Peptidylarginine deiminase inhibition is immunomodulatory and vasculoprotective in murine lupus
Jason S. Knight, Wenpu Zhao, Wei Luo, Venkataraman Subramanian, Alexander A. O’Dell, Srilakshmi Yalavarthi, Jeffrey B. Hodgin, Daniel T. Eitzman, Paul R. Thompson, Mariana J. Kaplan
Jason S. Knight, Wenpu Zhao, Wei Luo, Venkataraman Subramanian, Alexander A. O’Dell, Srilakshmi Yalavarthi, Jeffrey B. Hodgin, Daniel T. Eitzman, Paul R. Thompson, Mariana J. Kaplan
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Research Article Immunology

Peptidylarginine deiminase inhibition is immunomodulatory and vasculoprotective in murine lupus

Abstract

Recent evidence suggests that enhanced neutrophil extracellular trap (NET) formation activates plasmacytoid dendritic cells and serves as a source of autoantigens in SLE. We propose that aberrant NET formation is also linked to organ damage and to the premature vascular disease characteristic of human SLE. Here, we demonstrate enhanced NET formation in the New Zealand mixed 2328 (NZM) model of murine lupus. NZM mice also developed autoantibodies to NETs as well as the ortholog of human cathelicidin/LL37 (CRAMP), a molecule externalized in the NETs. NZM mice were treated with Cl-amidine, an inhibitor of peptidylarginine deiminases (PAD), to block NET formation and were evaluated for lupus-like disease activity, endothelial function, and prothrombotic phenotype. Cl-amidine treatment inhibited NZM NET formation in vivo and significantly altered circulating autoantibody profiles and complement levels while reducing glomerular IgG deposition. Further, Cl-amidine increased the differentiation capacity of bone marrow endothelial progenitor cells, improved endothelium-dependent vasorelaxation, and markedly delayed time to arterial thrombosis induced by photochemical injury. Overall, these findings suggest that PAD inhibition can modulate phenotypes crucial for lupus pathogenesis and disease activity and may represent an important strategy for mitigating cardiovascular risk in lupus patients.

Authors

Jason S. Knight, Wenpu Zhao, Wei Luo, Venkataraman Subramanian, Alexander A. O’Dell, Srilakshmi Yalavarthi, Jeffrey B. Hodgin, Daniel T. Eitzman, Paul R. Thompson, Mariana J. Kaplan

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Figure 7

PAD inhibition prolongs time to arterial thrombosis and reduces NET density in NZM mice.

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PAD inhibition prolongs time to arterial thrombosis and reduces NET dens...
20-week-old NZM mice were treated with Cl-amidine (10 mg/kg/d) or vehicle, by daily subcutaneous injection for 1 week. Carotid artery thrombosis was then induced by photochemical injury, with DNase administered just prior to injury in some vehicle-treated mice. (A) Both DNase and Cl-amidine change the content of thrombi, resulting in the capture of fewer citrullinated histone H3–positive (H3-Cit) and CRAMP-positive structures. H&E and Hoechst 33342 (DNA in blue) staining are also shown for these representative paraffin-embedded sections. Arrowheads highlight NET-like structures. (B) For each thrombus, at least 3 sections were quantified for H3-Cit–positive (white bars) and CRAMP-positive (black bars) structures (n = 5 mice per group). (C and D) Both DNase and Cl-amidine prolong time to vessel occlusion (n = 10 for vehicle and Cl-amidine groups; n = 5 for the DNase group), with representative carotid artery flow tracings in D. (E) Cl-amidine reduces the NET density of carotid thrombi. Frozen sections were stained with Hoechst 33342 (DNA in blue) and anti-CRAMP (green). Representative images are shown, with overlays to the right. V, vessel wall; T, thrombus. (F) For each thrombus, at least 3 sections were quantified for discrete areas of DNA/CRAMP overlap (n = 5 per group). Original magnification, ×1000. Scale bars: 50 microns. Volumes were determined by multiplying thrombus area by section depth. All quantification is presented as mean ± SEM. *P < 0.05; **P < 0.01.