No abstract

<p>Necroptosis is a form of regulated cell death that is characterized by membrane permeabilization. This permeabilization is responsible for the inflammatory properties of necroptosis and is critical for disease states involving this process. We previously showed that very long chain fatty acids (VLCFAs) are functionally involved in necroptosis, potentially through protein fatty acylation. Here, we define the scope of protein acylation by saturated VLCFAs during necroptosis. We show that mixed lineage kinase like protein (MLKL) and phosphoMLKL, key proteins for membrane permeabilization, are exclusively acylated during necroptosis. Reducing the levels of VLCFAs decreases their membrane recruitment, suggesting that acylation by VLCFAs contributes to their membrane localization. Acylation of phosphoMLKL occurs downstream of phosphorylation and oligomerization and appears to be, in part, mediated by ZDHHC5 (a palmitoyl transferase). We also show that disruption of the clathrin-mediated endocytosis increases cell viability during necroptosis, likely by removing phosphoMLKL from the plasma membrane. <br></p>

Neutrophils are an important part of the innate immune system and among the first cells to respond to infections and inflammation. Responses include chemotaxis towards stimuli, extravasation from the vasculature, and antimicrobial actions such as phagocytosis, granule release, reactive oxygen species (ROS) production, and neutrophil extracellular trap (NET) formation (NETosis). Studying how neutrophils respond to a variety of stimuli, from biomaterial interactions to microbial insults, is therefore an essential undertaking to fully comprehend the immune response. While there are some immortalized cell lines available that recapitulate many neutrophil responses, ex vivo or in vivo studies are required to fully understand the complete range of neutrophil phenotypes. Here we describe two protocols for neutrophil isolation for further ex vivo study: recovery of neutrophils from human peripheral blood, and isolation of neutrophils from the oral cavity. We also discuss an in vivo model of general inflammation with the murine air pouch that can be used to assess numerous parameters of neutrophil and immune activation, including neutrophil recruitment and biological activity. In these protocols, the cells are isolated to allow for a high degree of experimental control. The protocols are relatively straightforward and can be successfully used by labs with no prior primary cell experience.

<p>Necroptosis is a form of regulated cell death that is characterized by membrane permeabilization. This permeabilization is responsible for the inflammatory properties of necroptosis and is critical for disease states involving this process. We previously showed that very long chain fatty acids (VLCFAs) are functionally involved in necroptosis, potentially through protein fatty acylation. Here, we define the scope of protein acylation by saturated VLCFAs during necroptosis. We show that mixed lineage kinase like protein (MLKL) and phosphoMLKL, key proteins for membrane permeabilization, are exclusively acylated during necroptosis. Reducing the levels of VLCFAs decreases their membrane recruitment, suggesting that acylation by VLCFAs contributes to their membrane localization. Acylation of phosphoMLKL occurs downstream of phosphorylation and oligomerization and appears to be, in part, mediated by ZDHHC5 (a palmitoyl transferase). We also show that disruption of the clathrin-mediated endocytosis increases cell viability during necroptosis, likely by removing phosphoMLKL from the plasma membrane. <br></p>