**Acknowledgements**

*Cells of the Immune System*

by these leukocytes.

diabetic mice [110].

benefits to diabetic patients.

**5. Conclusion**

neutrophils derived from healthy patients produced more NETosis after preincubation with high glucose medium *in vitro*. In addition, large amounts of NETs were found in excisional skin sterile-wounds of streptozotocin-induced diabetic mice. Although the role of NETosis in wounds remains elusive, it has been confirmed that the inhibition of NETosis or degrading NETs improved sterile-wound healing and reduced NET-driven chronic inflammation in diabetic mice [105].

reduced LPS- or IL-6-induced NETosis *in vitro* [105, 107, 108].

Gram-positive bacteria cause more than half of cases of diabetes-related wound infections. Especially, *Staphylococcus aureus* is a major pathogen in these infections, and its presence correlates with significant delays in wound healing [106]. Wounds induced by *S. aureus* in T2DM mice showed delayed resolution compared to nondiabetic mice. Seven days after infection, the lesions of diabetic mice presented exacerbated NETosis, while nondiabetic mice had their inflammatory process already resolved and healing was nearly completed. Although neutrophils derived from both T1DM and T2DM patients produced greater amounts of NETs compared to healthy volunteer's neutrophils, the induction of NETosis cannot be explained just by hyperglycemia. In fact, some works showed that high glucose exposure

Some mechanisms that could also explain the increased neutrophil NETosis in diabetic patients are the elevated levels of zonulin and the overexpression of PAD4. Zonulin is a protein that modulates the permeability of tight junctions between cells of the digestive tract. Interestingly, the increased zonulin levels in diabetic patients revealed a strong correlation with neutrophil elastase concentration and NET formation in a glucose-independent way [109]. PAD4 is a calcium-dependent enzyme that mediates NETosis. In diabetes, PAD4 was upregulated in neutrophils from individuals with diabetes and was responsible for the unbalanced NET production

In T2DM mice, although neutrophil infiltration toward the lesion was augmented, the impaired wound healing upon surgical site infection with *S. aureus* was related to a significant reduction in phagocytic activity and bacterial killing by neutrophils. Consistently, *S. aureus*-induced phagolysosome maturation was abolished and PMA-stimulated superoxide production was decreased in neutrophils recovered from diabetic mice. In addition, treatment of neutrophils with insulin significantly restored neutrophil killing activities and increased phagocytosis. Interestingly, phagosome maturation and superoxide production restoring were dependent on glycemic control and not on a direct effect of insulin. These abnormalities in neutrophil functions were closely related with impaired wound healing in DM, once treatment with insulin restored normal wound healing in

The increased susceptibility to bacterial infections is one of the hallmarks of diabetic complications. Under comorbidity with diabetes, the high prevalence and severity of bacterial infections, as observed in tuberculosis, pneumonia, and sepsis, is closely associated to impairment in neutrophil functions, such as migration, phagocytosis, ROS production, and NET formation. The alterations in neutrophil functions noted in diabetics occur both dependently and independently of the glycemic control. Among the mechanisms that lead to neutrophil dysfunction in diabetic conditions not related to glycemic control, some targets have been highlighted, such as AGP, H2 receptor, IL-6, PAD4, resistin, and zonulin. These potential targets should be better explored in clinical studies concerning their putative

**22**

The authors thank Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Instituto Nacional de Ciência e Tecnologia-NIM, and Fundação Carlos Chagas de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ) for financial support.
