AKIRIN1 is a suitable reference gene in natural killer cells and granulocytes from patients with SIRS and septic shock [granulocytes]

Timely and reliable distinction of non-infectious systemic inflammatory response syndrome (SIRS), common in critically ill patients, from sepsis to support adequate antimicrobial therapy safes lives but is clinically challenging. Expeditious sepsis biomarkers are thus urgently sought. Blood transcriptional profiling provides insights into sepsis pathophysiology, but variability in leukocyte subtype composition complicates profile interpretation, and reliable reference genes to normalize gene expression in sepsis are lacking. Here, we identified AKIRIN1 as a reference gene, specifically, in peripheral NK cells and granulocytes for differential gene expression analysis between patients with SIRS and septic shock on intensive care unit admission. Discovery by a two-step probabilistic selection from microarray data followed by validation through branched DNA assays in independent patients revealed several candidate reference genes in NK cells, namely, AKIRIN1, PPP6R3, TAX1BP1, and ADRBK1. For in vitro priming of NK cells, GUSB however was confirmed as reference gene of choice. Initially, no candidate genes could be validated in granulocytes, an additional rescreen of known reference genes by RT-PCR included. By serendipity, we could determine equal AKIRIN1 expression levels also in SIRS and septic shock granulocytes and no change by in vitro challenge of granulocytes with LPS. Inspection of four external neutrophil transcriptome datasets further support unchanged AKIRIN1 expression in human systemic inflammation. Invariable AKIRIN1 expression in peripheral NK cells and granulocytes needs further validation in sepsis and other infectious and inflammatory diseases. As a reference gene in these cells and conditions, AKIRIN1 may further our understanding of innate immunity and lead to new biomarkers. We enrolled critically ill patients and collected blood on admission to our surgical intensive care unit if, within the preceding 24 hours, they had a diagnosis of post-traumatic systemic inflammatory response syndrome (SIRS) or septic shock. Hospitalized noncritically ill control patients were recruited on presurgical examination. To transcriptionally characterize the innate immune response to sterile (SIRS) and infectious (septic shock) systemic inflammation, granulocytes were isolated from patient blood for RNA extraction and hybridization on Affymetrix microarrays.

针对重症患者中高发的非感染性全身炎症反应综合征（SIRS）与脓毒症的及时精准鉴别，可通过合理的抗菌治疗挽救患者生命，但该临床鉴别极具挑战性。因此，亟需快速可靠的脓毒症生物标志物。血液转录组分析可为脓毒症病理生理学研究提供重要见解，但白细胞亚型组成的异质性会增加转录组结果解读的难度，且目前尚无适用于脓毒症基因表达标准化分析的可靠内参基因。本研究鉴定出AKIRIN1可作为内参基因，尤其适用于外周血自然杀伤细胞（NK细胞）与粒细胞，用于重症监护病房（ICU）入院时SIRS患者与脓毒性休克患者的差异基因表达分析。研究通过两步概率筛选法从微阵列数据中发掘候选基因，并通过支链DNA检测在独立队列患者中验证，最终在NK细胞中鉴定出AKIRIN1、PPP6R3、TAX1BP1与ADRBK1共4个候选内参基因。针对NK细胞的体外预刺激实验则证实，GUSB为该场景下的最优内参基因。最初，本研究未能在粒细胞中验证任何候选内参基因，后续通过逆转录聚合酶链反应（RT-PCR）对已知内参基因开展了额外的重新筛选。意外的是，我们发现SIRS与脓毒性休克患者的粒细胞中AKIRIN1表达水平无显著差异，且脂多糖（LPS）体外刺激粒细胞并不会改变AKIRIN1的表达水平。对4项公开中性粒细胞转录组数据集的分析进一步证实，在人类全身炎症反应中AKIRIN1表达保持稳定。外周血NK细胞与粒细胞中AKIRIN1表达稳定这一结论，仍需在脓毒症及其他感染性与炎症性疾病中进一步验证。作为上述细胞与临床场景下的内参基因，AKIRIN1可助力我们深入解析先天免疫机制，并有望助力新型生物标志物的开发。本研究纳入重症患者，若其在入院前24小时内被诊断为创伤后全身炎症反应综合征（SIRS）或脓毒性休克，则在其入住外科重症监护病房（SICU）入院时采集血液样本。同时招募术前检查的非重症住院患者作为对照。为从转录层面解析无菌性（SIRS）与感染性（脓毒性休克）全身炎症的先天免疫应答特征，本研究从患者血液中分离粒细胞，用于RNA提取及Affymetrix微阵列芯片杂交实验。