Biomarkers And Related Mechanisms In Patients With Anti-N-methyl-D-aspartate Receptor Encephalitis

Background:In recent years,the existence of autoantibodies against neuron cell surface or synaptic protein and its relationship with autoimmune encephalitis have been confirmed,among which the anti-N-methyl-D-aspartate receptor(anti-NMDAR)encephalitis is a kind of disorder whose autoantibodies aimed at structure of extracellular domain in GluN1(NR1)subunit of NMDAR,and for the first time in 2007 found in young women with ovarian teratoma characterized by diffuse encephalopathy,epilepsy and mental symptoms.Usually its clinical manifestations include mental symptoms and memory disorders,with varying degrees of movement disorders,epilepsy and autonomic dysfunction.These symptoms may develop into coma,in rare cases can lead to deathThyroid hormone exerts important influence on growth and physiological process.It can have an impact on many systems,including the most important targets,the central nervous system.In the process of human development,the changes of the supply of thyroid hormone can lead to irreversible mental retardation and neurologic deficit.In adults,thyroid hormones can affect mood and behavior.And thyroid disease may lead to mental symptoms.Several reports have revealed the intertwining relationships between systemic immune abnormalities,particularly autoimmune thyroid diseases,and immune-mediated neurological disorders,such as multiple sclerosis,and neuromyelitis optica spectrum disorder(NMOSD).Autoantibodies that are indicative of systemic immune disorders,such as antinuclear antibodies,have also been detected in patients with autoimmune encephalitis,but the literature in this regard is limited.Recent studies suggest that free tri-iodothyronine(fT3),an indicator of thyroid function,can serve as a prognostic factor in critical illness,as well as some autoimmune diseases(e.g.,systemic lupus erythematosus and NMOSD).Thyroid hormone is also closely involved in the immune system,such as cellular immunity.However,the association between thyroid function/autoimmunity and anti-NMDAR encephalitis has not been discussed.Does anti-NMDAR encephalitis concomitant with thyroid function changes?What are the clinical characteristics associated with these changes?Are there epigenetic changes such as IncRNA and mRNA changes in the thyroid function of anti-NMDAR encephalitis?Which functional pathways are primarily affected at the transcriptome level?These questions still need further discussion.Epigenetic regulation of immunity and inflammation is an emerging field.Long non-coding RNA(IncRNA)is non-protein coding RNA with more than 200 nucleotides in length.Through a variety of mechanisms,they play an important role in many biological processes,including adjusting the innate and adaptive immune response and immune cells development.The immune system provides a highly ordered biological environment in which cellular phenotypes and functions are finely delineated,cellular components are easily manipulated,and interference can be achieved at the molecular and cellular levels through in vitro and in vivo models.There is a significant gap of the role of IncRNA in the immune system and autoimmune diseases in future prospects,and it is expected to be a potential biomarker.Autoimmune encephalitis is a serious disease,which is increasingly being diagnosed and described in the literature,especially anti-NMDAR encephalitis,one of the most common autoimmune encephalitis.Anti-NMDAR encephalitis can appear a wide range of symptoms and signs,these signs and symptoms may be difficult to diagnosis.Current detection of anti-NMDAR encephalitis,including brain magnetic resonance imaging(MRI)and cerebrospinal fluid(CSF)studies,can be nonspecific or normal.Antibodies in both serum and cerebrospinal fluid are specific and tend to be more sensitive in cerebrospinal fluid,but the results are often delayed.Considering the critical importance of early diagnosis and treatment in anti-NMDAR encephalitis which is in relation to improved outcomes,it is necessary to use other objective tests to rapidly diagnose and identify and potentially monitor the course of treatment,and to further investigate the epigenetic mechanisms of anti-NMDAR encephalitis.Therefore,in the first part,we studied the relationship between thyroid function and immune-related indicators in anti-NMDAR encephalitis,and explored possible functional pathways,including thyroid function,affecting anti-NMDAR encephalitis from the transcriptome level in the second partObjective:To assess the prevalence of thyroid dysfunction and anti-thyroid antibodies in patients with NMDAR encephalitis and to investigate the relationship between thyroid dysfunction/autoimmunity and clinical features of anti-NMDAR encephalitis.To understand potential involvement of epigenetic mechanisms in pathogenesis of anti-NMDAR encephalitis,we initiated a study to compare the expression of lncRNAs and mRNAs in patients of anti-NMDAR encephalitis and healthy controls.Methods:1.This retrospective study enrolled 43 patients with anti-NMDAR encephalitis who were admitted to the Shandong Provincial Hospital affiliated to Shandong University from January 2014 to January 2018.Another 225 ordinary people from the health examination center at our hospital were selected as the contrast group randomly.Both of them were examined for thyroid function and antibodies.Examinations including brain magnetic resonance imaging(MRI),CT scan of the thorax,ultrasound of the abdomen and pelvic region,and lumbar puncture were done in all of the patients.Most of them were checked immunological related indications.We also evaluated the severity of illness through modified Rankin scale(mRS)scores on blood samples at admission,at discharge,and 3 months after discharge.Additional data comprised age,sex,time from symptom onset to treatment initiation,and treatments received.Patients were further classified into 2 subgroups based on their fT3 levels.And clinical characteristics,disease severity and prognosis were compared.Then correlation analysis was conducted to further analyze the factors related to anti-nmdar encephalitis and low T3 syndrome.Finally,by paired comparison of fT3 level and mRS before and after treatment,the relationship between disease improvement and fT3 level was studied.FT3 levels were also investigated after at least three months of follow-up.2.11 patients who were diagnosed as anti-NMDAR encephalitis were enrolled in our purely observational studies.Total RNA was extracted from patients' plasma and changes in IncRNAs and mRNAs expression levels were examined.Differential expression analysis via RNA sequencing,Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway analysis,as well as a co-expression network of lncRNA-mRNA were performed in 5 patients and 5 healthy controls to evaluate the changes.The expression levels of certain IncRNAs and mRNAs were further validated in 11 patients and 11 healthy controls using quantitative real-time polymerase chain reaction(qRT-PCR).Results:1.Serum levels of fT3 and thyroid-stimulating hormone(TSH)were found to be relatively lower in patients with anti-NMDAR encephalitis than in controls(both p<0.001).Low T3 syndrome also occurred more frequently in anti-NMDAR encephalitis(25.6 vs.0.4%,p<0.001).However,no statistical differences were detected between patients and CTL in terms of the positive rate of thyroid antibodies and other types of thyroid dysfunction.Patients with low T3 levels tended to have a longer hospital stay(p = 0.006),a higher rate of abnormal brain magnetic resonance imaging(MRI)findings(p = 0.033),a higher frequency of consciousness declination(p = 0.029).MRS score at admission and discharge was also higher(p =0.002,p = 0.022),but there was no significant difference in mRS score at 3 months after discharge.Low fT3 levels were also associated with abnormal brain MRI findings,a decline in consciousness,and the mRS score on admission(p = 0.002,p<0.001,p<0.001).In addition,flT3 seemed to gradually return to normal levels upon improvement of the mRS score(r ?-0.649,p p 0.002).2.It was found that a total of 83 IncRNAs and 2345 mRNAs were differentially expressed in five patients with anti-NMDAR encephalitis compared with five healthy controls.Of those IncRNAs,63 were upregulated and 20 downregulated,while 1509 mRNAs were upregulated and 836 downregulated.GO and KEGG pathway analyses showed that a wide range of biological functions were perturbed during acute anti-NMDAR encephalitis.Most differentially expressed genes were found to be involved in viral carcinogenesis,systemic lupus erythematosus,phagosome,chemokine signaling pathway,Alzheimer's disease and B cell receptor signaling pathway.qRT-PCR was conducted in 11 patients and 11healthy controls to further confirm the levels of six IncRNAs and four mRNAs,and the results were found to be consistent with those by RNA sequencing.The co-expression networks of IncRNA-mRNA were performed in some meaningful KEGG analyses,including chemokine signaling pathway,Long-term potentiation,B cell receptor signaling pathway and MAPK signaling pathway.Conclusions:Low T3 syndrome often copresents in anti-NMDAR encephalitis and indicates a higher clinical severity,more common in consciousness declination,a longer hospitalization and a higher abnormal MRI findings rate,and.However,fT3 levels do not seem to influence the prognosis of anti-NMDAR encephalitis.And in the second part,these findings suggest the involvement of a number of molecular pathways in anti-NMDAR encephalitis of which might serve as potential biomarkers to assist in diagnosis,treatment and prognosis.