RESUMO
BACKGROUND: Guillain-Barré syndrome (GBS) is an auto-inflammatory peripheral nerve disease. Dendritic cell-mediated T cell polarization is of pivotal importance in demyelinating lesions of peripheral nerves and nerve roots. However, the regulatory function of VX-509 (Decernotinib)-modified tolerogenic dendritic cells (VX-509-tolDCs) during immune remodeling following GBS remains unclear. Here, we used experimental autoimmune neuritis (EAN) as a model to investigate these aspects of GBS. METHODS: DCs were treated with varying concentrations of VX-509 (0.25, 1, and 4 µM) or served as a control using 10-8 M 1,25-(OH)2D3. Flow cytometry was employed to assess the apoptosis, phenotype, and capacity to induce T cell responses of the treated DCs. In the in vivo experiments, EAN mice received administration of VX-509-tolDCs or 1,25-(OH)2D3-tolDCs via the tail vein at a dose of 1x106 cells/mouse on days 5, 9, 13, and 17. RESULTS: VX-509 inhibited the maturation of DCs and promoted the development of tolDCs. The function of antigen-specific CD4 + T cells ex vivo was influenced by VX-509-tolDCs. Furthermore, the adoptive transfer of VX-509-tolDCs effectively alleviated inflammatory demyelinating lesions in EAN by promoting Th17/Treg (T helper 17 and regulatory T cells) rebalance. CONCLUSION: The adoptive transfer of VX-509-tolDCs alleviated inflammatory demyelinating lesions in a mouse model of GBS, known as the EAN mouse, by partially restoring the balance between Treg and Th17 cells.
Assuntos
Células Dendríticas , Camundongos Endogâmicos C57BL , Neurite Autoimune Experimental , Linfócitos T Reguladores , Células Th17 , Animais , Células Dendríticas/imunologia , Células Dendríticas/efeitos dos fármacos , Células Th17/imunologia , Células Th17/efeitos dos fármacos , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/efeitos dos fármacos , Neurite Autoimune Experimental/imunologia , Neurite Autoimune Experimental/tratamento farmacológico , Camundongos , Tolerância Imunológica/efeitos dos fármacos , Células Cultivadas , Feminino , Modelos Animais de Doenças , Masculino , HumanosRESUMO
BACKGROUND: The composition of gut microbiota plays a pivotal role in priming the immune system and thus impacts autoimmune diseases. Data on the effects of gut bacteria eradication via systemic antibiotics on immune neuropathies are currently lacking. This study therefore assessed the effects of antibiotics-induced gut microbiota alterations on the severity of experimental autoimmune neuritis (EAN), a rat model of Guillain-Barré Syndrome (GBS). Myelin P0 peptide 180-199 (P0 180-199)-induced EAN severity was compared between adult Lewis rats (12 weeks old) that received drinking water with or without antibiotics (colistin, metronidazole, vancomycin) and healthy rats, beginning antibiotics treatment immediately after immunization (day 0), and continuing treatment for 14 consecutive days. Neuropathy severity was assessed via a modified clinical score, and then related to gut microbiota alterations observed after fecal 16S rRNA gene sequencing at baseline and after EAN induction. Effectors of gut mucosal and endoneurial immunity were assessed via immunostaining. EAN rats showed increased gut mucosal permeability alongside increased mucosal CD8+ T cells compared to healthy controls. Antibiotics treatment alleviated clinical EAN severity and reduced endoneurial T cell infiltration, decreased gut mucosal CD8+ T cells and increased gut bacteria that may be associated with anti-inflammatory mechanisms, like Lactobacillus or Parasutterella. Our findings point out a relation between gut mucosal immunity and the pathogenesis of EAN, and indicate that antibiotics-induced intestinal immunomodulation might be a therapeutic approach to alleviate autoimmunity in immune neuropathies. Further studies are warranted to evaluate the clinical transferability of these findings to patients with GBS.
Assuntos
Antibacterianos , Microbioma Gastrointestinal , Imunomodulação , Neurite Autoimune Experimental , Ratos Endogâmicos Lew , Animais , Neurite Autoimune Experimental/imunologia , Neurite Autoimune Experimental/tratamento farmacológico , Ratos , Microbioma Gastrointestinal/efeitos dos fármacos , Antibacterianos/farmacologia , Imunomodulação/efeitos dos fármacos , MasculinoRESUMO
OBJECTIVE: We evaluated the potential neuro-regenerative effects of the mitochondrial uncoupler 2,4-Dinitrophenol in experimental autoimmune neuritis, an animal model for an acute autoimmune neuropathy. METHODS: Experimental autoimmune neuritis was induced in Lewis rats. Different concentrations of 2,4-Dinitrophenol (1 mg/kg, 0.1 mg/kg and 0.01 mg/kg) were applied during the recovery phase of the neuritis (at days 18, 22 and 26) and compared to the vehicle. Any effects were assessed through functional, electrophysiological, and morphological analysis via electron microscopy of all groups at day 30. Additional immune-histochemical analysis of inflammation markers and remyelination of the sciatic nerves were performed for the dosage of 1 mg/kg and control. RESULTS: No enhancement of functional or electrophysiological recovery was observed in all 2,4-Dinitrophenol-treated groups. Cellular inflammation markers of T cells (CD3+) were comparable to control, and an increase of macrophages (IbA1+) invasion in the sciatic nerves was observed. Treatment with 2,4-Dinitrophenol reduced axonal swelling in myelinated and unmyelinated fibers with an increased production of brain-derived neurotrophic factor. CONCLUSION: Our findings do not support the hypothesis that repurposing of the mitochondrial uncoupler 2,4-Dinitrophenol exerts functionally relevant neuro-regenerative effects in autoimmune neuritis.
Assuntos
Neurite Autoimune Experimental , Neurite (Inflamação) , Ratos , Animais , Ratos Endogâmicos Lew , Neurite Autoimune Experimental/tratamento farmacológico , 2,4-Dinitrofenol/farmacologia , Dinitrofenóis , InflamaçãoRESUMO
BACKGROUND: Autoimmune neuropathies can result in long-term disability and incomplete recovery, despite adequate first-line therapy. Kinesin-5 inhibition was shown to accelerate neurite outgrowth in different preclinical studies. Here, we evaluated the potential neuro-regenerative effects of the small molecule kinesin-5 inhibitor monastrol in a rodent model of acute autoimmune neuropathies, experimental autoimmune neuritis. METHODS: Experimental autoimmune neuritis was induced in Lewis rats with the neurogenic P2-peptide. At the beginning of the recovery phase at day 18, the animals were treated with 1 mg/kg monastrol or sham and observed until day 30 post-immunisation. Electrophysiological and histological analysis for markers of inflammation and remyelination of the sciatic nerve were performed. Neuromuscular junctions of the tibialis anterior muscles were analysed for reinnervation. We further treated human induced pluripotent stem cells-derived secondary motor neurons with monastrol in different concentrations and performed a neurite outgrowth assay. RESULTS: Treatment with monastrol enhanced functional and histological recovery in experimental autoimmune neuritis. Motor nerve conduction velocity at day 30 in the treated animals was comparable to pre-neuritis values. Monastrol-treated animals showed partially reinnervated or intact neuromuscular junctions. A significant and dose-dependent accelerated neurite outgrowth was observed after kinesin-5 inhibition as a possible mode of action. CONCLUSION: Pharmacological kinesin-5 inhibition improves the functional outcome in experimental autoimmune neuritis through accelerated motor neurite outgrowth and histological recovery. This approach could be of interest to improve the outcome of autoimmune neuropathy patients.
Assuntos
Células-Tronco Pluripotentes Induzidas , Neurite Autoimune Experimental , Ratos , Animais , Humanos , Neurite Autoimune Experimental/tratamento farmacológico , Neurite Autoimune Experimental/patologia , Cinesinas/uso terapêutico , Ratos Endogâmicos Lew , Células-Tronco Pluripotentes Induzidas/patologiaRESUMO
Objective: To investigate the expression of glycolytic genes in immune cells and the changes of related immune cells in experimental autoimmune neuritis (EAN), and deepen the understanding of pathogenesis of EAN. Methods: Twenty-four male C57BL/6 mice (6-8 weeks old, 18-20 g) were divided into four groups according to the random number table method: control group (P0180-199 was replaced by PBS during modeling and mice were sacrificed on the 16th day), EAN mice were sacrificed on the 8th day after the end of modeling (EAN 8 d), EAN mice were sacrificed on the 16th day after the end of modeling (EAN 16 d), and EAN mice received drug intervention and were sacrificed on the 16th day after the end of modeling (2-DG was intraperitoneally injected since the day of the first immunization, 550 mg/kg; EAN 16 d+2-DG), with 6 rats in each group. The clinical symptoms and clinical scores were observed and recorded daily. At the end of the experiment, the mice were sacrificed under chloral hydrate anesthesia, and the serum, spleen, sciatic nerve and other tissues of each group were collected. The degree of inflammatory cell infiltration and demyelination of sciatic nerve were observed by hematoxylin and eosin (HE) staining and luxol fast blue (LFB) staining. Flow cytometry was used to detect the proportion of M1 macrophages, Th17 cells and Tregs cells. The mRNA expression levels of glycolysis-related genes (mTORC1, HIF1α, GLUT1 and LDHA) were detected by RT-PCR. Western blotting was used to detect the level of pan-lysine lactate in macrophages and sciatic nerve tissue. Results: The expression of glycolysis-related genes (mTORC1, HIF1α, GLUT1 and LDHA) in spleen M1 macrophages and sciatic nerve was significantly up-regulated in EAN 16 d group, compared with control, EAN 8 d and EAN 16 d+2-DG groups (all P<0.05). The relative pan-lysine lactate (pankla) expression level of spleen M1 macrophages (1.25±0.02) and sciatic nerve tissue (1.23±0.26) significantly increased in EAN 16 d group, compared with control, EAN 8 d and EAN 16 d+2-DG groups (M1 macrophages: 0.12±0.10, 1.07±0.12 and 0.42±0.07; sciatic nerve: 0.10±0.12, 0.87±0.20 and 0.36±0.05) (all P<0.05). The expression of glycolytic genes in splenic CD4+T cells showed an increasing trend, but there were no statistically significant differences among the groups, and the expression of glycolytic genes did not decrease significantly after 2-DG treatment (all P>0.05). The proportion of spleen M1 macrophages in the control group, EAN 8 d group, EAN 16 d group and EAN 16 d+2-DG group was 4.28±0.13, 7.54±0.25, 13.16±0.33 and 4.13±0.38 respectively, which was significantly higher in the EAN 16 d group (all P<0.05). The proportion of spleen Th17 cells in the four groups was 3.78±0.03, 8.24±0.55, 12.30±1.34 and 4.83±0.01, respectively, which was significantly higher in the EAN 16 d group (all P<0.05). The proportion of spleen Tregs cells in the four groups was 10.01±1.05, 7.54±0.70, 3.82±0.47 and 8.22±1.21, respectively, which was significantly lower in the EAN 16 d group (all P<0.05). Conclusions: The expression of glycolytic genes in splenic macrophages significantly increases during EAN, but not in CD4+T cells. The proportion of M1 macrophages and Th17 cells in spleen gradually increases, while the proportion of Tregs cells gradually decreases.
Assuntos
Neurite Autoimune Experimental , Ratos , Camundongos , Masculino , Animais , Transportador de Glucose Tipo 1/metabolismo , Neurite Autoimune Experimental/tratamento farmacológico , Neurite Autoimune Experimental/patologia , Lisina/metabolismo , Lisina/uso terapêutico , Camundongos Endogâmicos C57BL , Nervo Isquiático/metabolismo , Nervo Isquiático/patologia , GlicóliseRESUMO
OBJECTIVE: Chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) is an immune-mediated disease that targets the myelin sheaths of the peripheral nerves. Fingolimod is a sphingosine 1 phosphate (S1P) receptor antagonist with a high affinity for S1P receptors through the Akt-mTOR pathway, and prior research has suggested that it might be helpful in autoimmune illnesses. METHODS: Chronic experimental autoimmune neuritis (c-EAN) was induced by immunizing Lewis rats with the S-palm P0(180-199) peptide, and then the treatment group was intraperitoneally injected with fingolimod (1 mg/kg) daily. Hematoxylin and eosin staining was used to assess the severity of nerve injury. Immunohistochemistry staining showed that fingolimod's anti-inflammatory effects on c-EAN rats might be realized through the NF-κB signaling pathway. Tumor necrosis factor-α (TNF-α), interferon-γ (INF-γ), interleukin-1beta (IL-1ß), interleukin 6 (IL-6), inducible nitric oxide synthase (iNOS), and intercellular adhesion molecule-1 (ICAM-1) were measured to evaluate the inflammation levels, and pAkt, p-S6, and p-p65 were used to measure the abundance of downstream activation markers to determine whether the Akt/mTOR/NF-κB signaling pathway was activated in the c-EAN model. RESULTS: Fingolimod treatment reduced the inflammatory reaction and the expression of NF-κB in sciatic nerves. It also decreased the mRNA levels of the proinflammatory cytokines TNF-α, IFN-γ, IL-1ß, IL-6, iNOS, and ICAM-1 and pAkt, p-S6, and p-p65, representing the Akt/mTOR/NF-κB signaling pathway. CONCLUSION: Our data showed that fingolimod could improve the disease course, alleviate the decrease in inflammation, and reduce proinflammatory cytokines through the Akt/mTOR/NF-κB axis in c-EAN rats, which could be beneficial for the development of CIDP-related research.
Assuntos
Neurite Autoimune Experimental , Polirradiculoneuropatia Desmielinizante Inflamatória Crônica , Ratos , Animais , Citocinas/metabolismo , NF-kappa B/metabolismo , Cloridrato de Fingolimode/efeitos adversos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Molécula 1 de Adesão Intercelular/efeitos adversos , Molécula 1 de Adesão Intercelular/genética , Fator de Necrose Tumoral alfa , Interleucina-6 , Neurite Autoimune Experimental/tratamento farmacológico , Ratos Endogâmicos Lew , Transdução de Sinais , Serina-Treonina Quinases TOR/efeitos adversos , Serina-Treonina Quinases TOR/metabolismo , Inflamação/tratamento farmacológicoRESUMO
BACKGROUND: Lysophosphatidic acid (LPA) is a pleiotropic lipid messenger that addresses at least six specific G-protein coupled receptors. Accumulating evidence indicates a significant involvement of LPA in immune cell regulation as well as Schwann cell physiology, with potential relevance for the pathophysiology of peripheral neuroinflammation. However, the role of LPA signaling in inflammatory neuropathies has remained completely undefined. Given the broad expression of LPA receptors on both Schwann cells and cells of the innate and adaptive immune system, we hypothesized that inhibition of LPA signaling may ameliorate the course of disease in experimental autoimmune neuritis (EAN). METHODS: We induced active EAN by inoculation of myelin protein 2 peptide (P255-78) in female Lewis rats. Animals received the orally available LPA receptor antagonist AM095, specifically targeting the LPA1 receptor subtype. AM095 was administered daily via oral gavage in a therapeutic regimen from 10 until 28 days post-immunization (dpi). Analyses were based on clinical testing, hemogram profiles, immunohistochemistry and morphometric assessment of myelination. RESULTS: Lewis rats treated with AM095 displayed a significant improvement in clinical scores, most notably during the remission phase. Cellular infiltration of sciatic nerve was only discretely affected by AM095. Hemogram profiles indicated no impact on circulating leukocytes. However, sciatic nerve immunohistochemistry revealed a reduction in the number of Schwann cells expressing the dedifferentiation marker Sox2 paralleled by a corresponding increase in differentiating Sox10-positive Schwann cells. In line with this, morphometric analysis of sciatic nerve semi-thin sections identified a significant increase in large-caliber myelinated axons at 28 dpi. Myelin thickness was unaffected by AM095. CONCLUSION: Thus, LPA1 signaling may present a novel therapeutic target for the treatment of inflammatory neuropathies, potentially affecting regenerative responses in the peripheral nerve by modulating Schwann cell differentiation.
Assuntos
Desdiferenciação Celular/fisiologia , Neurite Autoimune Experimental/imunologia , Receptores de Ácidos Lisofosfatídicos/imunologia , Células de Schwann/imunologia , Transdução de Sinais/fisiologia , Animais , Compostos de Bifenilo/farmacologia , Compostos de Bifenilo/uso terapêutico , Desdiferenciação Celular/efeitos dos fármacos , Feminino , Isoxazóis/farmacologia , Isoxazóis/uso terapêutico , Neurite Autoimune Experimental/tratamento farmacológico , Neurite Autoimune Experimental/metabolismo , Ratos , Ratos Endogâmicos Lew , Receptores de Ácidos Lisofosfatídicos/antagonistas & inibidores , Receptores de Ácidos Lisofosfatídicos/metabolismo , Células de Schwann/efeitos dos fármacos , Células de Schwann/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
Guillain-Barré syndrome (GBS) is an acute inflammatory and immune-mediated demyelinating disease of the peripheral nervous system (PNS). Macrophages play a central role in its animal model, experimental autoimmune neuritis (EAN), which has been well accepted. Additionally, nuclear factor (NF)-κB inhibitors have been used to treat cancers and have shown beneficial effects. Here, we investigated the therapeutic effect of M2 macrophage and the NF-κB pathway's correlation with macrophage activation in EAN in C57BL/6 mice. We demonstrate that M2 macrophage transfusion could alleviate the clinical symptoms of EAN by reducing the proportion of M1 macrophage in the peak period, inhibiting the phosphorylation of NF-κB p65. The NF-κB inhibitor (BAY-11-7082) could alleviate the clinical symptoms of EAN and shorten the duration of symptoms by reducing the proportion of M1 macrophages and the expression of proinflammatory cytokines. Consequently, BAY-11-7082 exhibits strong potential as a therapeutic strategy for ameliorating EAN by influencing the balance of M1/M2 macrophages and inflammatory cytokines.
Assuntos
Síndrome de Guillain-Barré , Macrófagos/imunologia , Neurite Autoimune Experimental , Nitrilas/farmacologia , Sulfonas/farmacologia , Fator de Transcrição RelA/antagonistas & inibidores , Animais , Síndrome de Guillain-Barré/tratamento farmacológico , Síndrome de Guillain-Barré/imunologia , Masculino , Camundongos , Neurite Autoimune Experimental/tratamento farmacológico , Neurite Autoimune Experimental/imunologia , Fator de Transcrição RelA/imunologiaRESUMO
BACKGROUND: Guillain-Barré syndrome (GBS) is an acquired immune-mediated inflammatory peripheral neuropathy. The immune regulation of ginkgolides have been revealed in recent years. We herein investigate the potential therapeutic effects of ginkgolides both on GBS and its animal model, experimental autoimmune neuritis (EAN). METHODS: EAN in C57BL/6 mice induced by subcutaneous injection with peripheral nerve myelin P0 protein peptide 180-199 (P0 peptide) were treated with ginkgolides at three different doses. GBS patients were randomly divided into two groups, the experimental group and the control group. The experimental group were treated with ginkgolides as soon as diagnosed. RESULTS: Our data indicated that ginkgolides administration daily ameliorated the score of EAN and delayed the peak of disease in EAN mice. Ginkgolides also down-regulated the proportions of T helper (Th) 17 cells in EAN spleens. Furthermore, we also found that administration of ginkgolides significantly decreased the levels of interferon (IFN)-γ and interleukin-12 (IL)-12 in GBS patients. CONCLUSIONS: Our results suggested that ginkgolides ameliorated the clinical score of EAN through down-regulating the proportions of Th 17 cells. Ginkgolides also suppressed inflammation response by decreasing pro-inflammatory cytokines IFN-γ and IL-12, suggesting ginkgolides had potential therapeutic effects on GBS patients and EAN in the future.
Assuntos
Ginkgolídeos/farmacologia , Síndrome de Guillain-Barré/tratamento farmacológico , Neurite Autoimune Experimental/tratamento farmacológico , Adulto , Idoso , Animais , Citocinas/metabolismo , Modelos Animais de Doenças , Feminino , Ginkgolídeos/metabolismo , Ginkgolídeos/uso terapêutico , Síndrome de Guillain-Barré/imunologia , Síndrome de Guillain-Barré/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Proteína P0 da Mielina , Neurite Autoimune Experimental/imunologia , Neurite Autoimune Experimental/metabolismo , Nervos Periféricos , Células Th17/imunologia , Células Th17/metabolismoRESUMO
Guillain-Barré Syndrome (GBS), characterized by peripheral nerve demyelination and axonal damage, is initiated and aggravated through various of immunopathogenesis. Ginsenoside Rd, main active components extracted from ginseng saponins, is known to exhibit immune-regulate functions in many immune-mediated diseases. However, the evidence of preventive effect of Ginsenoside Rd on GBS is lacking. Experimental autoimmune neuritis (EAN) mice, classic model of GBS, were established and treated with GSRd or vehicle. Clinical score and nerve tissue histomorphology were evaluated. Monocytes in peripheral blood and tissue were detected by flow cytometry analysis and immunofluorescence staining. For the in vitro study, GSRd and vehicle were added in the culture medium to assess their regulatory function on monocytes phenotype. In vivo data showed a protective role of GSRd on alleviating symptoms and tissue damage on Day 20 and 25. Administration of GSRd increased non-classical Ly6Clo monocytes in both peripheral blood and injured nerve tissue, and also switched tissue macrophages phenotype into resolution-phase. In vitro study indicated similar role of GSRd on monocytes differentiation status. Transcription factors like Nr4a1 were elevated after GSRd treatment. These findings revealed the protective role of GSRd against EAN, and potential preventive function on GBS patients.
Assuntos
Ginsenosídeos/uso terapêutico , Monócitos/efeitos dos fármacos , Monócitos/imunologia , Neurite Autoimune Experimental/tratamento farmacológico , Neurite Autoimune Experimental/imunologia , Panax , Sequência de Aminoácidos , Animais , Relação Dose-Resposta a Droga , Ginsenosídeos/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mycobacterium tuberculosis/imunologia , Neuropatia Ciática/tratamento farmacológico , Neuropatia Ciática/imunologiaRESUMO
This study aimed to investigate the therapeutic effect of fasudil on treating experimental autoimmune neuritis (EAN). Twenty-four EAN mice were randomly assigned to fasudil treatment (Fasudil group) or saline treatment (EAN model group) for 28 days. Clinical symptom score was evaluated every other day; inflammatory cell infiltration, demyelination, anti-myelin basic protein (MBP), inflammatory cytokines, inducible nitric oxide synthase (iNOS), and arginase-1 were detected in sciatic nerves at day 28. Th1, Th2, Th17, and Tregs proportions in splenocytes were detected at day 28. Clinical symptom score was found to be attenuated in the Fasudil group compared to the EAN model group from day 12 to day 28. Sciatic nerve inflammatory cell counts by HE staining and demyelination by luxol fast blue staining were both reduced, while MBP was increased in the Fasudil group compared to the EAN model group at day 28. Interferon γ (IFN-γ) and interleukin (IL)-17 were reduced, while IL-4 and IL-10 were elevated in the Fasudil group at day 28. Sciatic nerve M1 macrophages marker iNOS was decreased while M2 macrophages marker arginase-1 was increased in the Fasudil group at day 28. CD4+IFN-γ+ (Th1) and CD4+IL-17+ (Th17) cell proportions were both decreased, CD4+IL-4+ (Th2) cell proportion was similar, while CD25+FOXP3+ (Treg) cell proportion in splenocytes was increased in the Fasudil group. In summary, fasudil presented a good therapeutic effect for treating EAN by attenuating Th1/Th17 cells and promoting Tregs activation as well as M2 macrophages polarization.
Assuntos
Animais , Feminino , Coelhos , Interleucinas/sangue , Interferon gama/sangue , Linfócitos T Auxiliares-Indutores/efeitos dos fármacos , Neurite Autoimune Experimental/tratamento farmacológico , Nervo Isquiático/efeitos dos fármacos , Nervo Isquiático/metabolismo , Fatores de Tempo , Reação em Cadeia da Polimerase em Tempo Real , RNA Mitocondrial , Camundongos Endogâmicos C57BL , Neurite Autoimune Experimental/sangueRESUMO
This study aimed to investigate the therapeutic effect of fasudil on treating experimental autoimmune neuritis (EAN). Twenty-four EAN mice were randomly assigned to fasudil treatment (Fasudil group) or saline treatment (EAN model group) for 28 days. Clinical symptom score was evaluated every other day; inflammatory cell infiltration, demyelination, anti-myelin basic protein (MBP), inflammatory cytokines, inducible nitric oxide synthase (iNOS), and arginase-1 were detected in sciatic nerves at day 28. Th1, Th2, Th17, and Tregs proportions in splenocytes were detected at day 28. Clinical symptom score was found to be attenuated in the Fasudil group compared to the EAN model group from day 12 to day 28. Sciatic nerve inflammatory cell counts by HE staining and demyelination by luxol fast blue staining were both reduced, while MBP was increased in the Fasudil group compared to the EAN model group at day 28. Interferon γ (IFN-γ) and interleukin (IL)-17 were reduced, while IL-4 and IL-10 were elevated in the Fasudil group at day 28. Sciatic nerve M1 macrophages marker iNOS was decreased while M2 macrophages marker arginase-1 was increased in the Fasudil group at day 28. CD4+IFN-γ+ (Th1) and CD4+IL-17+ (Th17) cell proportions were both decreased, CD4+IL-4+ (Th2) cell proportion was similar, while CD25+FOXP3+ (Treg) cell proportion in splenocytes was increased in the Fasudil group. In summary, fasudil presented a good therapeutic effect for treating EAN by attenuating Th1/Th17 cells and promoting Tregs activation as well as M2 macrophages polarization.
Assuntos
1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/análogos & derivados , Interferon gama/sangue , Interleucinas/sangue , Neurite Autoimune Experimental/tratamento farmacológico , Linfócitos T Auxiliares-Indutores/efeitos dos fármacos , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/administração & dosagem , Animais , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Neurite Autoimune Experimental/sangue , RNA Mitocondrial , Reação em Cadeia da Polimerase em Tempo Real , Nervo Isquiático/efeitos dos fármacos , Nervo Isquiático/metabolismo , Fatores de TempoRESUMO
BACKGROUND: Accumulating evidence has pointed that T helper 17 cells and their cytokines are pathogenic in Guillain-Barré syndrome (GBS). However, little is known concerning the IL-17 expression change trend during the whole course of disease, and whether drugs specially targeting Th17 cells or their cytokines have potential effects on experimental autoimmune neuritis (EAN) is uncertain. METHODS: We explored the IL-17 and receptor-related orphan receptor-gamma-t (RORγt) expression change trends in EAN rats to identify the stage of effect of Th17 pathway in EAN, and further, we investigated the effect of RORγt inhibitors by assessing clinical score, histological staining, and IL-17 and RORγt expression change trends in serum and tissues. RESULTS: The expression level of IL-17 and RORγt in serum and tissues increased with the progression of the disease in the EAN group and decreased after the disease reaching its peak. RORγt-IN-1 treatment strikingly reduced the neurological deficits by ameliorating inflammatory cell infiltration, deceased the serum IL-17 and RORγt levels, and further downregulated the expression of IL-17 and RORγt mRNA in spleen, lymphnodes, and sciatic nerve. CONCLUSIONS: Th17 cells and their cytokines are closely associated with the onset of GBS and the novel RORγt inhibitors may be prospective strategies in treating GBS.
Assuntos
Interleucina-17/imunologia , Neurite Autoimune Experimental , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/antagonistas & inibidores , Células Th17/imunologia , Animais , Anti-Inflamatórios/farmacologia , Citocinas/imunologia , Síndrome de Guillain-Barré/tratamento farmacológico , Síndrome de Guillain-Barré/imunologia , Masculino , Neurite Autoimune Experimental/tratamento farmacológico , Neurite Autoimune Experimental/imunologia , Ratos , Ratos Endogâmicos Lew , Resultado do TratamentoRESUMO
Objective: Dimethyl fumarate (DMF) exerts immunomodulatory and neuroprotective effects in the animal model of experimental autoimmune neuritis (EAN) in the Lewis rat. DMF has been shown to modulate gut microbiota in veterinary medicine, however the effects of oral DMF on the gut-associated lymphoid tissue (GALT) remain unknown. Methods: Lewis rats were treated orally twice daily with DMF up to day 10 after immunization with immunogenic P2 peptide. Histological, flow cytometric and RT-PCR analyses of the GALT (intraepithelial layer, lamina propria, and Peyer patches) in duodenum, jejunum, and ileum were performed ex vivo. Moreover, cell transfer experiments were used to examine the protective effects of GALT regulatory T cells of the Peyer patches. Results: In the upper layers of duodenum, DMF induced a reduction of the toll-like receptor 4 (TLR4) mRNA expression. This was combined by a decrease of the pro-inflammatory lamina propria IFN-γ mRNA expression. In the ileum, we detected an immunoregulatory phenotype characterized by an increase of FoxP3 mRNA expression and of the nuclear factor (erythroid-derived-2)- like 2 (Nrf2) downstream molecule heme oxygenase-1 (HO-1) mRNA. Finally, CD4+ CD25+ regulatory T cells were increased in the Peyer patches. In vivo, the protective effect of these regulatory cells was verified by cell transfer into recipient EAN rats. Conclusions: Our results identified a novel immunomodulatory effect of DMF through the different regions and layers of the small intestine, which led to an increase of regulatory T cells, exerting a protective role in experimental neuritis.
Assuntos
Fumarato de Dimetilo/uso terapêutico , Fatores Imunológicos/uso terapêutico , Intestino Delgado/efeitos dos fármacos , Neurite Autoimune Experimental/tratamento farmacológico , Fármacos Neuroprotetores/uso terapêutico , Animais , Citocinas/genética , Fumarato de Dimetilo/farmacologia , Feminino , Fatores Imunológicos/farmacologia , Intestino Delgado/imunologia , Neurite Autoimune Experimental/imunologia , Fármacos Neuroprotetores/farmacologia , Nódulos Linfáticos Agregados/efeitos dos fármacos , Nódulos Linfáticos Agregados/imunologia , Ratos Endogâmicos Lew , Nervo Isquiático/efeitos dos fármacos , Nervo Isquiático/imunologia , Linfócitos T Reguladores/efeitos dos fármacos , Linfócitos T Reguladores/imunologiaRESUMO
BACKGROUND: Corticosteroids dominate in the treatment of chronic autoimmune neuropathies although long-term use is characterized by devastating side effects. METHODS: We introduce the intrathecal application of the synthetic steroid triamcinolone (TRIAM) as a novel therapeutic option in experimental autoimmune neuritis in Lewis rats RESULTS: After immunization with neuritogenic P2 peptide, we show a dose-dependent therapeutic effect of one intrathecal injection of 0.3 or 0.6 mg/kg TRIAM on clinical and electrophysiological parameters of neuritis with a lower degree of inflammatory infiltrates (T cells and macrophages) and demyelination in the sciatic nerve. In vitro studies in Schwann cell cultures showed an increased expression of IL-1 receptor antagonist and reduced expression of Toll-like receptor 4 after incubation with TRIAM as well as a protective effect of TRIAM against oxidative stress after H2O2 exposure. CONCLUSION: Intrathecal TRIAM application could be a novel immunomodulatory and potentially neuroprotective option for autoimmune neuropathies with a direct effect on Schwann cells.
Assuntos
Anti-Inflamatórios/administração & dosagem , Neurite Autoimune Experimental/tratamento farmacológico , Neurite Autoimune Experimental/patologia , Estresse Oxidativo/efeitos dos fármacos , Células de Schwann/efeitos dos fármacos , Triancinolona Acetonida/administração & dosagem , Animais , Antígenos CD/metabolismo , Técnicas de Cultura de Células , Modelos Animais de Doenças , Adjuvante de Freund/toxicidade , Regulação da Expressão Gênica/efeitos dos fármacos , Injeções Espinhais/métodos , Linfonodos/citologia , Masculino , Condução Nervosa/efeitos dos fármacos , Neurite Autoimune Experimental/induzido quimicamente , Ratos , Ratos Endogâmicos Lew , Fatores de Transcrição SOXE/metabolismo , Antígenos Thy-1/metabolismoRESUMO
BACKGROUND: Guillain-Barré syndrome is an acute inflammatory demyelinating polyneuropathy that is characterized histologically by demyelination of peripheral nerves and nerve roots, infiltrates of T lymphocytes, and an inflammatory response that includes macrophage infiltrates. The aim of this study was to evaluate the effects of vasoactive intestinal peptide (VIP) in a rat model of experimental autoimmune neuritis (EAN). METHODS: Forty male Lewis rats were divided into a control group (N=10), an EAN group (N=10), an EAN group treated with 15 nmol of VIP (N=10), and an EAN group treated with 30 nmol of VIP (N=10). The rat model was created by subcutaneous injection of P2 polypeptide (200 µg P257-81) into the base of the tail. Intraperitoneal injection of VIP was given on day 7. Rats were weighed and functionally evaluated using an EAN score (0-10). On day 16, the rats were euthanized. The sciatic nerve was examined histologically and using immunohistochemistry with antibodies against CD8, CD68, and forkhead box p3 (Foxp3). Serum concentrations of IL-17 and interferon-α (IFN-α) were measured by ELISA on day 16 after creating the EAN model. RESULTS: The VIP-treated EAN groups had increased body weight and improved EAN scores compared with the untreated EAN group. CD8-positive and CD68-positive cells were significantly reduced in the EAN group treated with 30 nmol of VIP compared with 15 nmol of VIP. Foxp3-positive cells were significantly decreased in both EAN groups treated with VIP, and serum concentrations of IL-17 and IFN-α were significantly lower compared with the untreated EAN group (P<0.05). CONCLUSION: In a rat model of EAN, treatment with VIP resulted in functional improvement, reduced nerve inflammation, and decreased serum levels of inflammatory cytokines.
Assuntos
Modelos Animais de Doenças , Síndrome de Guillain-Barré/tratamento farmacológico , Neurite Autoimune Experimental/tratamento farmacológico , Peptídeo Intestinal Vasoativo/uso terapêutico , Vasodilatadores/uso terapêutico , Animais , Relação Dose-Resposta a Droga , Síndrome de Guillain-Barré/sangue , Síndrome de Guillain-Barré/patologia , Interferon-alfa/sangue , Interleucina-17/sangue , Masculino , Neurite Autoimune Experimental/sangue , Neurite Autoimune Experimental/patologia , Ratos , Ratos Endogâmicos Lew , Peptídeo Intestinal Vasoativo/síntese química , Peptídeo Intestinal Vasoativo/química , Vasodilatadores/síntese química , Vasodilatadores/químicaRESUMO
The Class I phosphatidylinositol 3-kinase inhibitor, 2-(2-difluoromethy lbenzimidazol-1-yl)-4,6-dimorpholino-1,3,5-triazine (ZSTK474), has anti-inflammatory and immunoregulatory properties. However, whether it can be used to treat Guillain-Barré syndrome (GBS)-a neuroinflammatory disorder-is unknown. We induced experimental autoimmune neuritis (EAN) in Lewis rats, an established model of GBS. Orally administered ZSTK474 decreased neurological deficits in the GBS model, as demonstrated by diminished inflammatory cell infiltration, and ameliorated demyelination of sciatic nerves. Additionally, ZSTK474 decreased the number of Th1/Th17 cells and levels of the proinflammatory cytokines interleukin (IL)-1α, IL-1ß, IL-17, IL-23, interferon-γ, and tumor necrosis factor-α. We propose that the phosphoinositide 3-kinase/AKT/mammalian target of rapamycin complex 1 (PI3K/AKT/mTORC1) pathway likely contributed to the neuroprotective effect of ZSTK474. ZSTK474 effectively decreases the frequency of Th1/Th17 cells, thereby reducing the production of proinflammatory cytokines and successfully alleviating the symptoms of EAN. Thus, the neuroprotective effect of ZSTK474 indicates its potential utility as anti-inflammatory therapy for GBS.
Assuntos
Citocinas/efeitos dos fármacos , Neurite Autoimune Experimental/tratamento farmacológico , Triazinas/farmacologia , Animais , Doenças Desmielinizantes/tratamento farmacológico , Modelos Animais de Doenças , Síndrome de Guillain-Barré/tratamento farmacológico , Masculino , Fármacos Neuroprotetores/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Ratos , Ratos Endogâmicos Lew , Nervo Isquiático , Células Th1/efeitos dos fármacos , Células Th17/efeitos dos fármacos , Triazinas/metabolismoRESUMO
BACKGROUND: With the recognition of the key roles of cellular metabolism in immunity, targeting metabolic pathway becomes a new strategy for autoimmune disease treatment. Guillain-Barré syndrome (GBS) is an acute immune-mediated inflammatory demyelinating disease of the peripheral nervous system, characterized by inflammatory cell infiltration. These inflammatory cells, including activated macrophages, Th1 cells, and Th17 cells, generally undergo metabolic reprogramming and rely mainly on glycolysis to exert functions. This study aimed to explore whether enhanced glycolysis contributed to the pathogenesis of experimental autoimmune neuritis (EAN), a classic model of GBS. METHODS: Preventive and therapeutic treatments with glycolysis inhibitor, 2-deoxy-D-glucose (2-DG), were applied to EAN rats. The effects of treatments were determined by clinical scoring, weighting, and tissue examination. Flow cytometry and ELISA were used to evaluate T cell differentiation, autoantibody level, and macrophage functions in vivo and in vitro. RESULTS: Glycolysis inhibition with 2-DG not only inhibited the initiation, but also prevented the progression of EAN, evidenced by the improved clinical scores, weight loss, inflammatory cell infiltration, and demyelination of sciatic nerves. 2-DG inhibited the differentiation of Th1, Th17, and Tfh cells but enhanced Treg cell development, accompanied with reduced autoantibody secretion. Further experiments in vitro proved glycolysis inhibition decreased the nitric oxide production and phagocytosis of macrophages and suppressed the maturation of dendritic cells (DC). CONCLUSION: The effects of glycolysis inhibition on both innate and adaptive immune responses and the alleviation of animal clinical symptoms indicated that enhanced glycolysis contributed to the pathogenesis of EAN. Glycolysis inhibition may be a new therapy for GBS.
Assuntos
Glicólise/fisiologia , Neurite Autoimune Experimental/induzido quimicamente , Neurite Autoimune Experimental/metabolismo , Animais , Desoxiglucose/farmacologia , Desoxiglucose/uso terapêutico , Glicólise/efeitos dos fármacos , Imunidade Celular/efeitos dos fármacos , Imunidade Celular/fisiologia , Lipopolissacarídeos/toxicidade , Macrófagos Peritoneais/efeitos dos fármacos , Macrófagos Peritoneais/metabolismo , Camundongos , Neurite Autoimune Experimental/tratamento farmacológico , Células RAW 264.7 , Ratos , Ratos Endogâmicos LewRESUMO
Schwann cells (SCs), which form the peripheral myelin sheath, have the unique ability to dedifferentiate and to destroy the myelin sheath under various demyelination conditions. During SC dedifferentiation-associated demyelination (SAD) in Wallerian degeneration (WD) after axonal injury, SCs exhibit myelin and junctional instability, down-regulation of myelin gene expression and autophagic myelin breakdown. However, in inflammatory demyelinating neuropathy (IDN), it is still unclear how SCs react and contribute to segmental demyelination before myelin scavengers, macrophages, are activated for phagocytotic myelin digestion. Here, we compared the initial SC demyelination mechanism of IDN to that of WD using microarray and histochemical analyses and found that SCs in IDN exhibited several typical characteristics of SAD, including actin-associated E-cadherin destruction, without obvious axonal degeneration. However, autophagolysosome activation in SAD did not appear to be involved in direct myelin lipid digestion by SCs but was required for the separation of SC body from destabilized myelin sheath in IDN. Thus, lysosome inhibition in SCs suppressed segmental demyelination by preventing the exocytotic myelin clearance of SCs. In addition, we found that myelin rejection, which might also require the separation of SC cytoplasm from destabilized myelin sheath, was delayed in SC-specific Atg7 knockout mice in WD, suggesting that autophagolysosome-dependent exocytotic myelin clearance by SCs in IDN and WD is a shared mechanism. Finally, autophagolysosome activation in SAD was mechanistically dissociated with the junctional destruction in both IDN and WD. Thus, our findings indicate that SAD could be a common myelin clearance mechanism of SCs in various demyelinating conditions.
Assuntos
Desdiferenciação Celular/fisiologia , Neurite Autoimune Experimental/patologia , Neurite Autoimune Experimental/fisiopatologia , Células de Schwann/patologia , Neuropatia Ciática/patologia , Neuropatia Ciática/fisiopatologia , Animais , Proteína 7 Relacionada à Autofagia/genética , Proteína 7 Relacionada à Autofagia/metabolismo , Axotomia/efeitos adversos , Cloroquina/uso terapêutico , Doenças Desmielinizantes/tratamento farmacológico , Doenças Desmielinizantes/etiologia , Modelos Animais de Doenças , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/genética , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas da Mielina/genética , Proteínas da Mielina/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neurite Autoimune Experimental/tratamento farmacológico , Ratos , Ratos Endogâmicos Lew , Receptores de Ativador de Plasminogênio Tipo Uroquinase/genética , Receptores de Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Células de Schwann/metabolismo , Células de Schwann/ultraestrutura , Neuropatia Ciática/tratamento farmacológicoRESUMO
Intravenous human immunoglobulins dominate in the treatment of autoimmune neuropathies. We introduce intrathecal application as a new option for experimental autoimmune neuritis in Lewis rats. After immunisation with neuritogenic P2 peptide, we show a therapeutic and preventive effect of intrathecal human immunoglobulins (5-40mg/kg) on clinical and electrophysiological neuritis signs. Histology corroborated a lower degree of inflammation, demyelination, ICAM-1-dependent blood-nerve-barrier permeability and complement activation in the sciatic nerve. After preventive application, immunoglobulins induced a Th2 cytokine shift in the peripheral nerves already before clinical neuritis signs. Intrathecal immunoglobulin application could be a novel immunomodulatory option for autoimmune neuropathies.