Decidual lymphatic endothelial cell-derived GM-CSF induces M1 macrophage polarization via the NF-κB pathway in severe preeclampsia

국문 초록 (Abstract)

목적: 모체-태아 접촉면에서 손상된 면역 관용과 그로 인한 과도한 염증 반응은 자간전증을 발생시키는 원인이다. 최근까지 많은 연구에서 탈락막 대식세포가 자간전증에서 주로 염증성의 M1 표현형으로 분극화되며, 이는 염증 반응을 매개한다고 제시하고 있다. 탈락막 대식세포는 태반 형성 과정에서 자궁 주변의 면역 세포들과 밀접하게 접촉하므로, 탈락막 림프관 내피세포도 상주 대식세포와 상호 작용할 가능성이 높다. 더불어, 자간전증에서 아스피린과 엽산이 면역 관용을 유지하는데 도움이 될 수 있으므로, 탈락막 내피세포의 분극화 과정에도 영향을 미칠 수 있다. 이에, 본 논문에서는 탈락막 내피세포가 정상과 자간전증에서 탈락막 대식세포의 분극화에 미치는 영향을 조사하고, 대식세포 분극화에 영향을 미칠 가능성이 있는 자간전증의 탈락막 림프관 내피세포에서 유래된 싸이토카인을 찾고, 아스피린과 엽산이 자간전증 탈락막 림프관 내피세포와 대식세포 분화의 상호작용에 미치는 영향을 평가하고자 한다.
대상과 방법: 10명의 자간전증 산모와 10명의 정상 산모군에서 획득한 탈락막에서 림프관 내피세포를 분리하였다. 정상과 자간전증 탈락막 림프관 내피세포가 대식세포 분극화에 미치는 영향을 분석하기 위하여, PMA 처리한 THP-1 세포를 정상 및 자간전증 탈락막 림프관 내피세포 또는 획득한 상층액를 이용하여 배양하였다. M1 및 M2 표현형은 유세포 분석을 시행하여 분석하였으며, 분비된 싸이토카인의 발현은 마이크로어레이 분석을 통해 확인하였다. GM-CSF 단백의 양과 발현량은 효소결합면역흡착 어세이(ELISA)와 역전사 중합효소 연쇄반응(RT-PCR)을 통해 분석하였다. ERK의 인산화와 NF-κB의 활성화는 웨스턴 블랏과 면역화학 염색으로 분석하여 평가하였다. 그리고 정상과 자간전증 탈락막 림프관 내피세포를 여러 농도의 아스피린과 엽산에 24시간 노출시킨 후 GM-CSF 단백의 양과 mRNA 발현을 분석하였다.
결과 : 자간전증 산모에서 획득한 탈락막 림프관 내피세포와 그 상층액을 이용하여 대식세포를 분화시킨 후 유세포 분석을 시행한 결과, 대식세포는 주로 M1 표현형으로 주로 분화되었다. 자간전증 탈락막 림프관 내피세포에서 GM-CSF 의 분비와 발현양이 증가되어 있었다. 정상 탈락막 림프관 내피세포에서 획득한 상층액에 rhGM-CSF 를 추가한 경우 M1 표현형의 대식세포 분극화가 촉진되며, 반면에 전자간증 탈락막 림프관 내피세포를 shRNA를 이용하여 GM-CSF 발현을 억제한 경우 M1 표현형의 대식세포 분극화가 감소되었다. NF-κB 억제제인 PDTC 를 전자간증 탈락막 림프관 내피세포에 처리한 경우, NF-κB 기전이 억제되어 GM-CSF 발현이 감소됨을 확인할 수 있었고, 이 결과 NF-κB/GM-CSF 신호 경로가 탈락막의 M1 대식세포 분극화를 조율할 수 있을 것으로 보인다. 더불어, 아스피린과 엽산을 정상과 자간전증 탈락막 림프관 내피세포에 농도별로 전처리한 결과, GM-CSF의 전사와 분비가 용량 의존적으로 유의하게 감소하였다.
결론 : 자간전증의 림프관 내피세포에서 NF-κB 경로를 통해 유래된 GM-CSF 는 M1 표현형의 대식세포 분극화를 유도한다. 아스피린과 엽산은 자간전증 탈락막 림프관 내피세포에서 GM-CSF 의 분비를 조절할 수 있으므로, 자간전증의 진행을 완화시킬 수 있는 효과를 기대할 수 있다.

다국어 초록 (Multilingual Abstract)

Objective: The impaired immune tolerance and excessive inflammatory response at the maternal-fetal interface led to the development of preeclampsia (PE). Recent evidence suggests that decidual macrophages may be polarized into the proinflammatory M1 phenotype in PE and mediate inflammatory responses. Decidual macrophages are in close contact with uterine immune cells during placentation, and decidual lymphatic endothelial cells (DLEC) have a high probability of crosstalk with resident macrophages. It is likely to affect the decidual macrophage polarization, in addition to aspirin and folic acid that can contribute to the immune tolerance in PE. This thesis aimed to investigate whether DLECs influence decidual macrophage polarization in normal and preeclamptic pregnancy, find candidate cytokine expressed in PE-DLECs modulating macrophage polarization, and evaluate the effect of aspirin and folate on the interaction between decidual LECs and resident macrophage differentiation in PE.
Material and methods: Primary DLECs were isolated from decidua obtained from 10 pregnant women with PE and 10 gestational age-matched controls. To analyze the effects of normal and PE-DLECs on macrophage polarization, phorbol-12-myristate-13-acetate (PMA)-stimulated THP-1 cells were cultured in a DLEC-conditioned medium (CM) or co-cultured with DLECs from patients with PE and controls. Surface marker expression of M1 and M2 macrophages was detected by flow cytometry. Secreted cytokine expression levels from DLECs were determined by microarray. Granulocyte-macrophage colony-stimulating factor (GM-CSF) expression was measured by enzyme-linked immunosorbent assay, and transcripts for GM-CSF were detected by quantitative real-time reverse transcription polymerase chain reaction. Phosphorylation of extracellular signal-related kinase (ERK) and activation of nuclear factor-κB (NF-κB) were evaluated by western blotting and immunocytochemical analysis, respectively. Normal and PE-DLECs were exposed to four different concentrations of aspirin and folate during 24 hr, measuring protein and mRNA levels of GM-CSF.
Results: In a coculture and culture with DLEC-CM, the contribution of PE-DLECs to M1 polarization of macrophages was confirmed by M1-related marker expression using flow cytometry. GM-CSF secretion and expression levels were increased in PE-DLECs. The addition of rhGM-CSF to M0 macrophages with normal CM promoted M1 polarization, whereas knockdown of GM-CSF in PE-DLECs by shRNA significantly reduced M1 polarization. The results showed that GM-CSF expression from PE-DLECs is downstream of NF-κB activation using an NF-κB inhibitor (PDTC), and this NF-κB/GM-CSF signaling pathway can modulate the decidual M1 polarization. In addition, pretreatment of aspirin and folate significantly decreased the expression of GM-CSF of PE-DLECs at both protein and mRNA levels in a dose-dependent manner.
Conclusions: GM-CSF induces M1 macrophage polarization through NF-κB pathway in PE-DLECs. Since aspirin and folate modulate GM-CSF release, the present data suggest aspirin and folate may mitigate the progression of PE.

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