i3S - Instituto de Inovação e Investigação em Saúde, Universidade do Porto, Rua Alfredo Allen, 208, 4200-125 Porto, Portugal; INEB - Instituto de Engenharia Biomédica, Rua Alfredo Allen, 208, 4200-125 Porto, Portugal; ICBAS - Instituto de Ciências Biomédicas Abel Salazar, Universidade do Porto, Rua de Jorge Viterbo Ferreira, 228, 4050-313 Porto, Portugal.
Molecular Inflammation Group, Biomedical Research Institute of Murcia (IMIB-Arrixaca), Hospital Clínico Universitario Virgen de la Arrixaca, Carretera Buenavista, 30120 Murcia, Spain.
Acta Biomater. 2019 Jun;91:123-134. doi: 10.1016/j.actbio.2019.04.035. Epub 2019 Apr 17.
Chitosan (Ch) is used in different biomedical applications to promote tissue repair. However, tissue injury caused by biomaterial implantation lead to the release of danger signals that engage different inflammatory pathways on the host. Different implanted materials activate the inflammasome leading to the modulation of the immune response. Here we have studied how macroscopic biomaterials, Ch scaffolds with different chemical composition: 4% or 15% degree of acetylation (DA) modulate the activation of the NLRP3 inflammasome in vitro. For that, we assessed the NLRP3 inflammasome in bone marrow derived mouse macrophages (BMDM) and human macrophages cultured within 3D Ch scaffolds. We found that both Ch scaffolds did not trigger the NLRP3 inflammasome activation in macrophages. Furthermore, BMDMs and human macrophages cultured in both Ch scaffolds presented a reduction in the number of apoptosis-associated speck-like protein containing a caspase activating recruitment domain (ASC) specks and in IL-1β release upon classical NLRP3 inflammasome stimulation. We also found a decrease in proIL-1β in BMDMs after priming with LPS when cultured in Ch scaffolds with DA 4% DA after priming with LPS when compared to Ch scaffolds with 15% DA or to macrophages cultured in cell-culture plates. Our results shows that 3D Ch scaffolds with different DA impair NLRP3 inflammasome priming and activation. STATEMENT OF SIGNIFICANCE: In this research work we have assessed the role of the NLRP3 inflammasome in the macrophage response to 3D chitosan scaffolds with different degrees of acetylation (DA). To our knowledge this is the first work that demonstrates the modulatory capacity of 3D porous chitosan scaffolds in the NLRP3 inflammasome activation, because our results show that Ch scaffolds impair NLRP3 inflammasome assembly in macrophages. Interestingly, our results are in contrast with studies reported in the literature that indicate that chitosan is a powerful activator of the NLRP3 inflammasome in nanoscale chitosan products. Our studies that were performed in large scale chitosan scaffolds, stress out that the process of phagocytosis is pivotal in inflammasome assembly and activation, are rather important since they clearly illustrate the different role of the inflammasome in the biological response to large scale and nanoscale biomaterials.
壳聚糖（Chitosan）被用于不同的生物医学应用中，以促进组织修复。然而，生物材料植入引起的组织损伤会导致危险信号的释放，从而激活宿主的不同炎症途径。不同的植入材料会激活炎性小体，从而调节免疫反应。在这里，我们研究了不同化学组成的壳聚糖支架（4%或 15%的乙酰化程度（DA））如何在体外调节 NLRP3 炎性小体的激活。为此，我们评估了骨髓来源的小鼠巨噬细胞（BMDM）和在 3D 壳聚糖支架中培养的人巨噬细胞中的 NLRP3 炎性小体。我们发现，两种壳聚糖支架都不会引发巨噬细胞中 NLRP3 炎性小体的激活。此外，在两种壳聚糖支架中培养的 BMDM 和人巨噬细胞在经典 NLRP3 炎性小体刺激下，凋亡相关斑点样蛋白（ASC）斑点的数量和 IL-1β 的释放减少。我们还发现，与 15% DA 的壳聚糖支架或在细胞培养板中培养的巨噬细胞相比，用 LPS 预处理后，在含有 4% DA 的壳聚糖支架中培养的 LPS 预处理的 BMDM 中的 proIL-1β 减少。我们的结果表明，具有不同 DA 的 3D 壳聚糖支架会损害 NLRP3 炎性小体的启动和激活。 意义声明：在这项研究工作中，我们评估了 NLRP3 炎性小体在具有不同乙酰化程度（DA）的 3D 壳聚糖支架中的巨噬细胞反应中的作用。据我们所知，这是第一项证明 3D 多孔壳聚糖支架在 NLRP3 炎性小体激活中具有调节能力的工作，因为我们的结果表明壳聚糖支架会损害巨噬细胞中 NLRP3 炎性小体的组装。有趣的是，我们的结果与文献中报道的壳聚糖在纳米级壳聚糖产品中是 NLRP3 炎性小体的强大激活剂的研究结果相反。我们在大规模壳聚糖支架中进行的研究强调了吞噬作用过程在炎性小体组装和激活中的关键作用，这是相当重要的，因为它们清楚地说明了炎性小体在对大规模和纳米级生物材料的生物学反应中的不同作用。
