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.

<p><strong><span>Background: </span></strong><span>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.<strong> Methods: </strong>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<em> in vitro</em> and <em>in vivo</em> in <em>Hirudo verbana</em>.<strong> Results: </strong>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 <em>in vitro</em>, and reduced angiogenic ability <em>in vitro </em>and <em>in vivo</em>. Neuropathic NKs showed downregulation of angiogenic factors (IL-8, PDGF-B) and upregulation of factors related to monocyte/macrophage migration, effectively recruiting macrophages <em>in vivo</em>.<strong> Conclusion:</strong> 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.</span></p>