE. As shown in Fig. 2d–f, sham EAE group exhibited significant demyelination compared with BAY11-7082 prevention and treatment groups, which showed lesser degree of demyelination, especially in the prevention group.

Fig. 2.

a–c (40×): H&E staining of spinal cord sections. Infiltration of inflammatory cells in the BAY11-7082 prevention (20 mg/kg/i.p.) (b) and treatment (20 mg/kg/i.p.) (c) groups is less than that in the shame group (a). d–f (40×): LFB staining of the myelin sheath. There is significant demyelination and vacuolar degeneration in shame group (d). Demyelination and vacuolar degeneration is attenuated in the prevention (20 mg/kg/i.p.) (e) and treatment (20 mg/kg/i.p.) groups (f).

The expression of NLRP3 was inhibited in the BAY11-7082 group

The mRNA level of NLRP3 in spinal cord was detected in BAY11-7082 20 mg/kg/i.p. group and sham group. The mRNA levels of NLRP3 were reduced in the BAY11-7082 prevention and treatment groups. mRNA levels of NLRP3 in the prevention group reduced significantly both in onset phase and peak phase. The amount of NLRP3 mRNA in peak phase also decreased in the BAY11-7082 treatment group compared to that in shame group (Fig. 3a and b).

Fig. 3.

(a) NLRP3 mRNA level at onset phage of sham group and BAY11-7082 prevention group (20 mg/kg/i.p.). (b) NLRP3 mRNA level at peak phage of sham group, BAY11-7082 prevention group (20 mg/kg/i.p.), and treatment group (20 mg/kg/i.p.). The BAY11-7082 prevention group and treatment group showed significant decrease in NLRP3 expression. Mean clinical score ± SD are presented. ∗P < 0.01, ∗∗P < 0.01.

Protein level of NLRP3 in the prevention group

The semiquantitative analysis of NLRP3 amount was processed by western blot. Compared with EAE group, the mice in BAY11-7082 (20 mg/kg/i.p.) group showed lower NLRP3 level either in onset phase or peak phase. Besides, BAY11-7082 (20 mg/kg/i.p.) treatment also made the NLRP3 level declined significantly (Fig. 4a–d).

Fig. 4.

The semiquantitative analysis of NLRP3 content with western blot. (a) NLRP3 protein level at onset phage of sham group and BAY11-7082 prevention group (20 mg/kg/i.p.). (b) NLRP3 protein level at peak phage of sham group, BAY11-7082 prevention group (20 mg/kg/i.p.), and treatment group (20 mg/kg/i.p.). (c) Semiquantitative analysis of NLRP3 protein level at onset phage of sham group and BAY11-7082 prevention group (20 mg/kg/i.p.). (b) Semiquantitative analysis of NLRP3 protein level at peak phage of sham group, BAY11-7082 prevention group (20 mg/kg/i.p.), and treatment group (20 mg/kg/i.p.). Mean clinical score ± SD are presented. ∗P < 0.05, ∗∗P < 0.05.

BAY11-7082 inhibited the activity of NF-κB

To evaluate the activity of NF-κB, the level of phosphorylated NF-κB and total NF-κB was detected by western blot (Fig. 5a and b). The ratio of these two data was calculated and shown in Fig. 5c and d. The ratio decreased both at the onset and peak phase of the prevention group, and declined at peak phase in the treatment group.

Fig. 5.

Semiquantitative analysis of NF-κB activity. (a), representative western blot analysis of NF-κB p65 and phosphorylated p65 at onset phase of sham group and prevention group (20 mg/kg/i.p.). (b), representative western blot analysis of NF-κB p65 and phosphorylated p65 at peak phase of sham group, prevention group (20 mg/kg/i.p.), and treatment group (20 mg/kg/i.p.). The ratio of p-p65 and p65 was shown in (c) and (d). The ratio decreased in BAY11-7082 prevention group (20 mg/kg/i.p.) and treatment group (20 mg/kg/i.p.), compared with sham groups. ∗P < 0.05, ∗∗P < 0.05.

Serum levels of inflammatory cytokines

The inflammatory cytokines in serum were measured by CBA. The content of IL-6 and IL-1β experienced a fall with the intervention of BAY11-7082 (20 mg/kg/i.p.) in EAE mice (Fig. 6a and b).

Fig. 6.

The effect of BAY11-7082 on serum levels of inflammatory cytokines in EAE group and BAY11-7082 intervention group (20 mg/kg/i.p.). Serum levels of IL-1β and IL-6 in the BAY11-7082 prevention group (20 mg/kg/i.p.) were lower than those in the EAE peak group. ∗P < 0.05 (Kruskal–Wallis test).

Discussion

MS is an immune-mediated, inflammatory demyelinating disease of the CNS. It is the most common CNS demyelinating disease in the world [7]. Therefore, exploration of effective prevention and treatment methods for MS is a key imperative. This study emphasized the involvement of inflammasome NLRP3 in the development of EAE (animal model of MS). In addition, treatment with BAY11-7082 ameliorated EAE by inhibiting the activity of NF-κB and decreasing NLRP3.

The current study showed that the mRNA and protein level of NLRP3 inflammasome increases in spinal cord of EAE mice. This result is consistent with previous research. Gris et al. demonstrated that NLRP3^−/− EAE mice showed a significantly delayed disease course and less severe disease [8]. The expression of caspase-1, a component of NLRP3 inflammasome, is detected in MS plaques, and the level of IL-1β, a downstream effector of NLRP3 inflammasome, was found increased in the cerebrospinal fluid of MS patients before clinical relapse [9]. The above results prove that NLRP3 inflammasome is involved in the progression of EAE.

This is the first study to demonstrate that BAY11-7082 contributes to attenuation of peak severity and reduction in cumulative clinical score of EAE mouse. Several reagents have been reported which can release EAE by mediating NLRP3 inflammasomes activity, such as MCC950 [10] and JC-171 [11], and both of them are selective NLRP3 inflammasome inhibitors. BAY11-7082 is a synthetic IκB kinase-β antagonist. By inhibiting degradation of I-κB kinase-β, BAY11-7082 allows IκB to protect NF-κB and prevent its translocation into the nucleus. Besides, BAY11-7082 is also capable of inhibiting NLRP3 activation directly, independent of NF-κB [12, 13]. Thus, BAY11-7082 may potentially prevent inflammatory reaction and the progression of EAE. Our results demonstrate this conjecture. The clinical scores of EAE mice were decreased significantly after injection with BAY11-7082 before or at the same time of symptom appearing. The pathological change in spinal cord was correspondingly relieved.

The current study demonstrates the anti-inflammatory effect of BAY11-7082 in EAE. Decline of NF-κB and NLRP3 inflammasome levels after BAY11-7082 treatment in current study demonstrated that inflammation was inhibited by BAY11-7082. IL-1β is produced by NLRP3 inflammasome by cleaving pro-IL-1β [2]. Fluctuation of IL-1β level along with NLRP3 level enhanced the evidence that BAY11-7082 affects the function of NLRP3 inflammasome. IL-6 is the downstream regulator of NF-κB. It is involved in blood nerve barrier injury and immune cell infiltration and has been demonstrated related with development of EAE and MS [14, 15]. After BAY11-7082 treatment, the activity of NF-κB signaling pathway was declined in EAE mice, along with the decrease of its downstream regulatory factor IL-6. It further proves that BAY11-7082 effects NF-κB pathway.

Indeed, BAY11-7082 also exhibits a therapeutic effect on other autoimmune diseases. Zhao et al. [16] reported that BAY11-7082 successfully treated the lupus nephritis mouse model. Irrera et al. [17] demonstrated that BAY11-7082 inhibited the symptoms of imiquimod cream induced psoriasis-like dermatitis mice. Moreover, BAY11-7082 could ameliorate the severity of diabetic nephropathy [18], neuropathy [19], and cognitive deficits [20]. These studies reveal that BAY11-7082 may be a promising drug for curing MS and other inflammation-related diseases.

Actually, besides NF-κB pathway, BAY11-7082 also acts on other inflammatory pathways, such as ERK/JNK/AP-1, TBK1/IRF-3, and JAK-2/STAT-1 [21]. So, it is worthy further efforts to explore whether there are other mediators driven by BAY11-7082 to exert anti-EAE function.

The improvement effect of BAY11-7082 on EAE was related to the timing of administration. Compared with the prevention group and treatment group, the preventive application of BAY11-7082 showed a stronger effect on inhibiting the progress of EAE (Fig. 1e). It also suggests that the earlier the inflammatory response is controlled, the better the prognosis of EAE is.

The effect of BAY11-7082 on inhibiting EAE is positively correlated with the dose. Previous studies have applied BAY11-7082 to treat lupus nephritis mouse model, psoriasis mouse model, but the dosage was different (including 5 and 10 and 20 mg/kg/i.p.). Two dosage groups, 5 and 20 mg/kg/i.p., were established in current study. We found that the average score at peak of 20 mg/kg/i.p. group was significantly lower than that of the 5 mg/kg/i.p. group, showing that higher dose BAY11-7082 has better preventive and therapeutic effects on EAE mice. The dose-dependent effect has been reported in other research, e.g. BAY11-7082 can inhibit uveal melanoma cell proliferation in a dose-dependent manner by inhibiting NF-κB signaling pathway [22].

The present study, in agreement with the result of previous research, demonstrates that NF-κB plays a vital role in pathogenesis of EAE and targeting NF-κB pathway is a useful treatment mean for MS. Interesting, the relation between NLRP3 and NF-κB has not been fully elucidated. The NF-κB-associated pathway has been shown to be necessary for inflammasomes activation [4, 23]. But another study showed that NF-κB can restrict NLRP3 inflammasome activation by elimination of damaged mitochondria [6]. Besides, NLRP3 could in turn effect function of NF-κB since NLRP3 knockout hinders NF-κB activation [24]. Thus, the role of NF-κB in NLRP3 inflammasome activation seems to be conflicting. However, in the current study, the level of NF-κB increased parellelly with the level of NLRP3, indicating that NF-κB pathway is positively related with the NLRP3 inflammasome level in EAE mice. Besides, inhibiting NF-κB with BAY11-7082 did not make the NLRP3 level rise in this study; instead, there is a significant decrease of NLRP3 level. This might be caused by the offset of inhibition effect on NLRP3 activity from BAY11-7082. However, it also could not rule out the possibility that in EAE, NLRP3 inflammasome is positively related with NF-κB pathway and that these two factors may interact and enhance each other.

In conclusion, this study demonstrates that NLRP3 inflammasome is involved in the development of EAE via the NF-κB pathway. The NF-κB inhibitor, BAY11-7082, alleviated symptoms and attenuated pathological changes in EAE mice, along with reduction in NLRP3 inflammasome levels. Therefore, BAY11-7082 might be a potential treatment for MS.

Glossary

Abbreviations

ANSCs

α-synuclein on neural stem cells

ASC

apoptosis-associated speck-like protein containing a caspase recruitment domain

CBA

Cytometric Bead Array

CNS

central nervous system

DAMS

damage-associated molecular signals

EAE

experimental autoimmune encephalomyelitis

IFN

interferon

IL-1β

interleukin -1 β

MS

multiple sclerosis

NF-κB

NF-kappaB

NLRP3

NOD-like receptor pyrin domain-containing 3

NLRs

NOD-like receptors

PAMPs

pathogen-associated molecular patterns

PD

Parkinson’s disease

STING

stimulation of IFN genes

Funding

This study was supported by grants from the National Natural Science Foundation of China (no. 81471216, 81671177 81671186, and 82071351), the Swedish Research Council (Diarienummer: 2015-03005), and the grants from the First Hospital of Jilin University, Changchun, China

Conflict of interest

The authors declare that they have no competing interests.

Author contributions

All authors contributed to the study conception and design, especially J.Z. Material preparation, data collection, and analysis were performed by Y.L., L.C., F.C., L.L., C.Z., C.L., D.S., S.L., W.Z., and J.Z. The first draft of manuscript was written by Y.L.and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript. The figures in this manuscript were created with Printed 3D and Photoshop.

Ethical approval

All procedures performed in studies involving animals were in accordance with the ethical standards of Research Animal Ethics Committee, The First Hospital, Jilin Province, Changchun, China and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.

Informed consent

Not applicable.

Data availability

The data sets analyzed in the present study are available from the corresponding author on reasonable request.