All-Trans Rtinoic Acid Inhibits the Formation of Neutrophil Extracellular Traps by Activating BMP2
WANG Jinli1,2, LIU Zhidan3, WANG Xiaomin1, ZHONG Jingya1, CHENG Shuqun1,4, PAN Weiwei1, ZHAN Shuyu1*, ZHENG Yongxia1,2*
This study investigated the regulatory effect of ATRA (all-trans retinoic acid) on the release of NETs (neutrophil extracellular traps) and its possible mechanism. In vitro, neutrophils were extracted from C57BL/6 mice and pretreated with ATRA. Then, NETs were induced by PMA (phorbol 12-myristate 13-acetate). Afterwards, colocalization of MPO (myeloperoxidase) and DNA (deoxyribonucleotide) levels of NETs was detected by immunofluorescence, extracellular DNA was quantified by Sytox Green fluorescence, and MPO protein expression was determined by Western blot, and the level of H3Cit (citrullinated histone 3) in the supernatant was evaluated by ELISA. For the mechanism study, RNA-seq was used to screen the genes susceptible to ATRA, and qRT-PCR was used to verify the mRNA expression of ATRA related-genes (Arg1, Camkk1, BMP2, etc.). In order to study the effect of BMP2, the target gene of ATRA, on the formation of NETs, neutrophils were treated with BMP2 inhibitor (LDN193189), and Sytox Green fluorescence assay was used to verify the effect of LDN193189 on NETs. The results showed that all of detected values, including the colocalization of MPO and DNA, extracellular DNA, the expression of MPO protein, the H3Cit level in the supernatant of NETs, treated with ATRA were significantly lower than those in PMA group (P<0.000 1), indicating that ATRA inhibited the production of NETs in a dose-dependent manner; RNA-seq results showed that the expression of 456 genes changed, of which 274 genes were upregulated and 182 genes were down-regulated. qRT-PCR results showed that BMP2, Arg1, Camkk1, Abca1 and GSS were up-regulated in ATRA pretreated group compared with the PMA group, which was consistent with the RNAseq results. After LDN193189 treatment, the fluorescence value of Sytox Green quantitative NETs was significantly higher in ATRA pretreated group than that in PMA group (P<0.000 1). In summary, ATRA can inhibit the release of NETs, and its mechanism may be through the activation of BMP2 expression.
CN
EN