Supplementary MaterialsFigures S1-5. species during peritoneal inflammation significantly impairs M ingestion of apoptotic neutrophils and zymosan. Our data identify a key role of the chemerin peptide/ChemR23 axis in the efficient clearance of foreign material, efferocytosis, and, hence, the resolution of inflammation. Manipulation of the chemerin peptide/ChemR23 axis may represent a novel therapeutic approach for the treatment of inflammatory pathologies, especially if failure to efficiently clear phagocytic targets has been implicated in their pathogenesis. Macrophages (Ms) are innate immune cells that can recognize, phagocytose, and kill microbial pathogens; as such, they represent an important component of the bodys defense 1314890-29-3 against infection (1-3). Efficient clearance of pathogenic material by Ms is important in limiting the magnitude and duration of the ensuing inflammatory response, although recognition and engulfment of microbial particles by Ms typically results in their activation and the secretion of inflammatory cytokines (4, 5). In contrast, M ingestion of apoptotic cells is nonphlogistic (noninflammatory) because it does not provoke inflammatory mediator expression and is associated with active suppression of proinflammatory mediator release and upregulation of anti-inflammatory mediator expression, including TGF- (6-9). Thus, apoptotic cell phagocytosis (efferocytosis) plays an important role in the resolution of inflammation and the maintenance of peripheral immune MPH1 tolerance (1, 2, 7, 9, 10). Inefficient clearance of apoptotic cells, resulting in the accumulation of secondary necrotic cells, can result in the exacerbation of inflammation, because necrotic cell ingestion elicits M activation, and necrotic cell lysis releases cytotoxic, proinflammatory, and immunogenic material (11-17). Thus, failure to efficiently clear apoptotic cells favors inflammatory and autoimmune reactions rather than inflammatory resolution, and it promotes a persistent state of inflammation seen in systemic lupus erythematosus (SLE), as well as in atherosclerosis and diabetes mellitus (18-22). Studies in experimental animal models combined with clinical evidence from human inflammatory diseases, including SLE, highlight the importance of efficient phagocytosis of apoptotic material during inflammation and also suggest its potential as a therapeutic target for the treatment of certain inflammatory diseases (12, 19, 20, 22-24). We previously reported that picogram levels of C-terminal peptides produced from the chemoattractant chemerin, specifically chemerin15 (C15; AGEDPHGYFLPGQFA), inhibit M activation 1314890-29-3 and suppress peritonitis induced with the fungus cell wall structure component zymosan (25). We hypothesized that chemerin peptides might accomplish that impact, partly, by improving M phagocytosis from the inciting stimulus, zymosan, and/or modulating the nonphlogistic ingestion of apoptotic cells at the website of inflammation. In this scholarly study, we present for the very first time that chemerin peptides potently and profoundly enhance M clearance of microbial contaminants and apoptotic cells within a nonphlogistic and ChemR23-reliant manner, an activity that will require Syk-dependent adjustments in F-actin polymerization and phagosome development. Materials and Strategies Animals All pet studies were executed with ethical acceptance through the Dunn College of Pathology Regional Moral Review Committee and relative to the U.K. OFFICE AT HOME regulations (Help with the Procedure of Pets, Scientific Procedures Work, 1986). ChemR23?/? mice with an Sv129Ev history were a sort present of Takeda (Cambridge, U.K.). M lifestyle and zymosan phagocytosis assays Ms had been attained and cultured, as previously described (25). Briefly, Bio-Gel P100 polyacrylamide beads (1 ml 2% w/v in PBS; Bio-Rad, Hemel Hempstead, U.K.) were injected into the peritoneal cavities of 8C12-wk-old C57BL/6 mice. Mice were killed 4 d later, and peritoneal exudate cells (PECs) were collected in PBS 2 mM EDTA (Lonza, Slough, U.K.). Harvested cells were centrifuged and resuspended in OptiMEM supplemented with 2 mM L-glutamine, 50 U/ml penicillin, and 50 g/ml streptomycin (all from Invitrogen, Paisley, U.K.). Cells were plated in 24-well suspension plates (0.4 106/well; Greiner Bio-One, Stonehouse, U.K.) and allowed to adhere for 1 h at 37C in a humidified atmosphere made up of 5% CO2 to purify M populations by adherence. Nonadherent cells and Bio-Gel beads were removed by washing with PBS after 1 h. Ms were preincubated with 10?13C10?9 M C15 (Biosynthesis, Lewisville, TX), C15-S (scrambled C15 peptide; GLFHDQAGPPAGYEF; 1314890-29-3 Biosynthesis), or chemerin (R&D Systems, Minneapolis, MN) for 45 min at.