Macrophages are innate immune cells that interact with complex extracellular matrix environments, which have varied stiffness, composition, and structure, and such interactions can lead to the modulation of cellular activity. Collagen is often used in the culture of immune cells, but the effects of substrate functionalization conditions are not typically considered. Here, we show that the solvent system used to attach collagen onto a hydrogel surface affects its surface distribution and organization, and this can modulate the responses of macrophages subsequently cultured on these surfaces in terms of their inflammatory activation and expression of adhesion and mechanosensitive molecules. Collagen was solubilized in either acetic acid (Col-AA) or N-(2-hydroxyethyl)piperazine-N′-ethanesulfonic acid (HEPES) (Col-HEP) solutions and conjugated onto soft and stiff polyacrylamide (PA) hydrogel surfaces. Bone marrow-derived macrophages cultured under standard conditions (pH 7.4) on the Col-HEP-derived surfaces exhibited stiffness-dependent inflammatory activation; in contrast, the macrophages cultured on Col-AA-derived surfaces expressed high levels of inflammatory cytokines and genes, irrespective of the hydrogel stiffness. Among the collagen receptors that were examined, leukocyte-associated immunoglobulin-like receptor-1 (LAIR-1) was the most highly expressed, and knockdown of the Lair-1 gene enhanced the secretion of inflammatory cytokines. We found that the collagen distribution was more homogeneous on Col-AA surfaces but formed aggregates on Col-HEP surfaces. The macrophages cultured on Col-AA PA hydrogels were more evenly spread, expressed higher levels of vinculin, and exerted higher traction forces compared to those of cells on Col-HEP. These macrophages on Col-AA also had higher nuclear-to-cytoplasmic ratios of yes-associated protein (YAP) and transcriptional co-activator with PDZ-binding motif (TAZ), key molecules that control inflammation and sense substrate stiffness. Our results highlight that seemingly slight variations in substrate deposition for immunobiology studies can alter critical immune responses, and this is important to elucidate in the broader context of immunomodulatory biomaterial design.

中文翻译：

---

通过胶原沉积调节硬度依赖性巨噬细胞炎症反应

巨噬细胞是先天免疫细胞，与复杂的细胞外基质环境相互作用，这些环境具有不同的硬度、组成和结构，这种相互作用可以导致细胞活性的调节。胶原蛋白经常用于免疫细胞的培养，但通常不考虑底物功能化条件的影响。在这里，我们表明，用于将胶原蛋白附着到水凝胶表面的溶剂系统会影响其表面分布和组织，这可以调节随后在这些表面上培养的巨噬细胞在炎症激活以及粘附和机械敏感分子表达方面的反应。将胶原蛋白溶解在乙酸 (Col-AA) 或N- (2-羟乙基)哌嗪-N'-乙磺酸 (HEPES) (Col-HEP) 溶液中，并缀合到柔软和坚硬的聚丙烯酰胺 (PA) 水凝胶表面。在标准条件（pH 7.4）下在 Col-HEP 衍生表面上培养的骨髓衍生巨噬细胞表现出硬度依赖性炎症激活；相比之下，在 Col-AA 衍生表面上培养的巨噬细胞表达高水平的炎症细胞因子和基因，无论水凝胶硬度如何。在检测的胶原蛋白受体中，白细胞相关免疫球蛋白样受体-1 (LAIR-1) 的表达量最高，La​​ir-1基因的敲除增强了炎症细胞因子的分泌。我们发现胶原蛋白在 Col-AA 表面上分布更均匀，但在 Col-HEP 表面上形成聚集体。与 Col-HEP 上的细胞相比，在 Col-AA PA 水凝胶上培养的巨噬细胞分布更均匀，表达更高水平的纽蛋白，并施加更高的牵引力。 Col-AA 上的这些巨噬细胞还具有较高的 yes 相关蛋白 (YAP) 和具有 PDZ 结合基序 (TAZ) 的转录共激活因子（TAZ）的核质比，这是控制炎症和感知底物硬度的关键分子。我们的结果强调，免疫生物学研究中底物沉积的看似微小的变化可以改变关键的免疫反应，这对于在免疫调节生物材料设计的更广泛背景下阐明这一点非常重要。