David P. Sester

AIM2 and NLRP3 inflammasomes activate both apoptotic and pyroptotic death pathways via ASC.

Inflammasomes are protein complexes assembled upon recognition of infection or cell damage signals, and serve as platforms for clustering and activation of procaspase-1. Oligomerisation of initiating proteins such as AIM2 (absent in melanoma-2) and NLRP3 (NOD-like receptor family, pyrin domain-containing-3) recruits procaspase-1 via the inflammasome adapter molecule ASC (apoptosis-associated speck-like protein containing a CARD). Active caspase-1 is responsible for rapid lytic cell death termed pyroptosis. Here we show that AIM2 and NLRP3 inflammasomes activate caspase-8 and -1, leading to both apoptotic and pyroptotic cell death. The AIM2 inflammasome is activated by cytosolic DNA. The balance between pyroptosis and apoptosis depended upon the amount of DNA, with apoptosis seen at lower transfected DNA concentrations. Pyroptosis had a higher threshold for activation, and dominated at high DNA concentrations because it happens more rapidly. Gene knockdown showed caspase-8 to be the apical caspase in the AIM2- and NLRP3-dependent apoptotic pathways, with little or no requirement for caspase-9. Procaspase-8 localised to ASC inflammasome ‘specks’ in cells, and bound directly to the pyrin domain of ASC. Thus caspase-8 is an integral part of the inflammasome, and this extends the relevance of the inflammasome to cell types that do not express caspase-1.

The Molecular Basis for the Lack of Immunostimulatory Activity of Vertebrate DNA

Macrophages and B cells are activated by unmethylated CpG-containing sequences in bacterial DNA. The lack of activity of self DNA has generally been attributed to CpG suppression and methylation, although the role of methylation is in doubt. The frequency of CpG in the mouse genome is 12.5% of Escherichia coli, with unmethylated CpG occurring at ∼3% the frequency of E. coli. This suppression of CpG alone is insufficient to explain the inactivity of self DNA; vertebrate DNA was inactive at 100 μg/ml, 3000 times the concentration at which E. coli DNA activity was observed. We sought to resolve why self DNA does not activate macrophages. Known active CpG motifs occurred in the mouse genome at 18% of random occurrence, similar to general CpG suppression. To examine the contribution of methylation, genomic DNAs were PCR amplified. Removal of methylation from the mouse genome revealed activity that was 23-fold lower than E. coli DNA, although there is only a 7-fold lower frequency of known active CpG motifs in the mouse genome. This discrepancy may be explained by G-rich sequences such as GGAGGGG, which potently inhibited activation and are found in greater frequency in the mouse than the E. coli genome. In summary, general CpG suppression, CpG methylation, inhibitory motifs, and saturable DNA uptake combined to explain the inactivity of self DNA. The immunostimulatory activity of DNA is determined by the frequency of unmethylated stimulatory sequences within an individual DNA strand and the ratio of stimulatory to inhibitory sequences.

Phosphorothioate backbone modification modulates macrophage activation by CpG DNA.

Macrophages respond to unmethylated CpG motifs present in nonmammalian DNA. Stabilized phosphorothioate-modified oligodeoxynucleotides (PS-ODN) containing CpG motifs form the basis of immunotherapeutic agents. In this study, we show that PS-ODN do not perfectly mimic native DNA in activation of macrophages. CpG-containing PS-ODN were active at 10- to 100-fold lower concentrations than corresponding phosphodiester ODN in maintenance of cell viability in the absence of CSF-1, in induction of NO production, and in activation of the IL-12 promoter. These enhancing effects are attributable to both increased stability and rate of uptake of the PS-ODN. By contrast, PS-ODN were almost inactive in down-modulation of the CSF-1R from primary macrophages and activation of the HIV-1 LTR. Delayed or poor activation of signaling components may contribute to this, as PS-ODN were slower and less effective at inducing phosphorylation of the extracellular signal-related kinases 1 and 2. In addition, at high concentrations, non-CpG PS-ODN specifically inhibited responses to CpG DNA, whereas nonstimulatory phosphodiester ODN had no such effect. Although nonstimulatory PS-ODN caused some inhibition of ODN uptake, this did not adequately explain the levels of inhibition of activity. The results demonstrate that the phosphorothioate backbone has both enhancing and inhibitory effects on macrophage responses to CpG DNA.

Dengue virus NS1 protein activates cells via Toll-like receptor 4 and disrupts endothelial cell monolayer integrity

Dengue virus NS1 protein induces inflammatory responses via TLR4 and disrupts endothelial cell monolayer integrity. A leak in the dike Everyone knows how mosquitos can wreck an end-of-summer picnic. But in some climates, these pesky intruders persist and carry a variety of detrimental diseases—some with no preventative vaccines or targeted therapies. One such passenger is dengue virus (DENV), which infects up to 400 million people each year and comes in several serotypes (1 to 4) and disease presentations—from mild infection to severe disease and sometimes death. But to treat or prevent dengue requires that we have a more complete picture of the disease pathology. Now, Modhiran et al. and Beatty et al. describe the results of in vitro and in vivo experiments that point to circulating dengue virus non-structural protein 1 (NS1) and the innate immune Toll-like receptor 4 (TLR4) as a focus for basic scientists as well as vaccine and drug developers. DENV infection protects a patient from future reinfection with the same DENV serotype as well as producing temporary immune protection from severe dengue disease caused by a different DENV serotype. But unlike diamonds, this immune protection doesn’t last forever, and when the protected period passes, the patient becomes at increased risk of enhanced infection and progression to severe disease if he or she is infected with a second DENV serotype. This severe form of dengue infection is believed to result from immunopathogenic processes that induce cytokine storm and cause vascular leakage that leads to shock. Until now, no dengue viral proteins have been linked to vascular endothelium permeability (that is, vascular leakage). Beatty et al. show that inoculation of mice with DENV NS1 protein alone induces both vascular leak and secretion of inflammatory cytokines and that administration of NS1 with a sublethal dose of DENV2 leads to lethal vascular leak syndrome. In human endothelial cell monolayers in culture, NS1 from any of the four DENV serotypes triggered endothelial barrier permeability. NS1’s pathogenic effects were blocked by NS1-immune polyclonal mouse serum or monoclonal antibodies to NS1 (in vivo and in vitro), and immunization of mice with NS1 protected against lethal DENV2 challenge. In an independent study, Mondrian et al. explore the underlying mechanism of NS1’s effects. They show that highly purified NS1 acts as a pathogen-associated molecular pattern (PAMP) that activates mouse macrophages and human peripheral blood mononuclear cells (PBMCs) in culture via TLR4, resulting in release of inflammatory cytokines—an effect that was blocked by either a TLR4 antagonist or an anti-TLR4 antibody. Then, in an in vitro model of vascular leak, the authors found that NS1 fractured the integrity of endothelial cell monolayers through a TLR4-dependent pathway, a finding that was supported by the observation that a TLR4 antagonist quelled capillary leak in a mouse model of dengue virus infection. Together, these new findings highlight NS1 as an instigator of dengue-associated vascular leak and thus pinpoint a potential target for dengue drugs and component for dengue vaccines. Complications arising from dengue virus infection include potentially fatal vascular leak, and severe disease has been linked with excessive immune cell activation. An understanding of the triggers of this activation is critical for the development of appropriately targeted disease control strategies. We show here that the secreted form of the dengue virus nonstructural protein 1 (NS1) is a pathogen-associated molecular pattern (PAMP). Highly purified NS1 devoid of bacterial endotoxin activity directly activated mouse macrophages and human peripheral blood mononuclear cells (PBMCs) via Toll-like receptor 4 (TLR4), leading to the induction and release of proinflammatory cytokines and chemokines. In an in vitro model of vascular leak, treatment with NS1 alone resulted in the disruption of endothelial cell monolayer integrity. Both NS1-mediated activation of PBMCs and NS1-induced vascular leak in vitro were inhibited by a TLR4 antagonist and by anti-TLR4 antibody treatment. The importance of TLR4 activation in vivo was confirmed by the reduction in capillary leak by a TLR4 antagonist in a mouse model of dengue virus infection. These results pinpoint NS1 as a viral toxin counterpart of the bacterial endotoxin lipopolysaccharide (LPS). Similar to the role of LPS in septic shock, NS1 might contribute to vascular leak in dengue patients, which highlights TLR4 antagonists as a possible therapeutic option.

Pivotal Advance: Avian colony-stimulating factor 1 (CSF-1), interleukin-34 (IL-34), and CSF-1 receptor genes and gene products

Macrophages are involved in many aspects of development, host defense, pathology, and homeostasis. Their normal differentiation, proliferation, and survival are controlled by CSF‐1 via the activation of the CSF1R. A recently discovered cytokine, IL‐34, was shown to bind the same receptor in humans. Chicken is a widely used model organism in developmental biology, but the factors that control avian myelopoiesis have not been identified previously. The CSF‐1, IL‐34, and CSF1R genes in chicken and zebra finch were identified from respective genomic/cDNA sequence resources. Comparative analysis of the avian CSF1R loci revealed likely orthologs of mammalian macrophage‐specific promoters and enhancers, and the CSF1R gene is expressed in the developing chick embryo in a pattern consistent with macrophage‐specific expression. Chicken CSF‐1 and IL‐34 were expressed in HEK293 cells and shown to elicit macrophage growth from chicken BM cells in culture. Comparative sequence and co‐evolution analysis across all vertebrates suggests that the two ligands interact with distinct regions of the CSF1R. These studies demonstrate that there are two separate ligands for a functional CSF1R across all vertebrates.

The mononuclear phagocyte system of the pig as a model for understanding human innate immunity and disease

The biology of cells of the mononuclear phagocyte system has been studied extensively in the mouse. Studies of the pig as an experimental model have commonly been consigned to specialist animal science journals. In this review, we consider some of the many ways in which the innate immune systems of humans differ from those of mice, the ways that pigs may address the shortcomings of mice as models for the study of macrophage differentiation and activation in vitro, and the biology of sepsis and other pathologies in the living animal. With the completion of the genome sequence and the characterization of many key regulators and markers, the pig has emerged as a tractable model of human innate immunity and disease that should address the limited, predictive value of rodents in preclinical studies.

Colony-Stimulating Factor-1 Suppresses Responses to CpG DNA and Expression of Toll-Like Receptor 9 but Enhances Responses to Lipopolysaccharide in Murine Macrophages

During bacterial infections, the balance between resolution of infection and development of sepsis is dependent upon the macrophage response to bacterial products. We show that priming of murine bone marrow-derived macrophages (BMMs) with CSF-1 differentially regulates the response to two such stimuli, LPS and immunostimulatory (CpG) DNA. CSF-1 pretreatment enhanced IL-6, IL-12, and TNF-α production in response to LPS but suppressed the same response to CpG DNA. CSF-1 also regulated cytokine gene expression in response to CpG DNA and LPS; CpG DNA-induced IL-12 p40, IL-12 p35, and TNF-α mRNAs were all suppressed by CSF-1 pretreatment. CSF-1 pretreatment enhanced LPS-induced IL-12 p40 mRNA but not TNF-α and IL-12 p35 mRNAs, suggesting that part of the priming effect is posttranscriptional. CSF-1 pretreatment also suppressed CpG DNA-induced nuclear translocation of NF-κB and phosphorylation of the mitogen-activated protein kinases p38 and extracellular signal-related kinases-1/2 in BMMs, indicating that early events in CpG DNA signaling were regulated by CSF-1. Expression of Toll-like receptor (TLR)9, which is necessary for responses to CpG DNA, was markedly suppressed by CSF-1 in both BMMs and thioglycolate-elicited peritoneal macrophages. CSF-1 also down-regulated expression of TLR1, TLR2, and TLR6, but not the LPS receptor, TLR4, or TLR5. Hence, CSF-1 may regulate host responses to pathogens through modulation of TLR expression. Furthermore, these results suggest that CSF-1 and CSF-1R antagonists may enhance the efficacy of CpG DNA in vivo.

The JNK Are Important for Development and Survival of Macrophages

We report in this study that activation of the JNK by the growth factor, CSF-1 is critical for macrophage development, proliferation, and survival. Inhibition of JNK with two distinct classes of inhibitors, the pharmacological agent SP600125, or the peptide D-JNKI1 resulted in cell cycle inhibition with an arrest at the G2/M transition and subsequent apoptosis. JNK inhibition resulted in decreased expression of CSF-1R (c-fms) and Bcl-xL mRNA in mature macrophages and repressed CSF-1-dependent differentiation of bone marrow cells to macrophages. Macrophage sensitivity to JNK inhibitors may be linked to phosphorylation of the PU.1 transcription factor. Inhibition of JNK disrupted PU.1 binding to an element in the c-fms gene promoter and decreased promoter activity. Promoter activity could be restored by overexpression of PU.1. A comparison of expression profiles of macrophages with 22 other tissue types showed that genes that signal JNK activation downstream of tyrosine kinase receptors, such as focal adhesion kinase, Nck-interacting kinase, and Rac1 and scaffold proteins are highly expressed in macrophages relative to other tissues. This pattern of expression may underlie the novel role of JNK in macrophages.

Pig Bone Marrow-Derived Macrophages Resemble Human Macrophages in Their Response to Bacterial Lipopolysaccharide

Mouse bone marrow-derived macrophages (BMDM) grown in M-CSF (CSF-1) have been used widely in studies of macrophage biology and the response to TLR agonists. We investigated whether similar cells could be derived from the domestic pig using human rCSF-1 and whether porcine macrophages might represent a better model of human macrophage biology. Cultivation of pig bone marrow cells for 5–7 d in presence of human rCSF-1 generated a pure population of BMDM that expressed the usual macrophage markers (CD14, CD16, and CD172a), were potent phagocytic cells, and produced TNF in response to LPS. Pig BMDM could be generated from bone marrow cells that had been stored frozen and thawed so that multiple experiments can be performed on samples from a single animal. Gene expression in pig BMDM from outbred animals responding to LPS was profiled using Affymetrix microarrays. The temporal cascade of inducible and repressible genes more closely resembled the known responses of human than mouse macrophages, sharing with humans the regulation of genes involved in tryptophan metabolism (IDO, KYN), lymphoattractant chemokines (CCL20, CXCL9, CXCL11, CXCL13), and the vitamin D3-converting enzyme, Cyp27B1. Conversely, in common with published studies of human macrophages, pig BMDM did not strongly induce genes involved in arginine metabolism, nor did they produce NO. These results establish pig BMDM as an alternative tractable model for the study of macrophage transcriptional control.

Macrophage activation by immunostimulatory DNA.

Macrophage/dendritic cells and B cells remain the only cell types where direct responses to CpG DNA are well established. The role of macrophages in vivo in DNA clearance and the potent cytokine induction in macrophages and dendritic cells places them in the central role in the in vivo response to foreign DNA. Although responses to DNA are unlikely to evolve and be retained if they are not significant in the immune response to infection, the relative contributions of DNA and other stimulators of the innate immune recognition of foreign organisms is difficult to assess. Although CpG DNA and LPS have similar actions, significant differences are emerging that make the use of DNA as a therapeutic immunostimulatory molecule feasible. The macrophage response to DNA generates cytokines favouring the development of Th1-type immunity, and active oligonucleotides now show promise as Th1-promoting adjuvants and as allergy treatments.