The Protective Effect Of Molecular Hydrogen On Experimental Autoimmune Encephalomyelitis And Its Underlying Mechanism

Background and objectivesMultiple sclerosis?MS?is an autoimmune-mediated disorder of the central nervous system?CNS?,characterized by inflammation,demyelination,and axonal degeneration.The disease is associated with a variety of autoimmune reaction and inflammation that result in a range of neurological impairments and disabilities.Despite the neuropathological mechanism of MS has not been fully understood,evidence suggests that dysfunction of T helper?Th?cell regulation and relative signaling events may play an essential role in inducing tissue inflammation during the process.Therefore,experimental autoimmune encephalomyelitis?EAE?,established by immunization with myelin antigen or adoptive transfer of myelin-specific T cells,is widely used to define the sequence of immunopathogenic events in the development of autoimmune CNS-directed inflammatory disease.Four major subsets of Th cells,which are Th1,Th2,Th17,and regulatory T?Treg?cells,differentiated from na?ve CD4⁺T cells have been identified as mediating the adaptive immune system.Among them,Th17 cells are critically involved in the pathogenesis of autoimmune diseases,including MS.Several factors can regulate Th17 responses,and nucleotide-binding domain,leucine-rich repeat containing protein 3?NLRP3?is an important factor during Th17 cell differentiation.NLRP3 belongs to the NLR protein family,a group of intracellular proteins that regulate host inflammatory response.Mutations within the NLRP3 gene are associated with a spectrum of inflammatory disorders,such as fever,arthralgia,severe arthropathy,neurologic deficits,etc.Researchers have reported that the NLRP3 inflammasome is also involved in both MS pathogenicity and EAE development,suggesting that NLRP3 plays a critical role in the induction of autoimmune disease.Hydrogen gas?H₂?was once considered an inert gas in the living body.However,Ohsawa et al.reported that molecular hydrogen can protect the brain from ischemia and reperfusion injury and reduce oxidative stress level in animals.Since then,additional properties of molecular hydrogen have been elucidated by many researchers,including anti-oxidative,anti-apoptotic,and anti-inflammatory effects.H₂ is non-toxic,inexpensive,and easily accessed via either ingestion of hydrogen-rich water or inhalation of H₂ mixed with oxygen;therefore,it has great potentials in preventive and therapeutic applications.It has been demonstrated that drinking H₂-rich water could delay the onset and mitigate the severity of EAE,but the molecular mechanism of the protective effect is poorly understood.Although the specific mechanism underlying the beneficial properties of H₂ remain unclear,selective reduction of reactive oxygen species?ROS?is a widely accepted explanation.ROS are usually formed as a byproduct of the normal metabolism of oxygen;however,overproduction of ROS can stimulate the NLRP3 pathway,thus aggravating damage in EAE.Because H₂ may selectively block ROS,and the ROS/NLRP3 pathway is critical to the development of EAE,we hypothesize that H₂ may exert beneficial effects on EAE by inhibiting the ROS/NLRP3 pathway.The main aim of the present study was to explore the protective effect of molecular hydrogen on experimental autoimmune encephalomyelitis and its underlying mechanism.Inhalation of 67%H₂/33%O₂ mixture has previously been demonstrated to be safe and effective;therefore,we used a high dose of inhaled H₂?67%?for our experiments.The project is majorly consisted of three parts:Part 1 Inhalation of high concentration hydrogen exerts beneficial effects in EAE.Objective:The inflammatory infiltrates and demyelination are pathological characteristics of multiple sclerosis and experimental autoimmune encephalomyelitis,which leads to neurological disorders and weights loss in EAE mice.Hydrogen is a novel antioxidant,has showed its beneficial effects among many diseases.The aim of this part was to explore the beneficial effects of inhalation of high concentrated hydrogen on experimental autoimmune encephalomyelitis.Methods:Female 8-10 weeks mice were immunized with myelin oligodendrocyte glycoprotein?MOG 35-55?dissolved in complete Freund's adjuvant?CFA?mixed with heat-killed Mycobacterium tuberculosis H37Ra.The established EAE mice were further randomly divided into three group:EAE group,mice from which were without further treatment;EAE+HCH group,high concentration of hydrogen was applied to the mice 2hours per day start from day 12 after immunization till the end of sacrifice;EAE+HCN group,mice from which were administrated with 2 hours mixed-gas?33%O₂+67%nitrogen?start from day 12 till the end of sacrifice.The mice without immunization were belong to group Control.Neurological scores were assessed daily and body weight was recorded weekly.mice were sacrificed at day 21,and the spinal cords were collected for Hematoxylin and eosin staining and Luxol fast blue staining;Immunohistochemistry were performed for CD4⁺T cells assessment;cytokine of TNF-?,IFN?,IL-1?and IL-10 were assessed by ELISA.Results:Mice from EAE group showed progressive neurological scores and weight loss since day 9 and reach the peak at day 15.Mice from EAE+HCH group have the same results before treatment.But administration of HCH ameliorated clinical scores and weight loss after onset.Meanwhile,mice from EAE+HCN group showed no significant differences in clinical scores and body weights compared with EAE.Plenty of inflammatory infiltrates cells and mass demyelination were observed in spinal cord sections from EAE mice.However,the HCH administration could reduce the infiltrates cells and demyelinated area in central nervous system;there is no statistical differences in mice between EAE+HCN and EAE.Compared with mice from Ctrl,CD4⁺T cells were highly assessed in spinal cord from EAE mice and EAE+HCN mice;however,less CD4⁺T cells in spinal cord were observed in mice from EAE+HCH group.Inflammatory cytokines of TNF-?,IFN?,IL-1?were increased in spinal cord lysate of EAE mice;but reduced concentration of TNF-?,IFN?and IL-1?were recorded from EAE+HCH mice,along with increased IL-10.No major differences were observed between group EAE and group EAE+HCN.Conclusion:Administration of high concentration of hydrogen could exert beneficial effects on EAE.And less T cells and reduced inflammation in CNS were derived from HCH treatment.Part 2 Inhalation of high concentration hydrogen alters the CD4⁺and CD11b⁺cells population and reduce Th17 cells response.Objective:To explore the effects of hydrogen inhalation on different types of immune cells including CD4⁺,CD19⁺,and CD11b⁺cells in EAE.And further assess influences of molecular hydrogen on the four major subtypes of CD4⁺T cells.Methods:The established EAE mice were randomly allocated into two groups:EAE group and EAE+HCH group.Mice from EAE+HCH group received 2 hours high concentration of hydrogen since day 12 after immunization.Mice were sacrificed at day 19,and monocytes obtained from spinal cord and spleen were cultured with MOG-stimulation for 48 hours.FACS was applied to detect the population of CD4⁺,CD19⁺and CD11b⁺;and further assess the population of Th1/Th17,Th2 and Treg.The supernatant collected from culture medium were used to test cytokines of IFN?,IL-4,IL-17,and TGF-?.RT-PCR was applied to assess the mRNA expression of T-bet,ROR?t,GATA3 and Foxp3.Results:Compared with EAE,monocytes collected from EAE+HCH mice showed less CD4⁺T cells and CD11b⁺microglia/macrophagy both in spleen and spinal cord;but no differences were tested in CD19⁺cells.The percentages and numbers of Th17 cells were less both in spleen and spinal cord from EAE+HCH compared with EAE;no differences were recorded among other types of CD4⁺cell types.Less amount of IL-17 were assessed in group EAE+HCH compared to EAE;the other cytokines has no statistical differences within two groups.RT-PCR showed that ROR?t indicated a lower expression in EAE+HCH group,meanwhile T-bet,GATA3 and Foxp3 did not show major differences within two goups.Conclusion:Inhalation of high concentration of hydrogen could reduce the CD4⁺T cells and CD11b⁺cells population,along with decreased Th17 cells population.Meanwhile other types of CD4⁺T cells were not influenced by hydrogen administration.Part 3 Molecular hydrogen attenuates Th17 cell differentiation by inhibiting NLRP3 activationObjective:The pathogenetic Th17 cells have been shown its important role during the process of EAE and MS.The inflammasome NLRP3 play an essential role during the Th17cells differentiation.In this part,we are going to explore the underlying mechanism on the reduction of Th17 cells caused by hydrogen and the association of NLRP3 during this process.Methods:In vivo,established EAE mice were randomly divided into two groups:EAE group and EAE+HCH group.Mice without any treatment were belong to group Control.Mice from EAE+HCH group were administrated by high concentration of hydrogen since day 12 till the sacrifice.Splenocytes were collected since day 19,and further stimulated with MOG for 48 hours.FACS were used to test the DCFDA in CD4⁺T cells;and western blot was used to assess the expression of IL-1?,caspase-1 and NLRP3.In vitro,purified na?ve T cells were collected through negative magnetic methods from 8-10 weeks C57bl/6 mice,and then cultured under Th17 polarizaiton conditions for 4 days.ATP?5mM?with or without hydrogen rich medium were added to Th17 polarized culture.FACS were applied to assess the numbers and percentages of Th 17 cells.Western blot was used to assess the expression of IL-1?,caspase-1,and NLRP3.Results:Compared with EAE,the expression of ROS in CD4⁺T cells were significantly reduced in EAE+HCH group.Western blot shows that less expression of IL-1?,caspase-1and NLRP3 were observed in EAE+HCH group.ATP could increase the number of Th17cells during the Th17 differentiation,which correlated with enhanced expression of of IL-1?,caspase-1,and NLRP3 protein.Molecular hydrogen could inhibit the number of Th17cells during differentiation;and less expression of IL-1?,caspase-1,and NLRP were observed through administration of molecular hydrogen.Conclusion:Molecular hydrogen could inhibit NLRP3 activation by reducing ROS in CD4+T cells,which leads to a decreased population of Th17 cells during its differetiation.Statement:In summary,we found that inhalation of high concentration of hydrogen could provide beneficial effects against EAE.The ROS/NLRP3 pathway play a critical role in mediating the beneficial actions of H₂ against Th17-mediated EAE.As such,inhalation of high concentrated hydrogen may be a useful method in future clinical applications.