Table_1_Compliant Substrates Enhance Macrophage Cytokine Release and NLRP3 Inflammasome Formation During Their Pro-Inflammatory Response.docx

Immune cells process a myriad of biochemical signals but their function and behavior are also determined by mechanical cues. Macrophages are no exception to this. Being present in all types of tissues, macrophages are exposed to environments of varying stiffness, which can be further altered under pathological conditions. While it is becoming increasingly clear that macrophages are mechanosensitive, it remains poorly understood how mechanical cues modulate their inflammatory response. Here we report that substrate stiffness influences the expression of pro-inflammatory genes and the formation of the NLRP3 inflammasome, leading to changes in the secreted protein levels of the cytokines IL-1β and IL-6. Using polyacrylamide hydrogels of tunable elastic moduli between 0.2 and 33.1 kPa, we found that bone marrow-derived macrophages adopted a less spread and rounder morphology on compliant compared to stiff substrates. Upon LPS priming, the expression levels of the gene encoding for TNF-α were higher on more compliant hydrogels. When additionally stimulating macrophages with the ionophore nigericin, we observed an enhanced formation of the NLRP3 inflammasome, increased levels of cell death, and higher secreted protein levels of IL-1β and IL-6 on compliant substrates. The upregulation of inflammasome formation on compliant substrates was not primarily attributed to the decreased cell spreading, since spatially confining cells on micropatterns led to a reduction of inflammasome-positive cells compared to well-spread cells. Finally, interfering with actomyosin contractility diminished the differences in inflammasome formation between compliant and stiff substrates. In summary, we show that substrate stiffness modulates the pro-inflammatory response of macrophages, that the NLRP3 inflammasome is one of the components affected by macrophage mechanosensing, and a role for actomyosin contractility in this mechanosensory response. Thus, our results contribute to a better understanding of how microenvironment stiffness affects macrophage behavior, which might be relevant in diseases where tissue stiffness is altered and might potentially provide a basis for new strategies to modulate inflammatory responses.

免疫细胞可处理海量生化信号，但其功能与行为同时亦受机械信号调控。巨噬细胞亦不例外。巨噬细胞遍布各类组织，所处微环境的刚度存在差异，且在病理状态下该刚度还会发生进一步改变。尽管越来越多证据表明巨噬细胞具有机械敏感性，但目前学界对机械信号如何调控其炎症应答仍知之甚少。本研究发现，基质刚度可影响促炎基因的表达与NLRP3炎性小体（NLRP3 inflammasome）的组装，进而改变细胞因子IL-1β与IL-6的分泌蛋白水平。本研究使用弹性模量可调节（范围0.2~33.1 kPa）的聚丙烯酰胺水凝胶，发现相较于坚硬基质，骨髓来源巨噬细胞在柔软基质上的铺展程度更低、形态更趋圆形。经脂多糖（LPS）预刺激后，在弹性模量更低的水凝胶基质上，编码TNF-α的基因表达水平更高。当使用离子载体尼日利亚菌素对巨噬细胞进行二次刺激时，我们观察到在柔软基质上，NLRP3炎性小体的组装更为显著，细胞死亡水平升高，且IL-1β与IL-6的分泌蛋白水平也更高。柔软基质上炎性小体组装的上调并非主要源于细胞铺展程度降低：与充分铺展的细胞相比，通过微图案技术对细胞进行空间束缚后，炎性小体阳性细胞的比例反而降低。最后，干扰肌动蛋白-肌球蛋白收缩能力可削弱柔软与坚硬基质间炎性小体组装的差异。综上，本研究证实基质刚度可调控巨噬细胞的促炎应答，NLRP3炎性小体是巨噬细胞机械感知效应的靶点之一，且肌动蛋白-肌球蛋白收缩能力在该机械感知过程中发挥作用。因此，本研究结果有助于进一步阐明微环境刚度对巨噬细胞行为的调控机制，这在组织刚度发生改变的疾病中具有潜在研究价值，同时也为开发调控炎症应答的新策略提供了理论基础。