Clearance of invading microbes requires phagocytes of the innate immune system. However, successful pathogens have evolved sophisticated strategies to evade immune killing. The opportunistic human fungal pathogen Candida albicans is efficiently phagocytosed by macrophages, but causes inflammasome activation, host cytolysis, and escapes after hypha formation. Previous studies suggest that macrophage lysis by C. albicans results from early inflammasome-dependent cell death (pyroptosis), late damage due to glucose depletion and membrane piercing by growing hyphae. Here we show that Candidalysin, a cytolytic peptide toxin encoded by the hypha-associated gene ECE1, is both a central trigger for NLRP3 inflammasome-dependent caspase-1 activation via potassium efflux and a key driver of inflammasome-independent cytolysis of macrophages and dendritic cells upon infection with C. albicans. This suggests that Candidalysin-induced cell damage is a third mechanism of C. albicans-mediated mononuclear phagocyte cell death in addition to damage caused by pyroptosis and the growth of glucose-consuming hyphae.

Abdominal sepsis is associated with dysfunctional hemostasis. Thrombin generation (TG) is a rate-limiting step in systemic coagulation. Neutrophils can expell neutrophil extracellular traps (NETs) and/or microparticles (MPs) although their role in pathological coagulation remains elusive. Cecal ligation and puncture (CLP)-induced TG in vivo was reflected by a reduced capacity of plasma from septic animals to generate thrombin. Depletion of neutrophils increased TG in plasma from CLP mice. Sepsis was associated with increased histone 3 citrullination in neutrophils and plasma levels of cell-free DNA and DNA-histone complexes and administration of DNAse not only eliminated NET formation but also elevated TG in sepsis. Isolated NETs increased TG and co-incubation with DNAse abolished NET-induced formation of thrombin. TG triggered by NETs was inhibited by blocking factor XII and abolished in factor XII-deficient plasma but intact in factor VII-deficient plasma. Activation of neutrophils simultaneously generated large amount of neutrophil-derived MPs, which were found to bind to NETs via histone-phosphatidylserine interactions. These findings show for the first time that NETs and MPs physically interact, and that NETs might constitute a functional assembly platform for MPs. We conclude that NET-MP complexes induce TG via the intrinsic pathway of coagulation and that neutrophil-derived MPs play a key role in NET-dependent coagulation.

Clearance of cellular debris is required to maintain the homeostasis of multicellular organisms. It is intrinsic to processes such as tissue growth and remodeling, regeneration and resolution of injury and inflammation. Most of the removal of effete and damaged cells is performed by macrophages and neutrophils through phagocytosis, a complex phenomenon involving ingestion and degradation of the disposable particles. The study of the clearance of cellular debris has been strongly biased toward the removal of apoptotic bodies; as a result, the mechanisms underlying the removal of necrotic cells have remained relatively unexplored. Here, we will review the incipient but growing knowledge of the phagocytosis of necrotic debris, from their recognition and engagement to their internalization and disposal. Critical insights into these events were gained recently through the development of new in vitro and in vivo models, along with advances in live-cell and intravital microscopy. This review addresses the classes of "find-me" and "eat-me" signals presented by necrotic cells and their cognate receptors in phagocytes, which in most cases differ from the extensively characterized counterparts in apoptotic cell engulfment. The roles of damage-associated molecular patterns, chemokines, lipid mediators, and complement components in recruiting and activating phagocytes are reviewed. Lastly, the physiological importance of necrotic cell removal is emphasized, highlighting the key role of impaired debris clearance in autoimmunity.

C. albicans is the most common cause of nosocomial fungal infection, and over 3 million people acquire life-threatening invasive fungal infections every year. Even if antifungal drugs exist, almost half of these patients will die. Despite this, fungi remain underestimated as pathogens. Our study uses quantitative biophysical approaches to demonstrate that yeast-to-hypha transition occurs within the nutrient-deprived, acidic phagosome and that alkalinization is a consequence, as opposed to the cause, of hyphal growth.

Highlights d Phagosomes maintain integrity despite intraphagosomal growth of bacteria and fungi d Phagosomes expand their surface area by recruitment and insertion of lysosomes d Impairment of phagosomal expansion facilitates pathogen growth and escape d The associated phagosomal rupture leads to NLRP3 inflammasome activation