When the Phagosome Gets Leaky: Pore-Forming Toxin-Induced Non-Canonical Autophagy (PINCA).

Macrophages remove bacteria from the extracellular milieu via phagocytosis. While most of the engulfed bacteria are degraded in the antimicrobial environment of the phagolysosome, several bacterial pathogens have evolved virulence factors, which evade degradation or allow escape into the cytosol. To counter this situation, macrophages activate LC3-associated phagocytosis (LAP), a highly bactericidal non-canonical autophagy pathway, which destroys the bacterial pathogens in so called LAPosomes. Moreover, macrophages can also target intracellular bacteria by pore-forming toxin-induced non-canonical autophagy (PINCA), a recently described non-canonical autophagy pathway, which is activated by phagosomal damage induced by bacteria-derived pore-forming toxins. Similar to LAP, PINCA involves LC3 recruitment to the bacteria-containing phagosome independently of the ULK complex, but in contrast to LAP, this process does not require ROS production by Nox2. As last resort of autophagic targeting, macrophages activate xenophagy, a selective form of macroautophagy, to recapture bacteria, which evaded successful targeting by LAP or PINCA through rupture of the phagosome. However, xenophagy can also be hijacked by bacterial pathogens for their benefit or can be completely inhibited resulting in intracellular growth of the bacterial pathogen. In this perspective, we discuss the molecular differences and similarities between LAP, PINCA and xenophagy in macrophages during bacterial infections.

Macrophages target Listeria monocytogenes by two discrete non-canonical autophagy pathways.

Non-canonical autophagy pathways decorate single-membrane vesicles with Atg8-family proteins such as MAP1LC3/LC3 (microtubule-associated protein 1 light chain 3). Phagosomes containing the bacterial pathogen Listeria monocytogenes (L.m.) can be targeted by a non-canonical autophagy pathway called LC3-associated phagocytosis (LAP), which substantially contributes to the anti-listerial activity of macrophages and immunity. We here characterized a second non-canonical autophagy pathway targeting L.m.-containing phagosomes, which is induced by damage caused to the phagosomal membrane by the pore-forming toxin of L.m., listeriolysin O. This pore-forming toxin-induced non-canonical autophagy pathway (PINCA) was the only autophagic pathway evoked in tissue macrophages deficient for the NADPH oxidase CYBB/NOX2 that produces the reactive oxygen species (ROS) that are required for LAP induction. Similarly, also bone marrow-derived macrophages (BMDM) exclusively targeted L.m. by PINCA as they completely failed to induce LAP because of insufficient production of ROS through CYBB, in part, due to low expression of some CYBB complex subunits. Priming of BMDM with proinflammatory cytokines such as TNF and IFNG/IFNγ increased ROS production by CYBB and endowed them with the ability to target L.m. by LAP. Targeting of L.m. by LAP remained relatively rare, though, preventing LAP from substantially contributing to the anti-listerial activity of BMDM. Similar to LAP, the targeting of L.m.-containing phagosomes by PINCA promoted their fusion with lysosomes. Surprisingly, however, this did not substantially contribute to anti-listerial activity of BMDM. Thus, in contrast to LAP, PINCA does not have clear anti-listerial function suggesting that the two different non-canonical autophagy pathways targeting L.m. may have discrete functions.Abbreviations: actA/ActA: actin assembly-inducing protein A; ATG: autophagy-related; BMDM: Bone marrow-derived macrophages; CALCOCO2/NDP52: calcium-binding and coiled-coil domain-containing protein 2; CYBA/p22phox: cytochrome b-245 light chain; CYBB/NOX2: cytochrome b(558) subunit beta; E. coli: Escherichia coli; IFNG/IFNγ: interferon gamma; L.m.: Listeria monocytogenes; LAP: LC3-associated phagocytosis; LGALS: galectin; LLO: listeriolysin O; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; NCF1/p47phox: neutrophil cytosol factor 1; NCF2/p67phox: neutrophil cytosol factor 2; NCF4/p67phox: neutrophil cytosol factor 4; Peritoneal macrophages: PM; PINCA: pore-forming toxin-induced non-canonical autophagy; plc/PLC: 1-phosphatidylinositol phosphodiesterase; PMA: phorbol 12-myristate 13-acetate; RB1CC1/FIP200: RB1-inducible coiled-coil protein 1; ROS: reactive oxygen species; S. aureus: Staphylococcus aureus; S. flexneri: Shigella flexneri; SQSTM1/p62: sequestosome 1; S. typhimurium: Salmonella typhimurium; T3SS: type III secretion system; TNF: tumor necrosis factor; ULK: unc-51 like autophagy activating kinase; PM: peritoneal macrophages; WT: wild type.

Highly Efficient Transfection of Primary Macrophages with In Vitro Transcribed mRNA.

Macrophages are phagocytic cells specialized in detecting molecules of non-self origin. To this end, they are equipped with a large array of pattern recognition receptors (PRRs). Unfortunately, this also makes macrophages particularly challenging to transfect as the transfection reagent and the transfected nucleic acids are often recognized by the PRRs as non-self. Therefore, transfection often results in macrophage activation and degradation of the transfected nucleic acids or even in suicide of the macrophages. Here, we describe a protocol that allows highly efficient transfection of murine primary macrophages such as peritoneal macrophages (PM) and bone marrow-derived macrophages (BMDM) with mRNA in vitro transcribed from DNA templates such as plasmids. With this simple protocol, transfection rates of about 50-65% for PM and about 85% for BMDM are achieved without cytotoxicity or immunogenicity observed. We describe in detail the generation of mRNA for transfection from DNA constructs such as plasmids and the transfection procedure.

Mitochondrial reactive oxygen species enable proinflammatory signaling through disulfide linkage of NEMO.

A major function of macrophages during infection is initiation of the proinflammatory response, leading to the secretion of cytokines that help to orchestrate the immune response. Here, we identify reactive oxygen species (ROS) as crucial mediators of proinflammatory signaling leading to cytokine secretion in Listeria monocytogenes-infected macrophages. ROS produced by NADPH oxidases (Noxes), such as Nox2, are key components of the macrophage response to invading pathogens; however, our data show that the ROS that mediated proinflammatory signaling were produced by mitochondria (mtROS). We identified the inhibitor of κB (IκB) kinase (IKK) complex regulatory subunit NEMO [nuclear factor κB (NF-κB) essential modulator] as a target for mtROS. Specifically, mtROS induced intermolecular covalent linkage of NEMO through disulfide bonds formed by Cys54 and Cys347, which was essential for activation of the IKK complex and subsequent signaling through the extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) and NF-κB pathways that eventually led to the secretion of proinflammatory cytokines. We thus identify mtROS-dependent disulfide linkage of NEMO as an essential regulatory step of the proinflammatory response of macrophages to bacterial infection.