This article adds to the existing literature by strongly arguing for a causative role of NETs in atherosclerosis. previously described,18 with neutrophil-platelet aggregates (Ly-6G+CD61+) quantified in fresh heparinized blood. Neutrophil Depletion Neutrophils were depleted as described.4 In brief, depletion was with intraperitoneal injection of monoclonal antibody 1A8 (BioXCell, West Lebanon, NH). Mice were specifically treated with 100 g of the antibody every other day from weeks 8 to 18. The control antibody 2A3 was also from BioXCell. Statistical Analysis and Oversight Unless otherwise indicated, results are presented as the mean and standard error of the mean (SEM), and statistical analysis was performed using Student test in GraphPad Prism software version 5. All protocols were approved by the Committee on Use and Care of Animals of the University of Michigan. Results PAD Inhibition With Cl-amidine Reduces Atherosclerosis and Arterial Thrombosis in values that did not reach significance are indicated. B, Protein was prepared from aortic arches of the indicated gene with Cl-amidine treatment (Figure 6F). This repression was not seen in the spleens of the same animals (data not shown) nor was it seen for the gene. The only IFN-responsive gene to show a slight trend toward repression was (the tested gene most selective for IFN- as compared with IFN-),38 although this did not reach statistical significance (Figure 6F). Cl-amidine also downregulated H3-Cit protein by Western blot in the same samples for which quantitative PCR was performed (Figure 6G). To summarize, PAD inhibition represses IFN- synthesis, probably by blocking NET formation. Cl-Amidine Does Not Protect Against A-1155463 Atherosclerosis in Neutropenic or in Type I IFN Receptor-Deficient Mice It has previously been shown that neutrophil depletion with an anti-Ly-6G antibody protects against atherosclerosis in value that approaches significance is denoted. Our group has previously shown that atherosclerosis is reduced Rabbit Polyclonal to PLD1 (phospho-Thr147) in em Apoe /em ?/? mice that also carry a mutation in the type I IFN receptor gene.18 Similar to neutropenic mice, these em Apoe /em A-1155463 ?/? Ifnr?/? mice were not protected by treatment with Cl-amidine (Figure 7C). In summary, Cl-amidine does not protect against atherosclerosis in the background of neutrophil depletion or type I IFN receptor deletion, suggesting that Cl-amidine likely acts through a neutrophil-based pathway, such as NET formation, and the induction of type I IFN responses in the artery. Discussion Recent studies have observed the infiltration of netting neutrophils into the atheromatous lesions of mice.4-6 Indeed, in murine systems, depletion of either whole neutrophils or the NET component CRAMP can protect against atherosclerosis,4,6 whereas treatment with exogenously prepared CRAMP-DNA complexes can accelerate disease.5 Netting neutrophils can also be detected in the blood of patients with severe coronary atherosclerosis,39 as well as in the atherosclerotic plaques themselves.40 Furthermore, in human plaques, PAD4 has been observed deiminating fibrinogen to A-1155463 generate a novel rheumatoid arthritis autoantigen.41 Although the cellular sources of this PAD4 A-1155463 have not been explored,41 neutrophils are a prime candidate. Our group recently showed that PAD inhibition reduces NET formation, alters markers of autoimmunity, and potently mitigates vascular damage in a murine model of SLE, 26 a disease process that is highly dependent on type I IFNs like IFN-.42 Although disruption of PAD activity has been considered in a model of venous thrombosis,43 it has not been evaluated in a pure model of arterial damage or atherosclerosis. We now report that em Apoe /em ?/? mice are protected from atherosclerosis when treated with the PAD inhibitor Cl-amidine. We also show that PAD.