Hyperglycemia in diabetes mellitus induces modification of proteins by glucose and its derivative methylglyoxal (MG). Neutrophils perform their bactericidal activity mainly via reactive halogen (RHS) and oxygen (ROS) species generation catalyzed by myeloperoxidase (MPO) stored in neutrophil azurophilic granules (AGs) and membrane NADPH oxidase, respectively. Herein, we study the binding of human serum albumin (HSA) modified with MG (HSA-MG) to MPO and its effects on MPO activity and release by neutrophils. Peroxidase activity of MPO was registered by oxidation of 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt, and chlorinating activity by decolorization of Celestine blue B dye. Binding of HSA-MG to MPO was studied by affinity chromatography, disc-electrophoresis, ligand Western blotting and enzyme-linked solid phase immunoassay using monoclonal antibodies (mAbs) to MPO. ROS and RHS generation were detected by lucigenin (Luc) and luminol (Lum) chemiluminescence (CL), respectively. Neutrophil degranulation was assessed by flow cytometry using fluorescent labeled antibodies to the marker proteins CD63 from AGs and CD11b from peroxidase-negative granules (PNGs). NETosis was assayed by quantifying DNA network-like structures (NET-like structures) in blood smears stained by Romanowsky. HSA-MG bound to MPO, giving a stable complex (K_d = 1.5 nM) and competing with mAbs, and non-competitively inhibited peroxidase and chlorinating MPO activity and induced degranulation of PNGs but not of AGs. HSA-MG enhanced Luc-CL per se or following PMA, unlike Lum-CL, and did not affect spontaneous or PMA-stimulated NETosis. Thus, HSA modified under hyperglycemia-like conditions stimulated NADPH oxidase of neutrophils but dampened their functions dependent on activity of MPO, with no effect on its release via degranulation or NETosis. This phenomenon could underlie the downregulation of bactericidal activity of MPO and neutrophils, and hence of innate immunity, giving rise to wound healing impairment and susceptibility to infection in patients with hyperglycemia.

The application of vaterite microparticles for mucosal delivery depends on their interaction with mucin and immune cells. As we have shown previously, the binding of mucin onto particles enhances the generation of reactive oxygen species by neutrophils. The attenuation of the pro-oxidant effect of the bound mucin through the modification of vaterite could improve its biocompatibility. Hybrid microparticles composed of vaterite and pectin (CCP) were prepared using co-precipitation. In comparison with vaterite (CC), they had a smaller diameter and pores, a greater surface area, and a negative zeta-potential. We aimed to study the cytotoxicity and mucin-dependent neutrophil-activating effect of CCP microparticles. The incorporated pectin did not influence the neutrophil damage according to a lactate dehydrogenase test. The difference in the CC- and CCP-elicited luminol or lucigenin chemiluminescence of neutrophils was insignificant, with no direct pro- or antioxidant effects from the incorporated pectin. Unlike soluble pectin, the CCP particles were ineffective at scavenging radicals in an ABAP–luminol test. The fluorescence of SYTOX Green demonstrated a CCP-stimulated formation of neutrophil extracellular traps (NETs). The pre-treatment of CC and CCP with mucin resulted in a 2.5-times-higher CL response of neutrophils to the CC-mucin than to the CCP-mucin. Thus, the incorporation of pectin into vaterite microspheres enabled an antioxidant effect to be reached when the neutrophils were activated by mucin-treated microparticles, presumably via exposed ligands.

Hyperglycemia-induced protein glycation and formation of advanced glycation end-products (AGEs) plays an important role in the pathogenesis of diabetic complications and pathological biomineralization. Receptors for AGEs (RAGEs) mediate the generation of reactive oxygen species (ROS) via activation of NADPH-oxidase. It is conceivable that binding of glycated proteins with biomineral particles composed mainly of calcium carbonate and/or phosphate enhances their neutrophil-activating capacity and hence their proinflammatory properties. Our research managed to confirm this hypothesis. Human serum albumin (HSA) was glycated with methylglyoxal (MG), and HSA-MG was adsorbed onto mineral microparticles composed of calcium carbonate nanocrystals (vaterite polymorph, CC) or hydroxyapatite nanowires (CP). As scopoletin fluorescence has shown, H₂O₂ generation by neutrophils stimulated with HSA-MG was inhibited with diphenyleneiodonium chloride, wortmannin, genistein and EDTA, indicating a key role for NADPH-oxidase, protein tyrosine kinase, phosphatidylinositol 3-kinase and divalent ions (presumably Ca²⁺) in HSA-MG-induced neutrophil respiratory burst. Superoxide anion generation assessed by lucigenin-enhanced chemiluminescence (Luc-CL) was significantly enhanced by free HSA-MG and by both CC-HSA-MG and CP-HSA-MG microparticles. Comparing the concentrations of CC-bound and free HSA-MG, one could see that adsorption enhanced the neutrophil-activating capacity of HSA-MG.

BACKGROUND: Leukocyte myeloperoxidase catalyzes the formation of reactive halogen species, which oxidize and chlorinate biomolecules, thereby contributing to the development of halogenating stress. Myeloperoxidase is a key enzyme in neutrophil extracellular traps (NETs) during NETosis. There is reason to believe that under hyperglycemic conditions in patients with type 2 diabetes mellitus, halogenating stress and NETosis develop, which contribute to disease progression and complications. AIM: The work aimed to assess the levels of blood markers of halogenating stress (myeloperoxidase, chlorinated albumin) and NETosis (neutrophil extracellular traps) in patients with type 2 diabetes mellitus. METHODS: The study included patients with a previously established diagnosis of type 2 diabetes mellitus. Myeloperoxidase and chlorinated albumin in plasma were measured by enzyme-linked immunosorbent assay. The number of neutrophil extracellular traps was determined using light microscopy on standardized whole-blood smears stained according to Romanowsky. RESULTS: In patients with type 2 diabetes mellitus, blood levels of myeloperoxidase and chlorinated albumin were significantly higher than in the group of healthy volunteers, indicating the development of halogenating stress. At the same time, in the blood of patients with type 2 diabetes mellitus, a significant increase in the concentration of neutrophil extracellular traps was recorded compared to the control group of healthy volunteers, both in the absence of the activator—phorbol 12-myristate 13-acetate—and after its addition to the blood, indicating activation of NETosis in type 2 diabetes mellitus. CONCLUSION: The findings support the hypothesis that halogenating stress, caused by an excessive increase in blood myeloperoxidase concentration/activity, accompanies the development of type 2 diabetes mellitus and contributes to its progression and complications.

BACKGROUND: Leukocyte myeloperoxidase catalyzes the formation of HOCl, which, by oxidizing and chlorinating biomolecules, contributes to the development of oxidative/halogenative stress. The latter in hyperglycemia may interfere with wound healing in patients with diabetes mellitus complications. AIM: Evaluation of the concentration of markers of oxidative/halogenative stress and NETosis in the blood of experimental rats with hyperglycemia, its correction with lactoferrin, as well as elucidation of the effect of this multifunctional protein on skin wound healing. MATERIALS AND METHODS: For the experimental modeling of hyperglycemia, the animals were injected once with streptozotocin at a dosage of 43 mg/kg body weight. Blood samples were collected from the tail vein of anesthetized animals. Glucose was measured by the electrochemical method. Markers of oxidative/halogenative stress were detected by the immunoenzymatic and spectrophotometric methods. RESULTS: It was shown that in rats with streptozotocin-induced hyperglycemia, a “prophylactic+therapeutic” supplementation of lactoferrin (at a dose of 250 mg/kg on days 5, 3, and 1 before and days 2, 4, 6, and 8 after streptozotocin injection) significantly decreased blood concentration of glucose (fasting), myeloperoxidase, chlorinated ceruloplasmin, complexes of myeloperoxidase/DNA, and also prevented the decrease in thiols (SH-groups) and the activity of erythrocyte glutathione peroxidase. Moreover, lactoferrin administered according to the above regimen to rats with experimental hyperglycemia promoted wound healing, which was manifested by a 28% decrease in the wound area compared to the control animals. CONCLUSIONS: The results obtained indicate that lactoferrin has an ability in a hyperglycemia model in animals to reduce not only the hyperglycemia level, but also to prevent the development of oxidative/halogenative stress and NETosis, which leads to improved wound healing.