Lieskovská Jaroslava, Páleníková Jana, Langhansová Helena, Campos Chagas Andrezza, Calvo Eric, Kotsyfakis Michalis, Kopecký Jan
Faculty of Science, University of South Bohemia, Branišovská 1760, CZ-37005, České Budějovice, Czech Republic.
Institute of Parasitology, Biology Centre of the Academy of Sciences of the Czech Republic, Branišovská 31, CZ-37005, České Budějovice, Czech Republic.
Parasit Vectors. 2015 May 15;8:275. doi: 10.1186/s13071-015-0887-1.
Transmission of pathogens by ticks is greatly supported by tick saliva released during feeding. Dendritic cells (DC) act as immunological sentinels and interconnect the innate and adaptive immune system. They control polarization of the immune response towards Th1 or Th2 phenotype. We investigated whether salivary cystatins from the hard tick Ixodes scapularis, sialostatin L (Sialo L) and sialostatin L2 (Sialo L2), influence mouse dendritic cells exposed to Borrelia burgdorferi and relevant Toll-like receptor ligands.
DCs derived from bone-marrow by GM-CSF or Flt-3 ligand, were activated with Borrelia spirochetes or TLR ligands in the presence of 3 μM Sialo L and 3 μM Sialo L2. Produced chemokines and IFN-β were measured by ELISA test. The activation of signalling pathways was tested by western blotting using specific antibodies. The maturation of DC was determined by measuring the surface expression of CD86 by flow cytometry.
We determined the effect of cystatins on the production of chemokines in Borrelia-infected bone-marrow derived DC. The production of MIP-1α was severely suppressed by both cystatins, while IP-10 was selectively inhibited only by Sialo L2. As TLR-2 is a major receptor activated by Borrelia spirochetes, we tested whether cystatins influence signalling pathways activated by TLR-2 ligand, lipoteichoic acid (LTA). Sialo L2 and weakly Sialo L attenuated the extracellular matrix-regulated kinase (Erk1/2) pathway. The activation of phosphatidylinositol-3 kinase (PI3K)/Akt pathway and nuclear factor-κB (NF-κB) was decreased only by Sialo L2. In response to Borrelia burgdorferi, the activation of Erk1/2 was impaired by Sialo L2. Production of IFN-β was analysed in plasmacytoid DC exposed to Borrelia, TLR-7, and TLR-9 ligands. Sialo L, in contrast to Sialo L2, decreased the production of IFN-β in pDC and also impaired the maturation of these cells.
This study shows that DC responses to Borrelia spirochetes are affected by tick cystatins. Sialo L influences the maturation of DC thus having impact on adaptive immune response. Sialo L2 affects the production of chemokines potentially engaged in the development of inflammatory response. The impact of cystatins on Borrelia growth in vivo is discussed.
蜱虫在进食过程中释放的唾液极大地促进了病原体的传播。树突状细胞（DC）作为免疫哨兵，连接着先天性和适应性免疫系统。它们控制免疫反应向Th1或Th2表型的极化。我们研究了硬蜱肩突硬蜱的唾液半胱氨酸蛋白酶抑制剂，唾液抑素L（Sialo L）和唾液抑素L2（Sialo L2），是否会影响暴露于伯氏疏螺旋体和相关Toll样受体配体的小鼠树突状细胞。
通过GM-CSF或Flt-3配体从骨髓中获得的DC，在存在3μM Sialo L和3μM Sialo L2的情况下，用伯氏疏螺旋体或TLR配体激活。通过ELISA试验测量产生的趋化因子和IFN-β。使用特异性抗体通过蛋白质印迹法检测信号通路的激活。通过流式细胞术测量CD86的表面表达来确定DC的成熟度。
我们确定了半胱氨酸蛋白酶抑制剂对伯氏疏螺旋体感染的骨髓来源DC中趋化因子产生的影响。两种半胱氨酸蛋白酶抑制剂均严重抑制了MIP-1α的产生，而IP-10仅被Sialo L2选择性抑制。由于TLR-2是被伯氏疏螺旋体激活的主要受体，我们测试了半胱氨酸蛋白酶抑制剂是否会影响由TLR-2配体脂磷壁酸（LTA）激活的信号通路。Sialo L2和较弱的Sialo L减弱了细胞外基质调节激酶（Erk1/2）通路。磷脂酰肌醇-3激酶（PI3K）/Akt通路和核因子-κB（NF-κB）的激活仅被Sialo L2降低。响应伯氏疏螺旋体，Sialo L2损害了Erk1/2的激活。在暴露于伯氏疏螺旋体、TLR-7和TLR-9配体的浆细胞样DC中分析了IFN-β的产生。与Sialo L2相反，Sialo L降低了pDC中IFN-β的产生，并且还损害了这些细胞的成熟。
本研究表明，DC对伯氏疏螺旋体的反应受蜱半胱氨酸蛋白酶抑制剂的影响。Sialo L影响DC的成熟，从而对适应性免疫反应产生影响。Sialo L2影响可能参与炎症反应发展的趋化因子的产生。讨论了半胱氨酸蛋白酶抑制剂对体内伯氏疏螺旋体生长的影响。
