Calpain Participates in Cortical Cytoskeleton Modification and DNA Release during Neutrophil Extracellular Trap Formation.

INTRODUCTION: The formation of neutrophil extracellular traps (NETs) is a process in which several kinds of enzymes participate generating posttranslational modifications of proteins. NETs have been associated with infectious, autoimmune, and inflammatory diseases. Inhibition of several proteases reduces the formation of NETs. In the present work, we analyzed the role of several broad-acting and specific inhibitors of proteases in the formation of NETs. METHODS: Neutrophils were isolated from peripheral blood of healthy individuals by density gradient. The neutrophils were quantified and seeded into cell culture plates. Phorbol myristate acetate and A23187 were used as NETs inducers, and several specific inhibitors of proteases were used. The cells were stained for cytoskeleton or DNA. The cell-free supernatants were used to assess DNA release. Statistical analysis was carried out by a Kruskal-Wallis or ANOVA test. RESULTS: We observed marked changes in actin organization after the induction of NETs, suggesting that the cytoskeleton is being actively regulated. When we used protease inhibitors, the release of DNA was reduced, suggesting the participation of actin remodeling in the process. Further characterization of the specific proteases revealed that calpain modulates the reorganization of actin cytoskeleton and DNA release. Preservation of part of the actin cytoskeleton suggests that DNA release is not only a mechanic process associated to the chromatin decondensation; rather the process is highly regulated by active proteases that promote cytoskeleton reorganization and chromatin decondensation that culminates in DNA release. CONCLUSION: Calpain mediates the DNA release in the NET formation process by the modification of cortical actin cytoskeleton in a calcium-dependent manner.

Innate Immunity Alterations in Type 2 Diabetes Mellitus: Understanding Infection Susceptibility.

Diabetes is a chronic disease characterized by marked alterations in the metabolism of glucose and by high concentrations of glucose in the blood due to a decreased insulin production or resistance to the action of this hormone in peripheral tissues. The International Diabetes Federation estimates a global incidence of diabetes of about 10% in the adult population (20 - 79 years old), some 430 million cases reported worldwide in 2018. It is well documented that people with diabetes have a higher susceptibility to infectious diseases and therefore show higher morbidity and mortality compared to the non-diabetic population. Given that the innate immune response plays a fundamental role in protecting against invading pathogens through a myriad of humoral and cellular mechanisms, the present work makes a comprehensive review of the innate immune alterations in patients with type 2 diabetes mellitus (T2D) as well as a brief description of the molecular events leading or associated to such conditions. We show that in these patients a compromised innate immune response increases susceptibility to infections.