Macrophage Cell Death Research Articles

Palmitate induces apoptotic cell death and inflammasome activation in human placental macrophages

IntroductionThere is an increasing prevalence of non-communicable diseases worldwide. Metabolic diseases such as obesity and gestational diabetes mellitus (GDM) increasingly affect women during pregnancy, which can harm pregnancy outcomes and the long-term health and wellbeing of exposed offspring. Both obesity and GDM have been associated with proinflammatory effects within the placenta, the critical organ governing fetal development. MethodsThe purpose of these studies was to model, in vitro, the effects of metabolic stress (high levels of glucose, insulin and saturated lipids) on placental macrophage biology, since these cells are the primary innate immune phagocyte within the placenta with roles in governing maternofetal immune tolerance and antimicrobial host defense. Macrophages were isolated from the villous core of term, human placentae delivered through nonlaboring, elective Cesarean sections and exposed to combinations of elevated glucose (30 mM), insulin (10 nM) and the saturated lipid palmitic acid (palmitate, 0.4 mM). ResultsWe found that palmitate alone induced the activation of the nucleotide-binding oligomerization domain-like receptor (NLR) Family Pyrin Domain Containing 3 (NLRP3) inflammasome in placental macrophages, which was associated with increased interleukin 1 beta release and an increase in apoptotic cell death. Glucose and insulin neither provoked these effects nor augmented the impact of palmitate itself. DiscussionOur findings confirm an impact of saturated fat on placental macrophage immune activation and could be relevant to the impact of metabolic stress in vivo.

Relevance of Macrophage Extracellular Traps in C. albicans Killing.

Candida albicans causes systemic life-threatening infections, particularly in immunocompromised individuals, such as patients in intensive care units, patients undergoing chemotherapy, and post-surgical and neutropenic patients. The proliferation of invading Candida cells is mainly limited by the action of the human innate immune system, in which phagocytic cells play a fundamental role. This function is, however, limited in neutropenic patients, who rely mainly on the protective immunity mediated by macrophages. Macrophages have been shown to release extracellular DNA fibers, known as macrophage extracellular traps (METs), which can entrap and kill various microbes by a process called ETosis. In this study, we observed that, upon contact with C. albicans, macrophages became active in phagocyting and engulfing yeast cells. ETosis was induced in 6% of macrophages within the first 30 min of contact, and this percentage increased with the multiplicity of infection until a plateau was reached. After 2.5 h incubation, the presence of extracellular macrophage DNA was observed in approximately half of the cells. This study suggests that the formation of METs occurs before pyroptosis (first 6–8 h) and macrophage cell death (up to 24 h), and thus, METs could be included in models describing C. albicans–macrophage interactions. We also observed that macrophage ETosis and phagocytosis can occur simultaneously and that, in the first hours of infection, both processes are similarly important in controlling the proliferation of yeast cells, this being critical in neutropenic patients. Finally, it can also be concluded that, since C. albicans can degrade DNA, the structural component of METs, yeast extracellular DNase activity can be considered as an important virulence factor.

Erythrocyte-Coated Nanoparticles Block Cytotoxic Effects of Group B Streptococcus β-Hemolysin/Cytolysin.

Group B Streptococcus (GBS) emerged as a leading cause of invasive infectious disease in neonates in the 1970s, but has recently been identified as an escalating public health threat in non-pregnant adults, particularly those of advanced aged or underlying medical conditions. GBS infection can rapidly develop into life-threatening disease despite prompt administration of effective antibiotics and initiation of state-of-the-art intensive care protocols and technologies due to deleterious bacterial virulence factors, such as the GBS pore-forming toxin β-hemolysin/cytolysin (β-H/C). β-H/C is known to have noxious effects on a wide range of host cells and tissues, including lung epithelial cell injury, blood brain barrier weakening, and immune cell apoptosis. Neonatal and adult survivors of GBS infection are at a high risk for substantial long-term health issues and neurologic disabilities due to perturbations in organ systems caused by bacterial- and host- mediated inflammatory stressors. Previously engineered anti-virulence inhibitors, such as monoclonal antibodies and small molecular inhibitors, generally require customized design for each different pathogenic toxin and do not target deleterious host pro-inflammatory responses that may cause organ injury, septic shock, or death. By simply wrapping donor red blood cells (RBCs) around polymeric cores, we have created biomimetic “nanosponges.” Because nanoparticles retain the same repertoire of cell membrane receptors as their host cell, they offer non-specific and all-purpose toxin decoy strategies with a broad ability to sequester and neutralize various bacterial toxins and host pro-inflammatory chemokines and cytokines to attenuate the course of infectious disease. This proof-of-concept study successfully demonstrated that intervention with nanosponges reduced the hemolytic activity of live GBS and stabilized β-H/C in a dose-dependent manner. Nanosponge treatment also decreased lung epithelial and macrophage cell death following exposure to live GBS bacteria and stabilized β-H/C, improved neutrophil killing of GBS, and diminished GBS-induced macrophage IL-1β production. Our results, therefore, suggest biomimetic nanosponges provide a titratable detoxification therapy that may provide a first-in-class treatment option for GBS infection by sequestering and inhibiting β-H/C activity.

413. Differences in Inflammatory Mechanisms in Pseudomonas aeruginosa and Staphyloccoccus auereus Infections in Cystic Fibrosis

BackgroundChronic bacterial lung infections are the primary cause of morbidity and mortality in cystic fibrosis (CF). The most common CF pathogens, Pseudomonas aeruginosa (P. aeruginosa) or Staphylococcus aureus (S. aureus), are common commensal or environmental organisms that adapt to the CF lung. We sought to investigate whether adaptation from early lung colonizer to chronic pathogen alters the bacterial effects on host inflammation.Methods P. aeruginosa (n = 25) and S. aureus (n = 25) isolates from CF patients with early and chronic infections were acquired from Seattle Children’s CF. Environmental (n = 8) and clinical, non-CF P. aeruginosa (n = 8) isolates were obtained from the University of Ottawa. P. aeruginosa reference strain PA14 and PA14 transposon mutants for T3SS and flagellin were used to observe the relationship between cell death and cytokine production. We infected THP-1-derived macrophages (PMA differentiated) in vitro for 3 hours with various MOIs. We subsequently measured cell death of THP-1-derived macrophages using neutral red assay and cytokine production using ELISAs.ResultsInfections with PA14 mutants and non-CF P. aeruginosa isolates demonstrated that rapid cell death of THP-1-derived macrophages caused a reduction in cytokine production relative to strains that did not cause as much cell death. At 10 MOI, early P. aeruginosa isolates from CF patients induced more THP-1-derived macrophage cell death compared with chronic isolates (P < 0.0001). Chronic P. aeruginosa isolates induced greater production of TNF, IL-8, and IL-6 (P < 0.01, P < 0.0001, and P < 0.0001, respectively) compared with early strains. No difference in IL-1β production was observed. When controlling for cell death between the two groups by using heat-killed bacteria, the only difference maintained was in TNF production (P < 0.01). Between early and chronic S. aureus isolates, the one difference observed was greater IL-8 production among early isolates (P < 0.01).ConclusionChronic P. aeruginosa isolates from CF patients induce less cell death but more TNF, IL-8, and IL-6 production compared with early isolates. This suggests that P. aeruginosa producing chronic infections induce inflammatory signals that may contribute to increased morbidity among CF patients.Disclosures All authors: No reported disclosures.

Cutting Edge: Protein Arginine Deiminase 2 and 4 Regulate NLRP3 Inflammasome-Dependent IL-1β Maturation and ASC Speck Formation in Macrophages.

Protein arginine deiminase (PAD) enzymes catalyze the conversion of protein-bound arginine into citrulline, an irreversible posttranslational modification with loss of a positive charge that can influence protein-protein interactions and protein structure. Protein arginine deiminase activity depends on high intracellular calcium concentrations occurring in dying cells. In this study, we demonstrate that protein citrullination is common during pyroptotic cell death in macrophages and that inhibition of PAD enzyme activity by Cl-amidine, a pan-PAD inhibitor, blocks NLRP3 inflammasome assembly and proinflammatory IL-1β release in macrophages. Genetic deficiency of either PAD2 or PAD4 alone in murine macrophages does not impair IL-1β release; however, pharmacological inhibition or small interfering RNA knockdown of PAD2 within PAD4-/- macrophages does. Our results suggest that PAD2 and 4 activity in macrophages is required for optimal inflammasome assembly and IL-1β release, a finding of importance for autoimmune diseases and inflammation.

Cutting Edge: TAK1 Safeguards Macrophages against Proinflammatory Cell Death.

TGF-β-activated kinase 1 (TAK1) is known to play vital roles for innate and adaptive immunity; however, little is known about its potential role in limiting biological responses such as inflammation. In this study, we report that macrophage TAK1 participates in negatively regulating inflammation by restraining proinflammatory cell death. Macrophages from TAK1-deficient mice underwent cell death in response to LPS and poly(I:C), which took place in a manner dependent on TLR/TRIF-induced active Caspase8-mediated cleavage of gasdermin D, known as an executioner of pyroptosis. Likewise, TNF-α induced Caspase8-dependent gasdermin D processing following cell death in TAK1-deficient macrophages. Importantly, we demonstrated that this type of proinflammatory macrophage death is linked to susceptibility to septic shock in mice lacking TAK1 in macrophages in a TNF-α-independent fashion. Taken together, our data revealed that TAK1 acts as a signaling checkpoint to protect macrophages from unique proinflammatory cell death, ensuring the maintenance of innate immune homeostasis.

Abstract 792: Core Cell Survival Requirements of Hypoxia Inducible Factors in Macrophages Dominate Isoform-Specific Function during Cardiac Repair

Objective: Hypoxia inducible factors (HIF) regulate the adaptive response to ischemic injury and enhancement of HIF levels has been proposed as a therapeutic target to improve tissue repair after acute myocardial infarction (MI). HIFs also regulate the inflammatory function of macrophages, the main protagonists of tissue repair after MI, but it is unknown whether changes in macrophage HIF levels will affect outcomes after MI. Using a murine model of acute MI and conditional knockouts, we investigated a role for macrophage HIFs in tissue repair after MI. Methods and Results: In mice subjected to permanent occlusion MI, we observed an accumulation of hypoxic cardiac macrophages and sequential stabilization of HIF-2α followed by HIF-1α, which persisted into the development of heart failure. In patients with ischemic cardiomyopathy, flow cytometry and single-cell transcriptomics revealed a parallel stabilization of HIFs and hypoxia signaling in cardiac macrophages. Since HIFs were stabilized early after MI in cardiac macrophages, we selectively targeted macrophage HIFs using conditional knockouts. Selective deletion of macrophage HIF-2α promoted cardiac macrophage production of IL-10, activating fibroblasts and stimulating collagen deposition, culminating in improved ventricular remodeling and preservation of systolic function. Similarly, selective deletion of macrophage HIF-1α improved cardiac repair; however, this was mechanistically distinct from HIF-2α, inhibiting HIF-1α-dependent proteolytic destruction of MerTK and leading to improved efferocytosis by cardiac macrophages. Contrary to our expectations, loss of both HIF-1α and HIF-2α in hypoxic macrophages enhanced mitochondrial reactive oxygen species and activated necroptosis, leading to increased cell death and cardiac rupture after acute MI. Inhibition of necroptosis with necrostatin-1 attenuated hypoxic macrophage cell death and significantly improved survival after acute MI. Conclusions: These data imply strategies that broadly enhance or inhibit HIFs will lead to catastrophic outcomes after MI, while selective inhibition of macrophage HIF-1α or HIF-2α may enhance tissue repair, preserve systolic function, and limit progression to heart failure.

The Pyroptotic Cell Death Effector Gasdermin D Is Activated by Gout-Associated Uric Acid Crystals but Is Dispensable for Cell Death and IL-1β Release.

The pyroptotic cell death effector gasdermin D (GSDMD) is required for murine models of hereditary inflammasome-driven, IL-1β-dependent, autoinflammatory disease, making it an attractive therapeutic target. However, the importance of GSDMD for more common conditions mediated by pathological IL-1β activation, such as gout, remain unclear. In this study, we address whether GSDMD and the recently described GSDMD inhibitor necrosulfonamide (NSA) contribute to monosodium urate (MSU) crystal-induced cell death, IL-1β release, and autoinflammation. We demonstrate that MSU crystals, the etiological agent of gout, rapidly activate GSDMD in murine macrophages. Despite this, the genetic deletion of GSDMD or the other lytic effector implicated in MSU crystal killing, mixed lineage kinase domain-like (MLKL), did not prevent MSU crystal-induced cell death. Consequently, GSDMD or MLKL loss did not hinder MSU crystal-mediated release of bioactive IL-1β. Consistent with in vitro findings, IL-1β induction and autoinflammation in MSU crystal-induced peritonitis was not reduced in GSDMD-deficient mice. Moreover, we show that the reported GSDMD inhibitor, NSA, blocks inflammasome priming and caspase-1 activation, thereby preventing pyroptosis independent of GSDMD targeting. The inhibition of cathepsins, widely implicated in particle-induced macrophage killing, also failed to prevent MSU crystal-mediated cell death. These findings 1) demonstrate that not all IL-1β-driven autoinflammatory conditions will benefit from the therapeutic targeting of GSDMD, 2) document a unique mechanism of MSU crystal-induced macrophage cell death not rescued by pan-cathepsin inhibition, and 3) show that NSA inhibits inflammasomes upstream of GSDMD to prevent pyroptotic cell death and IL-1β release.

Lunar soil simulants alter macrophage survival and function.

Lunar regolith samples collected during previous Apollo missions were found to contain components that were established to be toxic to humans; however, the health effects due to inhalation of lunar soil as a whole are still unknown. Macrophages residing in the alveolar sacs of the lungs constitute one of the last lines of defense against inhaled particulates before entry into the bloodstream. Here, we examine the macrophage response to lunar simulants that are similar in chemical composition to the lunar regolith. We assess cytotoxicity, cellular morphology, phagocytosis of simulants and expression of inflammatory markers. Overall, the exposure of macrophages to lunar simulants results in moderate cytotoxicity and marked alteration of cell morphology and uptake of the simulants. Interestingly, simulant exposure decreased proinflammatory gene expression, but may induce an anti-inflammatory phenotype in the cells. These results illustrate that although macrophages phagocytose lunar simulants as a protective response, the simulants do induce a degree of macrophage cell death. Our study reveals some toxicity associated with lunar simulants and supports further evaluation of the inhalation of lunar regolith to understand the risks of exposure fully.

Conformational Switch Driven Membrane Pore Formation by Mycobacterium Secretory Protein MPT63 Induces Macrophage Cell Death

Virulent Mycobacterium tuberculosis (MTB) strains cause cell death of macrophages (Mϕ) inside TB granuloma using a mechanism which is not well understood. Many bacterial systems utilize toxins to induce host cell damage, which occurs along with immune evasion. These toxins often use chameleon sequences to generate an environment-sensitive conformational switch, facilitating the process of infection. The presence of toxins is not yet known for MTB. Here, we show that MTB-secreted immunogenic MPT63 protein undergoes a switch from β-sheet to helix in response to mutational and environmental stresses. MPT63 in its helical form creates pores in both synthetic and Mϕ membranes, while the native β-sheet protein remains inert toward membrane interactions. Using fluorescence correlation spectroscopy and atomic force microscopy, we show further that the helical form undergoes self-association to produce toxic oligomers of different morphology. Trypan blue and flow cytometry analyses reveal that the helical state can be utilized by MTB for killing Mϕ cells. Collectively, our study emphasizes for the first time a toxin-like behavior of MPT63 induced by an environment-dependent conformational switch, resulting in membrane pore formation by toxic oligomers and Mϕ cell death.

Targeting of M2-like tumor-associated macrophages with a melittin-based pro-apoptotic peptide

BackgroundTumor-associated macrophages (TAMs) are the major component of tumor-infiltrating immune cells. Macrophages are broadly categorized as M1 or M2 types, and TAMs have been shown to express an M2-like phenotype. TAMs promote tumor progression and contribute to resistance to chemotherapies. Therefore, M2-like TAMs are potential targets for the cancer immunotherapy. In this study, we targeted M2-like TAMs using a hybrid peptide, MEL-dKLA, composed of melittin (MEL), which binds preferentially to M2-like TAMs, and the pro-apoptotic peptide d (KLAKLAK)2 (dKLA), which induces mitochondrial death after cell membrane penetration.MethodsThe M1 or M2-differentiated RAW264.7 cells were used for mitochondrial colocalization and apoptosis test in vitro. For in vivo study, the murine Lewis lung carcinoma cells were inoculated subcutaneously in the right flank of mouse. The dKLA, MEL and MEL-dKLA peptides were intraperitoneally injected at 175 nmol/kg every 3 days. Flow cytometry analysis of tumor-associated macrophages and immunofluorescence staining were performed to investigate the immunotherapeutic effects of MEL-dKLA.ResultsWe showed that MEL-dKLA induced selective cell death of M2 macrophages in vitro, whereas MEL did not disrupt the mitochondrial membrane. We also showed that MEL-dKLA selectively targeted M2-like TAMs without affecting other leukocytes, such as T cells and dendritic cells, in vivo. These features resulted in lower tumor growth rates, tumor weights, and angiogenesis in vivo. Importantly, although both MEL and MEL-dKLA reduced numbers of CD206+ M2-like TAMs in tumors, only MEL-dKLA induced apoptosis in CD206+ M2-like TAMs, and MEL did not induce cell death.ConclusionTaken together, our study demonstrated that MEL-dKLA could be used to target M2-like TAMs as a promising cancer therapeutic agent.

SARS-Coronavirus Open Reading Frame-8b triggers intracellular stress pathways and activates NLRP3 inflammasomes

The SARS (severe acute respiratory syndrome) outbreak was caused by a coronavirus (CoV) named the SARS-CoV. SARS pathology is propagated both by direct cytotoxic effects of the virus and aberrant activation of the innate immune response. Here, we identify several mechanisms by which a SARS-CoV open reading frame (ORF) activates intracellular stress pathways and targets the innate immune response. We show that ORF8b forms insoluble intracellular aggregates dependent on a valine at residue 77. Aggregated ORF8b induces endoplasmic reticulum (ER) stress, lysosomal damage, and subsequent activation of the master regulator of the autophagy and lysosome machinery, Transcription factor EB (TFEB). ORF8b causes cell death in epithelial cells, which is partially rescued by reducing its ability to aggregate. In macrophages, ORF8b robustly activates the NLRP3 inflammasome by providing a potent signal 2 required for activation. Mechanistically, ORF8b interacts directly with the Leucine Rich Repeat domain of NLRP3 and localizes with NLRP3 and ASC in cytosolic dot-like structures. ORF8b triggers cell death consistent with pyroptotic cell death in macrophages. While in those cells lacking NLRP3 accumulating ORF8b cytosolic aggregates cause ER stress, mitochondrial dysfunction, and caspase-independent cell death.

Efferocytosis perpetuates substance accumulation inside macrophage populations.

In both cells and animals, cannibalism can transfer harmful substances from the consumed to the consumer. Macrophages are immune cells that consume their own dead via a process called cannibalistic efferocytosis. Macrophages that contain harmful substances are found at sites of chronic inflammation, yet the role of cannibalism in this context remains unexplored. Here we take mathematical and experimental approaches to study the relationship between cannibalistic efferocytosis and substance accumulation in macrophages. Through mathematical modelling, we deduce that substances which transfer between individuals through cannibalism will concentrate inside the population via a coalescence process. This prediction was confirmed for macrophage populations inside a closed system. We used image analysis of whole slide photomicrographs to measure both latex microbead and neutral lipid accumulation inside murine bone marrow-derived macrophages (104–) following their stimulation into an inflammatory state ex vivo. While the total number of phagocytosed beads remained constant, cell death reduced cell numbers and efferocytosis concentrated the beads among the surviving macrophages. As lipids are also conserved during efferocytosis, these cells accumulated lipid derived from the membranes of dead and consumed macrophages (becoming macrophage foam cells). Consequently, enhanced macrophage cell death increased the rate and extent of foam cell formation. Our results demonstrate that cannibalistic efferocytosis perpetuates exogenous (e.g. beads) and endogenous (e.g. lipids) substance accumulation inside macrophage populations. As such, cannibalism has similar detrimental consequences in both cells and animals.

480-P: Diabetes and Experimental Myocardial Infarction Synergize to Accelerate Atherosclerosis Destabilization

In addition to increasing the risk of an initial myocardial infarction (MI), diabetes increases the risk of a recurrent MI. Work by others suggests that an experimental MI can accelerate atherosclerosis via monocytosis. To test if diabetes and experimental myocardial infarction synergize to accelerate atherosclerosis, we induced atherosclerosis in Ldlr-/-; Gp+ mice, lethally irradiated them and transplanted with either wild type (WT) or LysM-Cre-mediated CD49D-deficient bone marrow. Following recovery, diabetes was induced using lymphocytic choriomeningitis virus, or saline was used as a control. The mice were subjected to ligation of the left anterior descending coronary artery to induce experimental MI or a sham surgery and then maintained for an additional 3 weeks. Diabetic (D) mice exhibited hyperglycemia and elevated plasma cholesterol and triglyceride levels compared to nondiabetic (ND) mice, but neither hematopoietic genotype or MI influenced that. All mice subjected to MI had significantly reduced left ventricular function. Neither diabetes nor MI resulted in monocytosis compared to ND sham mice. Despite not affecting total lesion size, the combination of diabetes and MI resulted in larger necrotic cores in the aortic sinus and in the brachiocephalic artery (9.8 ± 1% of total lesion in aortic sinus in ND sham, 11.9 ± 1% in ND MI, 15.4 ± 1% in D sham and 18.1 ± 2% in D MI, P&amp;lt;0.001 comparing ND Sham to D MI, n=14-24, ANOVA). The expansion of the necrotic cores was dependent on monocyte recruitment, since mice with myeloid cells deficient in the adhesion molecule CD49D were protected from necrotic core expansion. Diabetes and MI also synergized to accelerate macrophage cell death (6.1 ± 3% of peritoneal macrophages were dead in ND sham, 6.1 ± 3% in ND MI, 11± 1% in D sham and 20 ± 3% in D MI, n=3-6, p&amp;lt;0.05 ANOVA). In summary, diabetes and experimental MI destabilize the atherosclerotic lesion, potentially explaining why patients with diabetes are at an increased risk of recurrent MI. Disclosure F. Kramer: None. A.M. Martinson: None. T. Papayannopoulou: None. C.E. Murry: Other Relationship; Self; Cytocardia. J.E. Kanter: None. Funding American Diabetes Association (1-16-IBS-153 to J.E.K.)

The N-end rule ubiquitin ligase UBR2 mediates NLRP1B inflammasome activation by anthrax lethal toxin.

Anthrax lethal toxin (LT) is known to induce NLRP1B inflammasome activation and pyroptotic cell death in macrophages from certain mouse strains in its metalloprotease activity-dependent manner, but the underlying mechanism is unknown. Here, we establish a simple but robust cell system bearing dual-fluorescence reporters for LT-induced ASC specks formation and pyroptotic lysis. A genome-wide siRNA screen and a CRISPR-Cas9 knockout screen were applied to this system for identifying genes involved in LT-induced inflammasome activation. UBR2, an E3 ubiquitin ligase of the N-end rule degradation pathway, was found to be required for LT-induced NLRP1B inflammasome activation. LT is known to cleave NLRP1B after Lys44. The cleaved NLRP1B, bearing an N-terminal leucine, was targeted by UBR2-mediated ubiquitination and degradation. UBR2 partnered with an E2 ubiquitin-conjugating enzyme UBE2O in this process. NLRP1B underwent constitutive autocleavage before the C-terminal CARD domain. UBR2-mediated degradation of LT-cleaved NLRP1B thus triggered release of the noncovalent-bound CARD domain for subsequent caspase-1 activation. Our study illustrates a unique mode of inflammasome activation in cytosolic defense against bacterial insults.

Enrichment of ATP Binding Proteins Unveils Proteomic Alterations in Human Macrophage Cell Death, Inflammatory Response, and Protein Synthesis after Interaction with Candida albicans.

Macrophages are involved in the primary human response to Candida albicans. After pathogen recognition, signaling pathways are activated, leading to the production of cytokines, chemokines, and antimicrobial peptides. ATP binding proteins are crucial for this regulation. Here, a quantitative proteomic and phosphoproteomic approach was carried out for the study of human macrophage ATP-binding proteins after interaction with C.albicans. From a total of 547 nonredundant quantified proteins, 137 were ATP binding proteins and 59 were detected as differentially abundant. From the differentially abundant ATP-binding proteins, 6 were kinases (MAP2K2, SYK, STK3, MAP3K2, NDKA, and SRPK1), most of them involved in signaling pathways. Furthermore, 85 phosphopeptides were quantified. Macrophage proteomic alterations including an increase of protein synthesis with a consistent decrease in proteolysis were observed. Besides, macrophages showed changes in proteins of endosomal trafficking together with mitochondrial proteins, including some involved in the response to oxidative stress. Regarding cell death mechanisms, an increase of antiapoptotic over pro-apoptotic signals is suggested. Furthermore, a high pro-inflammatory response was detected, together with no upregulation of key mi-RNAs involved in the negative feedback of this response. These findings illustrate a strategy to deepen the knowledge of the complex interactions between the host and the clinically important pathogen C.albicans.

4-Phenylbutyrate protects against atherosclerotic lesion growth by increasing the expression of HSP25 in macrophages and in the circulation of Apoe-/- mice.

Endoplasmic reticulum stress plays an important role in cardiovascular disease (CVD) and atherosclerosis. We aimed to assess the ability of 4-phenylbutyrate (4-PBA), a small chemical chaperone administered via drinking water, to reduce atherosclerotic lesion size in chow-fed apolipoprotein (Apo) e-/- mice and to identify mechanisms that contribute to its antiatherogenic effect. Chow-fed 17-wk-old female Apoe-/- mice treated with 4-PBA-supplemented drinking water for 5 wk exhibited smaller lesions as well as increased plasma levels of heat shock protein (HSP) 25, the mouse homolog of human HSP27, compared with controls. In addition, 4-PBA inhibited cell death and increased HSP27 expression as measured by real-time PCR and immunoblotting, as well as induced nuclear localization of its transcription factor, heat shock factor 1, in human monocyte/macrophage (THP-1) cells. Furthermore, HSP27 small interfering RNA diminished the protective effect of 4-PBA on THP-1 macrophage attachment and differentiation. In summary, drinking water containing 4-PBA attenuated early lesion growth in Apoe-/- mice fed a chow diet and increased expression of HSP25 and HSP27 in macrophages and HSP25 in the circulation of Apoe-/- mice. Given that increased expression of HSP27 is inversely correlated with CVD risk, our findings suggest that 4-PBA protects against the early stages of atherogenesis in part by enhancing HSP27 levels, leading to inhibition of both macrophage cell death and monocyte-macrophage differentiation.-Lynn, E. G., Lhoták, Š., Lebeau, P., Byun, J. H., Chen, J., Platko, K., Shi, C., O'Brien, E. R., Austin, R. C. 4-Phenylbutyrate protects against atherosclerotic lesion growth by increasing the expression of HSP25 in macrophages and in the circulation of Apoe-/- mice.

Poly I:C Delivery into J774.1 and RAW264.7 Macrophages Induces Rapid Cell Death.

Cytosolic double-stranded RNA (dsRNA) is an important 'molecular signature' for the detection of intracellular viral infections. Although intracellular dsRNA is a known potent inducer of apoptosis, the optimal time and dose for the onset of dsRNA-mediated apoptosis have not been studied in detail. To perform an accurate temporal assessment of the cell death responses in dsRNA-dependent cytotoxicity. Poly I:C (PIC), a synthetic dsRNA molecule was delivered intracellularly into J774.1 and RAW264.7 murine macrophages via electroporation. Cell viability was measured using the MTT assay and apoptosis was determined by sub-G0/G1 DNA content using flow cytometry. Loss of cell viability was seen as early as 3h post-electroporation of macrophages. A significant increase in the sub-G0/G1 DNA content consistent with apoptosis was observed in PIC-electroporated macrophages as early as 3h post electroporation. Intracellular PIC delivery induces rapid macrophage cell death.

β-glucan suppresses cell death of ASC deficient macrophages invaded by periodontopathic bacteria through the caspase-11 pathway.

β-glucan is an abundant cell wall component of fungi and yeast. Dectin-1, a β-glucan receptor, plays an important regulatory role in the natural immunity. In the present study, we investigated the effect of β-glucan on mouse macrophages that had been invaded by the periodontopathic bacterium, Aggregatibacter actinomycetemcomitans. Exposure to curdlan, a type of β-glucan, suppressed cell death and led to the accumulation of a sub-G1-phase population upon A. actinomycetemcomitans invasion under conditions of constitutive expression of dectin-1. Members of the nucleotide-binding domain leucine-rich repeat-containing (NLR) protein family, such as NLR protein 3 (NLRP3), NLR family apoptosis inhibitory protein (NAIP), and NLR family CARD domain-containing protein 4 (NLRC4), as well as an associated protein, caspase-11, were clearly detected in A. actinomycetemcomitans-invaded control RAW cells (c-RAW cells; negative control). Interestingly, NAIP expression was upregulated and caspase-11 expression was downregulated by dectin-1 activity in A. actinomycetemcomitans-invaded dectin-1 overexpressing RAW 264.7 cells (d-RAW cells), suggesting that dectin-1 in macrophages regulates cell death upon A. actinomycetemcomitans invasion. These results support a potential correlation between dectin-1 and regulation of cell death in macrophages.

LXR Signaling Regulates Macrophage Survival and Inflammation in Response to Ionizing Radiation

To evaluate the role of liver X receptor (LXR) nuclear receptors on irradiation-induced cell death and polarization of macrophages and the potential implications in the context of radiation therapy treatment of cancer. Primary and immortalized murine bone marrow-derived macrophages (BMDMs) from wild type or LXR double knock-out mice were exposed to gamma irradiation. Subsequently, analysis of LXR signaling on cell proliferation and cytotoxicity induced by ionizing radiation was determined by time-lapse photomicroscopy. Genotoxic cell damage was evaluated by Western blot of γ-H2AX and p53. Pyroptosis was analyzed through cell viability assay, lactate dehydrogenase release assay, and Western blot of caspase-1 active protein. Expression of inflammatory markers was measured by real-time quantitative polymerase chain reaction. Genetic and pharmacologic inactivation of LXR induced radiosensitivity of macrophages. LXR deficiency decreased cell proliferation and enhanced cytotoxicity induced by ionizing radiation in both immortalized and primary BMDMs. Protein levels of γ-H2AX and p53, both involved in response to cell damage, were exacerbated in LXR-deficient macrophages exposed to irradiation. Cell membrane damage was augmented and cell viability was decreased in LXR-deficient macrophages compared with LXR wild type macrophages in response to irradiation. In addition, LXR deficiency enhanced both caspase-1 activation and lactate dehydrogenase release in BMDM exposed inflammasome activators. LXR inactivation or deficiency markedly increased the expression of proinflammatory markers IL-1β, IL-6, and inducible nitric oxide synthase in irradiated macrophages. The present work identifies LXR transcription factors as potential therapeutic targets to enhance the suppressive effects of radiation therapy on tumor growth through induction of macrophage cell death and activation of the inflammatory cascade.