Abstract
An understanding of the consequences of oxidative/halogenative stress triggered by neutrophil activation is impossible without considering NETosis. NETosis, formation of neutrophil extracellular traps (NETs), is known to promote microthrombus formation and impair wound healing in type 2 diabetes mellitus (T2DM) patients. Therefore, there is a need to search for drugs and treatment approaches that could prevent excessive NET formation. We aimed to evaluate the effect of vitamin D3 in combination with omega-3 polyunsaturated fatty acids (vitamin D3/omega-3 PUFAs) on NETosis in T2DM patients with purulent necrotizing lesions of the lower extremities. Patients and healthy subjects had vitamin D3 deficiency. Patients received, beyond standard treatment, 6000 IU of vitamin D3 and 480 mg of omega-3 PUFAs, and healthy subjects 1000 IU of vitamin D3 and 240 mg of omega-3 PUFAs daily for seven days. Neutrophil activation in ex vivo blood by phorbol-12-myristate-13-acetate (PMA) was used as a NETosis model. The percentage of blood NETs relative to leukocytes (NETbackground) before vitamin D3/omega-3 PUFA supplementation was 3.2%-4.9% in healthy subjects and 1.7%-10.8% in patients. These values rose, respectively, to 7.7%-9.1% and 4.0%-17.9% upon PMA-induced NETosis. In addition, the leukocyte count decreased by 700-1300 per 1 μL in healthy subjects and 700-4000 per 1 μL in patients. For both patients and healthy subjects, taking vitamin D3/omega-3 PUFAs had no effect on NETbackground but completely inhibited PMA-induced NET formation, though neutrophils exhibited morphological features of activation. Also, leukocyte loss was reduced (to 500 per 1 μL). For patients on standard treatment alone, changes occurred neither in background NETs and leukocytes nor in their amount after PMA stimulation. The decreased ability of neutrophils to generate NETs, which can be achieved by vitamin D3/omega-3 PUFA supplementation, could have a positive effect on wound healing in T2DM patients and reduce the incidence and severity of complications.
Highlights
Neutrophils, which are the largest population of circulating leukocytes (~60% in adults), play a key role in cellular innate immunity
Blood incubation for 2 h with 100 nM PMA resulted in neutrophil activation (Figures 1(a) and 1(c)) and neutrophil extracellular traps (NETs) formation, as evidenced by a significantly higher NET percentage (NETPMA) compared to control blood incubated with no added PMA (NETcontrol) (Figure 2(a))
Among NETs formed during PMA-stimulated NETosis were those that differed from background NETs by the presence of fibrillar structures (Figures 1(b) and 1(d)), and there were aggregates consisting of 3–4 activated neutrophils (Figure 1(e))
Summary
Neutrophils, which are the largest population of circulating leukocytes (~60% in adults), play a key role in cellular innate immunity. Neutrophil granules contain a rich antimicrobial “arsenal,” which allows these cells to efficiently destroy phagocytosed pathogens [1]. The functioning mainly of NADPH oxidase and MPO leads to the formation of reactive oxygen- and halogen-containing compounds, the socalled reactive oxygen species (ROS) and reactive halogen species (RHS), which are directly involved in the destruction of pathogens both inside and outside the cell [2]. Another antimicrobial mechanism of neutrophils, which was discovered relatively recently, has been termed NETosis [3]. In NETosis, neutrophils release the so-called neutrophil extracellular traps (NETs), which are tangles of decondensed chromatin fibers and attached bactericidal agents including
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