Background
The inflammatory interleukin (IL)-23/IL-17 axis plays an important role in the pathogenesis of ankylosing spondylitis (AS), but with an unknown regulatory mechanism. The aim of this study was to investigate the role of endoplasmic reticulum (ER) stress and autophagy pathway in the expression of IL-23 in peripheral blood-derived macrophages in AS patients.
Methods
Peripheral blood samples were obtained from 15 AS and 15 healthy control subjects. MACS was used to isolate monocytes from PBMCs. Then, M-CSF was used to differentiate monocytes to M2 macrophages. IFN-γ and/or LPS was used to activate macrophages and M2 polarization toward M1 macrophages. Thapsigargin was used to induce ER stress and 3-MA to inhibit autophagy. The purity of extracted monocytes and macrophage markers was evaluated by flow cytometry. mRNA expression of HLA-B and-B27, ER stress-related genes, autophagy-related genes, and IL-23p19 was performed using RT-qPCR. Soluble levels of IL-23p19 were measured using ELISA.
Results
Significant increase in mRNA expression of HLA-B, HLA-B27, BiP, XBP1, CHOP, and PERK mRNAs was observed in macrophages of AS patients before and after stimulation with IFN-γ and LPS. No significant change in autophagy gene expression was detected. mRNA and soluble levels of IL-23p19 demonstrated a significant increase in macrophages of AS patients compared to healthy subjects. ER stress induction led to a significant increase in IL-23p19 in macrophages. Inhibition of autophagy had no effect on IL-23 expression.
Conclusion
ER stress, unlike autophagy, in macrophages of AS patients is associated with increased IL-23 levels.