Lynda M. Stuart

CD36 ligands promote sterile inflammation through assembly of a Toll-like receptor 4 and 6 heterodimer

In atherosclerosis and Alzheimers disease, deposition of the altered self components oxidized low-density lipoprotein (LDL) and amyloid-β triggers a protracted sterile inflammatory response. Although chronic stimulation of the innate immune system is believed to underlie the pathology of these diseases, the molecular mechanisms of activation remain unclear. Here we show that oxidized LDL and amyloid-β trigger inflammatory signaling through a heterodimer of Toll-like receptors 4 and 6. Assembly of this newly identified heterodimer is regulated by signals from the scavenger receptor CD36, a common receptor for these disparate ligands. Our results identify CD36-TLR4-TLR6 activation as a common molecular mechanism by which atherogenic lipids and amyloid-β stimulate sterile inflammation and suggest a new model of TLR heterodimerization triggered by coreceptor signaling events.

CD36 coordinates NLRP3 inflammasome activation by facilitating intracellular nucleation of soluble ligands into particulate ligands in sterile inflammation

Particulate ligands, including cholesterol crystals and amyloid fibrils, induce production of interleukin 1β (IL-1β) dependent on the cytoplasmic sensor NLRP3 in atherosclerosis, Alzheimers disease and diabetes. Soluble endogenous ligands, including oxidized low-density lipoprotein (LDL), amyloid-β and amylin peptides, accumulate in such diseases. Here we identify an endocytic pathway mediated by the pattern-recognition receptor CD36 that coordinated the intracellular conversion of those soluble ligands into crystals or fibrils, which resulted in lysosomal disruption and activation of the NLRP3 inflammasome. Consequently, macrophages that lacked CD36 failed to elicit IL-1β production in response to those ligands, and targeting CD36 in atherosclerotic mice resulted in lower serum concentrations of IL-1β and accumulation of cholesterol crystals in plaques. Collectively, our findings highlight the importance of CD36 in the accrual and nucleation of NLRP3 ligands from within the macrophage and position CD36 as a central regulator of inflammasome activation in sterile inflammation.

Eater, a Transmembrane Protein Mediating Phagocytosis of Bacterial Pathogens in Drosophila

Phagocytosis is a complex, evolutionarily conserved process that plays a central role in host defense against infection. We have identified a predicted transmembrane protein, Eater, which is involved in phagocytosis in Drosophila. Transcriptional silencing of the eater gene in a macrophage cell line led to a significant reduction in the binding and internalization of bacteria. Moreover, the N terminus of the Eater protein mediated direct microbial binding which could be inhibited with scavenger receptor ligands, acetylated, and oxidized low-density lipoprotein. In vivo, eater expression was restricted to blood cells. Flies lacking the eater gene displayed normal responses in NF-kappaB-like Toll and IMD signaling pathways but showed impaired phagocytosis and decreased survival after bacterial infection. Our results suggest that Eater is a major phagocytic receptor for a broad range of bacterial pathogens in Drosophila and provide a powerful model to address the role of phagocytosis in vivo.

Inhibitory Effects of Apoptotic Cell Ingestion upon Endotoxin-Driven Myeloid Dendritic Cell Maturation

Dendritic cells (DCs) are the sentinels of the immune system, able to interact with both naive and memory T cells. The recent observation that DCs can ingest cells dying by apoptosis has raised the possibility that DCs may, in fact, present self-derived Ags, initiating both autoimmunity and tumor-specific responses, especially if associated with appropriate danger signals. Although the process of ingestion of apoptotic cells has not been shown to induce DC maturation, the exact fate of these phagocytosing DCs remains unclear. In this paper we demonstrate that DCs that ingest apoptotic cells are able to produce TNF-α but have a diminished ability to produce IL-12 in response to external stimuli, a property that corresponds to a failure to up-regulate CD86. By single-cell analysis we demonstrate that these inhibitory effects are restricted to those DCs that have engulfed apoptotic cells, with bystander DCs remaining unaffected. These changes were independent of the production of anti-inflammatory cytokines TGF-β1 and IL-10 and corresponded with a diminished capacity to stimulate naive T cells. Thus, the ingestion of apoptotic cells is not an immunologically null event but is capable of modulating DC maturation. These results have important implications for our understanding of the role of clearance of dying cells by DCs not only in the normal resolution of inflammation but also in control of subsequent immune responses to apoptotic cell-derived Ags.

Mannose-binding lectin-deficient mice display defective apoptotic cell clearance but no autoimmune phenotype

Mannose-binding lectin (MBL) is a circulating serum protein that is sequestered to sites of inflammation and infection. MBL is a member of the collectin family with structural similarities to the lung collectins and functional similarities to C1q. Both MBL and C1q activate complement; C1q activates the classical pathway and MBL the lectin pathway. Here we demonstrate that MBL binds apoptotic cells in vitro and confirm a role for MBL in clearance of apoptotic cells in vivo. Despite MBL null mice demonstrating defective apoptotic cell clearance they did not develop spontaneous autoimmunity, lymphoproliferation, or germinal center expansion although increased numbers of peritoneal B1 cells were detected. These data demonstrate an important in vivo role for MBL in clearance of dying cells and adds the MBL null animals to the few animals with demonstrable in vivo apoptotic cell clearance defects. Moreover, it demonstrates that failure of apoptotic cell clearance can be dissociated from autoimmunity.

Phagocytosis and comparative innate immunity: learning on the fly

Phagocytosis, the engulfment of material by cells, is a highly conserved process that arose before the development of multicellularity. Phagocytes have a key role in embryogenesis and also guard the portals of potential pathogen entry. They discriminate between diverse particles through the array of receptors expressed on their surface. In higher species, arguably the most sophisticated function of phagocytes is the processing and presentation of antigens derived from internalized material to stimulate lymphocytes and long-lived specific immunity. Central to these processes is the generation of a phagosome, the organelle that forms around internalized material. As we discuss in this Review, over the past two decades important insights into phagocytosis have been gleaned from studies in the model organism Drosophila melanogaster.

The neuroimmune guidance cue netrin-1 promotes atherosclerosis by inhibiting the emigration of macrophages from plaques

Atherosclerotic plaque formation is fueled by the persistence of lipid-laden macrophages in the artery wall. The mechanisms by which these cells become trapped, thereby establishing chronic inflammation, remain unknown. Here we found that netrin-1, a neuroimmune guidance cue, was secreted by macrophages in human and mouse atheroma, where it inactivated the migration of macrophages toward chemokines linked to their egress from plaques. Acting via its receptor, UNC5b, netrin-1 inhibited the migration of macrophages directed by the chemokines CCL2 and CCL19, activation of the actin-remodeling GTPase Rac1 and actin polymerization. Targeted deletion of netrin-1 in macrophages resulted in much less atherosclerosis in mice deficient in the receptor for low-density lipoprotein and promoted the emigration of macrophages from plaques. Thus, netrin-1 promoted atherosclerosis by retaining macrophages in the artery wall. Our results establish a causative role for negative regulators of leukocyte migration in chronic inflammation.

Requirements for Apoptotic Cell Contact in Regulation of Macrophage Responses

An important consequence of macrophage engulfment of apoptotic cells is suppression of inflammatory responses, which was first defined by assay of TNF-α release stimulated by LPS. These effects are apparently mediated in part by paracrine effects of TGF-β released by the subset of stimulated macrophages that ingest apoptotic cells, which suppresses neighboring cells. However, the apoptotic cell-derived signal that stimulates TGF-β release, and the nature of any additional signals required for the anti-inflammatory response remain poorly defined. In this study, we investigate the requirements for apoptotic cell engagement of macrophage surface receptors in these responses. We show that the apoptotic cell receptors CD36 and αvβ3 contribute to apoptotic cell phagocytosis by mouse macrophages, but are not essential for anti-inflammatory responses, suggesting that the mechanisms of response and phagocytosis are separate. In further defining requirements for response, we confirm the importance of TGF-β in suppression by apoptotic cells, and identify an additional level of control of these effects. We show that LPS-stimulated mouse macrophage TNF-α release is only suppressed if macrophages have first contacted apoptotic cells, and hence, bystander macrophages are refractory to TGF-β released by phagocytosing macrophages. We conclude that the profound suppression of LPS-driven TNF-α release by macrophage populations requires hitherto obscure contact-dependent licensing of macrophage responsiveness to TGF-β by apoptotic cells.

Nonphlogistic Clearance of Late Apoptotic Neutrophils by Macrophages: Efficient Phagocytosis Independent of β2 Integrins

Neutrophils undergo constitutive death by apoptosis, leading to safe nonphlogistic phagocytosis and clearance by macrophages. Recent work has shown that before secondary necrosis, neutrophils exhibiting classical features of apoptosis can progress to a morphologically defined late apoptotic state. However, whether such neutrophils could be safely cleared was unknown. We now report that human late apoptotic neutrophils could be purified from cultured neutrophil populations undergoing constitutive death and were subsequently ingested by human monocyte-derived macrophages by serum-independent mechanisms that did not trigger the release of IL-8 or TNF-α. Such ingestion was specifically inhibited by Abs to thrombospondin-1 and the αvβ3 vitronectin receptor. Murine bone marrow-derived macrophage phagocytosis of late and early apoptotic neutrophils occurred by similar mechanisms, proceeding with the same efficiency as that observed for wild-type controls when macrophages from αm−/− or β2−/− mice were used. We conclude that specific nonphlogistic, β2 integrin-independent mechanisms involving thrombospondin-1 and αvβ3 allow macrophages to ingest late apoptotic neutrophils without eliciting inflammatory cytokine secretion.

Activation of caspase-1 by the NLRP3 inflammasome regulates the NADPH oxidase NOX2 to control phagosome function

Phagocytosis is a fundamental cellular process that is pivotal for immunity as it coordinates microbial killing, innate immune activation and antigen presentation. An essential step in this process is phagosome acidification, which regulates many functions of these organelles that allow phagosomes to participate in processes that are essential to both innate and adaptive immunity. Here we report that acidification of phagosomes containing Gram-positive bacteria is regulated by the NLRP3 inflammasome and caspase-1. Active caspase-1 accumulates on phagosomes and acts locally to control the pH by modulating buffering by the NADPH oxidase NOX2. These data provide insight into a mechanism by which innate immune signals can modify cellular defenses and establish a new function for the NLRP3 inflammasome and caspase-1 in host defense.