Experimental lupus is aggravated in mouse strains with impaired induction of neutrophil extracellular traps
Deborah Kienhöfer, Jonas Hahn, Julia Stoof, Janka Zsófia Csepregi, Christiane Reinwald, Vilma Urbonaviciute, Caroline Johnsson, Christian Maueröder, Malgorzata J. Podolska, Mona H. Biermann, Moritz Leppkes, Thomas Harrer, Malin Hultqvist, Peter Olofsson, Luis E. Munoz, Attila Mocsai, Martin Herrmann, Georg Schett, Rikard Holmdahl, Markus H. Hoffmann
Deborah Kienhöfer, Jonas Hahn, Julia Stoof, Janka Zsófia Csepregi, Christiane Reinwald, Vilma Urbonaviciute, Caroline Johnsson, Christian Maueröder, Malgorzata J. Podolska, Mona H. Biermann, Moritz Leppkes, Thomas Harrer, Malin Hultqvist, Peter Olofsson, Luis E. Munoz, Attila Mocsai, Martin Herrmann, Georg Schett, Rikard Holmdahl, Markus H. Hoffmann
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Research Article Inflammation

Experimental lupus is aggravated in mouse strains with impaired induction of neutrophil extracellular traps

Abstract

Many effector mechanisms of neutrophils have been implicated in the pathogenesis of systemic lupus erythematosus (SLE). Neutrophil extracellular traps (NETs) have been assigned a particularly detrimental role. Here we investigated the functional impact of neutrophils and NETs on a mouse model of lupus triggered by intraperitoneal injection of the cell death–inducing alkane pristane. Pristane-induced lupus (PIL) was aggravated in 2 mouse strains with impaired induction of NET formation, i.e., NOX2-deficient (Ncf1-mutated) and peptidyl arginine deiminase 4–deficient (PAD4-deficient) mice, as seen from elevated levels of antinuclear autoantibodies (ANAs) and exacerbated glomerulonephritis. We observed a dramatically reduced ability to form pristane-induced NETs in vivo in both Ncf1-mutated and PAD4-deficient mice, accompanied by higher levels of inflammatory mediators in the peritoneum. Similarly, neutropenic Mcl-1ΔMyelo mice exhibited higher levels of ANAs, which indicates a regulatory function in lupus of NETs and neutrophils. Blood neutrophils from Ncf1-mutated and human individuals with SLE exhibited exuberant spontaneous NET formation. Treatment with specific chemical NOX2 activators induced NET formation and ameliorated PIL. Our findings suggest that aberrant NET is one of the factors promoting experimental lupus-like autoimmunity by uncontrolled release of inflammatory mediators.

Authors

Deborah Kienhöfer, Jonas Hahn, Julia Stoof, Janka Zsófia Csepregi, Christiane Reinwald, Vilma Urbonaviciute, Caroline Johnsson, Christian Maueröder, Malgorzata J. Podolska, Mona H. Biermann, Moritz Leppkes, Thomas Harrer, Malin Hultqvist, Peter Olofsson, Luis E. Munoz, Attila Mocsai, Martin Herrmann, Georg Schett, Rikard Holmdahl, Markus H. Hoffmann

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Figure 5

Treatment of pristane-induced lupus (PIL) with a NOX2 agonist.

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Treatment of pristane-induced lupus (PIL) with a NOX2 agonist. (A) Extra...
(A) Extracellular release of ROS (relative light units, RLU) from peritoneal cells isolated from WT or Ncf1** mice and treated with the NOX2 activator RE-02. Background values from vehicle-treated cells are subtracted. Line plots show means and SEM of 4 animals per group. (B) ROS production of cells isolated from mice treated s.c. twice weekly for 180 days with RE-02 or vehicle. Scatter plots show individual values, means, and SEM from 8 animals per group. ***P < 0.001 as determined by 2-tailed Student’s t test. (C) Quantitative analysis of neutrophil extracellular traps (NETs) in blood neutrophils treated for 3 hours with different concentrations of RE-02 or PMA. NETs were defined as SYTOX Green– and neutrophil elastase–positive (SYTOX Green+NE+) events with a 5-fold greater mean nuclear size. Scatter plot shows individual values, means, and SEM from 3 normal healthy control donors. (D) IgG autoantibodies against double-stranded DNA (dsDNA) and Sm/RNP throughout PIL in mice treated with RE-02 or vehicle were analyzed in sera (n = 8 per group) by ELISA. Shown are means ± SEM. (E) Representative immunofluorescence images of glomerular IgG and complement C3 deposits in mice 180 days after pristane injection that were treated with RE-02 or vehicle. Scale bar: 100 μm. Right panel shows scatter plots of glomerular deposition scores for IgG and complement factor C3. Depicted are individual values, means, and SEM (n = 4–5). *P < 0.05, **P < 0.01 as determined by 2-tailed Student’s t test. (F) Quantification of proteinuria in RE-02– or vehicle-treated mice at day 0, 90, and 180 after pristane injection. Bars show means and SEM (n = 8).