Purpose: Proteases play an essential role in the pathophysiology of inflammatory bowel disease (IBD), contributing to the intestinal mucosal lesions through the degradation of the extracellular matrix and alteration of the barrier function. Ulcerative colitis (UC) is characterized by an extensive infiltrate of neutrophils into the mucosa and hence, increased proteolytic activity. Human neutrophil elastase (HNE) is a serine protease that has been reported to be increased in UC patients’ intestinal mucosa. Based on our previous studies, we hypothesized that HNE might induce proteolytic degradation and loss of function of therapeutic monoclonal antibodies in IBD patients.
Patients and Methods: Elastase expression and elastinolytic activity were determined in mucosal explants from ulcerative colitis patients (n=6) and cultured ex vivo in the presence or absence of recombinant elafin. Enzymatic digestions of therapeutic monoclonal antibodies were performed using recombinant HNE and elafin. The integrity of the therapeutic antibodies was evaluated by immunoblotting and protein G binding assay, whereas their TNF-neutralizing activity was assessed with a reporter cell line.
Results: We found that HNE and its elastinolytic activity were increased in the gut mucosa of UC patients. We also demonstrated that HNE cleaved biological drugs, impairing the TNF-α neutralizing capacity of anti-TNF monoclonal antibodies. This proteolytic degradation was inhibited by the addition of the specific inhibitor, elafin.
Conclusion: Our results suggest that the high level of proteolytic degradation by mucosal neutrophil elastase, along with a potential imbalance with elafin, contributes to the loss of function of biologic agents, which are currently used in patients with IBD. These findings might explain the non-responsiveness of UC patients to therapeutic monoclonal antibodies and suggest the potential beneficial concomitant use of elafin in this treatment.

Keywords: elastinolytic activity, elafin, anti-TNF, inflammatory bowel disease, biological drugs


中文翻译：

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溃疡性结肠炎中的人中性粒细胞弹性蛋白酶蛋白水解活性有利于治疗性单克隆抗体的功能丧失。

目的：蛋白酶在炎症性肠病 (IBD) 的病理生理学中发挥重要作用，通过细胞外基质的降解和屏障功能的改变导致肠黏膜损伤。溃疡性结肠炎 (UC) 的特征是中性粒细胞广泛浸润到粘膜中，因此蛋白水解活性增加。人中性粒细胞弹性蛋白酶 (HNE) 是一种丝氨酸蛋白酶，据报道其在 UC 患者的肠粘膜中升高。基于我们之前的研究，我们假设 HNE 可能会诱导 IBD 患者中治疗性单克隆抗体的蛋白水解降解和功能丧失。
患者和方法：在来自溃疡性结肠炎患者（n = 6）的粘膜外植体中测定弹性蛋白酶表达和弹性蛋白溶解活性，并在存在或不存在重组弹性蛋白的情况下离体培养。使用重组 HNE 和弹性蛋白对治疗性单克隆抗体进行酶消化。通过免疫印迹和蛋白 G 结合测定评估治疗性抗体的完整性，而用报告细胞系评估其 TNF 中和活性。
结果：我们发现 HNE 及其弹性蛋白溶解活性在 UC 患者的肠粘膜中增加。我们还证明了 HNE 切割生物药物，削弱了抗 TNF 单克隆抗体的 TNF-α 中和能力。通过添加特异性抑制剂弹性蛋白来抑制这种蛋白水解降解。
结论：我们的研究结果表明，黏膜中性粒细胞弹性蛋白酶的高水平蛋白水解降解，以及与弹性蛋白的潜在失衡，导致目前用于 IBD 患者的生物制剂的功能丧失。这些发现可能解释了 UC 患者对治疗性单克隆抗体的无反应性，并表明在这种治疗中潜在有益的伴随使用 elafin。

关键词：弹性蛋白溶解活性，elafin，抗TNF，炎症性肠病，生物药物