泛素和来自系统性红斑狼疮患者的含有游离ISG15的中性粒细胞胞外陷阱诱导的髓过氧化物酶构象变化促进CD4 T细胞中的促炎细胞因子反应。
Conformational changes in myeloperoxidase induced by ubiquitin and NETs containing free ISG15 from systemic lupus erythematosus patients promote a pro-inflammatory cytokine response in CD4 T cells.
作者信息
Carrillo-Vázquez Daniel Alberto, Jardón-Valadez Eduardo, Torres-Ruiz Jiram, Juárez-Vega Guillermo, Maravillas-Montero José Luis, Meza-Sánchez David Eduardo, Domínguez-López María Lilia, Varela Jorge Carlos Alcocer, Gómez-Martín Diana
机构信息
Department of Internal Medicine, Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán, Vasco de Quiroga 15, Tlalpan, 14080, Mexico City, Mexico.
Department of Immunology and Rheumatology, Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán, Vasco de Quiroga 15, Tlalpan, 14080, Mexico City, Mexico.
出版信息
J Transl Med. 2020 Nov 11;18(1):429. doi: 10.1186/s12967-020-02604-5.
BACKGROUND
Neutrophil extracellular traps (NETs) from patients with systemic lupus erythematosus (SLE) are characterized by lower ubiquitylation and myeloperoxidase (MPO) as a substrate. The structural and functional effect of such modification and if there are additional post-translational modifications (PTMs) are unknown.
METHODS
To assess the expression and functional role of PTMs in NETs of patients with SLE; reactivation, proliferation and cytokine production was evaluated by flow cytometry using co-cultures with dendritic cells (DC) and CD4 from SLE patients and healthy controls. The impact of ubiquitylation on MPO was assessed by molecular dynamics. The expression of ISG15 in NETs was evaluated by immunofluorescence and Western Blot.
RESULTS
Fifteen patients with SLE and ten healthy controls were included. In the co-cultures of CD4 lymphocytes with DC stimulated with ubiquitylated MPO or recombinant MPO, a higher expression of IFNγ and IL-17A was found in CD4 from SLE patients (p < 0.05). Furthermore, with DC stimulated with ubiquitylated MPO a trend towards increased expression of CD25 and Ki67 was found in lupus CD4 lymphocytes, while the opposite was documented in controls (p < 0.05). Through molecular dynamics we found the K129-K488-K505 residues of MPO as susceptible to ubiquitylation. Ubiquitylation affects the hydration status of the HEME group depending on the residue to which it is conjugated. R239 was found near by the HEME group when the ubiquitin was in K488-K505. In addition, we found greater expression of ISG15 in the SLE NETs vs controls (p < 0.05), colocalization with H2B (r = 0.81) only in SLE samples and increased production of IFNγ in PBMCs stimulated with lupus NETs compared to healthy controls NETs.
CONCLUSION
The ubiquitylated MPO has a differential effect on the induction of reactivation of CD4 lymphocytes in patients with SLE, which may be related to structural changes by ubiquitylation at the catalytic site of MPO. Besides a lower ubiquitylation pattern, NETs of patients with SLE are characterized by the expression of ISG15, and the induction of IFNγ by Th1 cells.
背景
系统性红斑狼疮（SLE）患者的中性粒细胞胞外诱捕网（NETs）的特征是泛素化水平较低，且以髓过氧化物酶（MPO）作为底物。这种修饰的结构和功能影响以及是否存在其他翻译后修饰（PTM）尚不清楚。
方法
为评估PTM在SLE患者NETs中的表达和功能作用；通过与SLE患者和健康对照的树突状细胞（DC）和CD4进行共培养，利用流式细胞术评估再激活、增殖和细胞因子产生情况。通过分子动力学评估泛素化对MPO的影响。通过免疫荧光和蛋白质印迹法评估NETs中ISG15的表达。
结果
纳入15例SLE患者和10例健康对照。在用泛素化MPO或重组MPO刺激的DC与CD4淋巴细胞的共培养中，SLE患者CD4中IFNγ和IL-17A的表达较高（p<0.05）。此外，在用泛素化MPO刺激的DC作用下，狼疮CD4淋巴细胞中CD25和Ki67的表达有增加趋势，而在对照中则相反（p<0.05）。通过分子动力学，我们发现MPO的K129-K488-K505残基易发生泛素化。泛素化根据与其结合的残基影响血红素基团的水合状态。当泛素位于K488-K505时，在血红素基团附近发现R239。此外，我们发现SLE的NETs中ISG15的表达高于对照（p<0.05），仅在SLE样本中与H2B共定位（r=0.81），并且与健康对照的NETs相比，用狼疮NETs刺激的PBMC中IFNγ的产生增加。
结论
泛素化的MPO对SLE患者CD4淋巴细胞再激活的诱导有不同影响，这可能与MPO催化位点的泛素化导致的结构变化有关。除了较低的泛素化模式外，SLE患者的NETs还具有ISG15的表达以及Th1细胞诱导IFNγ产生的特征。
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