Tissue Eosinophilia is Effective for Studying Differential Expression of Genes by RNA sequencing, and may offer Superior Insights into Chronic Rhinosinusitis than Study by Polyp Status

RNA sequencing (RNAseq) is being used to study inflammatory pathways in chronic rhinosinusitis (CRS). Our goal was to probe validity of tissue eosinophilia as a metric to study RNAseq in CRS. The study was conducted on prospectively enrolled subjects undergoing sinonasal surgery. Subjects were categorized as control, CRS, CRS with nasal polyps (CRSwNP) and CRS without nasal polyps (CRSsNP). CRS was also categorized by tissue eosinophil levels per high power field (EOS/HPF) as <10 EOS/HPF or ≥10 EOS/HPF. Ethmoidal tissue samples were processed, differentially expressed (DE) genes were calculated and Ingenuity pathway analysis (IPA) performed.Controls separated clearly from CRS by both study criteria (polyp status, EOS/HPF). In both analyses, CRS differentiated into two distinct CRS subgroups. However, heatmaps showed greater homogeneity within each CRS subtype when studied by eosinophilia versus polyp status. Overall, high differential gene expression was found in CRS versus controls, with 736 statistically significant differentially expressed (DE) genes. On comparison between between CRSwNP and CRSsNP, 60 DE genes were found and on analyzing by tissue EOS/HPF, 110DE genes were statistically significant between CRS <10 EOS/HPF and CRS ≥10 EOS/HPF analyses. Tissue eosinophilia as a metric was further validated by finding of IL 17 signalling pathway between <10EOS/HPF (increased) versus ≥10 EOS/HPF samples on pathway analysis. As a metric, tissue eosinophilia is at least as effective as analysis by polyp status for RNA sequencing and may potentially offer superior insights into mechanistic pathways. 20 samples; 4 control, 16 CRS

RNA测序（RNA sequencing, RNAseq）目前被应用于慢性鼻-鼻窦炎（chronic rhinosinusitis, CRS）的炎症通路研究。本研究旨在探讨以组织嗜酸性粒细胞增多（tissue eosinophilia）作为指标，开展慢性鼻-鼻窦炎RNAseq研究的有效性。
研究对象为接受鼻鼻窦手术的前瞻性入组受试者，我们将其分为对照组、慢性鼻-鼻窦炎组、伴鼻息肉慢性鼻-鼻窦炎（CRSwNP）组以及不伴鼻息肉慢性鼻-鼻窦炎（CRSsNP）组。此外，依据每高倍视野（high power field, HPF）的组织嗜酸性粒细胞计数（EOS/HPF），将慢性鼻-鼻窦炎组进一步划分为<10 EOS/HPF组与≥10 EOS/HPF组。
对筛窦组织样本进行处理，计算差异表达（differentially expressed, DE）基因，并开展Ingenuity通路分析（Ingenuity pathway analysis, IPA）。
通过两项研究标准（息肉状态、EOS/HPF计数）均可清晰区分对照组与慢性鼻-鼻窦炎组。在两项分析中，慢性鼻-鼻窦炎组均分化为两个独立的亚组。不过相较于按息肉状态分组，按嗜酸性粒细胞增多情况分组时，热图显示各慢性鼻-鼻窦炎亚型内部的同质性更高。
整体而言，慢性鼻-鼻窦炎组与对照组相比存在显著的差异基因表达，共筛选得到736个具有统计学意义的差异表达基因。对比CRSwNP组与CRSsNP组，共发现60个差异表达基因；而按组织EOS/HPF计数分组时，<10 EOS/HPF组与≥10 EOS/HPF组之间存在110个具有统计学意义的差异表达基因。
通路分析显示，<10 EOS/HPF组中IL-17信号通路表达上调，这进一步验证了组织嗜酸性粒细胞增多作为指标的有效性。
综上，以组织嗜酸性粒细胞增多作为指标，其有效性至少等同于按息肉状态分组的分析方法，且可能为慢性鼻-鼻窦炎的机制通路研究提供更优质的视角。本研究共纳入20例样本，其中对照组4例，慢性鼻-鼻窦炎组16例。