Ryans Khamia, Omosun Yusuf, McKeithen Danielle N, Simoneaux Tankya, Mills Camilla C, Bowen Nathan, Eko Francis O, Black Carolyn M, Igietseme Joseph U, He Qing
Department of Microbiology, Biochemistry, and Immunology, Morehouse School of Medicine, 720 Westview Drive S.W., Atlanta, GA, 30310, USA.
Department of Biology, Clark Atlanta University, Atlanta, GA, 30314, USA.
BMC Immunol. 2017 May 19;18(1):27. doi: 10.1186/s12865-017-0212-1.
We have previously reported that interleukin-10 (IL-10) deficient dendritic cells (DCs) are potent antigen presenting cells that induced elevated protective immunity against Chlamydia. To further investigate the molecular and biochemical mechanism underlying the superior immunostimulatory property of IL-10 deficient DCs we performed proteomic analysis on protein profiles from Chlamydia-pulsed wild-type (WT) and IL-10 DCs to identify differentially expressed proteins with immunomodulatory properties.
The results showed that alpha enolase (ENO1), a metabolic enzyme involved in the last step of glycolysis was significantly upregulated in Chlamydia-pulsed IL-10 DCs compared to WT DCs. We further studied the immunoregulatory role of ENO1 in DC function by generating ENO1 knockdown DCs, using lentiviral siRNA technology. We analyzed the effect of the ENO1 knockdown on DC functions after pulsing with Chlamydia. Pyruvate assay, transmission electron microscopy, flow cytometry, confocal microscopy, cytokine, T-cell activation and adoptive transfer assays were also used to study DC function. The results showed that ENO1 knockdown DCs had impaired maturation and activation, with significant decrease in intracellular pyruvate concentration as compared with the Chlamydia-pulsed WT DCs. Adoptive transfer of Chlamydia-pulsed ENO1 knockdown DCs were poorly immunogenic in vitro and in vivo, especially the ability to induce protective immunity against genital chlamydia infection. The marked remodeling of the mitochondrial morphology of Chlamydia-pulsed ENO1 knockdown DCs compared to the Chlamydia-pulsed WT DCs was associated with the dysregulation of translocase of the outer membrane (TOM) 20 and adenine nucleotide translocator (ANT) 1/2/3/4 that regulate mitochondrial permeability. The results suggest that an enhanced glycolysis is required for efficient antigen processing and presentation by DCs to induce a robust immune response.
The upregulation of ENO1 contributes to the superior immunostimulatory function of IL-10 deficient DCs. Our studies indicated that ENO1 deficiency causes the reduced production of pyruvate, which then contributes to a dysfunction in mitochondrial homeostasis that may affect DC survival, maturation and antigen presenting properties. Modulation of ENO1 thus provides a potentially effective strategy to boost DC function and promote immunity against infectious and non-infectious diseases.
我们之前报道过，白细胞介素-10（IL-10）缺陷的树突状细胞（DCs）是强大的抗原呈递细胞，可诱导针对衣原体的增强的保护性免疫。为了进一步研究IL-10缺陷DCs卓越免疫刺激特性背后的分子和生化机制，我们对衣原体脉冲的野生型（WT）和IL-10 DCs的蛋白质谱进行了蛋白质组学分析，以鉴定具有免疫调节特性的差异表达蛋白。
结果显示，与WT DCs相比，参与糖酵解最后一步的代谢酶α烯醇化酶（ENO1）在衣原体脉冲的IL-10 DCs中显著上调。我们使用慢病毒小干扰RNA技术生成ENO1敲低的DCs，进一步研究ENO1在DC功能中的免疫调节作用。我们分析了衣原体脉冲后ENO1敲低对DC功能的影响。丙酮酸测定、透射电子显微镜、流式细胞术、共聚焦显微镜、细胞因子、T细胞活化和过继转移测定也用于研究DC功能。结果显示，与衣原体脉冲的WT DCs相比，ENO1敲低的DCs成熟和活化受损，细胞内丙酮酸浓度显著降低。衣原体脉冲的ENO1敲低DCs的过继转移在体外和体内免疫原性较差，尤其是诱导针对生殖器衣原体感染的保护性免疫的能力。与衣原体脉冲的WT DCs相比，衣原体脉冲的ENO1敲低DCs线粒体形态的显著重塑与调节线粒体通透性的外膜转位酶（TOM）20和腺嘌呤核苷酸转位酶（ANT）1/2/3/4的失调有关。结果表明，DCs进行有效的抗原加工和呈递以诱导强大的免疫反应需要增强糖酵解。
ENO1的上调有助于IL-10缺陷DCs卓越的免疫刺激功能。我们的研究表明，ENO1缺陷导致丙酮酸产生减少，进而导致线粒体稳态功能障碍，这可能影响DC的存活、成熟和抗原呈递特性。因此，调节ENO1提供了一种潜在有效的策略来增强DC功能并促进针对感染性和非感染性疾病的免疫。
