Supplementary Material for: Regulation of TLR10 Expression and Its Role in Chemotaxis of Human Neutrophils

Toll-like receptors are innate immune receptors that play a critical role in pathogen-associated molecular pattern recognition. TLR10 was recently identified and very limited data are available on its expression, mechanisms that regulate its expression, and its role in primary immune cells. To study the expression pattern of TLR10 in primary immune cells, we examined TLR10 protein expression in naive and Escherichia coli lipopolysaccharide (LPS)-activated human neutrophils. Human neutrophils challenged with LPS showed a decrease in total and surface TLR10 expression at 90 min. TLR10 in LPS-activated neutrophils colocalized with flotallin-1, a lipid raft marker, and EEA-1, an early endosomal marker, to suggest its endocytosis. There was increased colocalization of TLR10 with TLR4 at LPS 60 min followed by decrease at later LPS treatment times. Treatment with TLR4 neutralizing antibody decreased cytoplasmic localization of TLR10 in LPS-treated neutrophils. Reactive oxygen species (ROS) depletion and neutralization of p65 subunit of NF-κB in LPS-treated neutrophils decreased TLR10 expression. Live cell imaging of LPS-activated neutrophils showed TLR10 translocation in the leading edge and TLR10 knockdown in neutrophils reduced their fMLP-induced chemotaxis and the number of neutrophils with pseudopodia but without affecting the expression of key proteins of actin nucleation process, ARP-3 and Diap1. Taken together, our findings show that neutrophil activation alters TLR10 expression through ROS production and NF-κB regulation, and TLR10 knockdown reduced neutrophil chemotaxis.Toll样受体（Toll-like receptors）是一类在病原相关分子模式识别中发挥关键作用的固有免疫受体。Toll样受体10（TLR10）为近年鉴定得到的受体亚型，目前关于其表达特征、表达调控机制以及在原代免疫细胞中的功能相关数据仍十分有限。为探究TLR10在原代免疫细胞中的表达模式，本研究检测了未受刺激及大肠埃希菌脂多糖（LPS）激活的人中性粒细胞中TLR10蛋白的表达情况。经LPS刺激的人中性粒细胞在90分钟时，其总TLR10及膜表面TLR10的表达水平均出现下降。LPS激活的中性粒细胞内，TLR10可与脂筏标记物flotillin-1及早期内体标记物EEA-1发生共定位，提示TLR10发生了内吞过程。在LPS刺激60分钟时，TLR10与TLR4的共定位水平升高，而在后续的LPS处理时段中该共定位水平逐渐降低。使用TLR4中和抗体处理LPS刺激的中性粒细胞，可降低TLR10的胞质定位比例。在LPS刺激的中性粒细胞中，清除活性氧（ROS）或中和核因子κB（NF-κB）p65亚基，均可下调TLR10的表达水平。对LPS激活的中性粒细胞进行活细胞成像结果显示，TLR10发生了向细胞迁移前沿的转位；而在中性粒细胞中敲低TLR10后，其经fMLP诱导的趋化能力以及带有伪足的中性粒细胞数量均出现减少，但并未影响肌动蛋白成核过程关键蛋白肌动蛋白相关蛋白3（ARP-3）与凋亡抑制蛋白1（Diap1）的表达水平。综上，本研究结果表明，中性粒细胞激活可通过活性氧产生与核因子κB调控途径改变TLR10的表达水平，且TLR10敲低会削弱中性粒细胞的趋化能力。