Keeter W Coles, Moriarty Alina K, Akers Rachel, Ma Shelby, Mussbacher Marion, Nadler Jerry L, Galkina Elena V
Department of Microbiology and Molecular Cell Biology, Eastern Virginia Medical School, Norfolk, VA, United States.
Center for Integrative Neuroscience and Inflammatory Diseases, Eastern Virginia Medical School, Norfolk, VA, United States.
Front Cardiovasc Med. 2023 Jun 16;10:1175673. doi: 10.3389/fcvm.2023.1175673. eCollection 2023.
Neutrophils drive atheroprogression and directly contribute to plaque instability. We recently identified signal transducer and activator of transcription 4 (STAT4) as a critical component for bacterial host defense in neutrophils. The STAT4-dependent functions of neutrophils in atherogenesis are unknown. Therefore, we investigated a contributory role of STAT4 in neutrophils during advanced atherosclerosis.
We generated myeloid-specific , neutrophil-specific , and control mice. All groups were fed a high-fat/cholesterol diet (HFD-C) for 28 weeks to establish advanced atherosclerosis. Aortic root plaque burden and stability were assessed histologically by Movat pentachrome staining. Nanostring gene expression analysis was performed on isolated blood neutrophils. Flow cytometry was utilized to analyze hematopoiesis and blood neutrophil activation. homing of neutrophils to atherosclerotic plaques was performed by adoptively transferring prelabeled and bone marrow cells into aged atherosclerotic mice and detected by flow cytometry.
STAT4 deficiency in both myeloid-specific and neutrophil-specific mice provided similar reductions in aortic root plaque burden and improvements in plaque stability via reduction in necrotic core size, improved fibrous cap area, and increased vascular smooth muscle cell content within the fibrous cap. Myeloid-specific STAT4 deficiency resulted in decreased circulating neutrophils via reduced production of granulocyte-monocyte progenitors in the bone marrow. Neutrophil activation was dampened in HFD-C fed mice via reduced mitochondrial superoxide production, attenuated surface expression of degranulation marker CD63, and reduced frequency of neutrophil-platelet aggregates. Myeloid-specific STAT4 deficiency diminished expression of chemokine receptors CCR1 and CCR2 and impaired neutrophil trafficking to atherosclerotic aorta.
Our work indicates a pro-atherogenic role for STAT4-dependent neutrophil activation and how it contributes to multiple factors of plaque instability during advanced atherosclerosis in mice.
中性粒细胞推动动脉粥样硬化进展，并直接导致斑块不稳定。我们最近确定信号转导子和转录激活子4（STAT4）是中性粒细胞中细菌宿主防御的关键组成部分。中性粒细胞在动脉粥样硬化发生过程中依赖STAT4的功能尚不清楚。因此，我们研究了STAT4在晚期动脉粥样硬化过程中对中性粒细胞的作用。
我们构建了髓系特异性、中性粒细胞特异性和对照小鼠。所有组均喂食高脂/胆固醇饮食（HFD-C）28周以建立晚期动脉粥样硬化。通过Movat五色染色组织学评估主动脉根部斑块负担和稳定性。对分离的血液中性粒细胞进行纳米串基因表达分析。利用流式细胞术分析造血和血液中性粒细胞激活情况。通过将预先标记的野生型和STAT4缺陷型骨髓细胞过继转移到老龄动脉粥样硬化ApoE-/-小鼠中，并通过流式细胞术检测中性粒细胞向动脉粥样硬化斑块的归巢情况。
髓系特异性和中性粒细胞特异性小鼠中的STAT4缺陷均使主动脉根部斑块负担类似地降低，并通过减小坏死核心大小、改善纤维帽面积以及增加纤维帽内血管平滑肌细胞含量来改善斑块稳定性。髓系特异性STAT4缺陷通过减少骨髓中粒细胞-单核细胞祖细胞的产生导致循环中性粒细胞减少。在喂食HFD-C的STAT4缺陷小鼠中，中性粒细胞激活受到抑制，这是通过减少线粒体超氧化物产生、降低脱颗粒标志物CD63的表面表达以及减少中性粒细胞-血小板聚集体的频率实现的。髓系特异性STAT4缺陷减少了趋化因子受体CCR1和CCR2的表达，并损害了中性粒细胞向动脉粥样硬化主动脉的迁移。
我们的研究表明，依赖STAT4的中性粒细胞激活在小鼠晚期动脉粥样硬化过程中具有促动脉粥样硬化作用，以及它如何促成斑块不稳定的多个因素。
