INTRODUCTION 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. METHODS 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. RESULTS 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. CONCLUSION 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.

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TGFβ介导的SSc单核细胞中TGFβ激活整合素的表达：潜在TGFβ的激活受到干扰？

简介全身性硬化症（SSc）的病理生理与过度活跃的TGFβ信号传导密切相关。TGFβ产生并以潜伏形式循环，使其激活对于信号传导至关重要。该激活可以通过整联蛋白介导。我们研究了SSc患者血清中活性和潜在TGFβ之间的平衡，并研究了这是否与单核细胞上整联蛋白的表达有关。方法TGFβ/ SMAD3-或BMP / SMAD1 / 5-荧光素酶报告基因构建体在原代人皮肤成纤维细胞中表达。在这些细胞上测试了10名SSc患者的酸化和非酸化血清以及10个健康对照，分别测定了总TGFβ和活性TGFβ和BMP水平。使用泛特异性TGFβ1/ 2/3中和抗体来确认TGFβ信号传导。使用CD14 +阳性选择分离20例SSc患者的单核细胞，用qPCR检测整联蛋白基因表达。使用rhTGFβ1或TGFBR1的小分子抑制剂：SB-505124调节整联蛋白的表达。结果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的活性，但健康对照组和SSc血清之间不再存在差异，表明总TGFβ水平没有差异。加入泛特异性的TGFβ1/ 2/3中和抗体完全抑制了酸化和未酸化的对照和SSc血清的SMAD3报告基因活性。HC和SSc血清均诱导相似的SMAD1 / 5报道分子活性，酸化增加​​了该活性，但组间无差异。有趣的是，从SSc患者获得的单核细胞中两个整合素α亚基ITGA5和ITGAV的表达显着降低。此外，SSc单核细胞中ITGB3，ITGB5和ITGB8的表达也降低了。用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具有相反的作用。结论SSc患者和对照组的总TGFβ血清水平无差异，但TGFβ活性却不同。这与SSc患者单核细胞中激活TGFβ的整合素表达降低相吻合。由于TGFβ调节单核细胞中这些整合素的表达，因此负反馈机制可能是这些观察结果的基础。结论SSc患者和对照组的总TGFβ血清水平无差异，但TGFβ活性却不同。这与SSc患者单核细胞中激活TGFβ的整合素表达降低相吻合。由于TGFβ调节单核细胞中这些整合素的表达，因此负反馈机制可能是这些观察结果的基础。结论SSc患者和对照组的总TGFβ血清水平无差异，但TGFβ活性却不同。这与SSc患者单核细胞中TGFβ激活整合素的表达降低相吻合。由于TGFβ调节单核细胞中这些整合素的表达，因此负反馈机制可能是这些观察结果的基础。