*Department of Microbiology, All-India Institute of Medical Sciences, Patna, India; Rajendra Memorial Research Institute of Medical Sciences, Patna, India; Institute of Microbial Technology, Chandigarh, India; and National Centre for Cell Science, Pune, India
*Department of Microbiology, All-India Institute of Medical Sciences, Patna, India; Rajendra Memorial Research Institute of Medical Sciences, Patna, India; Institute of Microbial Technology, Chandigarh, India; and National Centre for Cell Science, Pune, India.
J Leukoc Biol. 2015 Feb;97(2):363-78. doi: 10.1189/jlb.4A0713-378RR. Epub 2014 Dec 3.
Regulation of macrophage PCD plays an important role in pathogenesis of leishmaniasis. However, the precise involvement of any parasite molecule in this process remains uncertain. In the current study, in silico wide analysis demonstrated that genes in the Leishmania donovani genome are highly enriched for CpG motifs, with sequence frequency of 8.7%. Here, we show that unmethylated species-specific CpG motifs in LdDNA significantly (P = 0.01) delay macrophage PCD by endosomal interaction with TLR9 via the adaptor protein MyD88. Importantly, LdDNA triggered high levels of luciferase activity (P = 0.001) under NF-κB-dependent transcription in HEK-TLR9 cells. Furthermore, the activation of caspases in macrophages was inhibited (P = 0.001) in the presence of LdDNA. Notably, the delay of PCD was mediated by modulation of the antiapoptotic proteins, Mcl-1 and Bfl-1, and impairment of loss of Δψm in macrophages through the neutralization of oxidative and nitrosative stress. The inhibition of caspase activation and up-regulation of Mcl-1 by LdDNA were TLR9 dependent. Analysis of the targets of LdDNA identified an early activation of the TLR9-dependent PI3K/Akt and SFK pathways, which were required for the observation of the antiapoptotic effects in macrophages. Moreover, we demonstrate that LdDNA modulates the TLR9-IκB-α pathway by promoting the tyrosine phosphorylation of TLR9 and the TLR9-mediated recruitment of Syk kinase. The results have identified a novel, TLR9-dependent antiapoptotic function of LdDNA, which will provide new opportunities for discovering and evaluating molecular targets for drug and vaccine designing against VL.
调控巨噬细胞程序性细胞死亡（PCD）在利什曼病的发病机制中起着重要作用。然而，寄生虫分子在这一过程中的具体作用尚不确定。在本研究中，通过计算机广泛分析表明，利什曼原虫 Donovan 基因组中的基因高度富含 CpG 基序，序列频率为 8.7%。在这里，我们表明，LdDNA 中未甲基化的种特异性 CpG 基序通过与 TLR9 的内体相互作用，通过衔接蛋白 MyD88 显著（P = 0.01）延迟巨噬细胞 PCD。重要的是，LdDNA 在 HEK-TLR9 细胞中，在 NF-κB 依赖性转录下触发高水平的荧光素酶活性（P = 0.001）。此外，LdDNA 的存在抑制了巨噬细胞中 caspase 的激活（P = 0.001）。值得注意的是，PCD 的延迟是通过调节抗凋亡蛋白 Mcl-1 和 Bfl-1 以及通过中和氧化和硝化应激来损害巨噬细胞中Δψm 的损失来介导的。LdDNA 对 caspase 激活的抑制和 Mcl-1 的上调是 TLR9 依赖性的。LdDNA 靶标的分析确定了 TLR9 依赖性 PI3K/Akt 和 SFK 途径的早期激活，这是观察巨噬细胞中抗凋亡作用所必需的。此外，我们证明 LdDNA 通过促进 TLR9 的酪氨酸磷酸化和 TLR9 介导的 Syk 激酶募集来调节 TLR9-IκB-α 途径。这些结果确定了 LdDNA 的一种新的、TLR9 依赖性的抗凋亡功能，这将为发现和评估针对 VL 的药物和疫苗设计的分子靶标提供新的机会。
