Background
Guillain–Barré syndrome (GBS) is an acute inflammatory polyneurogenic disease that can lead to motor dysfunction, sensory impairment, autonomic dysfunction, and respiratory failure [
1,
2]. Effective treatments for these ailments include plasma exchange and intravenous immunoglobulin infusion [
3]. However, 20% of patients continue to experience severe sequelae, and 5% die despite receiving immunotherapy [
4,
5]. The primary mechanism underlying disease development is molecular mimicry between microbial and neural antigens, resulting in an abnormal autoimmune response targeting peripheral nerves and their spinal roots [
6].
Experimental autoimmune neuritis (EAN) mimics the clinical, histopathologic, and electrophysiologic features of GBS [
7,
8]. Blood-nerve barrier breakdown is a characteristic feature of its pathophysiology, with the peripheral nervous system (PNS) being invaded by activated T cells and macrophages that demyelinate the peripheral nerves [
9]. The cellular immune response plays a considerable role in the onset of GBS. Macrophages and CD4
+ T cells enter the PNS and damage the nervous system either directly through phagocytic attack or indirectly through T cell-mediated cytotoxicity, as well as through cytokines and oxygen free radicals [
10,
11]. Activated T lymphocytes may also promote B cells growth and differentiation into plasma cells, which secrete antibodies against the phospholipid components of the peripheral medulla [
12]. The clinical scores of patients with GBS change in correlation with changes in CD4
+ T-lymphocyte subsets [
13,
14]. Regulating CD4
+ T cells differentiations can alleviate EAN symptoms [
14], but the endogenous molecular regulatory mechanism of CD4
+ T differentiation remains unclear.
The nucleotide receptor family (P2 receptors, P2Rs) comprises two subfamilies: G-protein-coupled metabotropic P2Y (P2YR) and the ligand (ATP)-gated ionotropic P2X (P2XR) receptors [
15]. P2X7R is a member of the P2X receptor subfamily of P2 receptors and is expressed in most innate and adaptive immune cells [
16], including macrophages, monocytes, dendritic cells, and T cells [
17,
18]. P2X7R is strongly correlated with inflammation and involved in inflammatory disorders, including inflammatory bowel disease [
19], rheumatoid arthritis [
20], acute pancreatitis-associated lung injury [
21], and Alzheimer’s disease [
22].
In this study, we used an EAN model to examine the role of P2X7R in neuroinflammation. We investigated the signaling pathways and molecular mechanisms underlying P2X7R-mediated immunomodulation in the PNS. Furthermore, we examined the anti-inflammatory effects of brilliant blue G (BBG), a safe and highly selective P2X7R antagonist [
23‐
25], in EAN rats. We found that BBG reduced neuroinflammatory reactions and neurological impairments by regulating CD4
+ T cell differentiation and NLRP3 inflammasome activation in an EAN rat model. These findings suggest that targeting CD4
+ T cell P2X7R may assist in the treatment of GBS.
Materials and methods
Patients and healthy control group
Fifteen patients with GBS who met the criteria outlined were recruited from Tianjin Medical University General Hospital and Tianjin Nankai Hospital. The inclusion criteria were the onset of weakness within 2 weeks, accompanied by an inability to walk independently for a distance of 10 m GBS disability score > 3) [
26,
27]. The exclusion criteria were under 18 years of age, a history of GBS, pregnancy, lactation, immunosuppressive therapy, antacid treatment, and severe concurrent complications [
28,
29]. As controls, 15 age- and sex-matched healthy volunteers were included. Blood samples from patients with GBS (collected prior to the initiation of treatment with intravenous immunoglobulin, or plasmapheresis) and healthy controls were utilized for qRT-PCR and flow cytometry analyses (Additional file 1: Table
S1). All samples were collected after obtaining informed consent and in accordance with the ethical guidelines of the Institutional Review Board at Tianjin Medical University General Hospital and Tianjin Nankai Hospital.
Animals
Male Lewis rats (6–8 weeks-old; 160–190 g) were purchased from the Vital River Corporation, Beijing, China. The rats were housed under specific pathogen-free conditions with a 12-h light/dark cycle, given free access to food and water, and randomly divided into prophylactic, therapeutic, and vehicle groups.
Induction of EAN and evaluation of clinical signs
A 300µL inoculum dose containing 300 µg of dissolved P0 peptide 180–199 (10 mg/mL; Bio-Synthesis) emulsified with an equivalent volume of complete Freund’s adjuvant (Sigma-Aldrich) with Mycobacterium tuberculosis H37RA (Becton, Dickinson and Company) at a final concentration of 1 mg/mL was administered to Lewis rats in both hind footpads.
Two investigators evaluated the rats’ neurological signs daily using the following scale: 0 (normal), 1 (reduced tail tonus), 2 (limp tail), 3 (absent righting), 4 (gait ataxia), 5 (mild hind limb paresis), 6 (moderate paraparesis), 7 (severe paraparesis or paraplegia of the hind limbs), 8 (tetraparesis), 9 (moribund), and 10 (death).
BBG treatment
Lyophilized powder of BBG (50 mg/kg, purity > 95%; MedChemExpress) was diluted in saline. Diluted BBG solution was administered intraperitoneally to EAN rats as follows: to the prophylactic group, BBG was administered continuously from the first day of immunization (day 0) until the peak of morbidity (day 18); to the treatment group, BBG was administered from the first day when neurological signs were observed (day 8) until the peak of morbidity; and the vehicle group received an equivalent dose of saline.
Histopathology
The rats from each group were anesthetized and perfused intracardially with cold PBS at the peak of the disease. Sciatic nerves were harvested and fixed in 4% paraformaldehyde overnight at 4 °C. The nerves were then dehydrated, vitrified, and embedded in paraffin. Transverse sections, 6 μm thick, were cut using a microtome (Leica RM2255) and stained with hematoxylin and eosin (Solarbio Science & Technology, China) and Luxol Fast Blue (LFB) (Abcam). Three random microscopic views were selected for sciatic nerve sections of each EAN rat and photographed using a Nikon microscope (200× magnification). Two independent observers (who were blinded to the treatment group) assessed inflammatory cell infiltration and demyelination. Inflammatory cell infiltration per square millimeter was quantified from three random microscopic views. Demyelinated lesions were identified as being stained with a lighter blue or remaining unstained under LFB staining at the same time. Histological scores were evaluated according to the following semi-quantitative pathological scale: 0 denoting a normal perivascular area; 1 indicating mild demyelination adjacent to the vessel; 2 representing moderate demyelination in proximity to the vessel; and 3 reflecting demyelination throughout the entire section.
Electrophysiologic analysis
Electromyography of the sciatic nerve was performed in each group of EAN rats on day 18 after immunization using a fully digital KeyPoint Compact EMG/NCS/EP recording system (Dantec). Rats were anesthetized with chloral hydrate (3 mg/kg), and the sciatic nerve was exposed from the hip (proximal) to the ankle (distal). A pair of needle electrodes was inserted into the proximal sciatic nerve notch and distal ankle joint to stimulate evoked compound muscle action potentials (CMAPs), and the motor nerve conduction velocity (MNCV), amplitude, and latency of the CMAPs were recorded. We used 1 Hz pulses with an average power of 5 mA and a pulse width of 0.3 ms to stimulate the nerves and induce CMAPs. The recording electrodes were positioned in the “belly” part of the gastrocnemius muscle to record evoked potentials from stimulated sciatic nerve. MNCV was determined by measuring the separation between the stimulated cathodes and the resulting latency difference. Using the obtained CMAP curves, the amplitude was determined from the baseline to the maximum peak. Once electrophysiological tests were completed, the incision was stitched in an aseptic environment. We maintained body temperatures above 34 °C during electrophysiologic tests using heating pads under the animals. For each animal, measurements were performed in triplicate.
Immunofluorescence staining
The sciatic nerves were fixed overnight in 4% paraformaldehyde at 4 °C, sequentially dehydrated in 15% and 30% sucrose, OCT-embedded, snap-frozen in liquid nitrogen, and then sectioned into 8-µm frozen slices using a cryostat (Leica Microsystems, Solms, Hessen, Germany). The sections were permeabilized with 0.3% Triton X-100 for 10 min and then washed. Non-specific binding sites were blocked with 3% BSA for 1 h. Next, the sections were incubated with the following antibodies overnight at 4 °C: rabbit anti-P2X7R (1:100, Alomone Labs), rabbit anti-IL-17 (1:200, Thermo Fisher Scientific, Waltham, MA, USA), mouse anti-CD4 (1:50, BioLegend), mouse anti-CD8 (1:50, BioLegend), mouse anti-CD68 (1:50, BioLegend), anti-NLRP3 (1:200, Thermo Fisher) and mouse anti-SOX10 (1:100, Thermo Fisher Scientific). Subsequently, the sections were washed with PBS and then incubated with Alexa Fluor 488-conjugated donkey anti-rabbit IgG (1:1000, Thermo Fisher Scientific) and Alexa Fluor 594-conjugated donkey anti-mouse IgG (1:1000; Thermo Fisher Scientific) at room temperature for 1 h. Following a PBS wash and covering with DAPI, images were captured using a fluorescence microscope (Nikon).
Flow cytometry
After isolating peripheral blood samples from patients with GBS and healthy controls, peripheral blood mononuclear cells (PBMCs) were separated using density gradient centrifugation. Next, the PBMCs were stained with PerCP-Cyanine5.5 anti-human CD45 antibody (BioLegend), APC anti-human CD4 antibody (BioLegend), PE anti-human CD14 antibody (BioLegend), and PE-cy7 anti-human CD8 antibody (BioLegend) for 40 min at room temperature. Following fixation and permeabilization, the cells were further stained with rabbit anti-human P2X7R antibody (Boster Biological Technology) or rabbit anti-human NLRP3 antibody (Thermo Fisher) for 40 min at room temperature, followed by incubation with FITC-conjugated donkey anti-rabbit IgG antibody (Thermo Fisher Scientific) for an additional 40 min at room temperature.
Single-cell suspensions were prepared from the rat spleens at the peak of the neurological course. Splenic mononuclear cells (MNCs) were divided into three groups and then blocked with 1% BSA before antibody incubation. One group was stimulated with a 1× Cell Stimulation Cocktail along with protein transport inhibitors (Thermo Fisher) for 6 h. Cultures were harvested, incubated with APC anti-rat CD4 antibody (BioLegend) for 40 min at room temperature, and subsequently fixed and permeabilized. They were then stained with FITC anti-rat IFN-γ antibody (BioLegend), PE anti-rat IL-4 antibody (BioLegend), and PerCP-Cyanine5.5 anti-rat IL-17 A (Thermo Fisher) for 30 min at 4 °C in the dark.
The second group of Splenic MNCs underwent surface staining with APC anti-rat CD4 antibody (BioLegend) and PE anti-rat CD25 antibody (BioLegend) for 40 min at room temperature. After fixation and permeabilization using the eBioscience Foxp3/Transcription Factor Staining Buffer Set (Thermo Fisher Scientific), the cells were stained with anti-rat Alexa Fluor 488 Foxp3 antibody (BioLegend).
The third set of MNCs were subjected to surface staining with PerCP-Cyanine5.5 anti-rat CD3 antibody (BioLegend), APC anti-rat CD4 antibody (BioLegend), PE anti-rat CD8 antibody (BioLegend), and PE-cy7 anti-rat CD68 antibody (BioLegend) for 40 min at room temperature. Following fixation and permeabilization, the cells were then stained with rabbit anti-rat P2X7R antibody (Boster Biological Technology) or rabbit anti-rat NLRP3 antibody (Thermo Fisher) for an additional 40 min at room temperature. Finally, the cells were incubated with FITC-conjugated donkey anti-rabbit IgG antibody (Thermo Fisher Scientific) at room temperature for 40 min. All procedures were performed per manufacturer’s instructions. Flow cytometry was carried out using a BD Accuri Cflow (BD Biosciences, San Jose, CA, USA), and the acquired data were analyzed with FlowJo® version 10.0 software (Ashland, OR, USA).
qRT-PCR
Total RNA was extracted from human PBMCs, EAN rat sciatic nerves and fresh rat splenic MNCs using TRIzol reagent (Glpbio, USA) according to the manufacturer’s guidelines. The cDNA was synthesized using TransScript First-Strand cDNA Synthesis Super Mix (TransGen Biotech) and qPCR was performed in triplicate using FastStart Universal SYBR Green Master Mix (Glpbio) on a CFX ConnectTM Real-Time PCR Detection System (Bio-Rad, USA). The primers used in the study are listed in Additional file 1: Table
S2. The qRT-PCR steps including incubation at 95 °C for 10 min, followed by 40 cycles at 95 °C for 15 s and 60 °C for 1 min and was performed in duplicate. Relative gene expression was normalized to β-actin gene expression and calculated using the 2
−ΔΔCt method.
Western blot analysis
Sciatic nerves were lysed in RIPA buffer (Solarbio) containing a protein phosphatase inhibitor cocktail. Proteins were separated by performing sodium dodecyl sulfate-polyacrylamide gel electrophoresis using 10% resolving gels and then transferred onto polyvinylidene difluoride membranes (Millipore, USA). The membranes were blocked with 5% non-fat milk and then incubated with the following primary antibodies overnight at 4 °C: anti-P2X7R (1:1000, Abcam), anti-NLRP3 (1:500, Thermo Fisher), anti-IL-1β (1:1000, Abcam), and anti-Caspase-1 (1:1000, Abcam). Next, the membranes were washed and incubated with donkey anti-rabbit IgG or donkey anti-mouse IgG conjugated with HRP at a 1:5000 dilution (Thermo Fisher Scientific) for 1 h at room temperature. Finally, the immunoreactive protein bands were detected using a Gel Doc imaging system (Bio-Rad), band intensity was expressed as the fraction corresponding to GAPDH and analyzed using ImageJ software.
Statistical analysis
Data were analyzed using GraphPad Prism (GraphPad Software, Version 9.4; La Jolla, CA, USA). Differences between the clinical scores were analyzed using a two-way ANOVA. The Mann–Whitney U test was used to compare the differences between the two groups. One-way ANOVA (Kruskal–Wallis test) followed by Dunnett’s multiple-comparison test was used to analyze the differences among multiple groups. Data are shown as mean ± SEM. Results with p < 0.05 were considered statistically significant.
Discussion
In our investigation with a highly selective P2X7R antagonist BBG, the anti-inflammatory effect was identified as a previously unknown function of P2X7R inhibition in EAN, a classical animal model for GBS. Our research indicates that blocking P2X7R with BBG reduces neurological impairment and neuroinflammation in EAN. BBG-led P2X7R modulation influences the differentiation of Th1 and Th17 subsets of CD4+ T lymphocytes and suppresses NLRP3 inflammasome-mediated inflammatory responses. These results underscore the crucial neuroprotective role of P2X7R in the EAN model.
Both prophylactic and therapeutic BBG treatments reduced the severity of EAN symptoms, while prophylactic administration also delayed the onset of clinical symptoms, as determined by clinical scores. Pathologically, demyelination and inflammatory cell infiltration are hallmarks of EAN [
8]. Compared to the vehicle group, the BBG-treated group exhibited significantly less inflammatory cell infiltration and demyelination in the sciatic nerve. Peripheral nerve damage in EAN results in decreased MNCV, prolonged CMAP latency, and reduced CMAP amplitude [
30]. Our electrophysiologic findings demonstrate that MNCV, CMAP amplitudes, and latencies improved in the BBG-treated group, suggesting that BBG-mediated P2X7R inhibition enhances nerve conduction function. The release of cytokines associated with inflammatory vesicles is closely linked to P2X7R activation and tissue inflammation onset [
31,
32]. Research studies have suggested a strong connection between IL-1β secretion and P2X7R inhibition [
33]. Consistent with previous research, rats treated with BBG exhibit lower IL-1β expression in their sciatic nerves [
34‐
36].
We observed a significant elevation in P2X7R levels in patients with GBS, primarily on CD4
+ T cells. We also found compelling evidence of a substantial increase in P2X7R expression on CD4
+ T cells in the EAN model. In comparison to CD8
+ T cells and macrophages, we identified the most significant upregulation of P2X7R expression in CD4
+ T cells of the EAN model, indicating that CD4
+ T cell P2X7R is strongly associated with improved outcomes in EAN rats treated with BBG. Spleen, the largest peripheral immune organ in the body, serves as a site for the settlement of mature T lymphocytes [
37], and the mobilization of splenic T cells and migration toward peripheral nerves may occur in EAN [
38,
39]. To further explore the immune modulation effects of BBG after EAN, we investigate both sciatic nerves and spleens to detect the change of CD4
+T cells subtype and inflammatory cytokine level. Flow cytometry data of spleen MNCs demonstrated a correlation between EAN improvement and a decrease in Th1 and Th17 CD4
+ T cell subsets. A reduction of Th17 cells infiltration was also observed in sciatic nerves in the group of rats treated with BBG. Th cells play a crucial role in the pathophysiology of EAN [
14,
40]. Th1 cells and Th1 cytokines, including IFN-γ and TNF-α, are considered the primary mediators of EAN [
41]. IFN-γ promotes T cell differentiation towards a Th1 phenotype and suppresses the growth of Th2 cells in GBS, shifting the immunological response to a Th1 phenotype [
42]. Moreover, GBS severity is correlated with serum TNF-α levels [
43,
44], which damage peripheral myelin sheaths and the blood-nerve barrier, and promote pro-inflammatory Th cell growth [
45]. Additionally, IL-17 A and Th17 cells may contribute to the EAN onset [
46‐
48]. In the present study, we found a reduction of mRNA levels of IL-2, IL-17, IFN-γ, and TNF-α in spleens which is consistent with the changes in sciatic nerve. These findings support the hypothesis that BBG reduces EAN by inhibiting the development of Th1 and Th17 cells.
P2X7R controls various cellular signaling pathways, such as the release of cytokines and chemokines, activation of the NLRP3 inflammasome, cell death, and autophagy [
49]. Of these pathways, the P2X7R-NLRP3 signaling pathway, has been associated with cognitive deficits in several neurological conditions, including Alzheimer’s disease [
50], vascular cognitive impairment [
51], and diabetes mellitus [
52]. However, its role in inflammatory neuropathies like GBS remains uncertain. P2X7R activation can reportedly promote the assembly and recruitment of the inflammatory vesicle component of NLRP3 [
53,
54]. NLRP3 is an intracellular polyprotein complex consisting of the carrier protein total caspase-1, the adaptor protein ASC, and the stimulus-detecting sensor NLRP3 [
55]. Physiologically active caspase-1 is activated by complete caspase-1 upon stimulation of NLRP3 [
56,
57]. Caspase-1 activation triggers the inflammatory response by cleaving pro-IL-1β and releasing mature IL-1β [
58]. Our findings support the hypothesis that antagonizing P2X7R with BBG, suppresses the expression of NLRP3, caspase-1, and IL-1β in EAN. The alteration of NLRP3 under P2X7R activation or inhibition was confirmed in CD4
+ T cells both in patients with GBS and EAN models. To the best of our knowledge, this is the first study to provide the evidence that P2X7R and the downstream inflammatory reactions mediated by the NLRP3 inflammasome contribute to EAN.
Additionally, research suggests that P2X7R may control the development of Th1 and Th17 cells via NLRP3 inflammatory activity in CD4
+T cells [
59,
60]. NLRP3 inflammasome assembles in human CD4
+ T cells and initiates caspase-1-dependent IL-1β secretion, thereby promoting IFN-γ production and Th1 differentiation in an autocrine fashion [
59]. We can’t exclude the potential protective effects of BBG through other cell types beyond CD4
+T cells, including CD8
+T cells, macrophages, and Schwann cells, the myelin-producing cells of the peripheral nervous system. Schwann cells play a role in the pathogenesis of GBS by inducing extracellular matrix proteins [
61], and membrane damage to Schwann cells can lead to secondary bystander axonal degeneration, resulting in acute axonal injury [
62]. P2X7R is reported to be expressed on Schwann cells [
63‐
65]. In our study, we found that P2X7R expression was mildly upregulated in Schwann cells in the EAN model, though the change was not significant (Additional file 1: Fig.
S2). P2X7R expression in Schwann cells may also contribute to the alleviation effect of BBG in EAN. Although remains unaltered under EAN induction, the P2X7R expression on CD8
+T cells and macrophages could also induce a downstream effect under BBG treatment. Further work needs to be conducted in mice conditional knockout of P2X7R in CD4
+T cells, which might further demonstrate the precise role of CD4
+T cells and P2X7R/NLRP3 pathway in BBG’s protective effects in GBS.
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