Background/Purpose: Immune complex (IC)-activated neutrophils appear to amplify inflammation in lupus through the release of neutrophil extracellular traps (NETosis).  There is presently no consensus as to how to most effectively inhibit NETosis in patients.  Recent studies have implicated the inositol-requiring enzyme 1 alpha (IRE1α) pathway (a mediator of the endoplasmic reticulum stress response) as a perpetuator of inflammation in infections, sepsis, and inflammatory disorders such as rheumatoid arthritis.  However, the IRE1α pathway has been little studied in relation to neutrophil function or lupus pathogenesis.

Methods: Total IgG was isolated from lupus patients and bound to ribonucleoprotein to generate lupus ICs, which were used to stimulate neutrophils.  IRE1α activity was measured by quantifying splicing of X-box binding protein 1 (XBP1) gene transcripts (spliced transcripts encode active XBP1 protein, recognized as a major facilitator of the IRE1α pathway).  We tested the impact of specific inhibition of neutrophil IRE1α on reactive oxygen species (ROS) production, caspase activation, mitochondrial function, and NETosis.  Finally, we assessed the therapeutic efficacy of inhibition of the IRE1α-XBP1 pathway in the toll-like receptor 7 (TLR7)-agonist mouse model of lupus.

Results: In vitro, control neutrophils stimulated with lupus ICs demonstrated increased IRE1α pathway activity as measured by the ratio of spliced XBP1 transcripts to total XBP1 transcripts.  Inhibition of IRE1α’s ribonuclease activity with the specific inhibitor 4µ8C alleviated lupus IC-triggered NETosis, as well as “spontaneous” NETosis of patient neutrophils.  Treatment of neutrophils with lupus ICs resulted in cytosolic ROS formation (mean 2-fold increase) and caspase 2 activation (mean 6-fold increase), both of which were significantly reversed by 4µ8C.  Mitochondrial dysfunction was also appreciated in IC-stimulated neutrophils (mean 8-fold increase in mitochondrial ROS), and could be neutralized by either traditional ROS scavengers or treatment with 4µ8C.  Finally, we induced a lupus-like phenotype in mice by treating BALB/c mice with the TLR7-agonist R848 (topical application, 10 mg/kg three times weekly) over six weeks.  R848 treatment resulted in activation of the neutrophil IRE1α-XBP1 pathway (mean 1.5-fold increase in XBP1 splicing).  Administration of 4µ8C (10 mg/kg, three times weekly over the duration of R848 treatment) not only reduced IRE1α pathway activation in neutrophils, but also significantly reduced mitochondrial ROS levels.  Furthermore, R848 treatment resulted in an elevation of plasma NET remnants (mean 2-fold increase), which returned to baseline levels with 4µ8C treatment.

Conclusion: These data are the first to identify a role for IRE1α-XBP1 in the activated state of lupus neutrophils, with this pathway apparently upstream of mitochondrial dysfunction and ROS generation.  Inhibition of IRE1α reduced mitochondrial ROS formation in peripheral blood neutrophils of lupus mice, and normalized plasma NET levels of the same mice.  Inhibition of the IRE1α pathway appears to be a potential strategy for neutralizing NETosis in lupus.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/the-ire1%ce%b1-pathway-mediates-neutrophil-stress-and-netosis-in-lupus/