In vitro silencing of RIP2 in naive CD4⁺ T cells from lupus-prone mice promotes pathogenic Th17 cell differentiation.

IF 2.9 3区医学 Q2 RHEUMATOLOGY

Clinical Rheumatology Pub Date : 2024-11-01 Epub Date: 2024-09-05 DOI:10.1007/s10067-024-07124-x

Zi-Cheng Song, Shu-Ting Liu, Xue-Ying Xia, Jia-Jia Hu, Rui-Xue Leng, Wei Zhao

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引用次数: 0

Abstract

Objective: This work aims to investigate whether RIP2 silencing in naive CD4⁺ T cells from lupus-prone mice impacts Th17 cell activity or differentiation in vitro.

Methods: Naive CD4⁺ T cells isolation from MRL/lpr mice's spleens. Three RNA interference target sequences of RIP2 were packaged with lentivirus and transfected into naive CD4⁺ T cells. The shRIP2 with the highest interference efficiency was selected and transfected into naive CD4⁺ T cells. Naive CD4⁺ T cells were cultured under conventional (TGF-β1 and IL-6) and pathogenic (IL-6, IL-23, IL-1β) differentiation environments, respectively. Then, RT-qPCR, Western blot or Flow Cytometry were used for measuring the amounts of RIP2 and IL-17 and the differentiation of Th17 cells in two settings.

Results: Under the conventional Th17 (cTh17) cell differentiation environment (TGF-β1 and IL-6), RIP2 deficiency is linked to decreased IL-17A levels (1.00 ± 0.03 vs 0.80 ± 0.03) and attenuated cTh17 cell (2.46 ± 0.08 vs 0.78 ± 0.03) differentiation (all, P < 0.05). Under the pathogenic Th17 (pTh17) cell environment (IL-1β, IL-23, IL-6), RIP2 deficiency is linked to elevated IL-17A levels (1.03 ± 0.05 vs 1.63 ± 0.07) and enhanced pTh17 cell (3.69 ± 0.19 vs 5.49 ± 0.10) differentiation (all, P < 0.05).

Conclusion: Our data suggest that RIP2 inhibition induces preferential differentiation of naive CD4⁺ T cells to pathogenic Th17 cells, while being able to upregulate IL-17A levels in the context of pTh17 cell differentiation. Our study opens up new research areas to reveal the underlying mechanisms and potential therapeutic targets for the prevention and treatment of SLE patients. Key Points • Silencing of RIP2 in naive CD4⁺ T cells from lupus-prone mice promotes pathogenic Th17 (pTh17) cell differentiation and IL-17A production under pTh17 cell (IL-1β, IL-23, and IL-6) conditions. • RIP2 deficiency in naive CD4⁺ T cells reduces conventional Th17 (cTh17) cell differentiation and IL-17A production under cTh17 cell (TGF-β1 and IL-6) conditions. • RIP2-deficient naive CD4⁺ T cells preferentially differentiate towards pTh17 cells rather than cTh17 cells in vitro. • Inhibition of RIP2 may be involved in the development of SLE via effects on Th17/IL-17.

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在狼疮易感小鼠的幼稚 CD4+ T 细胞中体外沉默 RIP2 可促进致病性 Th17 细胞分化。

目的本研究旨在探讨红斑狼疮易感小鼠的幼稚 CD4+ T 细胞中的 RIP2 沉默是否会影响体外 Th17 细胞的活性或分化：从MRL/lpr小鼠脾脏中分离出幼稚CD4+ T细胞。方法：从 MRL/lpr 小鼠脾脏中分离出幼稚 CD4+ T 细胞，用慢病毒包装 RIP2 的三个 RNA 干扰靶序列并转染到幼稚 CD4+ T 细胞中。筛选出干扰效率最高的 shRIP2 并转染至幼稚 CD4+ T 细胞。天真 CD4+ T 细胞分别在常规（TGF-β1 和 IL-6）和致病（IL-6、IL-23、IL-1β）分化环境下培养。然后，采用 RT-qPCR、Western 印迹或流式细胞术测定两种环境下 Th17 细胞的 RIP2 和 IL-17 含量及分化情况：结果：在传统的Th17（cTh17）细胞分化环境（TGF-β1和IL-6）下，RIP2的缺乏与IL-17A水平的降低（1.00 ± 0.03 vs 0.80 ± 0.03）和cTh17细胞分化的减弱（2.46 ± 0.08 vs 0.78 ± 0.03）有关（均为P 结论：我们的数据表明，RIP2的抑制与Th17细胞的分化有关：我们的数据表明，RIP2抑制可诱导幼稚CD4+ T细胞优先分化为致病性Th17细胞，同时还能在pTh17细胞分化的背景下上调IL-17A水平。我们的研究为揭示预防和治疗系统性红斑狼疮患者的潜在机制和治疗靶点开辟了新的研究领域。要点 - 在狼疮易感小鼠的幼稚 CD4+ T 细胞中沉默 RIP2 会促进致病性 Th17（pTh17）细胞分化，并在 pTh17 细胞（IL-1β、IL-23 和 IL-6）条件下产生 IL-17A。- 天真 CD4+ T 细胞缺乏 RIP2 会减少传统 Th17（cTh17）细胞分化和 cTh17 细胞（TGF-β1 和 IL-6）条件下 IL-17A 的产生。- RIP2 缺陷的天真 CD4+ T 细胞在体外更倾向于向 pTh17 细胞而非 cTh17 细胞分化。- 抑制RIP2可能会通过影响Th17/IL-17参与系统性红斑狼疮的发病。

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来源期刊

Clinical Rheumatology 医学-风湿病学

CiteScore

6.90

自引率

2.90%

发文量

441

审稿时长

3 months

期刊介绍： Clinical Rheumatology is an international English-language journal devoted to publishing original clinical investigation and research in the general field of rheumatology with accent on clinical aspects at postgraduate level. The journal succeeds Acta Rheumatologica Belgica, originally founded in 1945 as the official journal of the Belgian Rheumatology Society. Clinical Rheumatology aims to cover all modern trends in clinical and experimental research as well as the management and evaluation of diagnostic and treatment procedures connected with the inflammatory, immunologic, metabolic, genetic and degenerative soft and hard connective tissue diseases.