Lysozyme activates Enterococcus faecium to induce necrotic cell death in macrophages.

The 7-oxysterols are recognised as strong enhancers of inflammatory processes in foamy macrophages. Atheroma-relevant 7-oxysterol mixtures induce a mixed type of cell death in macrophages, and trigger cellular oxidative stress responses, which mimic oxidative exposures observed in atherosclerotic lesions. However, the macrophage proteome has not previously been determined in the 7-oxysterol treated cell model. The aim of the present study was to determine the specific effects of an atheroma-relevant 7-oxysterol mixture on human macrophage proteome. Human THP-1 macrophages were exposed to an atheroma-relevant mixture of 7β-hydroxycholesterol and 7-ketocholesterol. Two-dimensional gel electrophoresis and mass spectrometry techniques were used to analyse the alterations in macrophage proteome, which resulted in the identification of 19 proteins with significant differential expression upon oxysterol loading; 8 increased and 11 decreased. The expression patterns of 11 out of 19 identified significant proteins were further confirmed by tandem-mass spectrometry, including further validation of increased histone deacetylase 2 and macrophage scavenger receptor types I and II expressions by western blot analysis. Identified proteins with differential expression in the cell model have been associated with i) signalling imbalance in cell death and cellular longevity; ii) lipid uptake and metabolism in foam cells; and iii) inflammatory proteins. The presented findings highlight a new proteomic platform for further studies into the functional roles of macrophages in atherosclerosis, and present a cell model for future studies to modulate the macrophage proteome by potential anti-atherosclerotic agents.

Macrophages play a pivotal role in the pathogenesis of a variety of diseases. These studies were performed to characterize the mechanisms by which Toll-like receptor 4 (TLR4)-mediated NF-kappaB activation promotes resistance to cell death in macrophages. When NF-kappaB activation was inhibited by a super-repressor, IkappaBalpha, the TLR4 ligand lipopolysaccharide induced the activation of caspase 8, the loss of mitochondrial transmembrane potential (DeltaPsim), and apoptotic cell death in macrophages. The inhibition of caspase 8 activation suppressed DNA fragmentation but failed to protect macrophages against the loss of DeltaPsim and resulted in necrotic cell death. In contrast, the reduction of receptor-interacting protein 1 suppressed the loss of DeltaPsim and inhibited apoptotic cell death. Further, when caspase 8 activation was suppressed, the knock down of receptor-interacting protein inhibited the loss of DeltaPsim and necrotic cell death. These observations demonstrate that following TLR4 ligation by lipopolysaccharide, NF-kappaB is a critical determinant of macrophage life or death, whereas caspase 8 determines the pathway employed.

Interferon-γ-induced apoptosis and activation of THP-1 macrophages

Human respiratory syncytial virus (hRSV) is the most common cause of viral pneumonia in infants and children worldwide and creates significant airway disease in immunocompromised people and the elderly. Inflammation induced by hRSV infection is responsible for its hallmark manifestations of bronchiolitis and pneumonia. The cellular debris created secondary to lytic cell death of infected cells is a potent initiator of this inflammation. Macrophages are known to play a pivotal role in the early innate immune and inflammatory response to viral pathogens. While previous studies have investigated the mechanisms responsible for lytic cell death in Influenza A-infected macrophages and hRSV-infected neutrophils, the lytic cell death mechanisms associated with hRSV infection in macrophages remain unknown. In order to address this knowledge gap, we treated an hRSV-infected human THP-1 macrophage cell line with various inhibitors that block two lytic cell death pathways: pyroptosis and necroptosis. In addition, we used pyroptosis-deficient human macrophages to further clarify the lytic cell death mechanisms occurring during infection. Our studies revealed that hRSV induces lytic cell death in macrophages via both pyroptosis and necroptosis, specifically through caspase-1 and inflammasome-dependent pyroptotic mechanisms and through RIPK1-RIPK3-MLKL necroptotic mechanisms of cell death.

The mammalian Hippo kinases, MST1 and MST2, regulate organ development and suppress tumor formation by balancing cell proliferation and death. In macrophages, inflammasomes detect molecular patterns from invading pathogens or damaged host cells and trigger programmed cell death. In addition to lytic pyroptosis, the signatures associated with apoptosis are induced by inflammasome activation, but how the inflammasomes coordinate different cell death processes remains unclear. Here, we identify the crucial role of MST1/2 in inflammasome-triggered cell death. Macrophages proteolytically convert full-length MST1/2 into the MST1/2 N-terminal fragments (MST1/2-NT) when the NLRC4 inflammasome detects flagellin from the pathogenic bacterium, Legionella pneumophila. Activation of the NLRP3 inflammasome by the damage-associated molecular pattern, extracellular ATP, also produces MST1/2-NT. Caspase-1, the protease activated by these inflammasomes, directly cleaves MST1/2, and blockage of caspase-1 inhibits MST1/2-NT production in macrophages challenged with L. pneumophila. Importantly, MST1/2-NT production is critical for macrophages to trigger a set of death processes associated with apoptosis upon inflammasome activation and knocking out Mst1/2 causes dysregulated gasdermin protein cleavage for pyroptotic death. Furthermore, macrophages lacking MST1/2 have increased susceptibility to virulent L. pneumophila, revealing that the Hippo kinases are important restriction factors against the pathogen. These findings demonstrate that proteolytic cleavage of MST1/2 induced by inflammatory stimuli is an immune pathway to regulate programmed cell death in macrophages and uncover a unique link between the tumor-suppressive Hippo kinases and the inflammasomes in innate immunity.

Palmitate induces RIP1/RIP3-dependent necrosis via MLKL-mediated pore formation in the plasma membrane of RAW 264.7 cells

The cell survival protein kinase Akt/protein kinase B (PKB) is a known regulator of apoptosis but its role in Fas-mediated cell death and its regulatory mechanisms are unclear. Here, we show that stimulation of the Fas receptor by its ligand (FasL) induces rapid phosphorylation of Akt/PKB and a parallel increase in cell apoptosis in epidermal Cl41 cells. Inhibition of PI3K/Akt by overexpression of dominant-negative mutants of PI3K (Δp85) and Akt (Akt-T308A/S473A) protects the cells from apoptosis induced by FasL, indicating an unexpected pro-apoptotic role of PI3K/Akt in Fas signaling process. Electron spin resonance (ESR) studies also show that FasL treatment induces rapid generation of reactive oxygen species (ROS) and inhibition of ROS by antioxidants effectively inhibits Akt/PKB signaling, suggesting that FasL activation of Akt/PKB is redox-sensitive. In cells transfected with dominant-negative PI3K/Akt, Fas receptor expression is downregulated but FLICE-inhibitory protein (FLIP) expression is unaffected. We also investigate the role of TNF-α and NF-κB in FasL-mediated cell death in macrophages. Gene transfection studies using NF-κB-dependent reporter plasmid showed that FasL did indeed activate NF-κB promoter activity. Gel shift studies revealed that FasL mobilized the p50/p65 heterodimeric form of NF-κB. Inhibition of NF-κB by specific NF-κB inhibitor, caffeic acid phenylethyl ester, or by dominant expression of the NF-κB inhibitory subunit IκB caused an increase in FasL-induced apoptosis and a reduction in TNF-α expression. However, neutralization of TNF-α by specific anti-TNF-α antibody had no protective effective effect on FasL-induced apoptosis. These results indicate that FasL-mediated cell death in macrophages is regulated through NF-κB and is independent of TNF-α activation. The wild-type FLIPL and various N-terminal deletion plasmids were generated and used to study the role of FLIP in Fas-mediated NF-κB activation and apoptosis. NF-κB is activated by FLIPL and its deletion mutants except Δ7. Also, FLIPL and its deletion mutants Δ2-Δ6 (but not Δ7) inhibit FasL-induced apoptosis. We also tested whether our truncated mutants are capable of binding to TRAF2. We performed immunoprecipitation experiments and found that both DED1 and DED2 domains of FLIP are required for TRAF2 recruitment. These results are in good agreement with our apoptosis and NF-κB activation data.

Macrophages can impact the development of tumors and their responses to therapy. However, we lack a full understanding of how macrophage activation and survival are regulated and might be therapeutically modulated. To address these questions, we turned to a host-pathogen interaction that is likely to have impacted the evolution of these processes: infection by Mycobacterium tuberculosis (Mtb), the leading cause of death from infection. Once internalized by macrophages, Mtb resists killing by macrophages and replicates within them. Ultimately, Mtb infection results in death of macrophages, allowing dissemination of the pathogen to other cells. To explore how Mtb induces macrophage cell death and how macrophage cell death might impact host defense against Mtb, we performed a genome-wide CRISPR-Cas9 recessive genetic screen in RAW264.7 macrophages. We discovered that the absence of components of the type I interferon signaling pathway, including the IFN-α/β receptor (IFNAR), delays Mtb-induced cell death. It is known that Mtb infection induces macrophages to produce type I interferon. Our finding directly links type I interferon signaling pathway and Mtb-induced macrophage death. We are currently working on the mechanism of the type I IFN-induced death of bacterially-stimulated macrophages. Meanwhile, we are testing blockers of this pathway as host-directed therapy against TB and have seen striking protective effects of anti-IFNAR1 mAb in Mtb-infected mice, whether the mAb is administered before or after infection. Further exploration of the protective effect of anti-IFNAR mAb could point to an antibody-based, host-directed therapy. Acknowledgment: We thank Prof. R. Schreiber, Washington University, for facilitating access to anti-IFNAR mAb. Citation Format: Ryan Zander, David Schauder, Gang Xin, Christine Nguyen, Xiaopeng Wu, Weiguo Cui. Exploration and exploitation of macrophage death pathways: Infection by Mycobacterium tuberculosis as a model [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B201.

Genome scale mutagenesis identifies many genes required for mycobacterial infectivity and survival, but their contributions and mechanisms of action within the host are poorly understood. Using CRISPR interference, we created a knockdown of ppe31Mm gene in Mycobacterium marinum (M. marinum), which reduced the resistance to acid medium. To further explore the function of PPE31, the ppe31 mutant strain was generated in M. marinum and Mycobacterium tuberculosis (M. tuberculosis), respectively. Macrophages infected with the ppe31Mm mutant strain caused a reduced inflammatory mediator expressions. In addition, macrophages infected with M. marinum Δppe31Mm had decreased host cell death dependent on JNK signaling. Consistent with these results, deletion of ppe31Mtb from M. tuberculosis increased the sensitivity to acid medium and reduced cell death in macrophages. Furthermore, we demonstrate that both ppe31 mutants from M. marinum and M. tuberculosis resulted in reduced survival in macrophages, and the survivability of M. marinum was deceased in zebrafish due to loss of ppe31Mm. Our findings confirm that PPE31 as a virulence associated factor that modulates innate immune responses to mycobacterial infection.

Macrophage apoptosis is an important feature of atherosclerotic plaque development. Research directed at understanding the functional consequences of macrophage death in atherosclerosis has revealed opposing roles for apoptosis in atherosclerotic plaque progression. In early lesions, macrophage apoptosis limits lesion cellularity and suppresses plaque progression. In advanced lesions, macrophages apoptosis promotes the development of the necrotic core, a key factor in rendering plaques vulnerable to disruption and in acute lumenal thrombosis. The first section of this review will examine the role of phagocytic clearance of apoptotic macrophages, a process known as efferocytosis, in the dichotomous roles of macrophage apoptosis in early vs. advanced lesions. The second section will focus on the molecular and cellular mechanisms that are thought to govern macrophage death during atherosclerosis. Of particular interest is the complex and coordinated role that the endoplasmic reticulum (ER) stress pathway and pattern recognition receptors (PRRs) may play in triggering macrophage apoptosis.

The structure-activity relationships of lipopolysaccharide (LPS) in tumor necrosis factor-alpha (TNF-alpha) production and induction of macrophage cell death in the presence of cycloheximide (CHX) were examined in a murine macrophage-like cell line, J774.1. TNF-alpha production is one of the characteristic phenotypes of LPS-activated macrophages, and is observed upon incubation with LPS. On the contrary, macrophage cell death, which had been found in our laboratory (Amano F., Karahashi H., J. Endotoxin Res., 3, 415423 (1996)) and was examined as the release of lactate dehydrogenase (LDH) from cells into the culture supernatant, was observed 2.5 h after the addition of LPS in the presence of CHX. However, both TNF-alpha production and macrophage cell death were similarly dependent on the structures of LPS and lipid A. At more than 10 ng/ml, wild-type LPS from E.coli and S. minnesota exhibited the highest activity, and LPS as well as diphosphoryl lipid A from S. minnesota rough mutants also exhibited activity, but E. coli LPS detoxified by alkaline treatment or monophosphoryl lipid A from S. minnesota exhibited no activity even at 100 ng/ml. These results suggest that LPS-induced macrophage cell death in the presence of CHX shows similar requirements for LPS and/or lipid A structures as for the macrophage activation at higher doses than 10 ng/ml, although the former was not observed at 1 ng/ml LPS, while the latter was. However, TNF-alpha does not seem to be involved in the induction of macrophage cell death, because a neutralizing anti-TNF-alpha antibody failed to block the macrophage cell death and because recombinant TNF-alpha had little effect on the extent of LDH release in the presence or absence of LPS and/or CHX, and also because TNF-alpha production by LPS was abolished in the presence of CHX.

Clustering of the enteropathogenic Escherichia coli (EPEC) type III secretion system (T3SS) effector translocated intimin receptor (Tir) by intimin leads to actin polymerisation and pyroptotic cell death in macrophages. The effect of Tir clustering on the viability of EPEC-infected intestinal epithelial cells (IECs) is unknown. We show that EPEC induces pyroptosis in IECs in a Tir-dependent but actin polymerisation-independent manner, which was enhanced by priming with interferon gamma (IFNγ). Mechanistically, Tir clustering triggers rapid Ca2+ influx, which induces lipopolysaccharide (LPS) internalisation, followed by activation of caspase-4 and pyroptosis. Knockdown of caspase-4 or gasdermin D (GSDMD), translocation of NleF, which blocks caspase-4 or chelation of extracellular Ca2+, inhibited EPEC-induced cell death. IEC lines with low endogenous abundance of GSDMD were resistant to Tir-induced cell death. Conversely, ATP-induced extracellular Ca2+ influx enhanced cell death, which confirmed the key regulatory role of Ca2+ in EPEC-induced pyroptosis. We reveal a novel mechanism through which infection with an extracellular pathogen leads to pyroptosis in IECs.

Sterile solid microparticles (MPs) trigger an innate type 2 inflammatory response dependent on a SYK-BTK-IL33 signaling axis (Nature Materials,18, 289,2019). Inoculation with MPs trigger innate inflammation with influx of neutrophils, eosinophils and alternatively activated macrophages (M2). MPs also trigger cell death in macrophages leading to release of IL33 and the development of a type 2 response with increased IL4,IL5 and IL13 and expression of M2 markers including Arg1, Relmα and Chil3. Blockade of cell death by inhibiting necroptosis (necrox-2) or apoptosis (ZVED-FMK) alone or in combination inhibited the MP mediated release of IL33 by macrophages ex vivo. We also examined the role of adenosine in MP-mediated IL-33 release by inoculating LysMCreA2bfl/flwith MP.MP induced inflammation was blocked in LysMCreA2bfl/fmice compared to LysMCre controls, with significant decreases in type 2 cytokines and IL33. Adoptive transfer of wild type macrophages to LysMCreA2bfl/flrestored the MP-mediated type 2 inflammation. These findings indicate that programmed cell death and A2b receptor signaling pathways play a major role in particulate induced sterile inflammation.

Modulation of cell death is a powerful strategy employed by pathogenic bacteria to evade host immune clearance and occupy profitable replication niches during infection. Intracellular pathogens employ the type III secretion system (T3SS) to deliver effectors, which interfere with regulated cell death pathways to evade immune defenses. Here, we reveal that poly(ADP-ribose) polymerase-1 (PARP1)-dependent cell death restrains Edwardsiella piscicida’s proliferation in mouse monocyte macrophages J774A.1, of which PARP1 activation results in the accumulation of poly(ADP-ribose) (PAR) and enhanced inflammatory response. Moreover, E. piscicida, an important intracellular pathogen, leverages a T3SS effector YfiD to impair PARP1’s activity and inhibit PAR accumulation. Once translocated into the host nucleus, YfiD binds to the ADP-ribosyl transferase (ART) domain of PARP1 to suppress its PARylation ability as the pharmacological inhibitor of PARP1 behaves. Furthermore, the interaction between YfiD and ART mainly relies on the complete unfolding of the helical domain, which releases the inhibitory effect on ART. In addition, YfiD impairs the inflammatory response and cell death in macrophages and promotes in vivo colonization and virulence of E. piscicida. Collectively, our results establish the functional mechanism of YfiD as a potential PARP1 inhibitor and provide more insights into host defense against bacterial infection.

Programmed cell death is an essential host response to viral infection, but whether infected macrophages undergo inflammatory or non-inflammatory forms of death has important consequences for disease progression. In this study, we found that influenza A virus infection induces a modification of microtubules known as detyrosination, which stabilizes their structure. This change was driven by the host enzyme vasohibin-1 through activation of the inflammasome, a key signaling complex that normally promotes inflammatory cell death. Remarkably, enhanced detyrosination shifted dying cells away from inflammatory membrane rupture toward apoptosis, an immunologically silent cell death pathway that preserves membrane integrity. Our findings identify microtubule detyrosination as a stress-induced host response during influenza A virus infection, highlighting a novel mechanism by which cytoskeletal modification influences the outcome of infection.