Phagocytosis‐induced apoptosis of macrophages is linked to uptake, killing and degradation of bacteria

Abstract

Phagocytosis and intracellular destruction of pathogens by phagocytes is a crucial defense mechanism of the innate immune response during infection. It has been reported a number of times that the interaction with pyogenic, extracellular bacteria leads to the apoptotic death of phagocytes. The signaling events that cause this form of cell death are largely unknown. In this study, we demonstrate a link between uptake, killing and degradation of Escherichia coli bacteria and induction of apoptosis in macrophages. Treatment of murine RAW 264.7 macrophages with bafilomycin A 1 , a phagosome acidification inhibitor, reduced killing and degradation of phagocytosed bacteria and significantly decreased macrophage apoptosis. The stable overexpression of constitutively active or dominant-negative mutants of the small GTPase Rab5a increased bacterial phagocytosis and consecutively apoptosis. In these cells, relative killing and degradation were not affected, linking the increased apoptosis to enhanced uptake and suggesting that the apoptosis-inducing signal derives from the higher incidence of degradation events or an accumulation of phagosomes of a late maturation stage. These results thus provide a link between bacterial phagocytosis and degradation and the induction of apoptosis in macrophages. We propose that this form of apoptosis is the physiological conclusion of an innate immune response against pyogenic bacteria. IntroductionMacrophages and neutrophils are essential to the innate immune system's ability to remove and kill invading microorganisms during infection, as they combine potent anti-microbial functions with the ability to induce local inflammation and a systemic immune response. Pathogens are mainly eliminated through phagocytosis followed by intracellular digestion, and it is now established that this central anti-bacterial mechanism can also lead to apoptosis of the phagocyte. Although macrophage apoptosis has more frequently been reported during uptake of virulent pathogens that have adapted to intracellular life, including Mycobacteria, Legionella, Shigella and Salmonella (reviewed in [1][2][3]), several studies have reported macrophage death after phagocytosis of extracellular bacteria such as Staphylococci, Streptococci and Escherichia coli that are readily killed upon uptake [4][5][6][7]. The nature of the signals that induce this latter form of apoptosis, and how it may be linked to the process of phagocytosis is largely unknown.Macrophages sequester pathogens in phagosomal compartments, which gain bactericidal and digestive properties upon maturation (reviewed in [8]). In a series of sequential fusion events with early and late endosomes and lysosomes [9, 10], the maturing phagosome acquires acid hydrolases that are responsible for particle In the regulation of vesicle fusion during phagosome maturation, members of the Ras-related in brain (Rab) family of small GTPases have been shown to assume important functions. Rab proteins associate with organelle membranes and assemble factors involved in do...

The AIM2 inflammasome is critical for innate immunity to Francisella tularensis

“…6). The increased bacterial count in bafilomycin treated cells is consistent with previous observations linking phagosomal acidification to killing and degradation of phagocytosed bacteria 25.…”

“…Nevertheless, our results suggest that the DNA which activates AIM2, and likely type I interferon signaling, is produced by breakdown and digestion of killed Francisella by phagosomal enzymes. Supporting this conclusion, bafilomycin which can prevent killing and degradation of bacteria in the phagosome by inhibiting the activity of vacuolar-type ATPases that mediate lumen acidification 25, 41, completely inhibited AIM2 inflammasome activation and IFN-β production in wild-type macrophages by Francisella infection. Considering that bafilomycin does not prevent Francisella phagosomal permeabilization and escape into the cytosol 35, phagosomal escape by live Francisella and its replication in the cytosol is likely not the signal that activates AIM2 or type I interferon response.…”

Sensing Cytoplasmic Danger Signals by the Inflammasome

“…Supporting this hypothesis, inhibition of killing and degradation of F. tularensis in the phagosome using the phagosomal acidification inhibitors bafilomycin or NH 4 Cl completely inhibited these processes. Because phagosomal acidification inhibitors prevent killing and degradation of phagocytosed bacteria [33, 34] but do not prevent phagosomal permeabilization and escape of F. tularensis into the cytosol [35], these observations strongly suggest that phagosomal acidification is a critical event essential for providing a source for cytosolic DNA that subsequently activates both the AIM2 inflammasome and production of type I interferons.…”

Dynamic process of phagocytosis and forms of macrophage cell death induced by ingestion of apoptotic neutrophils

“…Phagocytosis-induced apoptosis has been well described in the setting of a bacterial infection [27]. MDC, a well-known specific fluorescent dye that accumulates in acidic vacuoles, is often used as a marker for autophagosomes [28].…”