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Abstract：
Developing a noninvasive imaging method to detect immune system activation with a high temporal resolution is key to improving inflammatory bowel disease (IBD) management. In this study, granzyme B (GZMB), typically released from cytotoxic T and natural killer cells, was targeted using PET with 68Ga-NOTA-GZP (where GZP is β-Ala-Gly-Gly-Ile-Glu-Phe-Asp-CHO) to detect early intestinal inflammation in murine models of colitis. Methods: Bioinformatic analysis was used to assess the potential of GZMB as a biomarker for detecting IBD and predicting response to treatment. Human active and quiescent Crohn disease and ulcerative colitis tissues were stained for GZMB. We used IL-10-/- mice treated with dextran sulfate sodium (DSS) as an IBD model, wild-type C57BL/6J mice as a control, and anti-tumor necrosis factor as therapy. We used a murine GZMB-binding peptide conjugated to a NOTA chelator (NOTA-GZP) labeled with 68Ga as the PET tracer. PET imaging was conducted at 1, 3, and 4 wk after colitis induction to evaluate temporal changes. Results: Bioinformatic analysis showed that GZMB gene expression is significantly upregulated in human ulcerative colitis and Crohn disease compared with the noninflamed bowel by 2.98-fold and 1.92-fold, respectively; its expression is lower by 2.16-fold in treatment responders than in nonresponders. Immunofluorescence staining of human tissues demonstrated a significantly higher GZMB in patients with active than with quiescent IBD (P = 0.032).68Ga-NOTA-GZP PET imaging showed significantly increased bowel uptake in IL-10-/- mice with DSS-induced colitis compared with vehicle-treated IL-10-/- mice (SUVmean, 0.75 vs. 0.24; P < 0.001) and both vehicle- and DSS-treated wild-type mice (SUVmean, 0.26 and 0.37; P < 0.001). In the IL-10-/- DSS-induced colitis model, the bowel PET probe uptake decreased in response to treatment with tumor necrosis factor-α (SUVmean, 0.32; P < 0.001). There was a 4-fold increase in colonic uptake of 68Ga-NOTA-GZP in the colitis model compared with the control 1 wk after colitis induction. The uptake gradually decreased to approximately 2-fold by 4 wk after IBD induction; however, the inflamed bowel uptake remained significantly higher than control at all time points (week 4 SUVmean, 0.23 vs. 0.08; P = 0.001). Conclusion: GZMB is a promising biomarker to detect active IBD and predict response to treatment. This study provides compelling evidence to translate GZMB PET for imaging IBD activity in clinical settings.
摘要：
开发一种具有高时间分辨率的非侵入性成像方法来检测免疫系统激活是改善炎症性肠病（IBD）管理的关键。在这项研究中,颗粒酶B（GZMB），通常从细胞毒性T细胞和自然杀伤细胞释放，使用具有68Ga-NOTA-GZP(其中GZP是β-Ala-Gly-Gly-Ile-Glu-Phe-Asp-CHO)的PET靶向,以检测结肠炎小鼠模型中的早期肠道炎症。方法：生物信息学分析用于评估GZMB作为检测IBD和预测治疗反应的生物标志物的潜力。将人活动性和静止性克罗恩病和溃疡性结肠炎组织进行GZMB染色。我们使用用葡聚糖硫酸钠（DSS）处理的IL-10-/-小鼠作为IBD模型，野生型C57BL/6J小鼠作为对照,和抗肿瘤坏死因子作为治疗。我们使用与用68Ga标记的NOTA螯合剂(NOTA-GZP)缀合的鼠GZMB结合肽作为PET示踪剂。在结肠炎诱导后1、3和4周进行PET成像以评估时间变化。结果：生物信息学分析表明，与非发炎的肠道相比，人类溃疡性结肠炎和克罗恩病的GZMB基因表达显着上调了2.98倍和1.92倍。分别；其表达在治疗应答者中比在无应答者中低2.16倍。人体组织的免疫荧光染色显示，活动性IBD患者的GZMB显着高于静态IBD患者（P=0.032）。68Ga-NOTA-GZPPET成像显示，患有DSS的IL-10-/-小鼠的肠摄取显着增加与载体治疗的IL-10-/-小鼠相比（SUVmean，0.75vs.0.24；P<0.001）和赋形剂和DSS处理的野生型小鼠（SUVmean，0.26和0.37；P<0.001）。在IL-10-/-DSS诱导的结肠炎模型中,对用肿瘤坏死因子-α治疗的肠PET探针摄取降低(SUVmean，0.32；P<0.001）。与结肠炎诱导后1周的对照相比,结肠炎模型中68Ga-NOTA-GZP的结肠摄取增加4倍。IBD诱导后4周，摄取逐渐降低至约2倍；然而，在所有时间点，发炎的肠摄取均显着高于对照组（第4周SUVmean，0.23vs.0.08；P=0.001）。结论：GZMB是检测活动性IBD和预测治疗反应的有前途的生物标志物。这项研究提供了令人信服的证据来翻译GZMBPET以在临床环境中对IBD活动进行成像。
