Calcineurin inhibitors exacerbate coronary arteritis via the MyD88 signalling pathway in a murine model of Kawasaki disease

Calcineurin inhibitors (CNIs) have been used off‐label for the treatment of refractory Kawasaki disease (KD). However, it remains unknown whether CNIs show protective effects against the development of coronary artery lesions in KD patients. To investigate the effects of CNIs on coronary arteries and the mechanisms of their actions on coronary arteritis in a mouse model of KD, we performed experiments with FK565, a ligand of nucleotide‐binding oligomerization domain‐containing protein 1 (NOD1) in wild‐type, severe combined immunodeficiency (SCID), caspase‐associated recruitment domain 9 (CARD9)^–/– and myeloid differentiation primary response gene 88 (MyD88)^–/– mice. We also performed in‐vitro studies with vascular and monocytic cells and vascular tissues. A histopathological analysis showed that both cyclosporin A and tacrolimus exacerbated the NOD1‐mediated coronary arteritis in a dose‐dependent manner. Cyclosporin A induced the exacerbation of coronary arteritis in mice only in high doses, while tacrolimus exacerbated it within the therapeutic range in humans. Similar effects were obtained in SCID and CARD9^–/– mice but not in MyD88^–/– mice. CNIs enhanced the expression of adhesion molecules by endothelial cells and the cytokine secretion by monocytic cells in our KD model. These data indicated that both vascular and monocytic cells were involved in the exacerbation of coronary arteritis. Activation of MyD88‐dependent inflammatory signals in both vascular cells and macrophages appears to contribute to their adverse effects. Particular attention should be paid to the development of coronary artery lesions when using CNIs to treat refractory KD.     

DEN2感染对MyD88沉默的小鼠树突状细胞相关膜分子表达及炎症因子分泌的影响

目的:探讨髓样分化因子88(MyD88)在DEN2感染BMDC后对其成熟及相关炎症因子分泌的影响.方法:利用rmIL-4、rmGM-CSF联合诱导培养BMDC;针对BMDC MyD88基因,设计并合成3对MyD88 siRNA,脂质体法转染BMDC;通过Western blot筛选一对高沉默效率的MyD88 siRNA;常规方法增殖及鉴定DEN2;直接免疫荧光及RT-PCR鉴定DEN2吸附BMDC;流式细胞术分析对照组、感染组、RNAi组、感染RNAi组、无义RNAi组BMDC膜表面分子CD86、MHCⅡ的表达,双抗体夹心ELISA法检测上清液中IP-10、TNF-α及IFN-α的分泌水平.结果:经细胞因子诱导培养后可获得CD11c表达为70.85％ ±2.66％的相对未成熟的BMDC;与空白对照组相比,序列1～3组的MyD88蛋白质表达率分别降低28.36％±14.26％、54.20％ ±22.93％、73.14％±11.80％,其中序列3的沉默效率最高,具有显著统计学意义(P ＜0.01);DEN2可吸附BMDC.与对照组比较,DEN2感染组DC膜表面分子CD86表达降低,MHCⅡ增高,感染RNAi组的MHCⅡ、CD86表达无明显变化;与对照组比较,DEN2感染组分泌TNF-α、IFN-α及IP-10的水平均增高,而感染RNAi组分泌的IP-10、IFN-α、TNF-α则无明显变化.结论:DEN2可吸附BMDC,并促使其成熟及细胞因子分泌;siRNA可沉默MyD88,有效阻断DEN2感染BMDC的胞内信号传导及其所引起的成熟及炎症因子分泌.     

Anti-β2GPI/β2GPI stimulates activation of THP-1 cells through TLR4/MD-2/MyD88 and NF-κB signaling pathways

Our previous study demonstrated that Toll-like receptor 4 (TLR4) could act as a co-receptor with annexin A2 (ANX2) mediating anti-β2-glycoprotein I/β2- glycoprotein I (anti-β₂GPI/β₂GPI) -induced tissue factor (TF) expression in human acute monocytic leukaemia cell line THP-1. In the current study, we further explored the roles of TLR4 and its adaptors, MD-2 and MyD88, as well as nuclear factor kappa B (NF-κB), in anti-β₂GPI/β₂GPI-induced the activation of THP-1 cells, especially on the expression of some proinflammatory molecules. The results showed that treatment of THP-1 cells with anti-β₂GPI (10 μg/ml)/β₂GPI (100 μg/ml) complex could increase IL-6 (interleukin-6), IL-8 (interleukin-8) as well as TNF-α (tumor necrosis factor alpha) expression (both mRNA and protein levels). These effects could be blocked by addition of TAK-242 (5 μM), a blocker of signaling transduction mediated by the intracellular domain of TLR4, and also by NF-κB inhibitor PDTC (20 μM). Overall, our results indicate that anti-β₂GPI/β₂GPI complex induced IL-6, IL-8 and TNF-α expression involving TLR4/MD-2/MyD88 and NF-κB signaling pathways and this might be associated with pathological mechanisms of antiphospholipid syndrome (APS).     

Cryptosporidium parvum antigens induce mouse and human dendritic cells to generate Th1-enhancing cytokines

Cryptosporidium parvum is an opportunistic intracellular parasite that causes mild to severe diarrhoea, which can be life‐threatening in an immunocompromised host. To increase our understanding of the mechanisms that play a role in host immune responses, we investigated the effects of C. parvum antigens on the phenotype of mouse and human dendritic cells (DCs). Cryptosporidium parvum antigens induced DC activation as indicated by upregulation of the maturation marker CD209, as well as by the production of the cytokines interleukin‐12 p70, IL‐2, IL‐1beta, IL‐6. In particular, significant increases in the expression of IL‐12 p70 were observed from mouse DCs derived from bone marrow in response to solubilized sporozoite antigen and the recombinant cryptosporidial antigens, Cp40 and Cp23. We observed a small but significant increase in IL‐18 expression following the exposure to Cp40. We found that the induction of Th1 cytokines was MyD88 dependent (MyD88 knockout mouse DCs were unresponsive). Additionally, both sporozoite preparations (solubilized and live) significantly induced IL‐12 production by human monocytic dendritic cells (MoDCs). This finding indicates that solubilized as well as recombinant antigens can induce the maturation of DCs and subsequently initiate an innate immune response.     

Evaluation of activated charcoal and lipid emulsion treatment in model of acute rivaroxaban toxicity

Aim

Reducing or reversing the toxicity effects of new oral anticoagulants is an important question.The purpose of the present study is to evaluate the effect of lipid emulsion (LE) and Activated Charcoal (AC) therapy on the intoxication of rivaroxaban, on mice.

IL-10 production by B cells is differentially regulated by immune-mediated and infectious stimuli and requires p38 activation

IL-10 is an immune suppressive cytokine with pleiotropic effects on B cell biology. IL-10 production has a pivotal role for the regulatory suppressive functions that B cells exert in many physiological and pathological settings. Several exogenous stimuli and endogenous immune mediators can trigger IL-10-producing B cell maturation. To clarify and gain a better insight into the mechanisms of IL-10 production by B cells, we first compared the effects of LPS, CpG, agonistic CD40 mAb and BAFF on IL-10 production, and then we investigated the signal transduction mechanisms responsible for these responses. While infectious/danger stimuli determine the rapid production and release of IL-10 by B cells, a limited subset of CD40-poised, IL-10-competent B cells produce IL-10 in response to a later antigenic or infectious signal. Although BAFF is able to induce a similar subset of IL-10-competent B cells, these cells do not similarly respond to the same antigenic or infectious signals. Importantly, by using specific inhibitors of the MAP kinase pathways, we found that while il-10 gene expression triggered by the TLR agonists LPS and CpG is strongly dependent on p38 activity, the induction of IL-10 competence in CD40-activated B cells does not depend on ERK1/2, p38 or JNK pathways.