Background/Purpose: The transcription factor nuclear factor erythroid 2-related factor (Nrf2) is a master regulator of genes involved in cellular defense against oxidative and electrophilic stresses. It forms a cytoplasmic complex with the ubiquitin ligase KEAP1 and CUL3, which in unstressed conditions promote the ubiquitin-mediated degradation of Nrf2. Modification of the reactive cysteine residues of KEAP1 by electrophiles or ROS reduces the ubiquitin ligase activity of KEAP1/CUL3, resulting in Nrf2 stabilization and binding to antioxidant response elements (AREs) in the promoters of target genes. As there is functional cross-talk between Nrf2 and NF-κB mediated inflammatory responses, we hypothesized that Nrf2 might downregulate chronic inflammation in mice with diffuse alveolar hemorrhage (DAH) associated with pristane-induced lupus, a disease driven by macrophages (Mϕ).

Methods: Murine Mϕ subsets were analyzed by flow cytometry and the cell subsets were flow-sorted. Binding of Nrf2 to its target sequence was determined in vitro. Gene expression in sorted Mϕ from pristane or mineral oil (MO) treated mice was determined using RNA-Seq and real-time PCR. Pristane-treated C57BL/6 mice received injections of CDDO-imidazolide every other day, a potent inducer of Nrf2/ARE signaling, or vehicle, starting 3-days after pristane treatment. DAH was assessed on day-14.

Results: Mϕ extracts from pristane-treated B6 mice exhibited lower Nrf2 binding in vitro to AREs vs. extracts from MO-treated controls. RNA-Seq showed that a series of Nrf2-regulated genes involved in the response to oxidative stress were expressed at lower levels in Mϕ from pristane vs. MO-treated mice. Low expression levels were confirmed by real-time PCR and Mϕ from pristane-treated mice had increased mitochondrial superoxide (MitoSox red staining), decreased mitochondrial membrane potential (tetramethylrhodamine staining), and increased cellular ROS (dichlorofluorescein diacetate staining) vs. controls. In vivo injection of CDDO-imidazolide decreased the number of CD11b+Ly6Chi (inflammatory) Mϕ, increased CD11b+CD138+ (pro-resolving) Mϕ, and reversed the pristane-induced changes in mitochondrial superoxide and cellular ROS while restoring mitochondrial membrane potential. Finally, CDDO-imidazolide treatment greatly attenuated the severity of DAH in pristane-treated mice (P<0.0001). A less potent Nrf2 activator, diethylmaleate, also was protective and reduced Mϕ TNFα production.

Conclusion: These observations suggest that oxidative stress is involved in the pathogenesis of chronic inflammation experimental lupus and that pharmacological Nrf2 activation can protect lupus mice from fatal lung hemorrhage.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/nrf2-downregulates-inflammation-and-protects-against-autoimmune-lung-disease-in-experimental-lupus/