Chronic inflammation is essential mechanism during the development of cardiovascular and metabolic diseases. The outcome of diseases depends on the balance between the migration/accumulation of pro-inflammatory (M1) and anti-inflammatory (M2) macrophages in damaged tissue. The mechanism of macrophage migration and subsequent accumulation is still not fully understood. Currently, the amoeboid adhesion-independent motility is considered essential for leukocyte migration in the three-dimensional environment. We challenge this hypothesis by studying the contribution of leukocyte adhesive receptors, integrins α_Mβ₂, and α_Dβ₂, to three-dimensional migration of M1-polarized, M2-polarized, and resident macrophages. Both integrins have a moderate expression on M2 macrophages, while α_Dβ₂ is upregulated on M1 and α_Mβ₂ demonstrates high expression on resident macrophages. The level of integrin expression determines its contribution to macrophage migration. Namely, intermediate expression supports macrophage migration, while a high integrin density inhibits it. Using in vitro three-dimensional migration and in vivo tracking of adoptively-transferred fluorescently-labeled macrophages during the resolution of inflammation, we found that strong adhesion of M1-activated macrophages translates to weak 3D migration, while moderate adhesion of M2-activated macrophages generates dynamic motility. Reduced migration of M1 macrophages depends on the high expression of α_Dβ₂, since α_D-deficiency decreased M1 macrophage adhesion and improved migration in fibrin matrix and peritoneal tissue. Similarly, the high expression of α_Mβ₂ on resident macrophages prevents their amoeboid migration, which is markedly increased in α_M-deficient macrophages. In contrast, α_D- and α_M-knockouts decrease the migration of M2 macrophages, demonstrating that moderate integrin expression supports cell motility. The results were confirmed in a diet-induced diabetes model. α_D deficiency prevents the retention of inflammatory macrophages in adipose tissue and improves metabolic parameters, while α_M deficiency does not affect macrophage accumulation. Summarizing, β₂ integrin-mediated adhesion may inhibit amoeboid and mesenchymal macrophage migration or support mesenchymal migration in tissue, and, therefore, represents an important target to control inflammation.

Background: The involvement of the autonomic nervous system in the regulation of inflammation is an emerging concept with significant potential for clinical applications. Recent studies demonstrate that stimulating the vagus nerve activates the cholinergic anti-inflammatory pathway that inhibits pro-inflammatory cytokines and controls inflammation. The α7 nicotinic acetylcholine receptor (α7nAChR) on macrophages plays a key role in mediating cholinergic anti-inflammatory effects through a downstream intracellular mechanism involving inhibition of NF-κB signaling, which results in suppression of pro-inflammatory cytokine production. However, the role of the α7nAChR in the regulation of other aspects of the immune response, including the recruitment of monocytes/macrophages to the site of inflammation remained poorly understood. Results: We observed an increased mortality in α7nAChR-deficient mice (compared with wild-type controls) in mice with endotoxemia, which was paralleled with a significant reduction in the number of monocyte-derived macrophages in the lungs. Corroborating these results, fluorescently labeled α7nAChR-deficient monocytes adoptively transferred to WT mice showed significantly diminished recruitment to the inflamed tissue. α7nAChR deficiency did not affect monocyte 2D transmigration across an endothelial monolayer, but it significantly decreased the migration of macrophages in a 3D fibrin matrix. In vitro analysis of major adhesive receptors (L-selectin, β1 and β2 integrins) and chemokine receptors (CCR2 and CCR5) revealed reduced expression of integrin αM and αX on α7nAChR-deficient macrophages. Decreased expression of αMβ2 was confirmed on fluorescently labeled, adoptively transferred α7nAChR-deficient macrophages in the lungs of endotoxemic mice, indicating a potential mechanism for α7nAChR-mediated migration. Conclusions: We demonstrate a novel role for the α7nAChR in mediating macrophage recruitment to inflamed tissue, which indicates an important new aspect of the cholinergic regulation of immune responses and inflammation.

Macrophage accumulation is a critical step during development of chronic inflammation, initiating progression of many devastating diseases. Leukocyte-specific integrin α_Dβ₂ (CD11d/CD18) is dramatically upregulated on macrophages at inflammatory sites. Previously we found that CD11d overexpression on cell surfaces inhibits in vitro cell migration due to excessive adhesion. In this study, we have investigated how inflammation-mediated CD11d upregulation contributes to macrophage retention at inflammatory sites during atherogenesis. Atherosclerosis was evaluated in CD11d^−/−/ApoE^−/− mice after 16 wk on a Western diet. CD11d deficiency led to a marked reduction in lipid deposition in aortas and isolated macrophages. Macrophage numbers in aortic sinuses of CD11d^−/− mice were reduced without affecting their apoptosis and proliferation. Adoptive transfer of fluorescently labeled wild-type and CD11d^−/− monocytes into ApoE^−/− mice demonstrated similar recruitment from circulation, but reduced accumulation of CD11d^−/− macrophages within the aortas. Furthermore, CD11d expression was significantly upregulated on macrophages in atherosclerotic lesions and M1 macrophages in vitro. Interestingly, expression of the related ligand-sharing integrin CD11b was not altered. This difference defines their distinct roles in the regulation of macrophage migration. CD11d-deficient M1 macrophages demonstrated improved migration in a three-dimensional fibrin matrix and during resolution of peritoneal inflammation, whereas migration of CD11b^−/− M1 macrophages was not affected. These results prove the contribution of high densities of CD11d to macrophage arrest during atherogenesis. Because high expression of CD11d was detected in several inflammation-dependent diseases, we suggest that CD11d/CD18 upregulation on proinflammatory macrophages may represent a common mechanism for macrophage retention at inflammatory sites, thereby promoting chronic inflammation and disease development.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease, with poor prognosis and no cure. Substantial evidence implicates inflammation and associated oxidative stress as a potential mechanism for ALS, especially in patients carrying the SOD1 mutation and, therefore, lacking anti-oxidant defense. The brain is particularly vulnerable to oxidation due to the abundance of polyunsaturated fatty acids, such as docosahexaenoic acid (DHA), which can give rise to several oxidized metabolites. Accumulation of a DHA peroxidation product, CarboxyEthylPyrrole (CEP) is dependent on activated inflammatory cells and myeloperoxidase (MPO), and thus marks areas of inflammation-associated oxidative stress. At the same time, generation of an alternative inactive DHA peroxidation product, ethylpyrrole, does not require cell activation and MPO activity. While absent in normal brain tissues, CEP is accumulated in the central nervous system (CNS) of ALS patients, reaching particularly high levels in individuals carrying a SOD1 mutation. ALS brains are characterized by high levels of MPO and lowered anti-oxidant activity (due to the SOD1 mutation), thereby aiding CEP generation and accumulation. Due to DHA oxidation within the membranes, CEP marks cells with the highest oxidative damage. In all ALS cases CEP is present in nearly all astrocytes and microglia, however, only in individuals carrying a SOD1 mutation CEP marks >90% of neurons, thereby emphasizing an importance of CEP accumulation as a potential hallmark of oxidative damage in neurodegenerative diseases.

A critical step in the development of chronic inflammatory diseases is the accumulation of proinflammatory macrophages in the extracellular matrix (ECM) of peripheral tissues. The adhesion receptor integrin α_Dβ₂ promotes the development of atherosclerosis and diabetes by supporting macrophage retention in inflamed tissue. We recently found that the end product of docosahexaenoic acid (DHA) oxidation, 2-(ω-carboxyethyl)pyrrole (CEP), serves as a ligand for α_Dβ₂. CEP adduct with ECM is generated during inflammation-mediated lipid peroxidation. The goal of this project was to identify a specific inhibitor for α_Dβ₂–CEP interaction that can prevent macrophage accumulation. Using a specially designed peptide library, Biacore-detected protein–protein interaction, and adhesion of integrin-transfected HEK 293 cells, we identified a sequence (called P5 peptide) that significantly and specifically inhibited α_D–CEP binding. In the model of thioglycollate-induced peritoneal inflammation, the injection of cyclic P5 peptide reduced 3-fold the macrophage accumulation in WT mice but had no effect in α_D-deficient mice. The tracking of adoptively transferred, fluorescently labeled WT and α_D^−/− monocytes in the model of peritoneal inflammation and in vitro two-dimensional and three-dimensional migration assays demonstrated that P5 peptide does not affect monocyte transendothelial migration or macrophage efflux from the peritoneal cavity but regulates macrophage migration through the ECM. Moreover, the injection of P5 peptide into WT mice on a high-fat diet prevents macrophage accumulation in adipose tissue in an α_Dβ₂-dependent manner. Taken together, these results demonstrate the importance of α_Dβ₂-mediated macrophage adhesion for the accumulation of infiltrating macrophages in the inflamed ECM and propose P5 peptide as a potential inhibitor of atherogenesis and diabetes.