Department of Medicine, Division of Allergy, Pulmonary and Critical Care Medicine, The University of Wisconsin-Madison School of Medicine and Public Health, K4/928 Clinical Science Center MC 9988, 600 Highland Avenue, Madison, WI, 53792, USA.
Department of Pediatrics, University of Wisconsin School of Medicine and Public Health, Madison, WI, 53792, USA.
Respir Res. 2017 Nov 10;18(1):188. doi: 10.1186/s12931-017-0669-8.
The association of eosinophils with inflammation and tissue remodeling is at least partially due to their release of toxic granule proteins and other mediators, including cytokines. Tissue remodeling and consequent functional defects are affected by activity of connective tissue fibroblasts. Exaggerated fibroblast activation, accumulation and change of phenotype may lead to fibrosis and loss of tissue function. So far, little information has been reported on how eosinophils affect inflammation and tissue remodeling via the activation of fibroblasts. We have recently shown that eosinophil activation with IL-3 led to a robust eosinophil degranulation on immunoglobin-G (IgG) coated plates. Thus, in the present study, we analyze the effects of IL-3-activated eosinophil degranulation products on primary human lung fibroblasts (HLF) using whole transcriptome sequencing.
Conditioned media was obtained from eosinophils that were pre-activated with IL-3 or IL-5 and subsequently cultured for 6 h on IgG to induce degranulation. This conditioned media was added on human lung fibroblasts (HLF) for 24 h and the cell lysates were then subjected to whole transcriptome sequencing to identify global changes in gene expression. Differentially expressed genes were analyzed using the Ingenuity Pathway Analysis (IPA), and validated by qPCR.
In HLF, the expression level of 300 genes was changed by conditioned media from IL-3-activated eosinophils compared to control fibroblast cultures. Among these 300 genes, the expression level of 35 genes coding for known proteins was upregulated by IL-3- versus IL-5-pre-activated eosinophils. Of the 35 upregulated genes, IPA identified C3, CH25H, CXCL1, CXCL8, CYP1A1, ICAM1, IL6 and UCN2 as having downstream functions on inflammation, tissue remodeling and lipid synthesis. This analysis combined with previous RNA sequencing analyses of eosinophils suggest IL-1ß, OSM and TNFSF12 as potential upstream regulators of fibroblasts.
This study has identified several novel pro-inflammatory and pro-remodeling mediators produced by fibroblasts in response to activated eosinophils. These findings may have significant implications on the role of eosinophil/fibroblast interactions in eosinophilic disorders.
嗜酸性粒细胞与炎症和组织重塑的关联至少部分归因于其释放的毒性颗粒蛋白和其他介质，包括细胞因子。组织重塑和随之而来的功能缺陷受结缔组织成纤维细胞的活性影响。成纤维细胞的过度激活、积累和表型改变可能导致纤维化和组织功能丧失。到目前为止，关于嗜酸性粒细胞如何通过激活成纤维细胞影响炎症和组织重塑的信息还很少。我们最近表明，IL-3 激活嗜酸性粒细胞导致在免疫球蛋白-G（IgG）包被的平板上嗜酸性粒细胞强烈脱颗粒。因此，在本研究中，我们使用全转录组测序分析 IL-3 激活的嗜酸性粒细胞脱颗粒产物对原代人肺成纤维细胞（HLF）的影响。
用 IL-3 或 IL-5 预先激活嗜酸性粒细胞，然后在 IgG 上培养 6 小时诱导脱颗粒，获得条件培养基。将这种条件培养基添加到人肺成纤维细胞（HLF）中 24 小时，然后对细胞裂解物进行全转录组测序，以确定基因表达的全局变化。差异表达基因使用 IPA 进行分析，并通过 qPCR 进行验证。
与对照成纤维细胞培养物相比，IL-3 激活的嗜酸性粒细胞的条件培养基使 HLF 中 300 个基因的表达水平发生变化。在这 300 个基因中，35 个编码已知蛋白的基因的表达水平被 IL-3-而非 IL-5-预激活的嗜酸性粒细胞上调。在这 35 个上调基因中，IPA 将 C3、CH25H、CXCL1、CXCL8、CYP1A1、ICAM1、IL6 和 UCN2 确定为具有炎症、组织重塑和脂质合成下游功能的基因。这项分析结合以前对嗜酸性粒细胞的 RNA 测序分析表明，IL-1β、OSM 和 TNFSF12 可能是成纤维细胞的潜在上游调节因子。
本研究鉴定了几种由激活的嗜酸性粒细胞产生的新型促炎和促重塑介质，这些发现可能对嗜酸性粒细胞/成纤维细胞相互作用在嗜酸性粒细胞疾病中的作用具有重要意义。
