Department of Biomedical Engineering, School of Engineering, Virginia Commonwealth University, Richmond, VA, United States.
Department of Biomedical Engineering, School of Engineering, Virginia Commonwealth University, Richmond, VA, United States.
Biomaterials. 2021 Apr;271:120715. doi: 10.1016/j.biomaterials.2021.120715. Epub 2021 Feb 11.
Neutrophils predominate the early inflammatory response to tissue injury and implantation of biomaterials. Recent studies have shown that neutrophil activation can be regulated by mechanical cues such as stiffness or surface wettability; however, it is not known how neutrophils sense and respond to physical cues, particularly how they form neutrophil extracellular traps (NET formation). To examine this, we used polydimethylsiloxane (PDMS) substrates of varying physiologically relevant stiffness (0.2-32 kPa) and examined the response of murine neutrophils to untreated surfaces or to surfaces coated with various extracellular matrix proteins recognized by integrin heterodimers (collagen, fibronectin, laminin, vitronectin, synthetic RGD). Neutrophils on higher stiffness PDMS substrates had increased NET formation and higher secretion of pro-inflammatory cytokines and chemokines. Extracellular matrix protein coatings showed that fibronectin induced the most NET formation and this effect was stiffness dependent. Synthetic RGD peptides induced similar levels of NET formation and pro-inflammatory cytokine release than the full-length fibronectin protein. To determine if the observed NET formation in response to substrate stiffness required focal adhesion kinase (FAK) activity, which is down stream of integrin activation, FAK inhibitor PF-573228 was used. Inhibition of FAK using PF-573228 ablated the stiffness-dependent increase in NET formation and pro-inflammatory molecule secretion. These findings demonstrate that neutrophils regulate NET formation in response to physical and mechanical biomaterial cues and this process is regulated through integrin/FAK signaling.
中性粒细胞在组织损伤和生物材料植入后的早期炎症反应中占主导地位。最近的研究表明，中性粒细胞的激活可以受到机械线索的调节，例如硬度或表面润湿性；然而，目前尚不清楚中性粒细胞如何感知和响应物理线索，特别是它们如何形成中性粒细胞胞外陷阱（NET 形成）。为了研究这一点，我们使用了具有不同生理相关硬度（0.2-32kPa）的聚二甲基硅氧烷（PDMS）基底，并研究了未处理表面或涂有各种细胞外基质蛋白的表面对小鼠中性粒细胞的反应，这些蛋白质被整合素异二聚体识别（胶原蛋白、纤维连接蛋白、层粘连蛋白、玻连蛋白、合成 RGD）。在较高硬度 PDMS 基底上的中性粒细胞具有更高的 NET 形成和更高的促炎细胞因子和趋化因子分泌。细胞外基质蛋白涂层表明，纤维连接蛋白诱导最多的 NET 形成，并且这种效果依赖于硬度。合成 RGD 肽诱导与全长纤维连接蛋白蛋白相似水平的 NET 形成和促炎细胞因子释放。为了确定观察到的对基底硬度的 NET 形成是否需要粘着斑激酶（FAK）活性，该活性是整合素激活的下游，使用了 FAK 抑制剂 PF-573228。使用 PF-573228 抑制 FAK 消除了 NET 形成和促炎分子分泌的硬度依赖性增加。这些发现表明，中性粒细胞调节 NET 形成以响应物理和机械生物材料线索，并且该过程通过整合素/FAK 信号传导进行调节。
