Experimental Rheumatology, Radboudumc, Geert Grooteplein 28, 6525 GA, Nijmegen, The Netherlands.
Department of Rheumatology, Radboudumc, Nijmegen, The Netherlands.
Arthritis Res Ther. 2020 Mar 6;22(1):42. doi: 10.1186/s13075-020-2130-5.
The pathophysiology of systemic sclerosis (SSc) is closely linked to overactive TGFβ signaling. TGFβ is produced and circulates in latent form, making its activation crucial for signaling. This activation can be mediated via integrins. We investigated the balance between active and latent TGFβ in serum of SSc patients and investigated if this correlates with integrin expression on monocytes.
A TGFβ/SMAD3- or BMP/SMAD1/5-luciferase reporter construct was expressed in primary human skin fibroblasts. Both acidified and non-acidified sera of ten SSc patients and ten healthy controls were tested on these cells to determine total and active TGFβ and BMP levels respectively. A pan-specific TGFβ1/2/3 neutralizing antibody was used to confirm TGFβ signaling. Monocytes of 20 SSc patients were isolated using CD14+ positive selection, and integrin gene expression was measured using qPCR. Integrin expression was modulated using rhTGFβ1 or a small molecule inhibitor of TGFBR1: SB-505124.
SSc sera induced 50% less SMAD3-reporter activity than control sera. Serum acidification increased reporter activity, but a difference between healthy control and SSc serum was no longer observed, indicating that total TGFβ levels were not different. Addition of a pan-specific TGFβ1/2/3 neutralizing antibody fully inhibited SMAD3-reporter activity of both acidified and not-acidified control and SSc sera. Both HC and SSc sera induced similar SMAD1/5-reporter activity, and acidification increased this, but not differently between groups. Interestingly, expression of two integrin alpha subunits ITGA5 and ITGAV was significantly reduced in monocytes obtained from SSc patients. Furthermore, ITGB3, ITGB5, and ITGB8 expression was also reduced in SSc monocytes. Stimulation of monocytes with TGFβ1 induced ITGA5 and ITGAV but lowered ITGB8 expression, whereas the use of the TGFβ receptor inhibitor SB-505124 had the opposite effect.
Total TGFβ serum levels are not different between SSc patients and controls, but TGFβ activity is. This coincides with a reduced expression of TGFβ-activating integrins in monocytes of SSc patients. Because TGFβ regulates expression of these integrins in monocytes, a negative feedback mechanism possibly underlies these observations.
系统性硬化症（SSc）的病理生理学与 TGFβ 信号的过度激活密切相关。TGFβ 以潜伏形式产生和循环，因此其激活对于信号传递至关重要。这种激活可以通过整合素介导。我们研究了 SSc 患者血清中活性和潜伏 TGFβ 之间的平衡，并研究了这是否与单核细胞上整合素的表达相关。
在原代人皮肤成纤维细胞中表达 TGFβ/SMAD3-或 BMP/SMAD1/5-报告基因构建体。用来自 10 例 SSc 患者和 10 例健康对照者的酸化和非酸化血清分别测试这些细胞，以分别确定总 TGFβ 和 BMP 水平及活性。使用泛特异性 TGFβ1/2/3 中和抗体来证实 TGFβ 信号转导。使用 CD14+阳性选择从 20 例 SSc 患者中分离单核细胞，并使用 qPCR 测量整合素基因表达。使用 rhTGFβ1 或 TGFBR1 的小分子抑制剂 SB-505124 调节整合素表达。
SSc 血清诱导的 SMAD3 报告基因活性比对照血清低 50%。血清酸化增加了报告基因活性，但健康对照组和 SSc 组之间不再观察到差异，表明总 TGFβ 水平没有差异。添加泛特异性 TGFβ1/2/3 中和抗体完全抑制了酸化和非酸化对照和 SSc 血清的 SMAD3 报告基因活性。HC 和 SSc 血清均诱导相似的 SMAD1/5 报告基因活性，酸化增加了该活性，但组间无差异。有趣的是，从 SSc 患者中获得的单核细胞中，两个整合素α亚基 ITGA5 和 ITGAV 的表达显著降低。此外，SSc 单核细胞中 ITGB3、ITGB5 和 ITGB8 的表达也降低。TGFβ1 刺激单核细胞诱导 ITGA5 和 ITGAV，但降低 ITGB8 表达，而使用 TGFβ 受体抑制剂 SB-505124 则产生相反的效果。
SSc 患者和对照组之间的血清总 TGFβ 水平没有差异，但 TGFβ 活性有差异。这与 SSc 患者单核细胞中 TGFβ 激活整合素的表达减少相一致。因为 TGFβ 调节单核细胞中这些整合素的表达，所以可能存在负反馈机制来解释这些观察结果。
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