Investigation Of Immune Modulation In Neurological Diseases

Objective: In classical neurological disease,like multiple sclerosis,immune system is activated first at periphery,then activated immune cells infiltrate into the CNS and iniate the inflammation,leading to disease progression.In the last decades,the altenative CNS immune activation pathway was gradually clarified,in almost all neurological diseases,like stroke,Alzheimer's disease,Parkinson disease,et al.immune system pariticipate in the pathological processes.In these conditions,CNS neuronal cells are damaged first,the the components of CNS are exposed to peripheral immune system,which subsequently activated the immune system,followed with the upregulation of inflammation in the brain.In this dissertation,my work consists of investigating the immune mechanism as well as related immune modulation strategy in both classical neuroimmune disease neuromyelitis optica spectrum disorders(NMOSD)and stroke.The objective of first part is: Modulation of sphingosine 1-phosphate receptor(S1PR)via fingolimod(FTY720)has shown efficacy in preclinical models and clinical trials in patients with acute ischemic stroke(AIS).While promising,fingolimod-induced bradycardia and the incomplete understanding of its mechanisms of action limit the design of new trials in AIS patients,also unknown were the time window of S1 PR modulation for ischemic stroke and the best responders for this treatment.The second-generation S1 PR modulator RP101075 selectively acts on S1PR1 and readily distributes in the brain,but minimizes cardiac side effects.We therefore tested the impact of RP101075 in animal models of ischemic stroke.To assess the translational significance,we determined whether neuroprotection can be obtained in multiple stroke models,whether delayed treatment was still effective,and whether RP101075 has effects on lymphocyte migration and preservation of brain vasculature post-ischemia.In 2nd part: Neuromtelitis optica spectrum disorders(NMOSDs)is a severe autoimmune central nervous system(CNS)disease which mainly targets cerebral spinal cord and optica nerves.The disease course of NMOSDs is oftern more severe than multiple sclerosis,another CNS autoimmune demyelinating disease.The incidence of NMOSD is higher in Asia than western world,with more patients are young women.The main pathological characteristics of NMOSD are deposit of complement companents and Ig Gs in the lesion area,which cause the destroy of astrocytes and demylination.Neural stem cells(NSCs)can self renew and differentiate,may have potential in regeneration therapy of serever CNS diseases.However,a major hurdle for effective stem cell therapy is ongoing inflammation in the target organ.Here we tested a new strategy to recondition the inflammatory microenvironment in the central nervous system to promote neural stem cell therapeutic effect in a mouse model of Neuromyelitis optica spectrum disorder(NMOSD).Methods: Several mouse models of brain ischemia with different infarct volumes and different lesion sites were used in this study,which were achieved by inducing middle cerebral artery occlusion(MCAO)with different time duration: 30 min,60 min,and 90 min;and also photothrombotic(PT)cortical infarction model.RP101075 were given via gavage once daily at a dosage of 0.6mg/kg or 0.3mg/kg,and 0.6mg/kg twice a day in some experiments.We first detected the effects of RP101075 on brain lesion and neurodeficit scores at different dosages and administration frequencies,fingolimod and a S1PR1 selective antagonist were used as controls.We also confirmed whether the efficacy of RP101075 was consistent to a relatively long duration after 3 doses.Then we determined the treatment time window via initiating administration of RP101075 to 60 min MCAO mice at different time point post ischemia.To determine which type of stroke responds best to RP101075 treatment,we then evaluated the efficacy of RP101075 on different stroke models and the effects sizes on infarct volume reduction.For the mechanical study,we detected the effects of RP101075 on rag2-/-gc-/-mice with MCAO to verify whether the efficacy of S1PR1 modulation is dependent on peripheral immune modulation.Flow cytometry was used to detect the lymphocytes counts in the peripheral blood and brain tissue after ischemia,real-time polymerase chain reaction(RT-PCR)was used to detect the brain inflammation level at different regions.Fibrinogen level leaked in the brain tissue was determined by western blot.Immunohistochemistry was used in the detection of fibrin and platelets deposition,blood brain barrier integrity,and micro vessel constriction.Mass spectrometry and liquid chromatography are used to determine the concentration of RP101075 in the brain and blood.In the second part: The NMOSD-like lesions in the brain were induced by injection of purified serum Ig G from NMOSD patients with strong NMO-Ig G positivity and human complement(h C),which mimics the complement-mediated pathological process of NMOSD patients.NSCs were separated from fetal mice,then were modified by letivirus contained with vetor complement factor H related protein 1(CFHR1)-green fluorescent protein(GFP),termed as CFHR1-NSCs.NSCs modified by GFP vector contained lentivirus were used as control(GFP-NSCs).The modified NSCs were transplanted to the brain immediately,3h,and 12 h after model induction.The therapeutic effects of engineered NSCs were evaluated at 7 day via MRI and histology.Mice primary astrocytes were used for in vitro mechanism experiments.Results: RP101075 reduced infarct volume and improved neurodeficits in C57BL/6 mice after brain ischemia.The beneficial effects of S1PR1 modulation was maintained up to12 hours after the onset of ischemia,and was effective with different sizes and locations of brain infarct.RP101075 inhibited the lymphocytic infiltration of the brain and reduced local inflammatory response with transient lymphopenia.Additionally,RP101075 improved the integrity of blood brain barrier and preserved brain microvasculature.In NMOSD study,we found that CFHR1-NSCs could attenuate inflammatory infiltration and immune-mediated damage of astrocytes,which is a cardinal pathogenic progress in NMOSD patients.Further,we demonstrated that transplantation of the CFHR1 modified NSCs effectively blocked the complement activation cascade and inhibited the formation of membrane attack complex(MAC),thus contributing to the protection of endogenous and transplanted NSC differentiated-astrocytes.Conclusions: The identification of RP101075 as a new S1PR1 modulator with an improved safety profile and time window,with its dual action on lymphocyte migration and brain vasculature,offers a new possibility of safe S1 PR modulation as a potential therapy for AIS.These findings suggest that manipulation of the lesion microenvironment contributes to a more effective cell replacement therapeutic strategy for autoimmune diseases of the central nervous system.