Wen-Bin Zhou

Possible involvement of toll-like receptors in the pathogenesis of myasthenia gravis.

Toll-like receptors (TLRs), the innate immunity components, have been demonstrated to participate in multiple autoimmune diseases. However, our knowledge of the roles of TLRs in myasthenia gravis (MG) is still scarce. In this study, we detected the mRNA expression of TLR1 to TLR10 in peripheral blood mononuclear cells (PBMCs) of MG patients and the healthy controls by quantitative real-time polymerase transcription chain reaction. Our data demonstrate that aberrant expressions of TLRs exist in the PBMCs of MG patients and of the total, expression level of TLR9 mRNA has significantly positive relation with the clinical severity of MG, which suggests that TLRs may be involved in the pathogenesis of MG.

Clinical and MRI Characteristics of Levamisole-Induced Leukoencephalopathy in 16 Patients

To study the clinical and radiological characteristics of levamisole‐induced leukoencephalopathy (LILE) in patients with recurrent aphthous ulcers (RAU) or infected with Ascaris.

Delivery of an miR155 inhibitor by anti-CD20 single-chain antibody into B cells reduces the acetylcholine receptor-specific autoantibodies and ameliorates experimental autoimmune myasthenia gravis

MicroRNA‐155 (miR155) is required for antibody production after vaccination with attenuated Salmonella. miR155‐deficient B cells generated reduced germinal centre responses and failed to produce high‐affinity immunoglobulin (Ig)G1 antibodies. In this study, we observed up‐regulation of miR155 in the peripheral blood mononuclear cells (PBMCs) of patients with myasthenia gravis (MG), and miR155 was also up‐regulated in torpedo acetylcholine receptor (T‐AChR)‐stimulated B cells. We used an inhibitor of miR155 conjugated to anti‐CD20 single‐chain antibody to treat both the cultured B cells and the experimental autoimmune MG (EAMG) mice. Our results demonstrated that silencing of miR155 by its inhibitor impaired the B cell‐activating factor (BAFF)‐R‐related signalling pathway and reduced the translocation of nuclear factor (NF)‐κB into the nucleus. Additionally, AChR‐specific autoantibodies were reduced, which may be related to the altered amounts of marginal zone B cells and memory B cells in the spleens of EAMG mice. Our study suggests that miR155 may be a promising target for the clinical therapy of MG.

Silencing miR-146a influences B cells and ameliorates experimental autoimmune myasthenia gravis

MicroRNAs have been shown to be important regulators of immune homeostasis as patients with aberrant microRNA expression appeared to be more susceptible to autoimmune diseases. We recently found that miR‐146a was up‐regulated in activated B cells in response to rat acetylcholine receptor (AChR) α‐subunit 97‐116 peptide, and this up‐regulation was significantly attenuated by AntagomiR‐146a. Our data also demonstrated that silencing miR‐146a with its inhibitor AntagomiR‐146a effectively ameliorated clinical myasthenic symptoms in mice with ongoing experimental autoimmune myasthenia gravis. Furthermore, multiple defects were observed after miR‐146a was knocked down in B cells, including decreased anti‐R97‐116 antibody production and class switching, reduced numbers of plasma cells, memory B cells and B‐1 cells, and weakened activation of B cells. Previously, miR‐146a has been identified as a nuclear factor‐κB‐dependent gene and predicted to base pair with the tumour necrosis factor receptor‐associated factor 6 (TRAF6) and interleukin‐1 receptor‐associated kinase 1 (IRAK1) genes to regulate the immune response. However, our study proved that miR‐146a inhibition had no effect on the expression of TRAF6 and IRAK1 in B cells. This result suggests that the function of miR‐146a in B cells does not involve these two target molecules. We conclude that silencing miR‐146a exerts its therapeutic effects by influencing the B‐cell functions that contribute to the autoimmune pathogenesis of myasthenia gravis.

Altered expression of miR-146a in myasthenia gravis

The purpose of this study was to analyze the expression of miR-146a in PBMCs obtained from patients with myasthenia gravis (MG) and healthy controls and to investigate the effect of the inhibition of miR-146a on the activation of AchR specific B cells obtained from mice. The expression of miR-146a levels in PBMCs obtained from patients with MG and healthy controls were determined by qRT-PCR. MiR-146as complementary fragment, AntagomiR-146a, was synthesized as inhibitor, and the nonfunctional fragment, which has similar construction to AntagomiR-146a, was synthesized as negative control inhibitor. The expression of miR-146a, CD40, CD80 and CD86 on AchR specific B cells were analyzed by qRT-PCR and flow cytometry. Western blotting was used to detect the expression of TLR4, NF-κB and Bcl-2 .The expression of miRNA-146a in PBMCs obtained from patients with MG was significantly upregulated compared to healthy controls (P<0.01). Transfection with miR-146a inhibitor dramatically decreased expression of miR-146a, CD40, CD80, TLR4 and NF-κB on AchR specific B cells compared to mock transfected cells. We conclude that abnormal expression/regulation of miR-146a may play an important role in the regulation of AchR specific B cells and contribute to the pathogenesis of MG.

FOXP3 −3279 and IVS9+459 polymorphisms are associated with genetic susceptibility to myasthenia gravis

Myasthenia gravis (MG) is an autoimmune disorder in which CD4(+)CD25(+) FOXP3(+)regulatory T cells (Tregs) are thought to play important roles in driving the ongoing autoimmune response. Although it is known that single-nucleotide polymorphisms (SNPs) in the fork head/winged-helix transcription factor 3 (FOXP3) gene contribute to some autoimmune diseases, information about the role of this gene in MG is limited. We therefore evaluated the association between FOXP3 gene SNPs and susceptibility to MG in a Han Chinese population. In a hospital-based, case-control study, two SNPs in the FOXP3 gene (-3279 and IVS9+459) were investigated in 118 MG and 124 healthy controls, and their relationship with the four parameters of gender, onset age, thymus pathology, and clinical classification of MG were performed with a stratified analysis. We found that the frequency of the FOXP3 IVS9+459 G allele was significantly lower in MG patients than in healthy controls (P=0.041), while the frequency of the FOXP3 -3279 polymorphisms was not significantly different between the two groups. Our results suggest that FOXP3 IVS9+459 polymorphisms appear to have an effect on the risk of MG in a Han Chinese population, and the G allele may be a genetic protective factor to MG.

Expression of Toll-Like Receptors 2, 4 and 9 in Patients with Guillain-Barré Syndrome

Objective: A myriad of transcription factors and inflammatory cytokines have been described to participate in the pathogenesis of Guillain-Barré syndrome (GBS). However, the innate immunity components – Toll-like receptors (TLRs) – have never been explored in this disease. We therefore investigated the expression of TLR2, 4 and 9 in the peripheral circulation of GBS patients as well as healthy controls. Methods: Twenty-one GBS patients and 21 healthy donors participated in this study. Peripheral blood mononuclear cells were used for mRNA and protein analysis of TLR-related molecules. Also, peripheral blood mononuclear cells from different subjects were incubated with different TLR agonists and the subsequent IFN-γ secretion was determined. Results: Expression of TLR2, 4 and 9 as well as their related signaling molecules were higher in GBS patients compared to healthy controls. Disability scores of GBS patients had a strong positive correlation with the high levels of expression of TLR2, 4 and 9. Conclusions: The TLR signaling pathway may be involved in the pathogenesis of GBS.

Inactivation of TLR9 by a Suppressive Oligodeoxynucleotides Can Ameliorate the Clinical Signs of EAN

Susceptible-strain animals immunized with P2 peptide could generate the disease of experimental autoimmune neuritis (EAN) with inflammation and demyelination of peripheral nerve. A myriad of transcription factors and inflammatory cytokines have been found to participate in this process; however, the roles of toll-like receptors (TLRs) are poorly understood in EAN. The aim of this study is to explore the role of TLR9 in the pathogenesis of EAN. The EAN was induced in Lewis rat by immunization with P253–78 and complete Freund’s adjuvant. CpG oligodeoxynucleotides (ODN) (cODN), a suppressive ODN (sODN) and a control non-specific ODN (nODN) were respectively administered to explore the role of TLR9 in EAN both in vivo and vitro. Following immunization up to the peak phase of EAN, EAN rats inoculated with sODN had remarkably better clinical score of EAN and expressed a significantly inhibited TLR9 signaling pathway. Our study suggests that TLR9 may be involved in the pathogenesis of EAN.

Macrophage migration inhibitory factor is necessary for the Lipo-oligosaccharide-induced response by modulation of Toll-like receptor 4 in monocytes from GBS patients

Previous studies have suggested that macrophage migration inhibitory factor (MIF) may play a critical role in the pathogenesis of Guillain-Barre syndrome (GBS); however, its definite mechanism remains unknown. In this study, we prepared the monocytes from the peripheral blood mononuclear cells (PBMCs) of GBS patients and the controls. Lipo-oligosaccharide (LOS) from Campylobacter jejuni was used as the stimulus of the monocytes in vitro and siRNA-MIF was used to explore the roles of MIF in LOS-induced response. Significantly, silencing of MIF attenuated the LOS-induced up-regulation of Toll-like receptor 4 (TLR4) and translocation of NF-кB into the nucleus; we also observed the up-regulation of IL-12p40, TNF-α, IL-6, CXCL8 and CCL5 in GBS monocytes with LOS stimulus; and siRNA-MIF overrided the effects of LOS on the production of the TNF-α, IL-6, and CXCL8. Conclusively, our study provides evidences that MIF may participate in the pathogenesis of GBS by modulating the LOS-induced response through TLR4 signaling pathway.

Transcriptional repressor Blimp1 regulates follicular regulatory T cells homeostasis and function

The B‐lymphocyte‐induced maturation protein 1 (Blimp1) regulates T‐cell homeostasis and function. Loss of Blimp1 could double the proportion of follicular regulatory T (Tfr) cells. However, the effects that Blimp1 may have on the function of Tfr cells remain unknown. Here we document the function for Blimp1 in Tfr cells in vitro and in vivo. Data presented in this study demonstrate that Tfr cells indirectly inhibit the activation and differentiation of B cells by negatively regulating follicular helper T cells, so lowering the secretion of antibody. Lack of Blimp1 makes the immune suppression function of Tfr cells impaired in vitro. In the in vivo study, adoptive transfer of Tfr cells could reduce immune responses in germinal centres and relieve the muscle weakness symptoms of mice with experimental autoimmune myasthenia gravis. Blimp1 deficiency resulted in reduced suppressive ability of Tfr cells. This study identifies that Tfr cells are potent suppressors of immunity and are controlled by Blimp1.