Oliver Soehnlein

Phagocyte partnership during the onset and resolution of inflammation.

Neutrophils, monocytes and macrophages are closely related phagocytic cells that cooperate during the onset, progression and resolution of inflammation. This Review highlights the mechanisms involved in the intimate partnership of phagocytes during each progressive phase of the inflammatory response. We describe how tissue-resident macrophages recognize tissue damage to promote the recruitment of neutrophils and the mechanisms by which infiltrating neutrophils can then promote monocyte recruitment. Furthermore, we discuss the phagocyte-derived signals that abrogate neutrophil recruitment and how the uptake of apoptotic neutrophils by macrophages leads to termination of the inflammatory response. Finally, we highlight the potential therapeutic relevance of these interactions.

Hyperlipidemia-Triggered Neutrophilia Promotes Early Atherosclerosis

Background— Inflammation and activation of immune cells are key mechanisms in the development of atherosclerosis. Previous data indicate important roles for monocytes and T lymphocytes in lesion formation, whereas the contribution of neutrophils remains to be firmly established. Here, we investigate the effect of hypercholesterolemia on peripheral neutrophil counts, neutrophil recruitment to atherosclerotic lesions, and the importance of neutrophils in atherosclerotic lesion formation in Apoe−/− mice. Methods and Results— Hypercholesterolemia induces neutrophilia, which was attributable to enhanced granulopoiesis and enhanced mobilization from the bone marrow. The degree of hypercholesterolemia-induced neutrophilia was positively correlated with the extent of early atherosclerotic lesion formation. In turn, neutropenic mice display reduced plaque sizes at early but not late stages of atherosclerotic lesion formation. Flow cytometry of enzymatically digested aortas further shows altered cellular plaque composition in neutropenic mice with reduced numbers of inflammatory monocytes and macrophages. Aortic neutrophil infiltration peaks 4 weeks after the start of a high-fat diet and decreases afterward. The recruitment of neutrophils to large arteries was found to depend on CCR1, CCR2, CCR5, and CXCR2, which contrasts to peripheral venous recruitment, which requires CCR2 and CXCR2 only. The involvement of CCR1 and CCR5 corresponded to the endothelial deposition of the platelet-derived chemokine CCL5 in arteries but not in veins. Conclusions— Our data provide evidence that hypercholesterolemia-induced neutrophilia is multifactorial and that neutrophils infiltrate arteries primarily during early stages of atherosclerosis. Collectively, these data suggest an important role of neutrophils in the initiation of atherosclerosis.

Protective Role of CXC Receptor 4/CXC Ligand 12 Unveils the Importance of Neutrophils in Atherosclerosis

The CXC ligand (CXCL)12/CXC receptor (CXCR)4 chemokine–receptor axis controls hematopoiesis, organ development, and angiogenesis, but its role in the inflammatory pathogenesis of atherosclerosis is unknown. Here we show that interference with Cxcl12/Cxcr4 by a small-molecule antagonist, genetic Cxcr4 deficiency, or lentiviral transduction with Cxcr4 degrakine in bone marrow chimeras aggravated diet-induced atherosclerosis in apolipoprotein E-deficient (Apoe−/−) or LDL receptor–deficient (Ldlr−/−) mice. Chronic blockade of Cxcr4 caused leukocytosis and an expansion of neutrophils and increased neutrophil content in plaques, associated with apoptosis and a proinflammatory phenotype. Whereas circulating neutrophils were recruited to atherosclerotic lesions, depletion of neutrophils reduced plaque formation and prevented its exacerbation after blocking Cxcr4. Disrupting Cxcl12/Cxcr4 thus promotes lesion formation through deranged neutrophil homeostasis, indicating that Cxcl12/Cxcr4 controls the important contribution of neutrophils to atherogenesis in mice

Resolution of inflammation: an integrated view

Resolution of inflammation is a coordinated and active process aimed at restoration of tissue integrity and function. This review integrates the key molecular and cellular mechanisms of resolution. We describe how abrogation of chemokine signalling blocks continued neutrophil tissue infiltration and how apoptotic neutrophils attract monocytes and macrophages to induce their clearance. Uptake of apoptotic neutrophils by macrophages reprograms macrophages towards a resolving phenotype, a key event to restore tissue homeostasis. Finally, we highlight the therapeutic potential that derives from understanding the mechanisms of resolution.

Neutrophil secretion products pave the way for inflammatory monocytes

The leukocyte response in inflammation is characterized by an initial recruitment of polymorphonuclear leukocytes (PMN) preceding a second wave of monocytes to the site of injury or infection. In the mouse, 2 populations of monocytes have been identified, Gr1(-)CCR2(-)CX3CR1(hi) resident monocytes and Gr1(+)CCR2(+)CX3CR1(lo) inflammatory monocytes. Here, intravital microscopy of the musculus cremaster and a subcutaneous air pouch model were used to investigate a possible link between PMN extravasation and the subsequent emigration of inflammatory monocytes in response to local stimulation with PAF. In mice that were made neutropenic by injection of a PMN-depleting antibody, the extravasation of inflammatory monocytes, but not resident monocytes, was markedly reduced compared with mice with intact white blood cell count but was restored by local treatment with secretion of activated PMN. Components of the PMN secretion were found to directly activate inflammatory monocytes and further examination revealed PMN-derived LL-37 and heparin-binding protein (HBP/CAP37/azurocidin) as primary mediators of the recruitment of inflammatory monocytes via activation of formyl-peptide receptors. These data show that LL-37 and HBP specifically stimulate mobilization of inflammatory monocytes. This cellular cross-talk functionally results in enhanced cytokine levels and increased bacterial clearance, thus boosting the early immune response.

Multiple Roles for Neutrophils in Atherosclerosis

Because of their rare detection in atherosclerotic lesions, the involvement of neutrophils in the pathophysiology of atherosclerosis has been largely denied. However, over the past couple of years, studies have provided convincing evidence for the presence of neutrophils in atherosclerotic plaques and further revealed the causal contribution of neutrophils during various stages of atherosclerosis. This review describes mechanisms underlying hyperlipidemia-mediated neutrophilia and how neutrophils may enter atherosclerotic lesions. It also highlights possible mechanisms of neutrophil-driven atherogenesis and plaque destabilization. Knowledge of the contribution of neutrophils to atherosclerosis will allow for exploration of new avenues in the treatment of atherogenesis and atherothrombosis.

Auto-Antigenic Protein-DNA Complexes Stimulate Plasmacytoid Dendritic Cells to Promote Atherosclerosis

Background— Inflammation has been closely linked to auto-immunogenic processes in atherosclerosis. Plasmacytoid dendritic cells (pDCs) are specialized to produce type-I interferons in response to pathogenic single-stranded nucleic acids, but can also sense self-DNA released from dying cells or in neutrophil extracellular traps complexed to the antimicrobial peptide Cramp/LL37 in autoimmune disease. However, the exact role of pDCs in atherosclerosis remains elusive. Methods and Results— Here we demonstrate that pDCs can be detected in murine and human atherosclerotic lesions. Exposure to oxidatively modified low-density lipoprotein enhanced the capacity of pDCs to phagocytose and prime antigen-specific T cell responses. Plasmacytoid DCs can be stimulated to produce interferon-&agr; by Cramp/DNA complexes, and we further identified increased expression of Cramp and formation of neutrophil extracellular traps in atherosclerotic arteries. Whereas Cramp/DNA complexes aggravated atherosclerotic lesion formation in apolipoprotein E–deficient mice, pDC depletion and Cramp-deficiency in bone marrow reduced atherosclerosis and anti–double-stranded DNA antibody titers. Moreover, the specific activation of pDCs and interferon-&agr; treatment promoted plaque growth, associated with enhanced anti–double-stranded–DNA antibody titers. Accordingly, anti–double-stranded DNA antibodies were elevated in patients with symptomatic versus asymptomatic carotid artery stenosis. Conclusions— Self-DNA (eg, released from dying cells or in neutrophil extracellular traps) and an increased expression of the antimicrobial peptide Cramp/LL37 in atherosclerotic lesions may thus stimulate a pDC-driven pathway of autoimmune activation and the generation of anti–double-stranded-DNA antibodies, critically aggravating atherosclerosis lesion formation. These key factors may thus represent novel therapeutic targets.

Platelet CD40L mediates thrombotic and inflammatory processes in atherosclerosis

CD40 ligand (CD40L), identified as a costimulatory molecule expressed on T cells, is also expressed and functional on platelets. We investigated the thrombotic and inflammatory contributions of platelet CD40L in atherosclerosis. Although CD40L-deficient (Cd40l(-/-)) platelets exhibited impaired platelet aggregation and thrombus stability, the effects of platelet CD40L on inflammatory processes in atherosclerosis were more remarkable. Repeated injections of activated Cd40l(-/-) platelets into Apoe(-/-) mice strongly decreased both platelet and leukocyte adhesion to the endothelium and decreased plasma CCL2 levels compared with wild-type platelets. Moreover, Cd40l(-/-) platelets failed to form proinflammatory platelet-leukocyte aggregates. Expression of CD40L on platelets was required for platelet-induced atherosclerosis as injection of Cd40l(-/-) platelets in contrast to Cd40l(+/+) platelets did not promote lesion formation. Remarkably, injection of Cd40l(+/+), but not Cd40l(-/-), platelets transiently decreased the amount of regulatory T cells (Tregs) in blood and spleen. Depletion of Tregs in mice injected with activated Cd40l(-/-) platelets abrogated the athero-protective effect, indicating that CD40L on platelets mediates the reduction of Tregs leading to accelerated atherosclerosis. We conclude that platelet CD40L plays a pivotal role in atherosclerosis, not only by affecting platelet-platelet interactions but especially by activating leukocytes, thereby increasing platelet-leukocyte and leukocyte-endothelium interactions.

Deficient CD40-TRAF6 signaling in leukocytes prevents atherosclerosis by skewing the immune response toward an antiinflammatory profile

The CD40–CD40 ligand (CD40L) signaling axis plays an important role in immunological pathways. Consequently, this dyad is involved in chronic inflammatory diseases, including atherosclerosis. Inhibition of CD40L in apolipoprotein E (Apoe)–deficient (Apoe−/−) mice not only reduced atherosclerosis but also conferred a clinically favorable plaque phenotype that was low in inflammation and high in fibrosis. Blockade of CD40L may not be therapeutically feasible, as long-term inhibition will compromise systemic immune responses. Conceivably, more targeted intervention strategies in CD40 signaling will have less deleterious side effects. We report that deficiency in hematopoietic CD40 reduces atherosclerosis and induces features of plaque stability. To elucidate the role of CD40–tumor necrosis factor receptor-associated factor (TRAF) signaling in atherosclerosis, we examined disease progression in mice deficient in CD40 and its associated signaling intermediates. Absence of CD40-TRAF6 but not CD40-TRAF2/3/5 signaling abolishes atherosclerosis and confers plaque fibrosis in Apoe−/− mice. Mice with defective CD40-TRAF6 signaling display a reduced blood count of Ly6Chigh monocytes, an impaired recruitment of Ly6C+ monocytes to the arterial wall, and polarization of macrophages toward an antiinflammatory regulatory M2 signature. These data unveil a role for CD40–TRAF6, but not CD40–TRAF2/3/5, interactions in atherosclerosis and establish that targeting specific components of the CD40–CD40L pathway harbors the potential to achieve therapeutic effects in atherosclerosis.

Mechanisms underlying neutrophil-mediated monocyte recruitment

Extravasation of polymorphonuclear leukocytes (PMNs) to the site of inflammation precedes a second wave of emigrating monocytes. That these events are causally connected has been established a long time ago. However, we are now just beginning to understand the molecular mechanisms underlying this cellular switch, which has become even more complex considering the emergence of monocyte subsets, which are affected differently by signals generated from PMNs. PMN granule proteins induce adhesion as well as emigration of inflammatory monocytes to the site of inflammation involving beta(2)-integrins and formyl-peptide receptors. Furthermore, modification of the chemokine network by PMNs and their granule proteins creates a milieu favoring extravasation of inflammatory monocytes. Finally, emigrated PMNs rapidly undergo apoptosis, leading to the discharge of lysophosphatidylcholine, which attracts monocytes via G2A receptors. The net effect of these mechanisms is the accumulation of inflammatory monocytes, thus promoting proinflammatory events, such as release of inflammation-sustaining cytokines and reactive oxygen species. As targeting PMNs without causing serious side effects seems futile, it may be more promising to aim at interfering with subsequent PMN-driven proinflammatory events.