Atherosclerosis is a chronic inflammatory disease characterized by the accumulation of oxidized lipoproteins and apoptotic cells. Adaptive immune responses to various oxidation-specific epitopes play an important role in atherogenesis. However, accumulating evidence suggests that these epitopes are also recognized by innate receptors, such as scavenger receptors on macrophages, and plasma proteins, such as C-reactive protein (CRP). Here, we provide multiple lines of evidence that oxidation-specific epitopes constitute a dominant, previously unrecognized target of natural Abs (NAbs) in both mice and humans. Using reconstituted mice expressing solely IgM NAbs, we have shown that approximately 30% of all NAbs bound to model oxidation-specific epitopes, as well as to atherosclerotic lesions and apoptotic cells. Because oxidative processes are ubiquitous, we hypothesized that these epitopes exert selective pressure to expand NAbs, which in turn play an important role in mediating homeostatic functions consequent to inflammation and cell death, as demonstrated by their ability to facilitate apoptotic cell clearance. These findings provide novel insights into the functions of NAbs in mediating host homeostasis and into their roles in health and diseases, such as chronic inflammatory diseases and atherosclerosis.

Missense mutations in the LRRK2 (Leucine-rich repeat protein kinase-2) and VPS35 genes result in autosomal dominant Parkinson's disease. The VPS35 gene encodes for the cargo-binding component of the retromer complex, while LRRK2 modulates vesicular trafficking by phosphorylating a subgroup of Rab proteins. Pathogenic mutations in LRRK2 increase its kinase activity. It is not known how the only thus far described pathogenic VPS35 mutation, [p.D620N] exerts its effects. We reveal that the VPS35[D620N] knock-in mutation strikingly elevates LRRK2-mediated phosphorylation of Rab8A, Rab10, and Rab12 in mouse embryonic fibroblasts. The VPS35[D620N] mutation also increases Rab10 phosphorylation in mouse tissues (the lung, kidney, spleen, and brain). Furthermore, LRRK2-mediated Rab10 phosphorylation is increased in neutrophils as well as monocytes isolated from three Parkinson's patients with a heterozygous VPS35[D620N] mutation compared with healthy donors and idiopathic Parkinson's patients. LRRK2-mediated Rab10 phosphorylation is significantly suppressed by knock-out or knock-down of VPS35 in wild-type, LRRK2[R1441C], or VPS35[D620N] cells. Finally, VPS35[D620N] mutation promotes Rab10 phosphorylation more potently than LRRK2 pathogenic mutations. Available data suggest that Parkinson's patients with VPS35[D620N] develop the disease at a younger age than those with LRRK2 mutations. Our observations indicate that VPS35 controls LRRK2 activity and that the VPS35[D620N] mutation results in a gain of function, potentially causing PD through hyperactivation of the LRRK2 kinase. Our findings suggest that it may be possible to elaborate compounds that target the retromer complex to suppress LRRK2 activity. Moreover, patients with VPS35[D620N] associated Parkinson's might benefit from LRRK2 inhibitor treatment that have entered clinical trials in humans.

Mutations that activate the LRRK2 (leucine-rich repeat protein kinase 2) protein kinase predispose to Parkinson's disease, suggesting that LRRK2 inhibitors might have therapeutic benefit. Recent work has revealed that LRRK2 phosphorylates a subgroup of 14 Rab proteins, including Rab10, at a specific residue located at the centre of its effector-binding switch-II motif. In the present study, we analyse the selectivity and sensitivity of polyclonal and monoclonal phospho-specific antibodies raised against nine different LRRK2-phosphorylated Rab proteins (Rab3A/3B/3C/3D, Rab5A/5B/5C, Rab8A/8B, Rab10, Rab12, Rab29[T71], Rab29[S72], Rab35 and Rab43). We identify rabbit monoclonal phospho-specific antibodies (MJFF-pRAB10) that are exquisitely selective for LRRK2-phosphorylated Rab10, detecting endogenous phosphorylated Rab10 in all analysed cell lines and tissues, including human brain cingulate cortex. We demonstrate that the MJFF-pRAB10 antibodies can be deployed to assess enhanced Rab10 phosphorylation resulting from pathogenic (R1441C/G or G2019S) LRRK2 knock-in mutations as well as the impact of LRRK2 inhibitor treatment. We also identify rabbit monoclonal antibodies displaying broad specificity (MJFF-pRAB8) that can be utilised to assess LRRK2-controlled phosphorylation of a range of endogenous Rab proteins, including Rab8A, Rab10 and Rab35. The antibodies described in the present study will help with the assessment of LRRK2 activity and examination of which Rab proteins are phosphorylated in vivo. These antibodies could also be used to assess the impact of LRRK2 inhibitors in future clinical trials.

Objectives We sought to assess the in vivo importance of scavenger receptor (SR)-mediated uptake of oxidized low density lipoprotein (OxLDL) in atherogenesis and test the efficacy of human antibody IK17-Fab or IK17 single chain Fv fragment (IK17-scFv), which lack immunological properties of intact antibodies other than the ability to inhibit uptake of OxLDL by macrophages, to inhibit atherosclerosis. Background The unregulated uptake of OxLDL by macrophage SR contributes to foam cell formation but the importance of this pathway in vivo is uncertain. Methods Cholesterol-fed LDLR−/− mice were treated with intraperitoneal infusion of human IK17-Fab (2.5mg/kg) 3 times/week for 14 weeks. Because these mice developed anti-human antibodies, LDLR−/−/Rag−/− mice (lacking ability to make immunoglobulins due to loss of T and B cell function) were treated with an adenoviral vector encoding Adv-IK17-scFv or control adenoviral-enhanced green fluorescent protein (adv-EGFP) vector intravenously every 2 weeks for 16 weeks. Results In LDLR−/− mice, infusion of IK17-Fab was able to sustain IK17 plasma levels for the first 8 weeks, but these diminished afterwards due to increasing murine anti-IK17 antibody titers. Despite this, after 14 weeks a 29% decrease in en face atherosclerosis was noted compared to PBS treated mice. In LDLR−/−/Rag−/− mice, sustained levels of plasma IK17-scFv was achieved by Adv-IK17-scFv mediated hepatic expression, which led to a 46% reduction (P<0.001) in en face atherosclerosis compared to adv-EGFP. Importantly, peritoneal macrophages isolated from Adv-IK17-scFv treated mice had decreased lipid accumulation compared to Adv-EGFP treated mice. Conclusion These data support an important role for SR-mediated uptake of OxLDL in the pathogenesis of atherosclerosis and demonstrate that oxidation-specific antibodies reduce the progression of atherosclerosis suggesting their potential in treating cardiovascular disease in humans.

Lipid peroxidation is a common event in health and is greatly accelerated in pro-inflammatory settings such as hypercholesterolemia. Consequently, oxidation-specific epitopes are generated, which are pro-inflammatory and immunogenic, leading to both adaptive and innate responses. Because innate immune mechanisms use conserved germline pattern recognition receptors (PRRs) that are preformed and present at birth, it is not obvious why they should bind to such epitopes. In this review, we put forward the hypothesis that because oxidation-specific epitopes are ubiquitous in both health and disease, and because they in essence represent "danger signals," they constitute a class of pathogenassociated molecular patterns leading to the natural selection of multiple innate PRRs that target such epitopes. We suggest that apoptotic cells, and the blebs and microparticles released from such cells, which are rich in oxidation-specific epitopes and thus pro-inflammatory, constitute an endogenous set of selecting antigens. In turn, natural antibodies, scavenger receptors, and soluble innate proteins, such as pentraxins, all represent PRRs that target such epitopes. We discuss the evidence for this hypothesis and the consequences of such responses in health and disease, such as

Background-Adaptive immunity and innate immunity play important roles in atherogenesis. Invariant chain (CD74)mediates antigen-presenting cell antigen presentation and T-cell activation. This study tested the hypothesis that CD74-deficient mice have reduced numbers of active T cells and resist atherogenesis.

It is now widely recognized that atherosclerosis is a chronic infl ammatory disease and that immune modulation Abstract Immunization with homologous malondialdehyde (MDA)-modifi ed LDL (MDA-LDL) leads to atheroprotection in experimental models supporting the concept that a vaccine to oxidation-specifi c epitopes (OSEs) of oxidized LDL could limit atherogenesis. However, modifi cation of human LDL with OSE to use as an immunogen would be impractical for generalized use. Furthermore, when MDA is used to modify LDL, a wide variety of related MDA adducts are formed, both simple and more complex. To defi ne the relevant epitopes that would reproduce the atheroprotective effects of immunization with MDA-LDL, we sought to determine the responsible immunodominant and atheroprotective adducts. We now demonstrate that fl uorescent adducts of MDA involving the condensation of two or more MDA molecules with lysine to form malondialdehyde-acetaldehyde (MAA)-type adducts generate immunodominant epitopes that lead to atheroprotective responses. We further demonstrate that a T helper (Th ) 2-biased hapten-specifi c humoral and cellular response is suffi cient, and thus, MAA-modifi ed homologous albumin is an equally effective immunogen. We further show that such Th2-biased humoral responses per se are not atheroprotective if they do not target relevant antigens. These data demonstrate the feasibility of development of a smallmolecule immunogen that could stimulate MAA-specifi c immune responses, which could be used to develop a vaccine approach to retard or prevent atherogenesis. -Gonen, A.,

Natural antibodies are preformed antibodies that are present even in naive germ-free mice in the absence of any exogenous antigenic exposure. Consistent with their specificities for microbial antigens, natural antibodies play an important non-redundant role in the first line defense against bacterial and viral infections. On the other hand natural antibodies have also been shown to have specificities for self antigens, and therefore have been proposed to provide important homeostatic "house-keeping" functions. Many of the recognized self-antigens may in fact be stress-induced self-antigens, such as oxidation-specific epitopes that accumulate during atherogenesis as well as in many other inflammatory settings, and natural antibodies could protect from the impact of the pathological accumulation of these self-antigens. In this review we will discuss the specific example of the prototypic natural antibody T15/EO6, which is increased in atherosclerotic mice and mediates atheroprotection, and discuss the potential role of natural antibodies in atherogenesis in general.