Caspase-8 is the molecular switch for apoptosis, necroptosis and pyroptosis.

Caspase-8 is the initiator caspase of extrinsic apoptosis1,2 and inhibits necroptosis mediated by RIPK3 and MLKL. Accordingly, caspase-8 deficiency in mice causes embryonic lethality3, which can be rescued by deletion of either Ripk3 or Mlkl4-6. Here we show that the expression of enzymatically inactive CASP8(C362S) causes embryonic lethality in mice by inducing necroptosis and pyroptosis. Similar to Casp8-/- mice3,7, Casp8C362S/C362S mouse embryos died after endothelial cell necroptosis leading to cardiovascular defects. MLKL deficiency rescued the cardiovascular phenotype but unexpectedly caused perinatal lethality in Casp8C362S/C362S mice, indicating that CASP8(C362S) causes necroptosis-independent death at later stages of embryonic development. Specific loss of the catalytic activity of caspase-8 in intestinal epithelial cells induced intestinal inflammation similar to intestinal epithelial cell-specific Casp8 knockout mice8. Inhibition of necroptosis by additional deletion of Mlkl severely aggravated intestinal inflammation and caused premature lethality in Mlkl knockout mice with specific loss of caspase-8 catalytic activity in intestinal epithelial cells. Expression of CASP8(C362S) triggered the formation of ASC specks, activation of caspase-1 and secretion of IL-1ß. Both embryonic lethality and premature death were completely rescued in Casp8C362S/C362SMlkl-/-Asc-/- or Casp8C362S/C362SMlkl-/-Casp1-/- mice, indicating that the activation of the inflammasome promotes CASP8(C362S)-mediated tissue pathology when necroptosis is blocked. Therefore, caspase-8 represents the molecular switch that controls apoptosis, necroptosis and pyroptosis, and prevents tissue damage during embryonic development and adulthood.

TRIM21 Is Targeted for Chaperone-Mediated Autophagy during Salmonella Typhimurium Infection.

Salmonella enterica serovar Typhimurium (S Typhimurium) is a Gram-negative bacterium that induces cell death of macrophages as a key virulence strategy. We have previously demonstrated that the induction of macrophage death is dependent on the host's type I IFN (IFN-I) response. IFN-I signaling has been shown to induce tripartite motif (TRIM) 21, an E3 ubiquitin ligase with critical functions in autoimmune disease and antiviral immunity. However, the importance and regulation of TRIM21 during bacterial infection remains poorly understood. In this study, we investigated the role of TRIM21 upon S Typhimurium infection of murine bone marrow-derived macrophages. Although Trim21 expression was induced in an IFN-I-dependent manner, we found that TRIM21 levels were mainly regulated posttranscriptionally. Following TLR4 activation, TRIM21 was transiently degraded via the lysosomal pathway by chaperone-mediated autophagy (CMA). However, S Typhimurium-induced mTORC2 signaling led to phosphorylation of Akt at S473, which subsequently impaired TRIM21 degradation by attenuating CMA. Elevated TRIM21 levels promoted macrophage death associated with reduced transcription of NF erythroid 2-related factor 2 (NRF2)-dependent antioxidative genes. Collectively, our results identify IFN-I-inducible TRIM21 as a negative regulator of innate immune responses to S Typhimurium and a previously unrecognized substrate of CMA. To our knowledge, this is the first study reporting that a member of the TRIM family is degraded by the lysosomal pathway.

Acid sphingomyelinase is a key regulator of cytotoxic granule secretion by primary T lymphocytes.

Granule-mediated cytotoxicity is the main effector mechanism of cytotoxic CD8+ T cells. We report that CD8+ T cells from acid sphingomyelinase (ASMase)-deficient (ASMase-KO) mice are defective in exocytosis of cytolytic effector molecules; this defect resulted in attenuated cytotoxic activity of ASMase-KO CD8+ T cells and delayed elimination of lymphocytic choriomeningitis virus from ASMase-KO mice. Cytolytic granules of ASMase-KO and wild-type CD8+ T cells were equally loaded with granzymes and perforin, and correctly directed to the immunological synapse. In wild-type CD8+ T cells, secretory granules underwent shrinkage by 82% after fusion with the plasma membrane. In ASMase-KO CD8+ T cells, the contraction of secretory granules was markedly impaired. Thus, ASMase is required for contraction of secretory granules and expulsion of cytotoxic effector molecules.

Reduced mitochondrial resilience enables non-canonical induction of apoptosis after TNF receptor signaling in virus-infected hepatocytes.

BACKGROUND & AIMS: Selective elimination of virus-infected hepatocytes occurs through virus-specific CD8 T cells recognizing peptide-loaded MHC molecules. Herein, we report that virus-infected hepatocytes are also selectively eliminated through a cell-autonomous mechanism. METHODS: We generated recombinant adenoviruses and genetically modified mouse models to identify the molecular mechanisms determining TNF-induced hepatocyte apoptosis in vivo and used in vivo bioluminescence imaging, immunohistochemistry, immunoblot analysis, RNAseq/proteome/phosphoproteome analyses, bioinformatic analyses, mitochondrial function tests. RESULTS: We found that TNF precisely eliminated only virus-infected hepatocytes independently of local inflammation and activation of immune sensory receptors. TNF receptor I was equally relevant for NF-kB activation in healthy and infected hepatocytes, but selectively mediated apoptosis in infected hepatocytes. Caspase 8 activation downstream of TNF receptor signaling was dispensable for apoptosis in virus-infected hepatocytes, indicating an unknown non-canonical cell-intrinsic pathway promoting apoptosis in hepatocytes. We identified a unique state of mitochondrial vulnerability in virus-infected hepatocytes as the cause for this non-canonical induction of apoptosis through TNF. Mitochondria from virus-infected hepatocytes showed normal biophysical and bioenergetic functions but were characterized by reduced resilience to calcium challenge. In the presence of unchanged TNF-induced signaling, reactive oxygen species-mediated calcium release from the endoplasmic reticulum caused mitochondrial permeability transition and apoptosis, which identified a link between extrinsic death receptor signaling and cell-intrinsic mitochondrial-mediated caspase activation. CONCLUSION: Our findings reveal a novel concept in immune surveillance by identifying a cell-autonomous defense mechanism that selectively eliminates virus-infected hepatocytes through mitochondrial permeability transition. LAY SUMMARY: The liver is known for its unique immune functions. Herein, we identify a novel mechanism by which virus-infected hepatocytes can selectively eliminate themselves through reduced mitochondrial resilience to calcium challenge.

Salmonella Typhimurium disrupts Sirt1/AMPK checkpoint control of mTOR to impair autophagy.

During intracellular infections, autophagy significantly contributes to the elimination of pathogens, regulation of pro-inflammatory signaling, secretion of immune mediators and in coordinating the adaptive immune system. Intracellular pathogens such as S. Typhimurium have evolved mechanisms to circumvent autophagy. However, the regulatory mechanisms targeted by S. Typhimurium to modulate autophagy have not been fully resolved. Here we report that cytosolic energy loss during S. Typhimurium infection triggers transient activation of AMPK, an important checkpoint of mTOR activity and autophagy. The activation of AMPK is regulated by LKB1 in a cytosolic complex containing Sirt1 and LKB1, where Sirt1 is required for deacetylation and subsequent activation of LKB1. S. Typhimurium infection targets Sirt1, LKB1 and AMPK to lysosomes for rapid degradation resulting in the disruption of the AMPK-mediated regulation of mTOR and autophagy. The degradation of cytosolic Sirt1/LKB1/AMPK complex was not observed with two mutant strains of S. Typhimurium, ΔssrB and ΔssaV, both compromising the pathogenicity island 2 (SPI2). The results highlight virulence factor-dependent degradation of host cell proteins as a previously unrecognized strategy of S. Typhimurium to evade autophagy.

Hypoxia-induced changes in plasma micro-RNAs correlate with pulmonary artery pressure at high altitude.

In vitro and animal studies revealed micro-RNAs (miRs) to be involved in modulation of hypoxia-induced pulmonary hypertension (HPH). However, knowledge of circulating miRs in humans in the context of HPH is very limited. Since symptoms of HPH are nonspecific and noninvasive diagnostic parameters do not exist, a disease-specific and hypoxemia-independent biomarker indicating HPH would be of clinical value. To examine whether plasma miR levels correlate with hypoxia-induced increase in pulmonary artery pressures, plasma miRs were assessed in a model of hypoxia-related pulmonary hypertension in humans exposed to extreme altitude. Forty healthy volunteers were repetitively examined during a high-altitude expedition up to an altitude of 7,050 m. Plasma levels of miR-17, -21, and -190 were measured by real-time quantitative PCR and correlated with systolic pulmonary artery pressure (SPAP), which was assessed by echocardiography. A significant altitude-dependent increase in circulating miR expression was found (all P values < 0.0001). Compared with baseline at 500 m, miR-17 changed by 4.72 ± 0.57-fold, miR-21 changed by 1.91 ± 0.33-fold, and miR-190 changed by 3.61 ± 0.54-fold at 7,050 m (means ± SD). Even after adjusting for hypoxemia, miR-17 and miR-190 were found to be independently correlated with increased SPAP. Progressive hypobaric hypoxia significantly affects levels of circulating miR-17, -21, and -190. Independently from the extent of hypoxemia, miR-17 and -190 significantly correlate with increased SPAP. These novel findings provide evidence for an epigenetic modulation of hypoxia-induced increase in pulmonary artery pressures by miR-17 and -190 and suggest the potential value of these miRs as biomarkers for HPH.

BID-dependent release of mitochondrial SMAC dampens XIAP-mediated immunity against Shigella.

The X-linked inhibitor of apoptosis protein (XIAP) is a potent caspase inhibitor, best known for its anti-apoptotic function in cancer. During apoptosis, XIAP is antagonized by SMAC, which is released from the mitochondria upon caspase-mediated activation of BID. Recent studies suggest that XIAP is involved in immune signaling. Here, we explore XIAP as an important mediator of an immune response against the enteroinvasive bacterium Shigella flexneri, both in vitro and in vivo. Our data demonstrate for the first time that Shigella evades the XIAP-mediated immune response by inducing the BID-dependent release of SMAC from the mitochondria. Unlike apoptotic stimuli, Shigella activates the calpain-dependent cleavage of BID to trigger the release of SMAC, which antagonizes the inflammatory action of XIAP without inducing apoptosis. Our results demonstrate how the cellular death machinery can be subverted by an invasive pathogen to ensure bacterial colonization.

p22phox C242T Single-Nucleotide Polymorphism Inhibits Inflammatory Oxidative Damage to Endothelial Cells and Vessels.

BACKGROUND: The NADPH oxidase, by generating reactive oxygen species, is involved in the pathophysiology of many cardiovascular diseases and represents a therapeutic target for the development of novel drugs. A single-nucleotide polymorphism, C242T of the p22(phox) subunit of NADPH oxidase, has been reported to be negatively associated with coronary heart disease and may predict disease prevalence. However, the underlying mechanisms remain unknown. METHODS AND RESULTS: With the use of computer molecular modeling, we discovered that C242T single-nucleotide polymorphism causes significant structural changes in the extracellular loop of p22(phox) and reduces its interaction stability with Nox2 subunit. Gene transfection of human pulmonary microvascular endothelial cells showed that C242T p22(phox) significantly reduced Nox2 expression but had no significant effect on basal endothelial O2 (.-) production or the expression of Nox1 and Nox4. When cells were stimulated with tumor necrosis factor-α (or high glucose), C242T p22(phox) significantly inhibited tumor necrosis factor-α-induced Nox2 maturation, O2 (.-) production, mitogen-activated protein kinases and nuclear factor κB activation, and inflammation (all P<0.05). These C242T effects were further confirmed using p22(phox) short-hairpin RNA-engineered HeLa cells and Nox2(-/-) coronary microvascular endothelial cells. Clinical significance was investigated by using saphenous vein segments from non-coronary heart disease subjects after phlebotomies. TT (C242T) allele was common (prevalence of ≈22%) and, in comparison with CC, veins bearing TT allele had significantly lower levels of Nox2 expression and O2 (.-) generation in response to high-glucose challenge. CONCLUSIONS: C242T single-nucleotide polymorphism causes p22(phox) structural changes that inhibit endothelial Nox2 activation and oxidative response to tumor necrosis factor-α or high-glucose stimulation. C242T single-nucleotide polymorphism may represent a natural protective mechanism against inflammatory cardiovascular diseases.

IAP antagonization promotes inflammatory destruction of vascular endothelium.

In this study, we show for the first time that the therapeutic antagonization of inhibitor of apoptosis proteins (IAPs) inhibits B16 melanoma growth by disrupting tumor vasculature. Specifically, the treatment of mice bearing B16 melanoma with an IAP antagonist compound A (Comp A) inhibits tumor growth not by inducing direct cytotoxicity against B16 cells but rather by a hitherto unrecognized antiangiogenic activity against tumor vessels. Our detailed analysis showed that Comp A treatment induces NF-κB activity in B16 tumor cells and facilitates the production of TNF. In the presence of Comp A, endothelial cells (ECs) become highly susceptible to TNF and undergo apoptotic cell death. Accordingly, the antiangiogenic and growth-attenuating effects of Comp A treatment were completely abolished in TNF-R knockout mice. This novel targeting approach could be of clinical value in controlling pathological neoangiogenesis under inflammatory condition while sparing blood vessels under normal condition.

Endogenous Il10 alleviates the systemic antiviral cellular immune response and T cell-mediated immunopathology in select organs of acutely LCMV-infected mice.

The immunoregulatory cytokine IL-10 suppresses T-cell immunity. The complementary question, whether IL-10 is also involved in limiting the collateral damage of vigorous T cell responses, has not been addressed in detail. Here, we report that the particularly strong virus-specific immune response during acute primary infection with the lymphocytic choriomeningitis virus (LCMV) in mice is significantly further increased in Il10-deficient mice, particularly regarding frequencies and cytotoxic activity of CD8(+) T cells. This increase results in exacerbating immunopathology in select organs, ranging from transient local swelling to an increased risk for mortality. Remarkably, LCMV-induced, T cell-mediated hepatitis is not affected by endogenous Il10. The alleviating effect of Il10 on LCMV-induced immunopathology was found to be operative in delayed-type hypersensitivity footpad-swelling reaction and in debilitating meningitis in mice of both the C57BL/6 and BALB/c strains. These strains are prototypic counterpoles for genetically imprinted type 1-biased versus type 2-biased T cell-mediated immune responses against various infectious pathogens. However, during acute LCMV infection, neither systemic cytokine patterns nor the impact of Il10 on LCMV-induced immunopathology differed conspicuously between these two strains of mice. This study documents a physiological role of Il10 in the regulation of a balanced T-cell response limiting immunopathological damage.

Riboflavin (vitamin B2 ) deficiency impairs NADPH oxidase 2 (Nox2) priming and defense against Listeria monocytogenes.

Riboflavin, also known as vitamin B2 , is converted by riboflavin kinase (RFK) into flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), which are essential cofactors of dehydrogenases, reductases, and oxidases including the phagocytic NADPH oxidase 2 (Nox2). Riboflavin deficiency is common in young adults and elderly individuals, who are at the coincidental risk for listeriosis. To address the impact of acute riboflavin deficiency on host defense against Listeria monocytogenes (L.m.), we generated conditional RFK knockout (KO) strains of mice. Phagocyte-specific RFK KO impaired the capability of phagocytes to control intracellular L.m., which corresponded to a greater susceptibility of mice to in vivo challenge with L.m. The oxidative burst of RFK-deficient phagocytes in response to L.m. infection was significantly reduced. Mechanistically, TNF-induced priming of Nox2, which is needed for oxidative burst, was defective in RFK-deficient phagocytes. Lack of riboflavin in wild-type macrophages for only 6 h shut down TNF-induced, RFK-mediated de novo FMN/FAD generation, which was accompanied by diminished ROS production and impaired anti-listerial activity. Vice versa, ROS production by riboflavin-deprived macrophages was rapidly restored by riboflavin supplementation. Our results suggest that acute riboflavin deficiency immediately impairs priming of Nox2, which is of crucial relevance for an effective phagocytic immune response in vivo.

Soluble ligands for NK cell receptors promote evasion of chronic lymphocytic leukemia cells from NK cell anti-tumor activity.

Natural killer (NK) cells are a major component of the anti-tumor immune response. NK cell dysfunctions have been reported in various hematologic malignancies, including chronic lymphocytic leukemia (CLL). Here we investigated the role of tumor cell-released soluble and exosomal ligands for NK cell receptors that modulate NK cell activity. Soluble CLL plasma factors suppressed NK cell cytotoxicity and down-regulated the surface receptors CD16 and CD56 on NK cells of healthy donors. The inhibition of NK cell cytotoxicity was attributed to the soluble ligand BAG6/BAT3 that engages the activating receptor NKp30 expressed on NK cells. Soluble BAG6 was detectable in the plasma of CLL patients, with the highest levels at the advanced disease stages. In contrast, NK cells were activated when BAG6 was presented on the surface of exosomes. The latter form was induced in non-CLL cells by cellular stress via an nSmase2-dependent pathway. Such cells were eliminated by lymphocytes in a xenograft tumor model in vivo. Here, exosomal BAG6 was essential for tumor cell killing because BAG6-deficient cells evaded immune detection. Taken together, the findings show that the dysregulated balance of exosomal vs soluble BAG6 expression may cause immune evasion of CLL cells.

Riboflavin kinase couples TNF receptor 1 to NADPH oxidase.

Reactive oxygen species (ROS) produced by NADPH oxidase function as defence and signalling molecules related to innate immunity and various cellular responses. The activation of NADPH oxidase in response to plasma membrane receptor activation depends on the phosphorylation of cytoplasmic oxidase subunits, their translocation to membranes and the assembly of all NADPH oxidase components. Tumour necrosis factor (TNF) is a prominent stimulus of ROS production, but the molecular mechanisms by which TNF activates NADPH oxidase are poorly understood. Here we identify riboflavin kinase (RFK, formerly known as flavokinase) as a previously unrecognized TNF-receptor-1 (TNFR1)-binding protein that physically and functionally couples TNFR1 to NADPH oxidase. In mouse and human cells, RFK binds to both the TNFR1-death domain and to p22(phox), the common subunit of NADPH oxidase isoforms. RFK-mediated bridging of TNFR1 and p22(phox) is a prerequisite for TNF-induced but not for Toll-like-receptor-induced ROS production. Exogenous flavin mononucleotide or FAD was able to substitute fully for TNF stimulation of NADPH oxidase in RFK-deficient cells. RFK is rate-limiting in the synthesis of FAD, an essential prosthetic group of NADPH oxidase. The results suggest that TNF, through the activation of RFK, enhances the incorporation of FAD in NADPH oxidase enzymes, a critical step for the assembly and activation of NADPH oxidase.

Factor associated with neutral sphingomyelinase activity mediates navigational capacity of leukocytes responding to wounds and infection: live imaging studies in zebrafish larvae.

Factor associated with neutral sphingomyelinase activity (FAN) is an adaptor protein that specifically binds to the p55 receptor for TNF (TNF-RI). Our previous investigations demonstrated that FAN plays a role in TNF-induced actin reorganization by connecting the plasma membrane with actin cytoskeleton, suggesting that FAN may impact on cellular motility in response to TNF and in the context of immune inflammatory conditions. In this study, we used the translucent zebrafish larvae for in vivo analysis of leukocyte migration after morpholino knockdown of FAN. FAN-deficient zebrafish leukocytes were impaired in their migration toward tail fin wounds, leading to a reduced number of cells reaching the wound. Furthermore, FAN-deficient leukocytes show an impaired response to bacterial infections, suggesting that FAN is generally required for the directed chemotactic response of immune cells independent of the nature of the stimulus. Cell-tracking analysis up to 3 h after injury revealed that the reduced number of leukocytes is not due to a reduction in random motility or speed of movement. Leukocytes from FAN-deficient embryos protrude pseudopodia in all directions instead of having one clear leading edge. Our results suggest that FAN-deficient leukocytes exhibit an impaired navigational capacity, leading to a disrupted chemotactic response.

On the antibiotic and antifungal activity of pestalone, pestalachloride A, and structurally related compounds.

Pestalone (1) is a prominent marine natural product first isolated by M. Cueto et al. in 2001 from a co-fermentation of a marine fungus with a marine bacterium. For more than 10 years, 1 had been considered as a promising new antibiotic compound, the reported MIC against methicillin-resistant Staphylococcus aureus (MRSA) being 37 ng/mL. After overcoming the limited availability of 1 by total synthesis (N. Slavov et al., 2010) we performed new biological tests, which did not confirm the expected degree of antibiotic activity. The observed activity of pestalone against different MRSA strains was 3-10 µg/mL, as determined independently in two laboratories. A number of synthetic derivatives of 1 including pestalachloride A and other isoindolinones (formed from 1 by reaction with amines) did not exhibit higher activities as compared to 1 against MRSA and a series of plant pathogens.

Development of an immunochromatographic lateral flow assay to rapidly detect OXA-23-, OXA-40-, OXA-58- and NDM-mediated carbapenem resistance determinants in Acinetobacter baumannii.

Introduction. Acinetobacter baumannii infections can be extremely challenging to treat owing to the worldwide prevalence of multidrug-resistant isolates, especially against carbapenems. Colonization with carbapenem-resistant A. baumannii (CRAb) requires rapid action from an infection control perspective because the organism is known for its propensity for epidemic spread. Hypothesis/Gap Statement. There is an unmet medical need to rapidly identify CRAb to enable appropriate antimicrobial treatment and to prevent transmission. Aim. Our aim was to expand the OXA-detection abilities of the rapid immunochromatographic test (ICT) OXA-23 K-SeT (Coris BioConcept) to include OXA-40- and OXA-58-like carbapenemases, which together confer carbapenem resistance to more than 94 % of CRAb isolates worldwide. Methodology. We used hybridoma technology to generate mAbs against OXA-40 and OXA-58 and selected them for productivity and specificity against recombinant and endogenous OXA-40 and OXA-58. Combinations of the resulting mAbs were analysed in ICT format for their ability to detect recombinant rOXA-40His6 or rOXA-58His6, respectively. Subsequently, selected antibody pairs were implemented into single-OXA-40 or single-OXA-58 prototypes and the final OXA-23/40/58/NDM ICT and were evaluated on clinical Acinetobacter spp. isolates with well-defined carbapenem resistance mechanisms. Results. Five anti-OXA-40 and anti-OXA-58 mAbs were selected. Competition ELISA with combinations of these antibodies revealed that the anti-OXA-40 antibodies bind to one of two binding clusters on OXA-40, while anti-OXA-58 antibodies bind to one of four binding clusters on OXA-58. Direct binding to the corresponding antigen in an ICT format has left only three antibodies against rOXA-40His6 and rOXA-58His6, respectively for the subsequent sandwich ICT selection procedure, which revealed that the anti-OXA-40 (#5) and anti-OXA-58 (#A8) mAbs in combination with the cross-reactive mAb #C8 performed best. They were implemented into single-OXA-40 and single-OXA-58 ICT prototypes and evaluated. These single ICT prototypes demonstrated 100 % specificity and sensitivity. Based on these results, an OXA-23/40/58/NDM-ICT was developed, complemented with OXA-23 and NDM-specific detection. An evaluation with selected carbapenem-resistant Acinetobacter spp. isolates (n=34) showed 100 % specificity. Conclusion. With this easy-to-use detection assay, one can save 12-48 h in diagnostics, which helps to treat patients earlier with appropriate antibiotics and allows immediate intervention to control transmission of CRAb.

Therapeutic concentrations of antibiotics inhibit Shiga toxin release from enterohemorrhagic E. coli O104:H4 from the 2011 German outbreak.

BACKGROUND: The shiga toxin-producing E. coli (STEC) O104:H4 caused a major outbreak in Germany in spring 2011. STEC are usually susceptible to common antibiotics. However, antibiotic treatment of STEC-infected patients is not recommended because STEC may enhance production and release of shiga toxins (STX) in response to antibiotics, which eventually enhances the frequency and severity of clinical symptoms, including haemolytic uraemic syndrome (HUS) and fatalities. RESULTS: We characterized the response to antibiotics of STEC O104:H4 isolates from two HUS patients during the German STEC outbreak in spring 2011 in comparison to the common STEC O157:H7. Liquid cultures of STEC O157:H7 and O104:H4 were incubated with graded dilutions of the antibiotics ciprofloxacin, meropenem, fosfomycin, gentamicin, rifampicin, and chloramphenicol. At defined times of antibiotic treatment, transcriptional activation of the STX2 gene, contents of STX and STX-activity in the culture supernatants were quantified. Unlike the common serotype O157:H7, STEC O104:H4 does not release STX in response to therapeutic concentrations of ciprofloxacin, meropenem, fosfomycin, and chloramphenicol. CONCLUSIONS: In future outbreaks, the response of the respective epidemiologic STEC strain to antibiotics should be rapidly characterized in order to identify antibiotics that do not enhance the release of STX. This will eventually allow clinical studies tackling the question whether antibiotic treatment impacts on the eradication of STEC, clinical course of disease, and frequency of carriers.

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.

Macrophage-intrinsic DUOX1 contributes to type 2 inflammation and mucus metaplasia during allergic airway disease.

The NADPH oxidase DUOX1 contributes to epithelial production of alarmins, including interleukin (IL)-33, in response to injurious triggers such as airborne protease allergens, and mediates development of mucus metaplasia and airway remodeling in chronic allergic airways diseases. DUOX1 is also expressed in non-epithelial lung cell types, including macrophages that play an important role in airway remodeling during chronic lung disease. We therefore conditionally deleted DUOX1 in either lung epithelial or monocyte/macrophage lineages to address its cell-specific actions in innate airway responses to acute airway challenge with house dust mite (HDM) allergen, and in chronic HDM-driven allergic airway inflammation. As expected, acute responses to airway challenge with HDM, as well as type 2 inflammation and related features of airway remodeling during chronic HDM-induced allergic inflammation, were largely driven by DUOX1 with the respiratory epithelium. However, in the context of chronic HDM-driven inflammation, DUOX1 deletion in macrophages also significantly impaired type 2 cytokine production and indices of mucus metaplasia. Further studies revealed a contribution of macrophage-intrinsic DUOX1 in macrophage recruitment upon chronic HDM challenge, as well as features of macrophage activation that impact on type 2 inflammation and remodeling.