Recent work from our group and others implicate aberrant neutrophil extracellular trap (NET) formation, the programmed release of chromatin fibers in complex with antimicrobial peptides, as a potentially crucial phenomenon involved in the induction of autoimmune responses, organ damage and premature cardiovascular disease (CVD) in systemic lupus erythematosus (SLE). Our data are supported by recent studies also linking NETosis to idiopathic atherosclerosis and to venous thrombosis. This competing renewal application will determine the in vivo importance of aberrant NETosis in CV damage and other manifestations of SLE, with obvious implications for medical therapy in patients affected by this condition.
Aim 1 : Explore the impact of NET inhibition on murine models of SLE vascular damage. While molecular events implicated in NET formation are incompletely understood, recent evidence indicates that histone citrullination is crucial for chromatin decondensation leading to NETosis6. Neutrophils express high levels of peptidylarginne deiminase-4 (PAD4), the enzyme that catalyzes histone citrullination7. Mice that lack PAD4 have decreased NETosis upon stimulation8. These observations indicate that PAD4 is crucial for NETosis and could be explored as a target in SLE and its associated vascular complications. Our collaborator, Dr. Paul Thompson (Scripps), has developed a novel PAD inhibitor N-?-benzoyl-N5-(2-chloro-1-iminoethyl-L-ornithine amide (Cl-amidine) that inhibits nuclear PAD4 with low ?M potency9,10. Cl-amidine inhibits NET formation in human and murine SLE neutrophils (see preliminary data) and has previously been well-tolerated in other murine inflammatory models. We will utilize chemical PAD4 inhibition and available PAD4 knockout (KO) models to block NETosis in murine SLE and in models of lupus with accelerated atherogenesis, allowing us, to assess the in vivo impact of NET inhibition on models of lupus-associated vascular damage: carotid thrombosis, neoangiogenesis, endothelial-dependent vasorelaxation and atherosclerosis. We predict that blocking NET formation will significantly reduce vascular damage in SLE.
Aim 2 : Determine if other SLE manifestations can be abrogated by NET inhibition. Recent data from various groups supports the concept that NETosis is a stimulus for IFN-? production, a phenomenon that may promote autoimmunity3,13,14;further, there are emerging hints that IFN-? may stimulate NETosis, thereby resulting in a harmful, self-perpetuating cycle. As NETs can damage the vasculature directly, Aim 1 is a logical place to begin our assessment. However, we anticipate that NET inhibition will also have a global impact on SLE disease activity and on type I IFN responses. We will assess the role of aberrant NETosis in autoantibody (autoAb) synthesis, induction of type I IFN-responses, adaptive immune responses and organ inflammation and damage in murine SLE, with a particular focus on kidney and skin damage.
Aim 3 : Determine the association of aberrant neutrophils, NETs and anti-neutrophil Abs in surrogate markers of endothelial dysfunction and CV risk in SLE patients. The association of neutrophil:platelet aggregates (NPAs), low-density granulocytes (LDGs), NETosis and autoAbs to neutrophil-derived immunostimulatory products, with endothelial dysfunction, subclinical atherosclerosis (carotid and coronary), type I IFN signatures and surrogate markers of vascular damage will be determined in cross-sectional and longitudinal analyses in a well-characterized CV Lupus Cohort established at the U. of Michigan. Results from this proposal will provide crucial information regarding the role of neutrophils and aberrant NETosis in the pathogenesis of autoimmune responses and organ damage in SLE, and may lead to changes in the therapeutic armamentarium in individuals with this devastating condition. If we confirm our hypothesis and extend the knowledge regarding mechanisms of effect in the studies proposed herein, Cl-amidine or other PAD-4 inhibitors may represent the first of a novel class of disease-modifying drugs for lupus that target a recently described process which may be crucial in autoimmune responses and organ damage in SLE.

Public Health Relevance

The results from this study will characterize novel mechanisms that may promote early cardiovascular disease and the development of autoimmune responses in patients with lupus and could contribute to the design of therapeutic interventions aimed at decreasing organ damage in this disease.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL088419-06
Application #
8666791
Study Section
Arthritis, Connective Tissue and Skin Study Section (ACTS)
Program Officer
Kirby, Ruth
Project Start
2007-04-01
Project End
2017-04-30
Budget Start
2014-05-22
Budget End
2015-04-30
Support Year
6
Fiscal Year
2014
Total Cost
$393,580
Indirect Cost
$115,091
Name
University of Michigan Ann Arbor
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
073133571
City
Ann Arbor
State
MI
Country
United States
Zip Code
48109
Knight, Jason S; Subramanian, Venkataraman; O'Dell, Alexander A et al. (2015) Peptidylarginine deiminase inhibition disrupts NET formation and protects against kidney, skin and vascular disease in lupus-prone MRL/lpr mice. Ann Rheum Dis 74:2199-206
Carmona-Rivera, Carmelo; Zhao, Wenpu; Yalavarthi, Srilakshmi et al. (2015) Neutrophil extracellular traps induce endothelial dysfunction in systemic lupus erythematosus through the activation of matrix metalloproteinase-2. Ann Rheum Dis 74:1417-24
Kahlenberg, J Michelle; Yalavarthi, Srilakshmi; Zhao, Wenpu et al. (2014) An essential role of caspase 1 in the induction of murine lupus and its associated vascular damage. Arthritis Rheumatol 66:152-62
Knight, Jason S; Luo, Wei; O'Dell, Alexander A et al. (2014) Peptidylarginine deiminase inhibition reduces vascular damage and modulates innate immune responses in murine models of atherosclerosis. Circ Res 114:947-56
Palanichamy, Arumugam; Bauer, Jason W; Yalavarthi, Srilakshmi et al. (2014) Neutrophil-mediated IFN activation in the bone marrow alters B cell development in human and murine systemic lupus erythematosus. J Immunol 192:906-18
Smith, Carolyne K; Vivekanandan-Giri, Anuradha; Tang, Chongren et al. (2014) Neutrophil extracellular trap-derived enzymes oxidize high-density lipoprotein: an additional proatherogenic mechanism in systemic lupus erythematosus. Arthritis Rheumatol 66:2532-44
Wang, H; Chen, Y E; Eitzman, Daniel T (2014) Imaging body fat: techniques and cardiometabolic implications. Arterioscler Thromb Vasc Biol 34:2217-23
Zhao, Wenpu; Berthier, Celine C; Lewis, Emily E et al. (2013) The peroxisome-proliferator activated receptor-? agonist pioglitazone modulates aberrant T cell responses in systemic lupus erythematosus. Clin Immunol 149:119-32
Carmona-Rivera, Carmelo; Kaplan, Mariana J (2013) Low-density granulocytes: a distinct class of neutrophils in systemic autoimmunity. Semin Immunopathol 35:455-63
Kahlenberg, J Michelle; Kaplan, Mariana J (2013) Little peptide, big effects: the role of LL-37 in inflammation and autoimmune disease. J Immunol 191:4895-901

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