Lupus-Prone Mice Demonstrate Enhanced Neutrophil Extracellular Trap Formation: Implications for Autoantibody Formation and Organ Damage

Jason S. Knight¹, Alexander A. O'Dell¹, Wenpu Zhao², Ritika Khandpur², Srilakshmi Yalavarthi² and Mariana J. Kaplan³, ¹Department of Internal Medicine, Division of Rheumatology, University of Michigan, Ann Arbor, MI, ²University of Michigan Rheumatology, Ann Arbor, MI, ³Systemic Autoimmunity Branch, National Institutes of Health/NIAMS, Bethesda, MD

Meeting: 2012 ACR/ARHP Annual Meeting

Keywords: Animal models, autoantibodies, interferons, neutrophils and systemic lupus erythematosus (SLE)

Session Information

Title: Systemic Lupus Erythematosus - Animal Models

Session Type: Abstract Submissions (ACR)

Background/Purpose: Recent evidence suggests that enhanced neutrophil extracellular trap (NET) formation—the programmed release of chromatin fibers decorated with granular antimicrobial peptides—is as an important activator of plasmacytoid dendritic cells in systemic lupus erythematosus (SLE). We have proposed that NETosis is also linked to skin, kidney, and premature vascular damage in human SLE. Understanding the role of NETosis in murine models of lupus will be crucial to further investigations of the pathogenic role of this process in autoimmune diseases.

Methods: The New Zealand Mixed (NZM) 2328 model of murine lupus was utilized, with BALB/c and C57BL/6 mice as non-autoimmune controls. Percentage of neutrophils undergoing NETosis was assayed both at baseline and upon exposure to NZM or control serum. Autoantibodies (autoAbs) to NET proteins were characterized by immunofluorescence, western blotting, and in-house ELISAs for Abs to the LL-37 mouse orthologue CRAMP and to NET proteins in general. Glomerular and skin NET-like material were detected by immunofluorescence analysis.

Results: NZM neutrophils demonstrated enhanced spontaneous NETosis (7.13% ± 1.16 vs. 1.83% ± 0.25; p=0.0008) as compared to control BALB/c neutrophils; further, incubation of control neutrophils with NZM serum increased NETosis percentage to levels observed in NZM neutrophils. The NET-stimulating effect of NZM serum was also robust in type I interferon receptor-knockout neutrophils, suggesting that serum factors other than these cytokines contribute to the phenotype. NZM mice develop anti-NET autoAbs. Immunofluorescence analysis using NZM serum as primary Ab demonstrated a granular pattern of reactivity with NETs, which in many places co-localized with anti-myeloperoxidase (MPO) and anti-neutrophil elastase (NE) staining. Using similar methodology, western blotting revealed NZM serum reactivity with specific proteins derived from NETs. By ELISA, NZM serum reacted more strongly with CRAMP (1.98 ± 0.19 vs. 1.00 ± 0.14; p=0.017) and NET proteins (1.85 ± 0.16 vs. 1.00 ± 0.090; p=0.014) than age-matched control serum. NET-like material—consisting of DNA, MPO, and NE—was detected in nephritic NZM kidneys and in non-affected NZM skin, but not in control mice.

Conclusion: The NZM2328 murine model of lupus replicates a number of the features of human SLE with regards to aberrant neutrophil function. These include enhanced NETosis, anti-NET autoAb formation, and the potential for organ damage attributable to NETs. Future studies should better dissect the role of NETosis both in driving lupus pathogenesis and in contributing to organ damage such as skin disease, nephritis, and accelerated atherosclerosis.

Disclosure:

J. S. Knight,
None;

A. A. O’Dell,
None;

W. Zhao,
None;

R. Khandpur,
None;

S. Yalavarthi,
None;

M. J. Kaplan,
None.

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