The Role Of HMGB1during The Progression Of Experimental Autoimmune Encephalomyelitis

Background:High mobility group box1(HMGB1) is a highly conserved nuclear nonhistone chromatin associated protein, which plays different roles according to the expression pattern such as the amount, cell location and subcellular location. It has been recently demonstrated that the systemic HMGB1is associated with autoimmune encephalomyelitis. HMGB1has also been reported to induce neuroinflammation after injury, such as lesions in the spinal cord and brain. However, the dynamic change of HMGB1expression pattern in the spinal cord that may be involved in the progression of disease is not folly understood. This study provides new evidence to reveal the role of HMGB1during the progression of EAE, but also provides a new therapeutic idea for blocking of HMGB1locally, which not only attenuated the clinical symptoms of EAE but also inhibited CNS inflammation.Objective:To unravel the amount, cell location and subcellular location of HMGB1in adult mice spinal cords during various stages of experimental autoimmune encephalomyelitis (EAE) and also to clarify the potential role of HMGB1in EAE following HMGB1antagonist.Methods:C57BL/6mice were used for induction of EAE by MOG35-55immunization. EAE disease course was divided into pre-onset, onset, peak and Chronic stage according to clinical score and disease development. Normal animals were used as control. Samples in four stages of EAE were collected on matching days and analyzed as following:1. HMGB1expression in the spinal cord was detected using western blot and immunohistochemical staining. EL1SA assay was used to analyze the level of HMGB1in different body fluids (sera, spinal cord homogenate and cerebrospinal fluid). The expression of endogenous HMGB1in the nuclear and cytoplasmic fractions in the spinal cord cells were assayed by immunohistochemical staining and western blot. The expression and CNS cellular distribution of TLR4in the spinal cord was detected by immunohistochemistry and immunofluorescence double staining. Meantime, the spinal cord inflammatory cells infiltration was stained by hematoxylin-eosin staining.2. Double Immunofluorescence staining was used to detect nuclear localization of HMGB1in astrocytes, microglia and neurons from normal adult mouse spinal cord and CNS cell lines (murine neuron-like N2a, murine microglia-like BV2, and human astrocyte-like U87) and primary cultured neurons and astrocytes. Double immunofluorescence staining was used to observe the dynamic change of HMGB1expression pattern in the spinal cord during the progression of EAE.3. The effects of HMGB1antagonist (glycyrrhizin (GL) or neutralizing anti-HMGB1monoclonal antibody (HMGB1Ab)) on EAE mice were investigated by assessing EAE clinical features, pathological characteristic, HMGB1expression, changes in CNS neurons, astrocytes and microglia and the level of HMGB1in different body fluids of both prophylactic and therperpeutic treatment with GL intraperitoneal (i.p.) injection or HMGB1Ab i.p. or intracerebroventricularly (i.c.v.) injection. The effects of HMGB1Ab i.p. treatment on spleen Thl and Th17cells amount were measured by Flow cytometry.Results:1. HMGB1increased during the progression of EAE:The amount of total HMGB1protein in the spinal cord was significantly increased at each stage and reached the highest in the onset stage as compared to that in the normal group. The level of HMGB1was significantly increased in different body fluids (sera, spinal cord homogenate and cerebrospinal fluid) during the progession of EAE. HMGB1translocated from the cell nucleus to the cytoplasm in the spinal cord cells at the peak period of EAE. TLR4was expressed in neurons, astrocytes and microglia and increased during the progression of EAE. Histopathological examination of the spinal cord revealed inflammatory cells infiltration, especially in the onset and peak period of EAE.2. HMGB1is expressed in the nuclei of spinal cord resident cells such as some astrocytes, microglia and a few neurons in normal situation. HMGB1is expressed in the nuclei of mouse neuroblastoma N2a cell line (N2a), mouse microglial cell line (BV2) and human glioma cell line (U87), and also HMGB1is normally present in the nuclei of primary cultured neurons and astrocytes. However, during EAE progression, the total and extracellular HMGB1in the spinal cord are increased, more HMGB1positive astrocytes and microglia are observed, and the intra-neurons HMGB1in the ventral horn and around the central canal localize majorly in the cytoplasm accompanied by the increasing extracellular HMGB1.3. In vivo, the preventive GL i.p. treatment had no significant influence on the progression of EAE. The therperpeutic GL treatment i.p. can alleviate the clinical symptoms of EAE. The therperpeutic HMGB1Ab treatment i.c.v. almost inhibited the progression of EAE totally. Therperpeutic GL or HMGB1Ab treatment exerts potent antiinflammatory effects that led to improved clinical score, which correlated with the reduced spinal cord inflammation and demyelination. It also decreased the level of HMGB1in different body fluids and the expression of HMGB1in the spinal cord. HMGB1Ab treatment also decreased the amount of peripheral spleen Thl and Th17cells, and downregulated the activation of astrocytes, microglia and improved the recovery of damaged neurons in the spinal cord of EAE mice.Conclusion:Our findings observe that the dynamic change of HMGB1expression pattern in the spinal cord during the progression of EAE. Blocking HMGB1can alleviate clinical symptoms in the onset of EAE. HMGB1may be a potential target for autoimmune encephalomyelitis (multiple sclerosis in human) therapy.