A Study Of The Triggering,Maintaining And Regulation Mechanism Of Humoral Immunity In The CNS Of Neurosyphilis

Part 1 Aberrant Humoral Immune Responses in Neurosyphilis: CXCL13/CXCR5 play a pivotal role for B cell recruitment to the CSF Background: Syphilis,a sexually transmitted disease caused by Treponema pallidum,can progress to affect the central nervous system,resulting in neurosyphilis(NS).Accumulating evidence suggest that the humoral immunity participate in neurological damage in neurosyphilis patients.However,the triggering and maintaining mechanisms of humoral immune responses involved in neurosyphilis have remained unknown.Methods: Using flow cytometry expression of B cells and B cell subsets was measures in CSF sample of 111 retrospectively indentified individuals.The sample represented neurosyphilis,including asymptomatic neurosyphilis(23 patients),symptomatic neurosyphilis(43 patients),and non-NS(45 patients).36 patients with the first follow-up and 17 patients with the second follow-up were available for follow-up requirement.Immunoglobulin(Ig M,Ig A,and IgG)and CXCL13 concentrations were detecteded by ELISA.A modified chamber assays were used to migration assays and inhibition assays.The migrated total B cells and B cell subsets were analysed and the migration was inhibited by preincubation of the CSF samples with neutralizing CXCL13 antibody.Section from one patient with the left parietal cortex syphiloma was screened using immunohistochemistry for the presence of spirochete,CD20+B cells,CD3+T cells,CD138+plasma cells and CD35+ follicular dendritic cells,and for the expression of lymphoid chemokine CXCL13 and its cognate receptor,CXCR5.Results: We found that enrichment of B cells were observed and activated in the cerebrospinal fluid(CSF)of NS patients compare to non-NS patients(P<0.001).The immunoglobulin indices in untreated NS patients were significantly higher than the indices in the non-NS patients(P<0.001,P<0.001,P<0.001;respectively)associated with the progress to neurosyphilis.The elevated levels of CSF CXCL13 were also found in CSF of patients with NS(P<0.001).Moreover,high expression of CSF CXCL13 mediated B cells migration both in vitro and in vivo.More importantly,there was a positive correlation between the CSF B cells,immunoglobulin indices,and CSF CXCL13 levels(P<0.05).Moreover,the chemotactic activity of NS CSF was significantly higher than that of non-NS and was reduced up to 53% after preincubation with a neutralizing CXCL13 antibody.Follicle-like structures containing B cells,T cells and plasma cells,and a network of follicular dendritic cells producing CXCL13,a key chemokine of CXCR5+ B cell migration,were observed in the intracranial syphilitic gumma.Conclusion: CXCL13/CXCR5 mediates aggregation of B cells,which directs the aberrant humoral immune response via formation of ectopic germinal centers(EGCs).These date reflect the mechanism of neurological damage in NS and also have implication for potential immunotherapy.Part 2 IgG Subclasses in Neurosyphilis: Regulation of the IgG Subclasses Distribution by Th1/Th2 CytokinesBackground: Neurosyphilis(NS)has shown a humoral immune response with high production of immunoglobulin in central nervous system.Since the cytokine environment seems to be important in the regulation of immunoglobulin production and in the switch between different isotypes and subclasses,and since the subclasses of IgG have different functions,we wanted to examine the IgG subclass distribution and its regulation mechanism in neurosyphilis.Methods:Innate immune cytokines(IL-1?,IL-6,IL-8,TNF-?),Th1 cytokines(IFN-?,IL-2,IL-12p70)and Th2 cytokines(IL-4,IL-10,IL-13)were detected by Meso Scale Discovery in CSF sample of 126 retrospectively indentified individuals.The sample represented healthy controls(15 donors),neurosyphilis,including asymptomatic neurosyphilis(23 patients),symptomatic neurosyphilis(43 patients),and non-NS(45 patients).31 patients with the first follow-up and 15 patients with the second follow-up were available for follow-up requirement.IgG subclasses(IgG1,IgG2,IgG3,IgG4)were measured by ELISA.Results:We found that high expression of cytokines(IL-1?,IL-6,IL-8,TNF-?,IFN-?,IL-2,IL-12p70,IL-4,IL-10,IL-13)were observed in the cerebrospinal fluid(CSF)of NS patients compare to non-NS patients and healthy controls(P<0.05).The IgG subclasses(IgG1,IgG2,IgG3,IgG4)in NS patients were significantly higher than that in non-NS(P<0.05)and healthy controls(P<0.001),but there was no difference between non-NS and healthy controls.Moreover,the situation of coexistence of Th1 and Th2 with secretion of IFN-? and IL-13 in CNS of neurosyphilis,suggests an association between those two cytokines and IgG subclasses(IgG1,IgG2,IgG3,IgG4),especially IgG1 and IgG3.For the longitudinal study,the IgG1,IgG2,and IgG3(P<0.05)consistently decreased after the first treatment,but IgG3 displayed no decrease after the second follow-up(P=0.147).Conclusion:IFN-? and IL-13 are involved in regulation of IgG subclasses distribution in CSF of patients with neurosyphilis.IgG3,as a protective immunoglubolin,might be used as an alternative surrogate marker for assessing the efficacy of clinical treatment of neurosyphilis patients.