Endoplasmic reticulum stress sensor IRE1α propels neutrophil hyperactivity in lupus
Gautam Sule, … , Mary X. O’Riordan, Jason S. Knight
Gautam Sule, … , Mary X. O’Riordan, Jason S. Knight
Published February 9, 2021
Citation Information: J Clin Invest. 2021;131(7):e137866. https://doi.org/10.1172/JCI137866.
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Research Article Autoimmunity Immunology

Endoplasmic reticulum stress sensor IRE1α propels neutrophil hyperactivity in lupus

Abstract

Neutrophils amplify inflammation in lupus through the release of neutrophil extracellular traps (NETs). The endoplasmic reticulum stress sensor inositol-requiring enzyme 1 α (IRE1α) has been implicated as a perpetuator of inflammation in various chronic diseases; however, IRE1α has been little studied in relation to neutrophil function or lupus pathogenesis. Here, we found that neutrophils activated by lupus-derived immune complexes demonstrated markedly increased IRE1α ribonuclease activity. Importantly, in neutrophils isolated from patients with lupus, we also detected heightened IRE1α activity that was correlated with global disease activity. Immune complex–stimulated neutrophils produced both mitochondrial ROS (mitoROS) and the activated form of caspase-2 in an IRE1α-dependent fashion, whereas inhibition of IRE1α mitigated immune complex–mediated NETosis (in both human neutrophils and a mouse model of lupus). Administration of an IRE1α inhibitor to lupus-prone MRL/lpr mice over 8 weeks reduced mitoROS levels in peripheral blood neutrophils, while also restraining plasma cell expansion and autoantibody formation. In summary, these data identify a role for IRE1α in the hyperactivity of lupus neutrophils and show that this pathway is upstream of mitochondrial dysfunction, mitoROS formation, and NETosis. We believe that inhibition of the IRE1α pathway is a novel strategy for neutralizing NETosis in lupus, and potentially other inflammatory conditions.

Authors

Gautam Sule, Basel H. Abuaita, Paul A. Steffes, Andrew T. Fernandes, Shanea K. Estes, Craig Dobry, Deepika Pandian, Johann E. Gudjonsson, J. Michelle Kahlenberg, Mary X. O’Riordan, Jason S. Knight

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Figure 4

Inhibition of IRE1α blocks lupus autoantibody–mediated NETosis.

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Inhibition of IRE1α blocks lupus autoantibody–mediated NETosis. (A–D) Co...
(A–D) Control neutrophils from healthy donors were stimulated with RNP–anti-RNP, with or without the indicated inhibitors (4μ8C or KIRA6 for IRE1α; NecroX-5 for mitoROS; Z-VDVAD-FMK for caspase-2). The ER stress–inducing agent thapsigargin was included in some experiments as a positive control. (A) NETosis was detected after 4 hours by microscopy via staining for neutrophil elastase (red) and DNA (blue). n = 4 independent biological replicates. ****P < 0.0001 and ####P < 0.0001, compared with the RNP–anti-RNP (DMSO) group, by 1-way ANOVA followed by Holm-Sidak’s multiple-comparison test. Scale bar: 50 μm. (B) NETosis was assayed by quantifying SYTOX Green–stained extracellular DNA. n = 5–9 independent experiments. ****P < 0.0001 and ####P < 0.0001, compared the with RNP–anti-RNP (DMSO) group, by 1-way ANOVA followed by Holm-Sidak’s multiple-comparison test. (C) Elastase activity was assayed by quantifying the cleavage of a fluorogenic elastase substrate. n = 5–9 independent experiments. ****P < 0.0001 and ####P < 0.0001, compared with the RNP–anti-RNP (DMSO) group, by 1-way ANOVA followed by Holm-Sidak’s multiple-comparison test. (D) Plasma NETs were measured in BALB/c mice treated with R848 and the IRE1α inhibitor 4μ8C, as described in Methods. n = 10 mice per group. ***P < 0.001 and ####P < 0.0001, by 1-way ANOVA followed by Holm-Sidak’s multiple-comparison test, compared with the DMSO control in R848 mice. (E and F) Control human neutrophils were stimulated with TL8-506 (TLR8 agonist) or R848 (TLR7/8 agonist), with or without the indicated inhibitors. (E) NETosis was assayed by quantifying SYTOX Green–stained extracellular DNA. n = 5 independent experiments. ****P < 0.0001, ###P < 0.001, and ####P < 0.0001, compared with the RNP–anti-RNP (DMSO) control of the respective groups, by 1-way ANOVA followed by Holm-Sidak’s multiple-comparison test. (F) Elastase activity was assayed by quantifying the cleavage of a fluorogenic elastase substrate. n = 5 independent experiments. **P < 0.01, ##P < 0.01, and ###P < 0.001, compared with the RNP–anti-RNP (DMSO) control of the respective groups, by 1-way ANOVA followed by Holm-Sidak’s multiple-comparison test.