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 2

Exacerbated pristane-induced lupus in the absence of NOX2-derived ROS.

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Exacerbated pristane-induced lupus in the absence of NOX2-derived ROS. (...
(A) BALB/c.Ncf1** mice produce higher levels of lupus autoantibodies and develop aggravated renal damage. IgG autoantibodies against double-stranded DNA (dsDNA), Sm/RNP, and histone were analyzed in sera of naive and pristane-primed mice (n =  13) by ELISA. Boundaries of boxes represent 25th/75th percentiles, lines within boxes indicate medians, and whiskers represent minimum/maximum values. Significant differences after testing by 2-way ANOVA with Bonferroni’s post-hoc test were detected beginning 60 days after pristane injection. (B) Box-and-whisker plots of IgG1, IgG2a, IgG2b, IgG3, and IgM anti-dsDNA autoantibodies in sera of pristane-injected BALB/c.Ncf1** and BALB/c WT mice (n = 8). **P < 0.01, ***P < 0.001, as determined by 2-way ANOVA with Bonferroni’s post-hoc test. (C) Representative immunofluorescence images of renal IgG deposits in BALB/c WT and BALB/c.Ncf1** mice before and 4 and 6 months after pristane injection. Scale bars: 100 μm. Scatter plot shows individual values, mean, and SEM of deposits at day 180 from 8 to 10 mice. *P < 0.05 as determined by 2-tailed Student’s t test. (D) Representative periodic acid–Schiff staining of kidneys from unchallenged and pristane-injected BALB/c WT and Ncf1** mice. Dramatically worsened mesangial cell and matrix expansion at day 180 after pristane injection can be seen in BALB/c.Ncf1** mice. Scale bars: 100 μm. Scatter plot shows individual values, mean, and SEM of renal damage at day 180 (n = 4). *P < 0.05 as determined by 2-tailed Student’s t test. (E) Monthly quantification of proteinuria in BALB/c.Ncf1** and WT mice before and after pristane injection. Shown are means and SEM (n = 7). **P < 0.01, ***P < 0.001, as determined by 2-way ANOVA using Bonferroni’s post-hoc test. (F) Box-and-whisker plots showing medians, 25th/75th percentiles, and minimum/maximum values of serum cytokine/chemokine concentrations from mice 3 months after pristane injection (n = 14). **P < 0.01, ***P < 0.001 as determined by 2-tailed Mann-Whitney U test.