Abstract Background: Epilepsy is one of the most common neurological disorders. The proepileptic and antiepileptic roles of microglia have recently garnered significant attention. Interleukin-1 receptor-associated kinase (IRAK)-M, an important kinases in the innate immune response, is mainly expressed in microglia and acts as a negative regulator of the TLR4 signaling pathway that mediates the anti-inflammatory effect. However, whether IRAK-M exerts a protective role in epileptogenesis as well as the molecular and cellular mechanisms underlying these processes are yet to be elucidated. Methods: An epilepsy mouse model induced by pilocarpine was used in this study. Real-time quantitative polymerase chain reaction and western blot analysis were used to analyze mRNA and protein expression levels, respectively. Whole-cell voltage-clamp recordings were employed to evaluate the glutamatergic synaptic transmission in hippocampal neurons. Immunofluorescence was utilized to show the glial cell activation and neuronal loss. Furthermore, the proportion of microglia was analyzed using flow cytometry. Results: Seizure dynamics influenced the expression of IRAK-M. Its knockout dramatically exacerbated the seizures and the pathology in epilepsy and increased the N-methyl-d-aspartate receptor (NMDAR) expression, thereby enhancing glutamatergic synaptic transmission in hippocampal CA1 pyramidal neurons in mice. Furthermore, IRAK-M deficiency augmented hippocampal neuronal loss via a possible mechanism of NMDAR-mediated excitotoxicity. IRAK-M deletion promotes microglia toward the M1 phenotype, which resulted in high levels of proinflammatory cytokines and was accompanied by a visible increase in the expressions of key microglial polarization-related proteins, including p-STAT1, TRAF6, and SOCS1. Conclusions: The findings demonstrate that IRAK-M dysfunction contributes to the progression of epilepsy by increasing M1 microglial polarization and glutamatergic synaptic transmission. This is possibly related to NMDARs, particularly Grin2A and Grin2B, which suggests that IRAK-M could serve as a novel therapeutic target for epilepsy.
Abstract The NOD-like receptor family pyrin domain-containing protein 3 (NLRP3) is a protein complex which mediates pro-inflammatory response and pyroptosis. The activation of NLRP3 inflammasome triggers pyroptosis via cleavage of the pore-forming protein gasdermin D. Previous studies have demonstrated that NLRP3 inflammasome activation in microglial contributes to the pathogenesis of EAE, while Inhibition of microglial NLRP3 inflammasome and the subsequent pyroptosis attenuates the severity of EAE. In addition, Interleukin-1 receptor-associated kinase (IRAK)-M, mainly expressed in microglial in the central nervous system, negatively regulates the TLR4/IL-1R signaling pathway through inhibiting the phosphorylation of IRAK1 and dissociation of IRAK1 from MyD88/IRAK-4/IRAK-1 complex as well as the formation of IRAK-1 and TRAF6 complex. Our recent study suggests the protective effect of microglial IRAK-M in EAE mice. Therefore, we hypothesize that IRAK-M may alleviate IRAK1-induced NLRP3 inflammasome activation and pyroptosis through inhibiting dissociation of IRAK1 from MyD88/IRAK-4/IRAK-1 complex and the following association of IRAK1 and TRAF6. Here, we used IRAK-M knockout mice and their microglia to clarify the role of IRAK-M in EAE. We demonstrated that IRAK-M decreased the incidence rate and improved the clinical symptoms accompanied by mild demyelination in EAE mice, and which was parallel to the inhibition of microglial NLRP3 inflammasome activation, pyroptosis, and IRAK1/TRAF6 formation in EAE. Our findings fill the gap in knowledge of IRAK-M on activation of NLRP3 inflammasome and pyroptosis and provide a potential novel target for the therapeutic strategy of multiple sclerosis and NLRP3-related diseases.
Abstract Background The pathway involving PTEN-induced putative kinase 1 (PINK1) and PARKIN plays a crucial role in mitophagy, a process activated by artesunate (ART). We propose that patients with anti-N-methyl-D-aspartate receptor (NMDAR) encephalitis exhibit insufficient mitophagy, and ART enhances mitophagy via the PINK1/PARKIN pathway, thereby providing neuroprotection. Methods Adult female mice aged 8–10 weeks were selected to create a passive transfer model of anti-NMDAR encephalitis. We conducted behavioral tests on these mice within a set timeframe. Techniques such as immunohistochemistry, immunofluorescence, and western blotting were employed to assess markers including PINK1, PARKIN, LC3B, p62, caspase3, and cleaved caspase3. The TUNEL assay was utilized to detect neuronal apoptosis, while transmission electron microscopy (TEM) was used to examine mitochondrial autophagosomes. Primary hippocampal neurons were cultured, treated, and then analyzed through immunofluorescence for mtDNA, mtROS, TMRM. Results In comparison to the control group, mitophagy levels in the experimental group were not significantly altered, yet there was a notable increase in apoptotic neurons. Furthermore, markers indicative of mitochondrial leakage and damage were found to be elevated in the experimental group compared to the control group, but these markers showed improvement following ART treatment. ART was effective in activating the PINK1/PARKIN pathway, enhancing mitophagy, and diminishing neuronal apoptosis. Behavioral assessments revealed that ART ameliorated symptoms in mice with anti-NMDAR encephalitis in the passive transfer model (PTM). The knockdown of PINK1 led to a reduction in mitophagy levels, and subsequent ART intervention did not alleviate symptoms in the anti-NMDAR encephalitis PTM mice, indicating that ART’s therapeutic efficacy is mediated through the activation of the PINK1/PARKIN pathway. Conclusions At the onset of anti-NMDAR encephalitis, mitochondrial damage is observed; however, this damage is mitigated by the activation of mitophagy via the PINK1/PARKIN pathway. This regulatory feedback mechanism facilitates the removal of damaged mitochondria, prevents neuronal apoptosis, and consequently safeguards neural tissue. ART activates the PINK1/PARKIN pathway to enhance mitophagy, thereby exerting neuroprotective effects and may achieve therapeutic goals in treating anti-NMDAR encephalitis.
Abstract Background Neuromyelitis optica (NMO), multiple sclerosis (MS) and autoimmune glial fibrillary acidic protein (GFAP) astrocytopathy belong to autoimmune diseases incentral nervous system mainly manifestate encephalomyelitis. The glycocalyx (GLX), consists of several membrane-bound macromolecules, is located on the luminal side of the endothelium and mediates the blood and the vessel interaction. Until now, there is still lacking a holistic understanding of the GLX degradation in autoimmune encephalomyelitis. Aim This study aimed to detect the shedding levels of GLX components, heparan sulfate (HS) and hyaluronic acid (HA) in serum and cerebrospinal fluid (CSF), correlate them with the severity and assess the diagnostic value of them, and evaluate their correlations with pro-inflammatory cytokines. Methods Serum and CSF samples were obtained from 24 NMO patients, 15 MS patients, 10 autoimmune GFAP astrocytopathy patients, and 18 controls without non-inflammatory neurological diseases. Soluble HS, HA and IFN-γ, IL-17A, matrix metalloproteinase (MMP)-1 were detected by enzyme linked immunosorbent assay ELISA. Results Besides levels of serum and CSF levels of HS, HA and related cytokines were significantly elevated in these diseases. Notably, HS, HA in NMO, MS patients, and autoimmune GFAP astrocytopathy diseases were widely correlated with EDSS scores. Importantly, the ROC curve analysis suggested a potential diagnostic role of HS or HA . Conclusions The results here suggested the GLX degradation and inflammation in NMO, MS and autoimmune GFAP astrocytopathy. Moreover, increased shedding of HS or HA may indicate worse clinical situation. Importantly, CSF HS and HA may be informative diagnostic biomarkers for telling autoimmune encephalomyelitis from the non-inflammatory neurological controls. Furthermore, therapeutic strategy for protecting GLX may be effective to these diseases.
The activation of the NOD-like receptor family pyrin domain-containing protein 3 (NLRP3) inflammasome triggers pyroptosis proinflammatory cell death in experimental autoimmune encephalomyelitis (EAE). However, the underlying mechanisms of the inflammatory processes of microglia in EAE remain unclear. Our previous studies suggested that interleukin-1 receptor-associated kinase (IRAK)-M down-regulates the toll-like receptor 4/interleukin-1 receptor signaling pathway. Here, we used IRAK-M knockout (IRAK-M-/-) mice and their microglia to dissect the role of IRAK-M in EAE. We found that deletion of IRAK-M increased the incidence rate and exacerbated the clinical symptoms in EAE mice. We then found that IRAK-M deficiency promoted the activation of microglia, activated NLRP3 inflammasomes, and enhanced GSDMD-mediated pyroptosis in the microglia of EAE. In contrast, over-expression of IRAK-M exerted inhibitory effects on neuroinflammation, NLRP3 activation, and pyroptosis. Moreover, IRAK-M deficiency enhanced the phosphorylation of IRAK1, while IRAK-M over-expression downregulated the level of phosphorylated IRAK1. Finally, we found upregulated binding of IRAK1 and TNF receptor-associated factor 6 (TRAF6) in IRAK-M-/- EAE mice compared to WT mice, which was blocked in AAVIRAK-M EAE mice. Our study reveals a complex signaling network of IRAK-M, which negatively regulates microglial NLRP3 inflammasomes and pyroptosis by inhibiting IRAK1 phosphorylation during EAE. These findings suggest a potential target for the novel therapeutic approaches of multiple sclerosis (MS)/EAE and NLRP3-related inflammatory diseases.
Lianhuaqingwen Capsule (LH-C) is a traditional Chinese medicine (TCM) formula used to treat respiratory tract infectious diseases in Chinese. The aim of this study was to determine the antiviral activity of LH-C and its immunomodulatory effects on viral infection.The in vitro cytotoxicity and antiviral activity of LH-C was determined by MTT and Plaque reduction assays. Time course study under single-cycle virus growth conditions were used to determine which stage of viral replication was blocked. The effect of LH-C on the nuclear export of the viral nucleoprotein was examined using an indirect immunofluorescence assay. The regulation to different signaling transduction events and cytokine/chemokine expression of LH-C was evaluated using Western blotting and real-time RT-PCR. After virus inoculation, BALB/c mice were administered with LH-C of different concentrations for 5 days. Body-weight, viral titers and lung pathology of the mice were measured, the level of inflammatory cytokines were also examined using real-time RT-PCR.LH-C inhibited the proliferation of influenza viruses of various strain in vitro, with the 50% inhibitory concentration (IC50) ranging from 0.35 to 2 mg/mL. LH-C blocked the early stages (0-2 h) of virus infection, it also suppressed virus-induced NF-kB activation and alleviated virus-induced gene expression of IL-6, IL-8, TNF-a, IP-10, and MCP-1 in a dose-dependent manner. LH-C treatment efficiently impaired the nuclear export of the viral RNP. A decrease of the viral titers in the lungs of mice were observed in groups administered with LH-C. The level of inflammatory cytokines were also decreased in the early stages of infection.LH-C, as a TCM prescription, exerts broad-spectrum effects on a series of influenza viruses, including the newly emerged H7N9, and particularly regulates the immune response of virus infection. Thus, LH-C might be a promising option for treating influenza virus infection.
Background: The activation of the NOD-like receptor family pyrin domain-containing protein 3 (NLRP3) inflammasome triggers pyroptosis proinflammatory cell death in experimental autoimmune encephalomyelitis (EAE). However, the underlying mechanisms of the inflammatory processes of microglia in EAE remain unclear. Our previous studies suggested that interleukin-1 receptor-associated kinase (IRAK)-M down-regulates the toll-like receptor 4/interleukin-1 receptor signaling pathway. Here, we used IRAK-M knockout (IRAK-M-/-) mice and their microglia to dissect the role of IRAK-M in EAE.Results: We found that deletion of IRAK-M increased the incidence rate and exacerbated the clinical symptoms in EAE mice. We then found that IRAK-M deficiency promoted the activation of microglia, activated NLRP3 inflammasomes, and enhanced GSDMD-mediated pyroptosis in the microglia of EAE. In contrast, Over-expression of IRAK-M exerted inhibitory effects on neuro-inflammation, NLRP3 activation, and pyroptosis. Moreover, IRAK-M deficiency enhanced the phosphorylation of IRAK1, while IRAK-M over-expression downregulated the level of phosphorylated IRAK1. Finally, we found upregulated binding of IRAK1 and TNF receptor-associated factor 6 (TRAF6) in IRAK-M-/- EAE mice compared to WT mice, which was blocked in AAVIRAK-M EAE mice.Conclusion: Our study reveals a complex signaling network of IRAK-M, which negatively regulates microglial NLRP3 inflammasomes and pyroptosis by inhibiting IRAK1 phosphorylation during EAE. These findings suggest a potential target for the novel therapeutic approaches of MS/EAE and NLRP3-related inflammatory diseases.Funding Information: This study was supported by the National Natural Science Foundation of China [81673950, 82060905, and 81760902], and Guangxi Natural Fund Project [2019GXNSFAA245079].Declaration of Interests: All authors declare no potential conflict of interest relevant to this article.Ethics Approval Statement: All of the animal care and experimental procedures were performed in accordance with the NIH Guide for the Care and Use of Laboratory Animals (NIH, revised 1996) and approved by the Laboratory Animal Ethics Committee of Southern Medical University (Guangzhou, China).
'/%3e%3cuse xlink:href='%23a' transform='rotate(23.036 .093 25.536)'/%3e%3cuse xlink:href='%23a' transform='rotate(45.87 1.273 16.18)'/%3e%3cuse xlink:href='%23a' transform='rotate(69.945 .996 12.078)'/%3e%3cuse xlink:href='%23a' transform='rotate(20.66 -19.689 31.932)'/%3e%3c/svg%3e)