Macrophage Cell Death Research Articles

PSS332 - Investigation of the Role of ROS in Silica Induced Phago-Lysosomal Leakage and Cell Death in Alveolar Macrophages

PSS332 - Investigation of the Role of ROS in Silica Induced Phago-Lysosomal Leakage and Cell Death in Alveolar Macrophages

Confounding roles for type I interferons during bacterial and viral pathogenesis

Although type I interferons (IFN-I) were initially defined as potent antiviral agents, they can also cause decreased host resistance to some bacterial and viral infections. The many antiviral functions of the IFN-I include direct suppression of viral replication and activation of the immune response against viruses. In addition to their antiviral effects, IFN-I are also protective against several extracellular bacterial infections, in part, by promoting the induction of TNF-α and nitric oxide. In contrast, there is a negative effect of IFN-I on host resistance during chronic infection with lymphocytic choriomeningitis virus (LCMV) and acute infections with intracellular bacteria. In the case of LCMV, chronic IFN-I signaling induces adaptive immune system suppression. Blockade of IFN-I signaling removes the suppression and allows CD4 T-cell- and IFN-γ-mediated resolution of the infection. During acute intracellular bacterial infection, IFN-I suppress innate immunity by at least two defined mechanisms. During Francisella infection, IFN-I prevent IL-17 upregulation on γδ T cells and neutrophil recruitment. Following Listeria infection, IFN-I promote the cell death of macrophages and lymphocytes, which leads to innate immune suppression. These divergent findings for the role of IFN-I on pathogen control emphasize the complexity of the interferons system and force more mechanistic evaluation of its role in pathogenesis. This review evaluates IFN-I during infection with an emphasis on work carried out IFN-I-receptor-deficient mice.

Crosstalk Between Autophagy and Apoptosis in RAW 264.7 Macrophages Infected With Ectromelia Orthopoxvirus

Several studies have provided evidence that complex relationships between autophagic and apoptotic cell death pathways occur in cancer and virus-infected cells. Previously, we demonstrated that infection of macrophages with Moscow strain of ectromelia virus (ECTV-MOS) induces apoptosis under in vitro and in vivo conditions. Here, we found that autophagy was induced in RAW 264.7 cells during infection with ECTV-MOS. Silencing of beclin 1, an autophagy-related gene, reduced the percentage of late apoptotic cells in virus-infected RAW 264.7 macrophages. Pharmacological modulation of autophagy by wortmannin (inhibitor) or rapamycin (inductor) did not affect or cause increased apoptosis in ECTV-MOS-infected RAW 264.7 cells, respectively. Meantime, blocking apoptosis by a pan-caspase inhibitor, Z-VAD-FMK, increased the formation of autophagosomes in infected macrophages. Taken together, three important points arise from our study. First, autophagy may co-occur with apoptosis in RAW 264.7 cells exposed to ECTV-MOS. Second, at later stages of infection, autophagy may partially participate in the execution of macrophage cell death by enhancing apoptosis. Third, when apoptosis is blocked infected macrophages undergo increased autophagy. Our results provide new information about the relationship between autophagy and apoptosis in ECTV-MOS-infected macrophages.

Triglyceride enhances susceptibility to TNF-α-induced cell death in THP-1 cells

Monocytes and macrophages play a major role in atherosclerosis development. Previously, we found that triglyceride (TG) promoted cell death of PMA-differentiated THP-1 macrophages. In this study, we compared the responsiveness of THP-1 monocytes and PMA-differentiated THP-1 macrophages to TNF-α-induced cell death. We found that, whereas THP-1 monocytes were TNF-α-resistant, THP-1 macrophages were sensitive to TNF-α-induced cell death. THP-1 monocytes treated with TG underwent cell death beginning at 24 h and addition of TNF-α further increased cell death. Based on these observations, we hypothesized that TG-induced differentiation of THP-1 monocytes into THP-1 macrophages, subsequently allowing sensitivity to TNF-α. To determine if TG could induce differentiation of THP-1 monocytes into THP-1 macrophages, we examined the mRNA expression levels of the macrophage-specific markers, CD11b, CD18, CD36 and CD68, by RT-PCR analysis. Our results show that expression of CD11b, CD36 and CD68 increased in TG-treated THP-1 monocytes in a dose- and time-dependent manner; furthermore, TNF-α expression was upregulated in TG-treated THP-1 monocytes. We have concluded that TG induces differentiation of THP-1 monocytes into macrophages concomitant with the production of TNF-α and increased sensitivity to TNF-α-dependent cell death.

Repression of Inflammasome by Francisella tularensis during Early Stages of Infection

Francisella tularensis is an important human pathogen responsible for causing tularemia. F. tularensis has long been developed as a biological weapon and is now classified as a category A agent by the Centers for Disease Control because of its possible use as a bioterror agent. F. tularensis represses inflammasome; a cytosolic multi-protein complex that activates caspase-1 to produce proinflammatory cytokines IL-1β and IL-18. However, the Francisella factors and the mechanisms through which F. tularensis mediates these suppressive effects remain relatively unknown. Utilizing a mutant of F. tularensis in FTL_0325 gene, this study investigated the mechanisms of inflammasome repression by F. tularensis. We demonstrate that muted IL-1β and IL-18 responses generated in macrophages infected with F. tularensis live vaccine strain (LVS) or the virulent SchuS4 strain are due to a predominant suppressive effect on TLR2-dependent signal 1. Our results also demonstrate that FTL_0325 of F. tularensis impacts proIL-1β expression as early as 2 h post-infection and delays activation of AIM2 and NLRP3-inflammasomes in a TLR2-dependent fashion. An enhanced activation of caspase-1 and IL-1β observed in FTL_0325 mutant-infected macrophages at 24 h post-infection was independent of both AIM2 and NLRP3. Furthermore, F. tularensis LVS delayed pyroptotic cell death of the infected macrophages in an FTL_0325-dependent manner during the early stages of infection. In vivo studies in mice revealed that suppression of IL-1β by FTL_0325 early during infection facilitates the establishment of a fulminate infection by F. tularensis. Collectively, this study provides evidence that F. tularensis LVS represses inflammasome activation and that F. tularensis-encoded FTL_0325 mediates this effect.

Notch signaling regulates expression of Mcl-1 and apoptosis in PPD-treated macrophages

Macrophages are cellular targets for infection by bacteria and viruses. The fate of infected macrophages plays a key role in determining the outcome of the host immune response. Apoptotic cell death of macrophages is considered to be a protective host defense that eliminates pathogens and infected cells. In this study, we investigated the involvement of Notch signaling in regulating apoptosis in macrophages treated with tuberculin purified protein derivative (PPD). Murine bone marrow-derived macrophages (BMMs) treated with PPD or infected with Mycobacterium bovis Bacillus Calmette-Guérin (BCG) induced upregulation of Notch1. This upregulation correlated well with the upregulation of the anti-apoptotic gene mcl-1 both at the transcriptional and translational levels. Decreased levels of Notch1 and Mcl-1 were observed in BMM treated with PPD when a gamma secretase inhibitor (GSI), which inhibits the processing of Notch receptors, was used. Moreover, silencing Notch1 in the macrophage-like cell line RAW264.7 decreased Mcl-1 protein expression, suggesting that Notch1 is critical for Mcl-1 expression in macrophages. A significant increase in apoptotic cells was observed upon treatment of BMM with PPD in the presence of GSI compared to the vehicle-control treated cells. Finally, analysis of the mcl-1 promoter in humans and mice revealed a conserved potential CSL/RBP-Jκ binding site. The association of Notch1 with the mcl-1 promoter was confirmed by chromatin immunoprecipitation. Taken together, these results indicate that Notch1 inhibits apoptosis of macrophages stimulated with PPD by directly controlling the mcl-1 promoter.

TB: the Yin and Yang of lipid mediators

There is a growing appreciation of the diverse roles that lipid mediators play in modulating inflammatory responses during infection. In the case of tuberculosis, virulent mycobacteria induce host production of anti-inflammatory mediators, including lipoxins, which limit the host inflammatory response and lead to necrotic cell death of infected macrophages. Recent work using the zebrafish model suggests that, while excess anti-inflammatory lipoxins are host detrimental during mycobacterial infections, excess pro-inflammatory lipids also drive host susceptibility. The balance of these inflammatory states is influenced by common human genetic variation in Asia. Fuller understanding of the mechanisms of eicosanoid-mediated inflammatory imbalance during tuberculosis infection has important implications for the development of adjunctive therapies.

Catastrophic Inflammatory Death of Monocytes and Macrophages by Overtaking of A Critical Dose of Endocytosed Synthetic Amorphous Silica Nanoparticles/Serum Protein Complexes

We tested whether phagocytic monocytes/macrophages are more susceptible than nonphagocytes to nanoparticle (NP) toxicity. We compared in vitro cell death and proinflammatory cytokine production in human monocytes, macrophages, lymphocytes and HeLa cells due to synthetic amorphous silica (SiO2)-NPs in different serum concentrations and correlated them with cellular uptake and distribution. Phagocytes were approximately ten-times more sensitive than nonphagocytes to SiO2-NPs and more effectively endocytosed SiO2-NP-serum protein nanoagglomerates, so determining their accumulation in acidic endocytic compartments well beyond a critical/cytotoxic threshold. Monocyte/macrophage death was paralleled by cytokine secretion. The physiological specialization of monocytes/macrophages to effectively capture NPs may expose them to the risk of catastrophic inflammatory death upon saturation of their maximal storage capacity.

Modulation of Apoptotic Pathways of Macrophages by Surface-Functionalized Multi-Walled Carbon Nanotubes

Biomedical applications of carbon nanotubes (CNTs) often involve improving their hydrophilicity and dispersion in biological media by modifying them through noncovalent or covalent functionalization. However, the potential adverse effects of surface-functionalized CNTs have not been well characterized. In this study, we functionalized multi-walled CNTs (MWCNTs) via carboxylation, to produce MWCNTs-COOH, and via poly (ethylene glycol) linking, to produce MWCNTs-PEG. We used these functionalized MWCNTs to study the effect of surface functionalization on MWCNTs-induced toxicity to macrophages, and elucidate the underlying mechanisms of action. Our results revealed that MWCNTs-PEG were less cytotoxic and were associated with less apoptotic cell death of macrophages than MWCNTs-COOH. Additionally, MWCNTs-PEG induced less generation of reactive oxygen species (ROS) involving less activation of NADPH oxidase compared with MWCNTs-COOH, as evidenced by membrane translocation of p47phox and p67phox in macrophages. The less cytotoxic and apoptotic effect of MWCNTs-PEG compared with MWCNTs-COOH resulted from the lower cellular uptake of MWCNTs-PEG, which resulted in less activation of oxidative stress-responsive pathways, such as p38 mitogen-activated protein kinases (MAPK) and nuclear factor (NF)-κB. These results demonstrate that surface functionalization of CNTs may alter ROS-mediated cytotoxic and apoptotic response by modulating apoptotic signaling pathways. Our study thus provides new insights into the molecular basis for the surface properties affecting CNTs toxicity.

Distribution of Six Effector Protein Virulence Genes Among Salmonella enterica enterica Serovars Isolated from Children and their Correlation with Biofilm Formation and Antimicrobial Resistance

Salmonella enterica enterica encodes a variety of virulence factors. Among them, the type III secretion system (TTSS) encoded in the Salmonella pathogenicity islands (SPIs) is required for induction of proinflammatory responses, invasion of intestinal epithelial cells, induction of cell death in macrophages, and elicitation of diarrhea. The presence of the effector protein genes sopB, sopD, sopE, sopE2, avrA, and sptP of the SPIs was analyzed in 194 S. enterica enterica strains belonging to 19 serovars. S. enterica enterica strains were collected from children with gastroenteritis, either hospitalized or attending the outpatient clinic, aged 1-14years. Nineteen different serotypes were included in the study. Serotyping, biofilm formation determination, and antimicrobial resistance of the planktonic as well as the biofilm forms of the strains have been reported previously. At least one virulence gene was present in all Salmonella isolates. Biofilm formation was statistically independent of any of the six genes. Strains lacking sopE and sopE2 were more resistant to all the antimicrobials. The association of the virulence genes with the antimicrobial resistance of Salmonella in general has been previously reported and is a matter of further investigation. For the clinical expression of pathogenicity in humans, the contribution of these genes is questionable, as some strains bearing only a single gene (either sptP or avrA) were still capable of causing gastroenteritis.

IL-18 provided in dying bacterial-infected macrophages induces IFN-γ production in functional T-cell hybridoma B6HO3 through cell conjugates

We have previously reported that the co-culture of functional T-cell hybridoma B6HO3 with dying J774 macrophage cells infected with Listeria monocytogenes (LM) results in the production of IFN-γ by B6HO3 cells. Here, we explore the mechanism underlying this phenomenon. We found that IFN-γ production was dependent on IL-18, but that the dying LM-infected macrophages produced no more than 100 pg/ml of IL-18, much less than the amount of IL-18 required for stimulating B6HO3 cells to produce IFN-γ. Furthermore, IL-18 binding protein added to the co-culture was unable to easily gain access to IL-18 for neutralisation. B6HO3 cells formed cell conjugates with J774 macrophages, and IFN-γ-producing B6HO3 cells were spatially and temporally associated with LM-infected macrophage cell death that exhibited neither pyroptosis nor pyronecrosis. These results suggest that the IL-18 produced by dying LM-infected macrophages is released to the interface of the cell conjugates, thereby inducing B6HO3 cells to produce IFN-γ. Based on the present and also previous findings, we propose that IL-18 released from macrophages because of cell death caused by bacteria may be the primary cytokine that triggers the innate IFN-γ production that is required for activating the bactericidal functions of macrophages at early stages of bacterial infection.

Hydrogen Peroxide Produced by Oral Streptococci Induces Macrophage Cell Death

Hydrogen peroxide (H2O2) produced by members of the mitis group of oral streptococci plays important roles in microbial communities such as oral biofilms. Although the cytotoxicity of H2O2 has been widely recognized, the effects of H2O2 produced by oral streptococci on host defense systems remain unknown. In the present study, we investigated the effect of H2O2 produced by Streptococcus oralis on human macrophage cell death. Infection by S. oralis was found to stimulate cell death of a THP-1 human macrophage cell line at multiplicities of infection greater than 100. Catalase, an enzyme that catalyzes the decomposition of H2O2, inhibited the cytotoxic effect of S. oralis. S. oralis deletion mutants lacking the spxB gene, which encodes pyruvate oxidase, and are therefore deficient in H2O2 production, showed reduced cytotoxicity toward THP-1 macrophages. Furthermore, H2O2 alone was capable of inducing cell death. The cytotoxic effect seemed to be independent of inflammatory responses, because H2O2 was not a potent stimulator of tumor necrosis factor-α production in macrophages. These results indicate that streptococcal H2O2 plays a role as a cytotoxin, and is implicated in the cell death of infected human macrophages.

Oxidized low density lipoprotein induced caspase-1 mediated pyroptotic cell death in macrophages: implication in lesion instability?

BackgroundMacrophage death in advanced lesion has been confirmed to play an important role in plaque instability. However, the mechanism underlying lesion macrophage death still remains largely unknown.Methods and ResultsImmunohistochemistry showed that caspase-1 activated in advanced lesion and co-located with macrophages and TUNEL positive reaction. In in-vitro experiments showed that ox-LDL induced caspase-1 activation and this activation was required for ox-LDL induced macrophages lysis, IL-1β and IL-18 production as well as DNA fragmentation. Mechanism experiments showed that CD36 and NLRP3/caspase-1/pathway involved in ox-LDL induced macrophage pyroptosis.ConclusionOur study here identified a novel cell death, pyroptosis in ox-LDL induced human macrophage, which may be implicated in lesion macrophages death and play an important role in lesion instability.

Mechanisms of Mycobacterium avium subsp. paratuberculosis induced apoptosis and necrosis in bovine macrophages

The interaction between Mycobacterium avium subsp. paratuberculosis (Map) and macrophages is a complex process to maximize the chances of their respective survival. Previous studies have shown that Map induces cell death in macrophages, but the mechanism is not known. In the present study, we investigated the mechanism by which Map induces cell death in bovine macrophages using the fluorescent and electron microscopic techniques. The macrophages infected with an equal number of Map (i.e., multiplicity of infection, MOI=1) showed no changes of cell death, but those macrophages infected at MOI=10 showed the morphological changes consistent with apoptosis. Strikingly, the macrophages infected by Map at MOI=50 showed the changes of apoptosis and necrosis. The Map-induced apoptosis was a caspase-dependent mechanism at MOI=10 while it was caspase- and nitric oxide-independent at MOI=50. The results of the present study suggest that the mitochondrial damage following Map infection initiates the cell death processes in macrophages.

Macrophage destruction and loss of immunoregulatory function contributes to the pathology associated with MRSA pneumonia

Methicillin‐resistant Staphylococcus aureus (MRSA) USA300 produce numerous toxins that lyse specific population of leukocytes, including macrophages. We hypothesized that MRSA leads to immune dysregulation due to macrophage killing. We quantified macrophage gene products by qRT‐PCR and cytokine array after a 4‐hour exposure to MRSA USA300. THP‐1 cells significantly upregulated pro‐inflammatory cytokines including IL‐1β, IL‐1α, IL‐12 and MIP‐1α 4 hours after exposure to MRSA (≥2 fold each). Anti‐inflammatory cytokines IL‐27 (over 100‐fold, P<0.01) and IL‐10 (over 10‐fold, P<0.01) were induced 24 hours post‐MRSA infection. The cytotoxicity induced by MRSA was measured by LDH assay, cell viability by propidium iodide staining and flow cytometry analysis. Low dose (MOI 10) MRSA killed 35% of THP‐1 cells after 2 hours and over 70% after 4 hours. Macrophage cell death was not associated with annexin‐V staining, but was caspase‐dependent, as Z‐VAD‐FMK, a pan‐caspase inhibitor, significantly reduced THP‐1 cells’ cytotoxicity (40% reduction; P<0.001). A α‐hemolysin null mutant of MRSA USA300 killed significantly less macrophages than wild‐type MRSA (19% vs 28%; P<0.01), consistent with the role of Hla in activating the NLRC3 inflammasome. These results suggest the leukocidal activity of MRSA targeting macrophages can result in loss of their immunoregulatory activity.

Interaction of macrophages with a cytotoxic Serratia liquefaciens human isolate

Macrophages play key roles in host defense by recognizing, engulfing, and killing microorganisms. Understanding the response of macrophages to pathogens may provide insights into host defenses and the tactics used by pathogens to circumvent these defenses. In the present study, we investigated the interaction between a clinical isolate of Serratia liquefaciens and macrophages. S. liquefaciens strain HUMV-3250 triggers a fast and potent cytotoxic effect upon infection. This process requires the presence of live bacteria, adherence, and protein synthesis but not phagocytosis/bacterial internalization. Moreover, cytotoxicity assays, analysis of DNA integrity, immunofluorescence, and confocal, scanning, and time-lapse microscopy revealed that macrophage viability decreased rapidly with time upon challenge, and depends on the MOI used. Treatment of macrophages with caspase-1 inhibitors, or with specific inhibitors of phagocytosis, did not alter the infection outcome. Moreover, human macrophages exhibited similar cytotoxic changes after infection with this strain. Macrophages responded to this cytotoxic strain with a robust pattern of pro-inflammatory gene expression. However, phagocytosis attempts to engulf live bacteria were unsuccessful, and the phagocytes were unable to kill the bacteria. We conclude that macrophage cell death occurs rapidly as a result of necrotic events after close contact with S. liquefaciens. These results likely have important implications for understanding Serratia pathogenesis and host response to infection.

Characterization of P2X7R and Its Function in the Macrophages of ayu, Plecoglossus altivelis

P2X purinoceptor 7 (P2X7R), an ATP-gated ion channel, plays an important role during the innate immune response in mammals. However, relatively little is known about the role of P2X7R in the fish immune system. Here, we cloned a cDNA sequence encoding ayu (Plecoglossus altivelis) P2X7R (aP2X7R). The predicted protein was composed of 574 amino acid residues with a P2X family signature, two transmembrane domains, and a long C-terminal. aP2X7R transcripts were mainly distributed in ayu immune tissues and significantly increased in all tested tissues and in macrophages after Listonella anguillarum infection. The aP2X7R protein was upregulated significantly in macrophages upon bacterial challenge. An antibody against the ectodomain of aP2X7R (aEPAb) and an antagonist (oATP) were used to block aP2X7R. aP2X7R siRNA was also used to knockdown the receptor expression in ayu macrophages. Cell death induced by ATP was significantly inhibited in ayu macrophages after aEPAb, oATP, or siRNA treatment. Moreover, aP2X7R ablation also resulted in suppression of phagocytic activity and ATP-induced bacterial killing in ayu macrophages. Our results indicated that aP2X7R was upregulated after infection and mediated cell death, phagocytosis, and bacterial killing of ayu macrophages.

Glucose 6P Binds and Activates HlyIIR to Repress Bacillus cereus Haemolysin hlyII Gene Expression

Bacillus cereus is a Gram-positive spore-forming bacterium causing food poisoning and serious opportunistic infections. These infections are characterized by bacterial accumulation despite the recruitment of phagocytic cells. We have previously shown that B. cereus Haemolysin II (HlyII) induces macrophage cell death by apoptosis. In this work, we investigated the regulation of the hlyII gene. We show that HlyIIR, the negative regulator of hlyII expression in B. cereus, is especially active during the early bacterial growth phase. We demonstrate that glucose 6P directly binds to HlyIIR and enhances its activity at a post-transcriptional level. Glucose 6P activates HlyIIR, increasing its capacity to bind to its DNA-box located upstream of the hlyII gene, inhibiting its expression. Thus, hlyII expression is modulated by the availability of glucose. As HlyII induces haemocyte and macrophage death, two cell types that play a role in the sequestration of nutrients upon infection, HlyII may induce host cell death to allow the bacteria to gain access to carbon sources that are essential components for bacterial growth.

Identification of Novel Host-Targeted Compounds That Protect from Anthrax Lethal Toxin-Induced Cell Death

Studying how pathogens subvert the host to cause disease has contributed to the understanding of fundamental cell biology. Bacillus anthracis, the causative agent of anthrax, produces the virulence factor lethal toxin to disarm host immunity and cause pathology. We conducted a phenotypic small molecule screen to identify inhibitors of lethal toxin-induced macrophage cell death and used an ordered series of secondary assays to characterize the hits and determine their effects on cellular function. We identified a structurally diverse set of small molecules that act at various points along the lethal toxin pathway, including inhibitors of endocytosis, natural product inhibitors of organelle acidification (e.g., the botulinum neurotoxin inhibitor, toosendanin), and a novel proteasome inhibitor, 4MNB (4-methoxy-2-[2-(5-methoxy-2-nitrosophenyl)ethyl]-1-nitrosobenzene). Many of the compounds, including three drugs approved for use in humans, also protected against the related Clostridium difficile toxin TcdB, further demonstrating their value as novel tools for perturbation and study of toxin biology and host cellular processes and highlighting potential new strategies for intervening on toxin-mediated diseases.

TLR4 Activation Under Lipotoxic Conditions Leads to Synergistic Macrophage Cell Death through a TRIF-Dependent Pathway

Macrophage dysfunction in obesity and diabetes may predispose to the development of diabetic complications, such as infection and impaired healing after tissue damage. Saturated fatty acids, such as palmitate, are present at elevated concentrations in the plasma of patients with metabolic disease and may contribute to the pathogenesis of diabetes and its sequelae. To examine the effect of lipid excess on macrophage inflammatory function, we determined the influence of palmitate on LPS-mediated responses in peritoneal macrophages. Palmitate and LPS led to a profound synergistic cell death response in both primary and RAW 264.7 macrophages. The cell death had features of apoptosis and necrosis and was not dependent on endoplasmic reticulum stress, ceramide generation, or reactive oxygen species production. Instead, we uncovered a macrophage death pathway that required TLR4 signaling via TRIF but was independent of NF-κB, MAPKs, and IRF3. A significant decrease in macrophage lysosomal content was observed early in the death pathway, with evidence of lysosomal membrane damage occurring later in the death response. Overexpression of the transcription factor TFEB, which induces a lysosomal biogenic program, rescued the lysosomal phenotype and improved viability in palmitate- and LPS-treated cells. Our findings provide new evidence for cross-talk between lipid metabolism and the innate immune response that converges on the lysosome.