gms | German Medical Science

Introduction: Granulomatosis with polyangiitis (GPA) is characterized by extravascular necrotizing granulomatous inflammation and systemic anti-neutrophil cytoplasmic autoantibody (ANCA) – associated vasculitis [1]. Dysregulated cell death and tissue damage has major impact on perpetuating the chronic inflammation in GPA [2]. Preliminary work has shown that microRNA-184 (miR-184) is upregulated in GPA. In this study, we aimed to identify potential cellular sources of miRNA-184 in GPA. Furthermore, we investigated the role of miR-184 in the context of inflammatory cell death and potential regulation of the autoantigen proteinase 3 in granulocyte cell models.

Methods: NB4 cells were differentiated by all-trans retinoic acid for 24 h. Granulocyte-like differentiation was analyzed by FACS, qPCR and western blot. Endogenous miR-184 expression was examined by qPCR. miR-184 and GPADH miRNA control was transfected using lipofection. Downregulation of target genes and proteins were analyzed by qPCR after 24h and by western blot after 48h. In U937 cells apoptosis and necroptosis was induced and miR-184 expression was analyzed by qPCR. Expression of cell death markers was confirmed by western blot. The effect of miR-184 inhibition on cell death was investigated by transfection of miRNA-184-inhibitor and confirmation of cell death marker expression by western blot.

Results: NB4 cells were differentiated into a CD11b+/CD14- granulocyte-like phenotype expressing PR3 mRNA and protein. Endogenous miR-184 expression was not found. Downregulation of GAPDH by control miRNA was validated on protein level. However, no downregulation of PR3 on mRNA level or protein level by miR-184 transfection was observed. In contrast, downregulation of AKT2 on mRNA and protein level by miR-184 was detected. We found expression of miR-184 in circulating mononuclear cells and granulocytes of the peripheral blood, but there was no significant difference between GPA and healthy controls. MiR-184 expression was increased in U937 cells after induction of necroptosis but not following apoptosis. In contrast, inhibition of miR-184 had no direct effect on cell death induction.

Conclusion: In this study we identified peripheral mononuclear cells and granulocytes as a source of miR-184. In addition, we showed that miR-184 regulates AKT2, but not PR3 mRNA in a granulocyte model. Moreover, we found a necroptosis-associated increase of miR-184 expression suggesting a potential link between up-regulated miR-184 and inflammatory cell death in GPA.

Disclosure: The authors have nothing to declare.