Macrophages respond to chemical/metabolic and physical stimuli, but their effects cannot be readily decoupled in vivo during pro-inflammatory activation. Here, we show that preventing macrophage spreading by spatial confinement, as imposed by micropatterning, microporous substrates or cell crowding, suppresses late lipopolysaccharide (LPS)-activated transcriptional programs (biomarkers IL-6, CXCL9, IL-1β, and iNOS) by mechanomodulating chromatin compaction and epigenetic alterations (HDAC3 levels and H3K36-dimethylation). Mechanistically, confinement reduces actin polymerization, thereby lowers the LPS-stimulated nuclear translocation of MRTF-A. This lowers the activity of the MRTF-A–SRF complex and subsequently downregulates the inflammatory response, as confirmed by chromatin immunoprecipitation coupled with quantitative PCR and RNA sequencing analysis. Confinement thus downregulates pro-inflammatory cytokine secretion and, well before any activation processes, the phagocytic potential of macrophages. Contrarily, early events, including activation of the LPS receptor TLR4, and downstream NF-κB and IRF3 signalling and hence the expression of early LPS-responsive genes were marginally affected by confinement. These findings have broad implications in the context of mechanobiology, inflammation and immunology, as well as in tissue engineering and regenerative medicine.

巨噬细胞对化学/代谢和物理刺激有反应，但在促炎性激活过程中，它们的作用在体内不易分离。在这里，我们显示出通过微图案化，微孔底物或细胞拥挤施加的空间限制来防止巨噬细胞扩散，可以抑制脂多糖（LPS）激活的转录程序（生物标记IL-6，CXCL9，IL-1β和iNOS）），通过机械调节染色质的紧实度和表观遗传学改变（HDAC3水平和H3K36-二甲基化）。从机理上讲，限制作用降低了肌动蛋白的聚合反应，从而降低了LPTF刺激的MRTF-A的核易位。染色质免疫沉淀结合定量PCR和RNA测序分析证实，这会降低MRTF-A-SRF复合物的活性并随后下调炎症反应。因此，限制作用下调了促炎性细胞因子的分泌，并在任何激活过程之前很远地下调了巨噬细胞的吞噬潜能。相反，早期事件，包括LPS受体TLR4的激活，下游NF-κB和IRF3信号传导以及早期LPS反应基因的表达受到限制的影响。