【目的】

在临床工作中，我们发现SLE患者常常伴随中性粒细胞减少，但是中性粒细胞数量减少的原因尚无明确结论。另外，中性粒细胞减少是SLE发病过程中的伴随表象，还是导致SLE起病或者进展加重的病因，也尚不清楚。因此，本研究旨在从临床现象-细胞活动-分子机制-动物表型的四个层面进行逐一探索，希望在这一研究过程中挖掘新的诊断标记物、治疗靶点和开发狼疮动物模型，以指导后续的临床实践。

【方法】

我们构建了126人的狼疮队列，并对临床现象和实验室检查进行分析。同时收集患者原代中性粒细胞进行测序、验证、培养和鉴定。此外，用CRISPR-Cas9构建条件基因敲除鼠模型，对提出的科学问题进行体内验证。

【结果】

这里，我们发现了系统性红斑狼疮（SLE）患者和狼疮易感小鼠的中性粒细胞会发生大量的铁死亡，分子层面表现为谷胱甘肽过氧化物酶4（GPX4，铁死亡关键负调控分子）下降和过氧化脂质（Lipid-ROS）增加；在对患者进行有效的系统治疗后，这种细胞死亡模式会恢复到正常水平。从发病机制上讲，SLE患者血清中的自身抗体和I型干扰素-α通过增强转录抑制因子CREMα与GPX4的结合，抑制了GPX4的转录，从而导致Lipid-ROS的积累最终诱发中性粒细胞铁死亡。此外，我们繁育了中性粒细胞特异性GPX4单倍体不足的小鼠，该种小鼠模拟了SLE患者的主要临床特征，包括自身抗体阳性、中性粒细胞减少、皮损和蛋白尿。同时，我们用铁死亡抑制剂治疗可以明显延缓狼疮易感小鼠（MRL/lpr）的病情进展。

【结论】

1. 活动性系统性红斑狼疮（SLE）患者的中性粒细胞发生大量铁死亡。

2. 自身反应性IgG和I型干扰素诱导中性粒细胞铁死亡。

3. 抑制铁死亡可明显缓解狼疮易感模型MRL/lpr小鼠的疾病进展。

4. 骨髓细胞特异性的GPX4的单倍体功能不足可导致小鼠中出现狼疮样表型。

5. 自身反应性IgG和I型干扰素通过CaMKIV-CREMα途径抑制SLE患者中性粒细胞中GPX4的表达，导致细胞内脂质过氧化物的积累和中性粒细胞铁死亡。

【亮点】

尽管适应性免疫系统在诱发系统性自身免疫疾病方面被赋予了重要的作用，但我们提出了一个新的理论，即先天免疫系统细胞的异常可以单独导致系统性自身免疫的发展。具体来说，由GPX4缺陷引起的中性粒细胞的铁死亡会诱发系统性自身免疫疾病。铁死亡以前没有在自身免疫性疾病中被研究过，目前的研究结果表明，铁死亡不仅在系统性红斑狼疮这种疾病的白细胞减少中起着突出的作用，而且在导致狼疮发展的一系列事件中也起着重要作用。

Objective:

In clinical work, we found that systemic lupus erythematosus (SLE) patients are often accompanied by neutropenia, but the reasons have not been clearly concluded. Furthermore, it is also unclear whether the neutropenia is a concomitant phenotype in the pathogenesis of SLE or an etiological factor leading to the onset or progression of SLE exacerbation. Therefore, this study aimed to explore these questions with the four aspects of lupus: the clinical phenomena, cellular activities, molecular mechanisms, and animal phenotypes. Hoping to explore new diagnostic markers, therapeutic targets and develop new animal models of lupus. And eventually guide the subsequent clinical practice.

Methods:

More than one hundred patients with SLE were included in the analysis, established the correlation between clinical phenomena and laboratory results. Primary neutrophils from patients were collected for sequencing, validation, culture and identification. In addition, a conditional knockout mouse model was constructed using CRISPR-Cas9 to validate the scientific hypothesis in vivo.

Results:

We demonstrate that neutrophils from patients with systemic lupus erythematosus (SLE) undergo ferroptosis, a unique form of iron-dependent programmed cell death, which primarily accounts for the neutropenia observed in patients with SLE. In SLE, autoantibodies and interferon-α present in the sera increase the production of lipid-reactive oxygen species and induce neutrophil ferroptosis. Mechanistically, the increased binding of the transcriptional repressor CREMα to the glutathione peroxidase 4 (GPX4) promoter leads to inhibition of the transcription of GPX4, a key regulator of ferroptosis. Different lupus-prone mice display enhanced neutrophil ferroptosis and treatment with a specific inhibitor for cell ferroptosis significantly ameliorates disease severity by modulating lipid-reactive oxygen species generation. Importantly, mice with neutrophil-specific Gpx4 haploinsufficiency recapitulate many key clinical features of human SLE, including neutropenia, skin lesions, the production of autoantibodies and the development of proteinuria. Therefore, our results, reported herein, identify a central cellular defect and provide the missing link between neutropenia and lupus pathogenesis.

Conclusion:

1.  Neutrophils from patients with active systemic lupus erythematosus undergo spontaneous ferroptosis and this returns to normal levels following effective systemic treatment of patients.

2.  Autoreactive IgG and type 1 interferon induced neutrophil ferroptosis.

3.  Inhibition of ferroptosis significantly alleviates disease in lupus-prone MRL/lpr mice.

4.  Myeloid cell specific haploinsufficiency of GPX4, a regulator of ferroptosis, results in the development of a lupus-like disease in otherwise normal mice.

5.  Serum autoreactive IgG and type 1 interferon inhibit GPX4 expression in neutrophils from SLE patients through the CaMKIV/CREM pathway, resulting in the accumulation of intracellular lipid-reactive oxygen species and neutrophil ferroptosis.

Highlights:

Whereas the adaptive immune system has been assigned important roles in the induction of systemic autoimmune diseases, we unveil a new paradigm whereby abnormalities in cells of the innate immune system alone can lead to the development of systemic autoimmunity. Specifically, ferroptosis of neutrophils resulting from GPX4-deficiency induces systemic autoimmune disease. Ferroptosis has not previously been studied in autoimmune disease, and the current findings indicate a prominent role not only the leukopenia of this disease, but also in the induction of a cascade of events resulting in the development of SLE.