MLKL Modulates Necroptosis and Neuroinflammation in a Mouse Model of MS

Abstract

MLKL not only plays an important role in necroptosis but also regulates many diseases through non-necrotizing apoptotic function. Many studies have shown that MLKL plays an important role in neuroinflammation, degenerative diseases, and infectious diseases. Multiple sclerosis is an immune-mediated neurodegenerative disease characterized mainly by inflammatory demyelinating lesions of the central nervous system (CNS). At present, few studies have investigated the role of MLKL in regulating necroptosis and neuroinflammation in MS. We aimed to explore the role of MLKL in regulating necroptosis and neuroinflammation and to further elucidate the regulatory mechanisms in a mouse model of MS. An experimental autoimmune encephalomyelitis (EAE) MS mouse model was established, and the mice were divided into two groups (the EAE + NSA group and the EAE group). The two groups of mice were injected with the MLKL inhibitor necrosulfonamide(NSA) or control solution at the same time. We measured the severity of disease using a clinical EAE scoring system. HE staining and LFB staining of the spinal cord were used to observe the infiltration of CNS inflammatory cells and myelination in the two groups of mice. Western blotting was performed to assess the expression of proteins in the NLRP3/caspase-1/GSDMD pathway, the MyD88/NF-κB pathway and the RIPK3/MLKL pathway. Immunofluorescence experiments were performed to observe changes in microglia, astroglia, oligodendrocytes, and related factors. Finally, we analyzed the changes in inflammation in both groups via an ELISA. Blockade of MLKL alleviates clinical symptoms, demyelination and inflammatory cell infiltration in EAE mice. Furthermore, it increased the number of oligodendrocytes, protected axons, decreased the number of activated astrocytes and microglia and reduced inflammation. MLKL inhibitors may ameliorate necroptosis through the RIPK3/MLKL pathway. Blockade of MLKL may exert therapeutic effects by inhibiting the activation of the NLRP3/caspase-1/GSDMD pathway and the MyD88/NF-κB pathway in EAE mice. Blocking MLKL plays a therapeutic role in EAE not only through regulating necroptosis via the RIPK3/MLKL pathway but also through regulating neuroinflammation via the NLRP3/caspase-1/GSDMD pathway and the MyD88/NF-κB pathway.

Keywords: Experimental autoimmune encephalomyelitis (EAE); Mixed lineage kinase domain-like protein(MLKL); Multiple sclerosis (MS); MyD88/NF-κB signaling pathway; NLRP3/caspase-1/GSDMD pathway; Necroptosis; Necrosulfonamide (NSA); RIPK3/MLKL signaling pathway.

Fig. 1

NSA reduced the clinical scores of EAE mice. A Experimental design of NSA treatment in an EAE model. B Daily fluctuations in clinical scores between the two cohorts throughout the disease course. (mean ± SEM; n = 6 each; * P < 0.05, ** P < 0.01, and *** P < 0.001). C. Analysis of the mean scores, cumulative scores, maximal scores, and onset days for both the EAE group and NSA + EAE group. (n = 6 each; * P < 0.05, ** P < 0.01, and *** P < 0.001)

Fig. 2

HE and LFB staining of the spinal cord was performed in the NSA + EAE and EAE groups. Inflammatory infiltration was observed through HE, and demyelination was detected by LFB staining with corresponding inflammation scores. A Inflammatory infiltration was observed via HE staining, and corresponding inflammatory scoring was performed (n = 6 each; *P＜0.05, **P＜0.01, and ***P＜0.001). B Demyelination was detected using LFB staining (n = 6 each; *P＜0.05, **P＜0.01, and ***P＜0.001)

Fig. 3

NSA inhibited the RIPK3/MLKL pathway in EAE mice. A‒C Immunofluorescence staining was used to detect the protein expression of P-MLKL, RIPK3, and MLKL in the lumbar spinal cord of both the EAE and EAE + NSA groups. Each Group n = 3. The data are expressed as the mean ± SEM. *P < 0.05, **P < 0.01. D The protein expression levels of P-MLKL, RIPK3, and MLKL in lumbar spinal lysates were detected by immunoblotting. Each Group n = 6. The data are expressed as the mean ± SEM. * p < 0.05, **P < 0.01

Fig. 4

NSA inhibited microglia and astrocyte activation and activated oligodendrocytes in EAE mice. A-C Immunofluorescence staining was used to detect the protein expression of anti-Olig2, anti-Iba-1, and anti-GFAP in the lumbar spinal cords of the EAE and EAE + NSA groups. (Each Group n = 3. The data are expressed as the mean ± SEM. *P < 0.05, *** p < 0.001). D The protein expression levels of Olig2, Iba-1, and GFAP in the lumbar spinal lysate were detected by immunoblotting (n = 6 per group). The data are expressed as the mean ± SEM. **P < 0.01, ***p < 0.001, ****p < 0.0001)

Fig. 5

NSA inhibited the MyD88/NF-κB pathway in EAE mice. A Immunofluorescence staining was used to detect the protein expression of IL-1β in the lumbar spinal cord of the EAE group and the EAE + NSA group. (Each Group n = 3. The data are expressed as the mean ± SEM. * P < 0.05, *** P < 0.001). B The protein expression levels of MyD88, IL-1β, and NF-κB P65 in lumbar spine lysates were detected by immunoblotting. (Each Group n = 6. The data are expressed as the mean ± SEM. * p < 0.05, **** p < 0.0001). C-D An ELISA kit was used to measure IL-1β levels (n = 6 for each group). Serum samples were used for the ELISA assay. The data are expressed as the mean ± SEM. * p < 0.05)

Fig. 6

NSA alleviates the inflammatory development of Th1 and Th17. A Immunofluorescence staining was used to detect the expression of Th1 in the lumbar spinal cords of the EAE group and the EAE + NSA group. (Each Group n = 3. The data are expressed as the mean ± SEM. *** P < 0.001). B The expression levels of Th17 in the lumbar spinal cord were detected by immunofluorescence staining. (Each Group n = 3. The data are expressed as the mean ± SEM. **p < 0.01)

Fig. 7

NSA inhibited the NLRP3/caspase-1/GSDMD pathway in EAE mice. A Immunofluorescence staining was used to detect the protein expression of NLRP3 in the lumbar spinal cords of the EAE group and the EAE + NSA group. (Each Group n = 3. The data are expressed as the mean ± SEM. * P < 0.05, *** P < 0.001). B The protein expression levels of NLRP3, procaspase-1‒20, cleaved caspase-1‒20, GSDMD, and GSDMD‒N in the lumbar spinal cord tissue lysate were detected by immunoblotting. (Each Group n = 6. The data are expressed as the mean ± SEM. * p < 0.05, **p < 0.01, *** p < 0.001)