Skeletal muscle of patients with sporadic inclusion body myositis (sIBM) presents with inflammation, including upregulation of inflammatory cytokines such as interferon γ (IFNγ). Non-inflammatory features are also observed, like the sarcoplasmic accumulation of proteins including TDP-43 and p62. This study aimed to investigate the effect of IFNγ and interleukin 1-β (IL-1β) on TDP-43 and p62 aggregation in vitro. Primary human myotubes were treated with IL-1β (20 ng/mL) and IFNγ (750 ng/mL) separately or combined for 48 hr. Sarcoplasmic TDP-43 aggregates and p62 puncta were assessed using image analysis for size, frequency, and colocalization with each other. Total protein expression of TDP-43, p62 and LC3 was assessed using western blotting. The subcellular localization of TDP-43 was also analyzed using image analysis. Combined IL-1β and IFNγ treatment increased puncta size of p62 compared to control (0.49 ± 0.13 µm² versus 0.28 ± 0.06 µm²), without affecting puncta frequency or p62 expression but with an increased LC3II/LC3I ratio, suggesting autophagic alterations. IL-1β or IFNγ did not alter p62 puncta size or frequency, suggesting a combined insult of multiple inflammatory mediators is necessary to cause p62 alterations. IL-1β increased p62 protein expression in an autophagy-independent manner. None of the cytokine treatments affected TDP-43 protein expression, size, or frequency of TDP-43 aggregates or localization, suggesting IL-1β and IFNγ may influence TDP-43 processing in human skeletal muscle cells. TDP-43 was localized to the sarcoplasm under control conditions, suggesting this may not be a pathological feature. Overall, sIBM-like TDP-43/p62 features were not triggered by IL-1β and/or IFNγ.

中文翻译：

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炎症细胞因子 IL-1β 和 IFNγ 对肌管中 p62 和 TDP-43 肌浆聚集的影响

散发性包涵体肌炎 (sIBM) 患者的骨骼肌出现炎症，包括干扰素γ (IFN γ ) 等炎症细胞因子的上调。还观察到非炎症特征，例如 TDP-43 和 p62 等蛋白质的肌浆积聚。本研究旨在体外研究IFN γ和白细胞介素1- β（IL-1β ）对TDP-43和p62聚集的影响。原代人肌管分别用 IL-1 β (20 ng/mL) 和 IFN γ (750 ng/mL)处理或组合处理 48 小时。使用图像分析评估肌浆 TDP-43 聚集体和 p62 斑点的大小、频率和彼此的共定位。使用蛋白质印迹法评估 TDP-43、p62 和 LC3 的总蛋白表达。还使用图像分析对 TDP-43 的亚细胞定位进行了分析。与对照相比，IL-1 β和 IFN γ联合治疗增加了 p62 的斑点大小（0.49 ± 0.13  µ ​​m ²与 0.28 ± 0.06  µ m ²），不影响斑点频率或 p62 表达，但 LC3II/LC3I 比率增加，表明自噬改变。IL-1 β或 IFN γ不会改变 p62 斑点的大小或频率，表明多种炎症介质的联合损伤是引起 p62 改变所必需的。IL-1β以不依赖自噬的方式增加p62蛋白表达。所有细胞因子治疗均不影响 TDP-43 蛋白表达、TDP-43 聚集体的大小或频率或定位，表明 IL-1 β和 IFN γ可能影响人骨骼肌细胞中的 TDP-43 加工。在对照条件下，TDP-43 定位于肌浆，表明这可能不是病理特征。总体而言，类似 sIBM 的 TDP-43/p62 特征不是由 IL-1 β和/或 IFN γ触发的。