The Pathogenic Roles Of Toll-like Receptor2in Systemic Lupus Erythematosus And Its Molecular Mechanisms

Systemic lupus erythematosus (SLE) is a prototypical autoimmune disease with diverse and nonspecific presentations. The exact molecular mechanisms underlying SLE remain elusive. However, the dysregulation of innate immune system are generally believed to play an important role in the pathogenesis of SLE. Toll-like receptor2(TLR2) belongs to pattern recognition receptors, it can recognize both exogenous pathogen-associated molecular patterns (PAMPs) and endogenous damage-associated molecular patterns (DAMPs). The stimulation of TLR2by PAMPs or DAMPs can contribute to the initiation and maintenance of the perpetuated inflammatory reactions in autoimmune diseases, including SLE. It was reported that, the mRNA level of TLR2elevated in the peripheral blood mononuclear cells (PBMCs) in SLE patients, which indicated that TLR2may play a role in the development of SLE. To determine the pathogenic roles of TLR2in SLE and its molecular mechanisms, we designed the following studies. Firstly, we detected the expressional level of TLR2in the CD4+T cells from SLE patients and normal controls. Secondly, we investigated the relationship between TLR2stimulation and autoimmunity. Lastly, we explored the epigenetic mechanisms by which TLR2stimulation increased the expressional levels of IL-17a and IL-17f.In our study, we found that the expressional level of TLR2increased in CD4+T cells from SLE patients. The stimulation of TLR2could enhance the self-response of CD4+T cells and promote the production of inflammatory cytokines. In addition, the ligation of TLR2could mediate the histone acetylation and methylation of the promoter region of IL-17a and IL-17f, thus enhances the IL-17response. Our studies revealed the role of TLR2in the pathogenesis of SLE, thereby providing a theoretical basis for more effective therapy of SLEPart1Expression of TLR2in CD4+T cells from SLE patientsObjective:To explore the expressional level of TLR2in CD4+T cells from SLE patients.Methods:Peripheral blood mononuclear cells (PBMCs) from six healthy controls and eight SLE patients were isolated by Ficoll-Hypaque density gradient centrifugation. CD4+T cells were then isolated by positive selection using magnetic beads. TLR2mRNA level were determined by real-time PCR (RT-PCR). Flow cytometry was used to distinguish TLR2+CD4+T cells from PBMCs of19SLE patients and14normal controls.Results:Compared to normal controls, the mRNA level of TLR2elevated in CD4+T cells from SLE patients (3.684±0.3849vs17.53±2.795,P=0.0012). And the percentage of TLR2+CD4+T cells in PBMC significantly increased in both treated (9.581±1.3728) and untreated (8.867±1.2992) SLE groups (P=0.0002; P=0.032). However, the drugs showed no effects on the expression of TLR2in CD4+T cells from SLE patients (P=0.803)Conclusion:Compared to normal controls, TLR2expression significantly increased in CD4+T cells from SLE patients.Part2The association between abnormal TLR2expression and autoimmunitySection1TLR2stimulation enhances the production of cytokines and self-reactivity of CD4+T cells Objective:To investigate the effects of TLR2stimulation on the production of cytokines and self-reactivity of CD4+T cells.Methods:Peripheral blood mononuclear cells (PBMCs) from three SLE patients were isolated by Ficoll-Hypaque density gradient centrifugation. CD4+T cells were then isolated by positive selection using magnetic beads. CD4+T cells were divided into Pam3CSK4and control groups, and respectively treated with Pam3CSK4(1mg/ml) and PBS for72hours. Flow cytometry was used to detect the expression of CD40L、 CD70and CD11a. ELISA was used to determine the levels of IL-6,IL-10, IL-17a, TNF-a and IFN-y in the supernatant.Results:Compared to control group, the expression of CD40L (0.017±0.0015vs0.031±0.0030, P=0.0163) and CD70(0.033±0.0032vs0.050±0.0052,P=0.0088) significantly upregulated in Pam3CSK4group, but the expression of CD11a (0.917±0.0088vs0.963±0.0088,P=0.1181) exhibit no difference between the two groups. The levels of IL-6(353.69±10.66vs1171.09±15.18, P=0.0005), IL-17a (30.06±0.32vs106.45±6.64, P=0.007)and TNF-a (2503.16±35.51vs4906.58±33.65, P=0.000) elevated after treated with Pam3CSK4, while the IL-10protein level (151.2±6.535vs195.6±18.52,P=0.087) and the level of IFN-y (61.76±0.13vs61.18±0.21, P=0.0786) did not change significantly (P=0.0786).Conclusion:The expression of autoimmune-related genes CD40L and CD70increased in CD4+T cells after stimulated by Pam3CSK4, which enhanced the autoreactivity of CD4+T cells from SLE patients. The levels of cytokines IL-6, IL-10, IL-17a and TNF-a also elevated after treated with Pam3CSK4which contributed to the perpetuation of inflammatory response in SLE.Section2Stimulation of TLR2inhibits Foxp3expression and promotes IL-17productionObjective:To investigate the effects of TLR2stimulation on foxp3and IL-17expression. Methods:PBMCs from three SLE patients were isolated by Ficoll-Hypaque density gradient centrifugation. CD4+T cells were then isolated by positive selection using magnetic beads. CD4+T cells were divided into Pam3CSK4and control groups, and respectively treated with Pam3CSK4(1mg/ml) and PBS for72hours. RT-PCR was applied to detect the mRNA levels of foxp3, IL-17a and IL-17f.Results:After treated with Pam3CSK4, the mRNA level of foxp3decreased in CD4+T cells from SLE patients (P=0.0255), while the mRNA levels of IL-17a (P=0.0234) and IL-17f (P=0.0432) increased.Conclusion:TLR2stimulation could down-regulated foxp3expression and up-regulated IL-17production, which may lead to the inhibition of regulatory T cells and enhancement of Th17response.Part3The epigenetic mechanisms of IL-17a and IL-17f production regulated by TLR2stimulation in CD4+T cellsObjectives:To explore the effects of TLR2stimulation on the histone modification status of IL-17a and IL-17f promoter regions.Methods:PBMCs from three SLE patients were isolated by Ficoll-Hypaque density gradient centrifugation. CD4+T cells were then isolated by positive selection using magnetic beads. CD4+T cells were divided into Pam3CSK4and control groups, and respectively treated with Pam3CSK4(1mg/ml) and PBS for72hours. ChIP-PCR was used to determine the histone modification status.Results:TLR2stimulation could increase H3K4tri-methylation status (P=P=0.0196) as well as H4acetylation status (P=0.0317), decrease H3K9tri-methylation status (P=0.0158) of IL-17a promoter, but it had no impact on H3acetylation status (P>0.05). It also increase H4acetylation status (P=0.0002) and decrease H3K9tri-methylation status (P=0.0044) of IL-17f promoter but no significant changes of H3K4tri-methylation status and H3acetylation status of IL-7f promoter (P>0.05). Conclusion:TLR2stimulation could regulate the expression of IL-17a and IL-17f through histone modifications.