Alexandra Kadl

Nucleotides released by apoptotic cells act as a find-me signal to promote phagocytic clearance

Phagocytic removal of apoptotic cells occurs efficiently in vivo such that even in tissues with significant apoptosis, very few apoptotic cells are detectable. This is thought to be due to the release of ‘find-me’ signals by apoptotic cells that recruit motile phagocytes such as monocytes, macrophages and dendritic cells, leading to the prompt clearance of the dying cells. However, the identity and in vivo relevance of such find-me signals are not well understood. Here, through several lines of evidence, we identify extracellular nucleotides as a critical apoptotic cell find-me signal. We demonstrate the caspase-dependent release of ATP and UTP (in equimolar quantities) during the early stages of apoptosis by primary thymocytes and cell lines. Purified nucleotides at these concentrations were sufficient to induce monocyte recruitment comparable to that of apoptotic cell supernatants. Enzymatic removal of ATP and UTP (by apyrase or the expression of ectopic CD39) abrogated the ability of apoptotic cell supernatants to recruit monocytes in vitro and in vivo. We then identified the ATP/UTP receptor P2Y2 as a critical sensor of nucleotides released by apoptotic cells using RNA interference-mediated depletion studies in monocytes, and macrophages from P2Y2-null mice. The relevance of nucleotides in apoptotic cell clearance in vivo was revealed by two approaches. First, in a murine air-pouch model, apoptotic cell supernatants induced a threefold greater recruitment of monocytes and macrophages than supernatants from healthy cells did; this recruitment was abolished by depletion of nucleotides and was significantly decreased in P2Y2-/- (also known as P2ry2-/-) mice. Second, clearance of apoptotic thymocytes was significantly impaired by either depletion of nucleotides or interference with P2Y receptor function (by pharmacological inhibition or in P2Y2-/- mice). These results identify nucleotides as a critical find-me cue released by apoptotic cells to promote P2Y2-dependent recruitment of phagocytes, and provide evidence for a clear relationship between a find-me signal and efficient corpse clearance in vivo.

Lymphocyte recruitment into the aortic wall before and during development of atherosclerosis is partially L-selectin dependent

Atherosclerosis is an inflammatory disease of large arteries. Flow cytometry of aortic cell suspensions showed that B and T lymphocytes and some macrophages and dendritic cells are already present in the adventitia of normal/noninflamed mouse aortas. Adoptively transferred lymphocytes constitutively homed to the aorta and resided within the adventitia up to 7 d after transfer. Lymphocyte trafficking into normal/noninflamed or atherosclerosis-prone aortas was partially L-selectin dependent. Antigen-activated dendritic cells induced increased T lymphocyte proliferation within the aorta 72 h after adoptive transfer. During progression of atherosclerosis in apolipoprotein-E–deficient mice, the total number of macrophages, T cells, and dendritic cells, but not B cells, increased significantly. This alteration in immune cell composition was accompanied by the formation of tertiary lymphoid tissue in the adventitia of atherosclerotic aortas. These results demonstrate that lymphocytes already reside within the normal/noninflamed aorta before the onset atherosclerosis as a consequence of constitutive trafficking. Atherosclerosis induces the recruitment of macrophages and dendritic cells that support antigen presentation.

Protective role of phospholipid oxidation products in endotoxin-induced tissue damage

Lipopolysaccharide (LPS), an outer-membrane component of Gram-negative bacteria, interacts with LPS-binding protein and CD14, which present LPS to toll-like receptor 4 (refs 1, 2), which activates inflammatory gene expression through nuclear factor κB (NFκB) and mitogen-activated protein-kinase signalling. Antibacterial defence involves activation of neutrophils that generate reactive oxygen species capable of killing bacteria; therefore host lipid peroxidation occurs, initiated by enzymes such as NADPH oxidase and myeloperoxidase. Oxidized phospholipids are pro-inflammatory agonists promoting chronic inflammation in atherosclerosis; however, recent data suggest that they can inhibit expression of inflammatory adhesion molecules. Here we show that oxidized phospholipids inhibit LPS-induced but not tumour-necrosis factor-α-induced or interleukin-1β-induced NFκB-mediated upregulation of inflammatory genes, by blocking the interaction of LPS with LPS-binding protein and CD14. Moreover, in LPS-injected mice, oxidized phospholipids inhibited inflammation and protected mice from lethal endotoxin shock. Thus, in severe Gram-negative bacterial infection, endogenously formed oxidized phospholipids may function as a negative feedback to blunt innate immune responses. Furthermore, identified chemical structures capable of inhibiting the effects of endotoxins such as LPS could be used for the development of new drugs for treatment of sepsis.

Identification of a Novel Macrophage Phenotype That Develops in Response to Atherogenic Phospholipids via Nrf2

Rationale: Macrophages change their phenotype and biological functions depending on the microenvironment. In atherosclerosis, oxidative tissue damage accompanies chronic inflammation; however, macrophage phenotypic changes in response to oxidatively modified molecules are not known. Objective: To examine macrophage phenotypic changes in response to oxidized phospholipids that are present in atherosclerotic lesions. Methods and Results: We show that oxidized phospholipid-treated murine macrophages develop into a novel phenotype (Mox) that is strikingly different from the conventional M1 and M2 macrophage phenotypes. Compared to M1 and M2, Mox macrophages show a different gene expression pattern, as well as decreased phagocytotic and chemotactic capacity. Treatment with oxidized phospholipids induces both M1 and M2 macrophages to switch to the Mox phenotype. Whole-genome expression array analysis and subsequent gene ontology clustering revealed that the Mox phenotype was characterized by abundant overrepresentation of Nrf2-mediated expression of redox-regulatory genes. In macrophages isolated from Nrf2−/− mice, oxidized phospholipid-induced gene expression and regulation of redox status were compromised. Moreover, we found that Mox macrophages comprise 30% of all macrophages in advanced atherosclerotic lesions of low-density lipoprotein receptor knockout (LDLR−/−) mice. Conclusions: Together, we identify Nrf2 as a key regulator in the formation of a novel macrophage phenotype (Mox) that develops in response to oxidative tissue damage. The unique biological properties of Mox macrophages suggest this phenotype may play an important role in atherosclerotic lesion development as well as in other settings of chronic inflammation.

Expression of Heme Oxygenase-1 in Human Vascular Cells Is Regulated by Peroxisome Proliferator-Activated Receptors

Objective—Activation of peroxisome proliferator-activated receptors (PPARs) by lipid-lowering fibrates and insulin-sensitizing thiazolidinediones inhibits vascular inflammation, atherosclerosis, and restenosis. Here we investigate if the vasculoprotective and anti-inflammatory enzyme heme oxygenase-1 (HO-1) is regulated by PPAR ligands in vascular cells. Methods and Results—We show that treatment of human vascular endothelial and smooth muscle cells with PPAR ligands leads to expression of HO-1. Analysis of the human HO-1 promoter in transient transfection experiments together with mutational analysis and gel shift assays revealed a direct transcriptional regulation of HO-1 by PPAR&agr; and PPAR&ggr; via 2 PPAR responsive elements. We demonstrate that a clinically relevant polymorphism within the HO-1 promoter critically influences its transcriptional activation by both PPAR isoforms. Moreover, inhibition of HO-1 enzymatic activity reversed PPAR ligand-mediated inhibition of cell proliferation and expression of cyclooxygenase-2 in vascular smooth muscle cells. Conclusion—We demonstrate that HO-1 expression is transcriptionally regulated by PPAR&agr; and PPAR&ggr;, indicating a mechanism of anti-inflammatory and antiproliferative action of PPAR ligands via upregulation of HO-1. Identification of HO-1 as a target gene for PPARs provides new strategies for therapy of cardiovascular diseases and a rationale for the use of PPAR ligands in the treatment of other chronic inflammatory diseases.

Apoptotic cell clearance by bronchial epithelial cells critically influences airway inflammation

Lung epithelial cells can influence immune responses to airway allergens. Airway epithelial cells also undergo apoptosis after encountering environmental allergens; yet, relatively little is known about how these are cleared, and their effect on airway inflammation. Here we show that airway epithelial cells efficiently engulf apoptotic epithelial cells and secrete anti-inflammatory cytokines, dependent upon intracellular signalling by the small GTPase Rac1. Inducible deletion of Rac1 expression specifically in airway epithelial cells in a mouse model resulted in defective engulfment by epithelial cells and aberrant anti-inflammatory cytokine production. Intranasal priming and challenge of these mice with house dust mite extract or ovalbumin as allergens led to exacerbated inflammation, augmented Th2 cytokines and airway hyper-responsiveness, with decreased interleukin (IL)-10 in bronchial lavages. Rac1-deficient epithelial cells produced much higher IL-33 upon allergen or apoptotic cell encounter, with increased numbers of nuocyte-like cells. Administration of exogenous IL-10 ‘rescued’ the airway inflammation phenotype in Rac1-deficient mice, with decreased IL-33. Collectively, these genetic and functional studies suggest a new role for Rac1-dependent engulfment by airway epithelial cells and in establishing the anti-inflammatory environment, and that defects in cell clearance in the airways could contribute to inflammatory responses towards common allergens.

Oxidized Phospholipids Trigger Atherogenic Inflammation in Murine Arteries

Objective—Lipoprotein-derived phospholipid oxidation products have been implicated as candidate triggers of the inflammatory process in atherosclerosis. However, in vivo evidence regarding the impact of oxidized phospholipids on the artery wall thus far has been elusive. Therefore, the aim of this study was to investigate if structurally defined oxidized phospholipids induce expression of atherogenic chemokines and monocyte adhesion in intact murine arteries. Methods and Results—To model the accumulation of oxidized phospholipids in the arterial wall, oxidized 1-palmitoyl-2-arachidonoyl-sn-3-glycero-phosphorylcholine (OxPAPC) was topically applied to carotid arteries in mice using pluronic gel. Using quantitative reverse-transcriptase polymerase chain reaction (PCR) and immunohistochemistry, we show that OxPAPC induced a set of atherosclerosis-related genes, including monocyte chemotactic protein 1 (MCP-1) and keratinocyte-derived chemokine (KC), tissue factor (TF), interleukin 6 (IL-6), heme oxygenase 1 (HO-1), and early growth response 1 (EGR-1). OxPAPC-regulated chemokines were also expressed in atherosclerotic lesions of apolipoprotein E-deficient (ApoE−/−) mice. In isolated perfused carotid arteries, OxPAPC triggered rolling and firm adhesion of monocytes in a P-selectin and KC-dependent manner. Conclusion—Oxidized phospholipids contribute to vascular inflammation in murine arteries in vivo, initiating atherogenic chemokine expression that leads to monocyte adhesion; therefore, they can be regarded as triggers of the inflammatory process in atherosclerosis.

Oxidized Phospholipids Stimulate Angiogenesis Via Autocrine Mechanisms, Implicating a Novel Role for Lipid Oxidation in the Evolution of Atherosclerotic Lesions

Angiogenesis is a common feature observed in advanced atherosclerotic lesions. We hypothesized that oxidized phospholipids (OxPLs), which accumulate in atherosclerotic vessels can stimulate angiogenesis. We found that oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (OxPAPC) stimulated the formation of sprouts from endothelial cell spheroids and promoted growth of capillaries into Matrigel plugs in mice. OxPLs stimulated expression of vascular endothelial growth factor (VEGF) in vivo and in several normal and tumor cell types in vitro. In addition, OxPAPC upregulated cyclooxygenase (COX)-2 and interleukin (IL)-8. COX-2 inhibitors, as well as blocking antibodies to IL-8 suppressed activation of sprouting by OxPAPC. We conclude that OxPAPC stimulates angiogenesis via autocrine mechanisms involving VEGF, IL-8, and COX-2–generated prostanoids. Our data suggest that accumulation of OxPLs may contribute to increased growth of blood capillaries in advanced lesions, thus leading to progression and destabilization of atherosclerotic plaques.

Oxidized phospholipid-induced inflammation is mediated by Toll-like receptor 2.

Oxidative tissue damage is a hallmark of many chronic inflammatory diseases. However, the precise mechanisms linking oxidative changes to inflammatory reactions remain unclear. Herein we show that Toll-like receptor 2 (TLR2) translates oxidative tissue damage into inflammatory responses by mediating the effects of oxidized phospholipids. Intraperitoneal injection of oxidized 1-palmitoyl-2-arachidonyl-sn-3-glycerophosphorylcholine (OxPAPC) resulted in upregulation of inflammatory genes in wild-type, but not in TLR2(-/-) mice. In vitro, OxPAPC induced TLR2 (but not TLR4)-dependent inflammatory gene expression and JNK and p38 signaling in macrophages. Induction of TLR2-dependent gene expression required reducible functional groups on sn-2 acyl chains of oxidized phospholipids, as well as serum cofactors. Finally, TLR2(-/-) mice were protected against carbon tetrachloride-induced oxidative tissue damage and inflammation, which was accompanied by accumulation of oxidized phospholipids in livers. Together, our findings demonstrate that TLR2 mediates cellular responses to oxidative tissue damage and they provide new insights into how oxidative stress is linked to acute and chronic inflammation.

Multi-Hit Inhibition of Circulating and Cell-Associated Components of the Toll-Like Receptor 4 Pathway by Oxidized Phospholipids

Objective—Oxidized phospholipids (OxPLs) that are abundant in atherosclerotic lesions are increasingly recognized as context-dependent lipid mediators demonstrating both pro- and antiinflammatory activities. Molecular mechanisms of their effects are largely unknown. Here we present novel information on the mechanisms whereby OxPLs modulate activation of TLR4 by lipopolysaccharide (LPS). Methods and Results—We show, using several cell types and various inflammatory genes as readouts, that different classes and molecular species of OxPLs do not stimulate TLR4 but exert prominent inhibitory effects on LPS-induced reactions. Our data demonstrate that binding of OxPLs to the LPS-binding protein (LBP) and CD14 prevents recognition of LPS by these proteins, thus impairing activation of TLR4. In addition, OxPLs inhibited LBP- and CD14-independent activation of TLR4 by the synthetic TLR4 agonist E6020 indicating that in parallel with LBP and CD14, OxPLs target cell-associated steps in TLR4 cascade. Conclusions—Our data suggest that OxPLs inhibit action of LPS via a multi-hit mechanism. These results support the notion that OxPLs are endogenous inhibitors of TLR4 produced in response to oxidative stress.