Department of Medicine, University of Wisconsin-Madison, 1685 Highland Avenue, Madison, WI 53705, USA.
Department of Pathology and Laboratory Medicine, University of Wisconsin-Madison, 1685 Highland Avenue, Madison, WI 53705, USA.
J Immunol Res. 2019 Mar 12;2019:2160192. doi: 10.1155/2019/2160192. eCollection 2019.
In rheumatoid arthritis, an autoimmune inflammatory arthritis, citrullinated proteins are targeted by autoantibodies and thus thought to drive disease. Neutrophil extracellular traps (NETs) are a source of citrullinated proteins and are increased in rheumatoid arthritis and therefore also implicated in disease pathogenesis. However, not all NETs are citrullinated. One theory aiming to clarify the intersection of citrullination, NETs, and rheumatoid arthritis suggests that specific stimuli induce different types of NETs defined by citrullination status. However, most studies do not evaluate uncitrullinated NETs, only citrullinated or total NETs. Further, the requirement for peptidylarginine deiminase (PAD) 2 and 4, two important citrullinating enzymes in neutrophils and rheumatoid arthritis, in the formation of different NETs has not been clearly defined. To determine if specific stimulants induce citrullinated or uncitrullinated NETs and if those structures require PAD2 or PAD4, human and murine neutrophils, including from PAD4 and PAD2 mice, were stimulated and NETs imaged and quantified. In humans, phorbol myristate acetate (PMA), ionomycin, monosodium urate (MSU), and induced NETs with MSU and inducing primarily citrullinated, PMA primarily uncitrullinated, and ionomycin a mix of NETs. Only ionomycin and were strong inducers of NETs in mice with ionomycin-induced NETs mostly citrullinated and -induced NETs a mix of citrullinated and uncitrullinated. Interestingly, no stimulus induced exclusively citrullinated or uncitrullinated NETs. Further, PAD4 was required for citrullinated NETs only, whereas PAD2 was not required for either NET in mice. Therefore, specific stimuli induce varying proportions of both citrullinated and uncitrullinated NETs with different requirements for PAD4. These findings highlight the complexity of NET formation and the need to further define the mechanisms by which different NETs form and their implications for autoimmune disease.
在类风湿关节炎中，一种自身免疫性炎症性关节炎，瓜氨酸化蛋白被自身抗体靶向，因此被认为是驱动疾病的原因。中性粒细胞胞外诱捕网（NETs）是瓜氨酸化蛋白的来源，在类风湿关节炎中增加，因此也与疾病发病机制有关。然而，并非所有的 NETs 都是瓜氨酸化的。一个旨在阐明瓜氨酸化、NETs 和类风湿关节炎之间的交集的理论表明，特定的刺激会诱导不同类型的 NETs，这些 NETs由瓜氨酸化状态定义。然而，大多数研究并没有评估非瓜氨酸化的 NETs，只评估瓜氨酸化或总 NETs。此外，中性粒细胞和类风湿关节炎中两种重要的瓜氨酸化酶——肽基精氨酸脱亚氨酶（PAD）2 和 4 在不同 NETs 形成中的作用尚未明确界定。为了确定特定的刺激是否诱导瓜氨酸化或非瓜氨酸化的 NETs，以及这些结构是否需要 PAD2 或 PAD4，我们刺激了人源和鼠源中性粒细胞，包括 PAD4 和 PAD2 敲除小鼠的中性粒细胞，并对 NETs 进行成像和定量。在人类中，佛波醇肉豆蔻酸酯（PMA）、离子霉素、单钠尿酸盐（MSU）和热休克蛋白 70（HSP70）诱导 NETs，其中 MSU 和 主要诱导瓜氨酸化的 NETs，PMA 主要诱导非瓜氨酸化的 NETs，而离子霉素诱导混合的 NETs。只有离子霉素和 HSP70 是小鼠中 NETs 的强诱导剂，其中离子霉素诱导的 NETs 主要是瓜氨酸化的，而 HSP70 诱导的 NETs 是瓜氨酸化和非瓜氨酸化的混合。有趣的是，没有刺激物能诱导完全瓜氨酸化或非瓜氨酸化的 NETs。此外，PAD4 仅对瓜氨酸化的 NETs 有需求，而 PAD2 对小鼠中的任何一种 NETs 都没有需求。因此，特定的刺激物诱导不同比例的瓜氨酸化和非瓜氨酸化的 NETs，需要不同的 PAD4 来诱导。这些发现突出了 NET 形成的复杂性，需要进一步定义不同 NETs 形成的机制及其对自身免疫性疾病的影响。
