N-acetylcysteine reduces reactive oxygen species and pro-inflammatory cytokine production in HLA-B27 positive macrophages and shapes them towards a quiescent metabolic state

Abstract

Abstract HLA-B27, which predisposes to spondyloarthritis (SpA), has a tendency to misfold, generate endoplasmic reticulum (ER) stress, and activate the unfolded protein response (UPR). The UPR may contribute to SpA pathogenesis by promoting the production of pro-inflammatory cytokines like IL-23 and TNF. During ER stress, cells produce more reactive oxygen species (ROS), which can further worsen the UPR and promote inflammation. Here, we examined whether reducing oxidative stress in HLA-B27+ macrophages affects pro-inflammatory cytokine production and UPR gene expression. Bone marrow-derived macrophages from HLA-B27-transgenic (Tg) treated with IFNg and LPS (stimulated) showed increased ROS production compared to HLA-B7-Tg and wild type (WT) control rats. This was accompanied by robust activation of the UPR only in HLA-B27-expressing cells. Treatment with N-acetylcysteine (NAC), a ROS scavenger, significantly reduced ROS levels in HLA-B27+ macrophages and strongly decreased the transcription of many pro-inflammatory cytokines including Il-23, Il-12, Tnf, Il-6 and Il-1b. The effect of NAC was further verified by ELISA assays available for rat IL-1b, IL-6, and TNFa. NAC treatment also partially reduced UPR markers including spliced Xbp1, and BiP, Chop, Manf and Derlin-3 in HLA-B27+ macrophages. Interestingly, NAC reduced metabolic activity in stimulated macrophages to levels similar to untreated control cells as measured by the Seahorse Assay. Our data demonstrate that reducing ROS levels with NAC ameliorates ER stress-related pro-inflammatory effects of HLA-B27 misfolding. Alteration of the oxidative stress pathway should be further explored for therapeutic intervention in SpA.