Background/Purpose: HLA-B27 is associated with the development of spondyloarthritis (SpA) and has a tendency to generate ER stress due to misfolding which can activate the unfolded protein response (UPR). UPR may contribute to SpA pathogenesis by promoting the production of pro-inflammatory cytokines like IL-23 and TNF. Under ER stress cells produce more reactive oxygen species (ROS), which further can worsen the UPR and promote inflammation. Our preliminary data indicate that IFNg and LPS treatment increases ROS and cytokine production to a greater extent in HLA-B27-expressing macrophages compared to wild type cells or cells expressing the non-disease associated allele, HLA-B7. Another important aspect of inflammation is that upon inflammatory activation immune cells have to change their metabolic requirements in order to meet the adequate response like producing pro-inflammatory cytokines. Here we examined whether reducing oxidative stress in HLA-B27+ macrophages affects pro-inflammatory cytokine production and UPR gene expression. Furthermore, we were interested whether targeting the oxidative stress pathway will modulate the metabolic requirements of the cells upon inflammatory activation.

Methods: Bone marrow derived macrophages from HLA-B27 and human b₂m (hb₂m) transgenic rats were examined with and without IFNg and LPS treatment. ROS levels were meseared with the ROS-sensitive dye MitoSOX for mitochondrial ROS production and 2’,7’-dichlorofluorescein diacetate (DCFDA) for cellular ROS level detection. N-acetylcysteine (NAC) was used as an antioxidant, and  differential gene expression was determined by RNASeq. For cytokine secretion, ELISAs were used and in order to measure metabolic activity the Seahorse Assay was performed. Wild type (WT) and HLA-B7/hb₂m transgenic rat macrophages were used as controls.

Results: Bone marrow-derived macrophages from HLA-B27-transgenic (Tg) showed increased baseline mitochondrial ROS production in the absence of UPR compared to HLA-B7-Tg and wild type (WT) control rats. Further stimulation of HLA-B27-Tg macrophages with IFNg and LPS (stimulated) showed increased ROS production compared to HLA-B7-Tg and WT control rats.This was accompanied by robust activation of the UPR only in HLA-B27-expressing cells. Treatment with NAC, an 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, Il-1a and Il-1b. The effect of NAC was further verified by ELISA assays available for rat IL-6 and TNF. NAC treatment had minimal effect on UPR gene expression, with partially reduction of BiP expression and no changes in HLA-B27 expression. Interestingly, NAC reduced metabolic activity in stimulated macrophages to levels similar to untreated control cells as measured by the Seahorse Assay.

Conclusion: 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.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/targeting-the-oxidative-stress-pathway-in-experimental-spondyloarthritis-reduces-pro-inflammatory-response-in-rat-macrophages-and-modulates-their-metabolic-requirements/