No abstract

Zinc plays an important physiological role in the entire body, especially in the immune system. It is one of the most abundant microelements in our organism and an essential component of enzymes and antibacterial proteins. Zinc levels were reported to be correlated with the intensity of innate immunity responses, especially those triggered by neutrophils. However, as the results are fragmentary, the phenomenon is still not fully understood and requires further research. In this study, we aimed to perform a comprehensive assessment and study the impact of zinc on several basic neutrophils’ functions in various experimental setups. Human and murine neutrophils were preincubated in vitro with zinc, and then phagocytosis, oxidative burst, degranulation and release of neutrophil extracellular traps (NETs) were analyzed. Moreover, a murine model of zinc deficiency and zinc supplementation was introduced in the study and the functions of isolated cells were thoroughly studied. We showed that zinc inhibits NETs release as well as degranulation in both human and murine neutrophils. Our study revealed that zinc decreases NETs release by inhibiting citrullination of histone H3. On the other hand, studies performed in zinc-deficient mice demonstrated that low zinc levels result in increased release of NETs and enhanced neutrophils degranulation. Overall, it was shown that zinc affects neutrophils’ functions in vivo and in vitro. Proper zinc level is necessary to maintain efficient functioning of the innate immune response.

Neutrophils apply several antimicrobial strategies including degranulation, phagocytosis, the generation of reactive oxygen species (ROS) and the release of neutrophil extracellular traps (NETs) to fight pathogens. Iron is considered to be an invaluable constituent of host immune defense and plays a dual role in immunity. It is a well-known component of antimicrobial proteins and is a necessary microelement for pathogen survival. The aim of this study was to broaden the knowledge regarding the impact of iron on the function of neutrophils. Neutrophils from healthy blood donors and patients with mild irondeficiency anemia and HL-60 cells differentiated toward granulocyte-like cells were incubated with Fe 2+ , Fe 3+ or holo-transferrin (holo-Tf). Moreover, we isolated murine neutrophils of HFE gene knockout (KO) mice and mice fed irondeficient, iron-equivalent and high-iron diets. We analyzed the release of NETs, phagocytosis, degranulation of azurophilic granules, ROS release, bactericidal activity of granulocytes against Escherichia coli and neutrophil elastase (NE) activity. We show that holo-Tf inhibits the release of NETs stimulated by phorbol 12-myristate 13-acetate by inhibiting NE activity. Studies performed in mice models reveal that iron overload inhibits the release of NETs and ROS production in neutrophils isolated from HFE KO mice and mice fed a high-iron diet. No impact of a low-iron diet on neutrophil phagocytosis, ROS production or release of NETs was observed. Our study underscores the physiological significance of iron in neutrophil function, specifically in the release of NETs.

Neutrophils represent the first line of defense against pathogens using various strategies, such as phagocytosis, production of reactive oxygen species (ROS) and neutrophil extracellular traps (NETs) formation. Recently, an autophagy-independent role of autophagy related (ATG) gene 5 in immune cells, including neutrophils, was emphasized. Our aim was to investigate the role of ATG5 protein in neutrophils’ antimicrobial functions, proliferation and apoptosis. To this end, we used genetically modified human promyelocytic leukemia (HL-60) cells overexpressing ATG5, differentiated toward granulocyte-like cells with all-trans retinoic acid (ATRA) and dimethylformamide. The level of differentiation, phagocytosis, proliferation and apoptosis were determined by flow cytometry. ROS production and NETs release was assessed by fluorometry and fluorescent microscopy. ATG5 gene expression was evaluated by real-time PCR, whereas the protein level of ATG5 and LC3-II was determined by Western blot. We did not observe the induction of autophagy in differentiated HL-60 cells overexpressing ATG5. The increased expression of ATG5 affects the differentiation of HL-60 cells with ATRA, ROS production and phagocytosis. However, we did not detect changes in NETs release. Moreover, ATG5 protects differentiated HL-60 cells from apoptosis but does not cause changes in proliferation rate.

ImportanceThe incidence of diabetes in childhood has increased during the COVID-19 pandemic. Elucidating whether SARS-CoV-2 infection is associated with islet autoimmunity, which precedes type 1 diabetes onset, is relevant to disease etiology and future childhood diabetes trends.ObjectiveTo determine whether there is a temporal relationship between SARS-CoV-2 infection and the development of islet autoimmunity in early childhood.Design, Setting, and ParticipantsBetween February 2018 and March 2021, the Primary Oral Insulin Trial, a European multicenter study, enrolled 1050 infants (517 girls) aged 4 to 7 months with a more than 10% genetically defined risk of type 1 diabetes. Children were followed up through September 2022.ExposureSARS-CoV-2 infection identified by SARS-CoV-2 antibody development in follow-up visits conducted at 2- to 6-month intervals until age 2 years from April 2018 through June 2022.Main Outcomes and MeasuresThe development of multiple (≥2) islet autoantibodies in follow-up in consecutive samples or single islet antibodies and type 1 diabetes. Antibody incidence rates and risk of developing islet autoantibodies were analyzed.ResultsConsent was obtained for 885 (441 girls) children who were included in follow-up antibody measurements from age 6 months. SARS-CoV-2 antibodies developed in 170 children at a median age of 18 months (range, 6-25 months). Islet autoantibodies developed in 60 children. Six of these children tested positive for islet autoantibodies at the same time as they tested positive for SARS-CoV-2 antibodies and 6 at the visit after having tested positive for SARS-CoV-2 antibodies. The sex-, age-, and country-adjusted hazard ratio for developing islet autoantibodies when the children tested positive for SARS-CoV-2 antibodies was 3.5 (95% CI, 1.6-7.7; P = .002). The incidence rate of islet autoantibodies was 3.5 (95% CI, 2.2-5.1) per 100 person-years in children without SARS-CoV-2 antibodies and 7.8 (95% CI, 5.3-19.0) per 100 person-years in children with SARS-CoV-2 antibodies (P = .02). Islet autoantibody risk in children with SARS-CoV-2 antibodies was associated with younger age (<18 months) of SARS-CoV-2 antibody development (HR, 5.3; 95% CI, 1.5-18.3; P = .009).Conclusion and relevanceIn young children with high genetic risk of type 1 diabetes, SARS-CoV-2 infection was temporally associated with the development of islet autoantibodies.