Diabetic peripheral neuropathy is associated with changes in the frequency and function of circulating Natural Killer cells: results of a pilot study in patients with diabetic foot.

Background: Diabetic critical limb-threatening ischemia (CLTI) is a severe vascular disease with a high incidence of amputation and death. Circulating inflammatory markers may represent early diagnostic and prognostic tools. We previously showed that patients with diabetes mellitus and CLTI bear low abundance and impaired functionality of pro-angiogenic CD34+ hematopoietic stem and progenitor cells (HSPCs) and high frequency of natural killer cells (NKs). However, no study assessed the frequency, phenotype, and function of NKs in the early stage of CLTI characterized by neuropathy. Methods: We enrolled 177 subjects sorted into 5 groups reflecting CLTI progression: 1) non-diabetic healthy controls (n=25), diabetic patients 2) without neurovascular complications (n=27), 3) with peripheral neuropathy (n=39), 4) with peripheral neuropathy and foot lesions (n=42), 5) with peripheral neuroischemia and foot lesions (n=44). We assessed the frequency of circulating B-, CD4+, and CD8+ T-cells, NKs, and CD34+HSPCs, by a flow cytometry (FC)-based diagnostic kit. We investigated by FC the frequency of circulating NK subpopulations in donors with diabetic neuropathy (n=16) and controls (n=16). We characterized FACS-sorted circulating NKs secretome by antibody membrane arrays and we tested its pro-angiogenic potential in vitro and in vivo in Hirudo verbana. Results: Using age and sex-adjusted logistic multivariate models, we observed a decrease in CD34+HSPCs in all diabetic groups compared to control. Interestingly, NK frequency was increased in diabetic patients with neuropathy. Although the frequency of angiogenic NKs was similar in all groups, functionally, NKs from patients with diabetic neuropathy showed impaired degranulation in vitro, and reduced angiogenic ability in vitro and in vivo. Neuropathic NKs showed downregulation of angiogenic factors (IL-8, PDGF-B) and upregulation of factors related to monocyte/macrophage migration, effectively recruiting macrophages in vivo. Conclusion: By a routine laboratory test, we identified a specific cellular signature for patients with diabetes mellitus and peripheral neuropathy that includes low CD34+HSPCs and high NK frequency. Moreover, we show for the first time a functional impairment of NKs in neuropathic patients. If confirmed in larger cohorts, FACS detection of the circulating anti-angiogenic NK cell populations could be used for the early diagnosis of diabetic CLTI, a still unmet clinical need.

研究背景：糖尿病性重症肢体缺血（Critical Limb-Threatening Ischemia, CLTI）是一类高截肢率、高病死率的重症血管疾病。循环炎症标志物或可作为早期诊断及预后评估的潜在指标。本课题组既往研究发现，糖尿病合并CLTI患者体内促血管生成CD34+造血干细胞及祖细胞（hematopoietic stem and progenitor cells, HSPCs）丰度低下且功能受损，而自然杀伤细胞（natural killer cells, NKs）的频率升高。但目前尚无研究针对以神经病变为特征的早期CLTI患者体内NK细胞的频率、表型及功能展开评估。

研究方法：本研究纳入177名受试者，依据CLTI疾病进展阶段分为5组：1）非糖尿病健康对照组（n=25）；2）无神经血管并发症的糖尿病患者组（n=27）；3）合并周围神经病变的糖尿病患者组（n=39）；4）合并周围神经病变及足部病变的糖尿病患者组（n=42）；5）合并周围神经缺血性病变及足部病变的糖尿病患者组（n=44）。采用基于流式细胞术（flow cytometry, FC）的诊断试剂盒，检测受试者外周血B细胞、CD4+T细胞、CD8+T细胞、NK细胞及CD34+HSPCs的频率。针对16名糖尿病周围神经病变患者及16名健康对照者，通过FC分析其外周血NK细胞亚群的频率。通过抗体膜阵列对荧光激活细胞分选（Fluorescence Activated Cell Sorting, FACS）获取的外周血NK细胞分泌组进行表征，并利用医蛭（Hirudo verbana）模型体外及体内验证其促血管生成潜能。

研究结果：经年龄与性别校正的多因素logistic回归模型分析显示，相较于健康对照组，所有糖尿病组的CD34+HSPCs水平均显著降低。值得关注的是，合并周围神经病变的糖尿病患者体内NK细胞频率显著升高。尽管各组间促血管生成NK细胞的频率无显著差异，但功能学分析发现，糖尿病周围神经病变患者的NK细胞体外脱颗粒功能受损，且体内外促血管生成能力均下降。糖尿病周围神经病变患者的NK细胞中，促血管生成因子（IL-8、PDGF-B）表达下调，而与单核细胞/巨噬细胞迁移相关的因子表达上调，可在体内有效招募巨噬细胞。

研究结论：通过常规实验室检测手段，本课题组明确了糖尿病合并周围神经病变患者的特异性细胞特征谱：CD34+HSPCs水平低下且NK细胞频率升高。此外，本研究首次证实了周围神经病变患者体内NK细胞存在功能受损现象。若在更大规模队列中得到验证，通过荧光激活细胞分选（FACS）检测外周血抗血管生成NK细胞群，或可用于糖尿病性CLTI的早期诊断，填补当前未被满足的临床需求。