Curriculum in Toxicology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States of America.
Division of Pharmacotherapy and Experimental Therapeutics, UNC Eshelman School of Pharmacy, Chapel Hill, North Carolina, United States of America.
PLoS One. 2019 Jan 2;14(1):e0208958. doi: 10.1371/journal.pone.0208958. eCollection 2019.
Hepatic fibrosis develops from a series of complex interactions among resident and recruited cells making it a challenge to replicate using standard in vitro approaches. While studies have demonstrated the importance of macrophages in fibrogenesis, the role of Kupffer cells (KCs) in modulating the initial response remains elusive. Previous work demonstrated utility of 3D bioprinted liver to recapitulate basic fibrogenic features following treatment with fibrosis-associated agents. In the present study, culture conditions were modified to recapitulate a gradual accumulation of collagen within the tissues over an extended exposure timeframe. Under these conditions, KCs were added to the model to examine their impact on the injury/fibrogenic response following cytokine and drug stimuli. A 28-day exposure to 10 ng/mL TGF-β1 and 0.209 μM methotrexate (MTX) resulted in sustained LDH release which was attenuated when KCs were incorporated in the model. Assessment of miR-122 confirmed early hepatocyte injury in response to TGF-β1 that appeared delayed in the presence of KCs, whereas MTX-induced increases in miR-122 were observed when KCs were incorporated in the model. Although the collagen responses were mild under the conditions tested to mimic early fibrotic injury, a global reduction in cytokines was observed in the KC-modified tissue model following treatment. Furthermore, gene expression profiling suggests KCs have a significant impact on baseline tissue function over time and an important modulatory role dependent on the context of injury. Although the number of differentially expressed genes across treatments was comparable, pathway enrichment suggests distinct, KC- and time-dependent changes in the transcriptome for each agent. As such, the incorporation of KCs and impact on baseline tissue homeostasis may be important in recapitulating temporal dynamics of the fibrogenic response to different agents.
肝纤维化是由一系列复杂的细胞间相互作用引起的，这使得使用标准的体外方法来复制它成为一个挑战。虽然研究已经证明了巨噬细胞在纤维化形成中的重要性，但库普弗细胞（KCs）在调节初始反应中的作用仍然难以捉摸。以前的工作表明，3D 生物打印肝脏在使用纤维化相关试剂处理后，具有重现基本纤维化特征的用途。在本研究中，修改了培养条件，以在延长的暴露时间内重现组织内胶原的逐渐积累。在这些条件下，向模型中添加了 KCs，以研究它们在细胞因子和药物刺激后对损伤/纤维化反应的影响。在 10ng/mL TGF-β1 和 0.209μM 甲氨蝶呤（MTX）的 28 天暴露下，导致持续的 LDH 释放，当 KCs 被纳入模型时，这种释放被减弱。miR-122 的评估证实了 TGF-β1 引起的早期肝细胞损伤，而在 KCs 存在的情况下，这种损伤似乎延迟，而当 KCs 被纳入模型时，MTX 诱导的 miR-122 增加。虽然在模拟早期纤维化损伤的条件下，胶原反应很轻微，但在 KC 修饰的组织模型中，治疗后观察到细胞因子的总体减少。此外，基因表达谱表明，KCs 随着时间的推移对组织的基线功能有显著影响，并且在损伤的背景下具有重要的调节作用。尽管在不同处理中差异表达基因的数量是可比的，但途径富集表明，每种试剂的转录组中都存在 KC 和时间依赖性的明显变化。因此，KCs 的纳入及其对基线组织稳态的影响可能在不同试剂对纤维化反应的时间动态的重现中很重要。
