Ragupathy Sakthikumar, Esmaeili Farnaz, Paschoud Serge, Sublet Emmanuelle, Citi Sandra, Borchard Gerrit
School of Pharmaceutical Sciences; University of Geneva; University of Lausanne; Geneva, Switzerland.
Department of Molecular Biology; Institute of Genetics and Genomics; University of Geneva; Geneva, Switzerland ; Department of Cell Biology; Institute of Genetics and Genomics; University of Geneva; Geneva, Switzerland.
Tissue Barriers. 2014 May 12;2:e29166. doi: 10.4161/tisb.29166. eCollection 2014.
We investigated the role of Toll-like receptor (TLR) 2 in maintaining the integrity of the airway epithelial barrier using the human bronchial epithelial cell line Calu-3. Activation of TLR2 by its ligands, Pam3CysSK4 and Peptidoglycan showed a concentration dependent increase in epithelial barrier function, as measured by transepithelial electrical resistance (TEER). This was confirmed by a decrease in paracellular flux of fluorescein sodium. This TLR2 induced increase in TEER was significantly reduced by pretreatment with polyclonal anti-human TLR2-neutralizing antibody. TLR2 stimulation in Calu-3 cell monolayers resulted in an increased expression of the tight junction proteins claudin-1 and ZO-1, and a decreased expression of occludin, at both the mRNA and protein levels. A pseudosubstrate inhibitor to PKCζ significantly prevented the TLR2 mediated increase in barrier function. It also prevented the increase in claudin-1 in a concentration dependent manner up to 1 µM. TLR2 stimulation led to an increase in phosphorylation of atypical PKC ζ, which was prevented by the pseudosubstrate inhibitor in a concentration dependent manner. Taken together, our observations support a model whereby increased tight junction barrier function induced by activation of TLR2 occurs through increased expression of claudin-1, and through modulation of PKC ζ activity.
我们使用人支气管上皮细胞系Calu-3研究了Toll样受体（TLR）2在维持气道上皮屏障完整性中的作用。用其配体Pam3CysSK4和肽聚糖激活TLR2后，经跨上皮电阻（TEER）测量，上皮屏障功能呈浓度依赖性增加。荧光素钠的细胞旁通量降低证实了这一点。用多克隆抗人TLR2中和抗体预处理可显著降低TLR2诱导的TEER增加。在Calu-3细胞单层中刺激TLR2导致紧密连接蛋白claudin-1和ZO-1的表达增加，而occludin的表达在mRNA和蛋白质水平均降低。PKCζ的假底物抑制剂显著阻止了TLR2介导的屏障功能增加。它还以浓度依赖性方式阻止claudin-1的增加，最高可达1µM。TLR2刺激导致非典型PKCζ的磷酸化增加，而假底物抑制剂以浓度依赖性方式阻止了这种增加。综上所述，我们的观察结果支持一种模型，即TLR2激活诱导的紧密连接屏障功能增加是通过claudin-1表达增加以及PKCζ活性调节实现的。
