Objective

To analyze how monocyte and macrophage exposure to CXCL4 induces inflammatory and fibrotic processes observed in Systemic sclerosis (SSc) patients.

Methods

In six independent experiments, monocytes of healthy controls (HC) and SSc patients were stimulated with CXCL4, TLR-ligands, IFNɑ or TGFβ and the secretion of cytokines in the supernatant was assessed by multiplex immunoassays. PDGF-BB production by monocyte-derived macrophages was quantified using immunoassays. The number of monocytes and PDGF-BB in circulation was quantified in HC and SSc patients with the Sysmex XT-1800i haematology counter and immunoassays. Intracellular PDGF-BB was quantified in monocytes by Western blot. PDGF-receptor inhibition was achieved using siRNA-mediated knockdown or treatment with Crenolanib. The production of inflammatory mediators and extracellular matrix (ECM) components by dermal fibroblasts was analyzed by qPCR, ELISA and ECM deposition assays.

Results

SSc and HC monocytes released PDGF-BB upon stimulation with CXCL4. Conversely, TLR ligands, IFNɑ or TGFβ did not induce PDGF-bb release. PDGF-BB plasma levels were significantly (P = 0.009) higher in diffuse SSc patients (n = 19), compared with HC (n = 21). In healthy dermal fibroblasts, PDGF-BB enhanced TNFɑ-induced expression of inflammatory cytokines and increased ECM production. Comparable results were observed in fibroblasts cultured in supernatant taken from macrophages stimulated with CXCL4. This effect was almost completely abrogated by inhibition of the PDGF-receptor using Crenolanib.

Conclusion

Our findings demonstrate that CXCL4 can drive fibroblast activation indirectly via PDGF-BB production by myeloid cells. Hence, targeting PDGF-BB or CXCL4-induced PDGF-BB release could be clinically beneficial for patients with SSc.

中文翻译：

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CXCL4 触发单核细胞和巨噬细胞产生 PDGF-BB，最终导致成纤维细胞活化：对系统性硬化症的影响。

客观的

分析单核细胞和巨噬细胞暴露于 CXCL4 如何诱导在系统性硬化症 (SSc) 患者中观察到的炎症和纤维化过程。

方法

在六个独立实验中，健康对照 (HC) 和 SSc 患者的单核细胞用 CXCL4、TLR 配体、IFNɑ 或 TGFβ 刺激，并通过多重免疫测定评估上清液中细胞因子的分泌。使用免疫测定法量化单核细胞衍生的巨噬细胞产生的 PDGF-BB。使用 Sysmex XT-1800i 血液计数器和免疫测定法量化 HC 和 SSc 患者循环中的单核细胞和 PDGF-BB 的数量。通过蛋白质印迹在单核细胞中定量细胞内 PDGF-BB。使用 siRNA 介导的敲低或用克诺拉尼治疗实现 PDGF 受体抑制。通过 qPCR、ELISA 和 ECM 沉积测定分析了真皮成纤维细胞产生的炎症介质和细胞外基质 (ECM) 成分。

结果

SSc 和 HC 单核细胞在用 CXCL4 刺激后释放 PDGF-BB。相反，TLR 配体、IFNɑ 或 TGFβ 不诱导 PDGF-bb 释放。 与 HC（n = 21）相比，弥漫性 SSc 患者（n = 19）的PDGF-BB 血浆水平显着（P = 0.009）更高。在健康的真皮成纤维细胞中，PDGF-BB 增强了 TNFɑ 诱导的炎性细胞因子的表达并增加了 ECM 的产生。在取自用 CXCL4 刺激的巨噬细胞的上清液中培养的成纤维细胞中观察到了类似的结果。使用 Crenolanib 抑制 PDGF 受体几乎完全消除了这种作用。

结论

我们的研究结果表明，CXCL4 可以通过骨髓细胞产生 PDGF-BB 间接驱动成纤维细胞活化。因此，靶向 PDGF-BB 或 CXCL4 诱导的 PDGF-BB 释放可能对 SSc 患者具有临床益处。