Halpern Melissa D, Gupta Akash, Zaghloul Nahla, Thulasingam Senthilkumar, Calton Christine M, Camp Sara M, Garcia Joe G N, Ahmed Mohamed
Division of Neonatology, Department of Pediatrics, College of Medicine, University of Arizona, Tucson, AZ 85724, USA.
Center for Inflammation Science and Systems Medicine, University of Florida Scripps Research Institute, Jupiter, FL 33458, USA.
Biomedicines. 2024 Apr 28;12(5):970. doi: 10.3390/biomedicines12050970.
Necrotizing enterocolitis (NEC) is the most common gastrointestinal emergency of prematurity. Postulated mechanisms leading to inflammatory necrosis of the ileum and colon include activation of the pathogen recognition receptor Toll-like receptor 4 (TLR4) and decreased levels of transforming growth factor beta (TGFβ). Extracellular nicotinamide phosphoribosyltransferase (eNAMPT), a novel damage-associated molecular pattern (DAMP), is a TLR4 ligand and plays a role in a number of inflammatory disease processes. To test the hypothesis that eNAMPT is involved in NEC, an eNAMPT-neutralizing monoclonal antibody, ALT-100, was used in a well-established animal model of NEC. Preterm Sprague-Dawley pups delivered prematurely from timed-pregnant dams were exposed to hypoxia/hypothermia and randomized to control-foster mother dam-fed rats, injected IP with saline (vehicle) 48 h after delivery; control + mAB-foster dam-fed rats, injected IP with 10 µg of ALT-100 at 48 h post-delivery; NEC-orally gavaged, formula-fed rats injected with saline; and NEC + mAb-formula-fed rats, injected IP with 10 µg of ALT-100 at 48 h. The distal ileum was processed 96 h after C-section delivery for histological, biochemical, molecular, and RNA sequencing studies. Saline-treated NEC pups exhibited markedly increased fecal blood and histologic ileal damage compared to controls ( < 0.0001), and findings significantly reduced in ALT-100 mAb-treated NEC pups ( < 0.01). Real-time PCR in ileal tissues revealed increased NAMPT in NEC pups compared to pups that received the ALT-100 mAb ( < 0.01). Elevated serum levels of tumor necrosis factor alpha (TNFα), interleukin 6 (IL-6), interleukin-8 (IL-8), and NAMPT were observed in NEC pups compared to NEC + mAb pups ( < 0.01). Finally, RNA-Seq confirmed dysregulated TGFβ and TLR4 signaling pathways in NEC pups that were attenuated by ALT-100 mAb treatment. These data strongly support the involvement of eNAMPT in NEC pathobiology and eNAMPT neutralization as a strategy to address the unmet need for NEC therapeutics.
坏死性小肠结肠炎（NEC）是早产最常见的胃肠道急症。导致回肠和结肠炎症性坏死的假定机制包括病原体识别受体Toll样受体4（TLR4）的激活以及转化生长因子β（TGFβ）水平的降低。细胞外烟酰胺磷酸核糖转移酶（eNAMPT）是一种新型的损伤相关分子模式（DAMP），是一种TLR4配体，在许多炎症性疾病过程中起作用。为了验证eNAMPT参与NEC的假说，在一个成熟的NEC动物模型中使用了一种eNAMPT中和单克隆抗体ALT-100。从定时怀孕的母鼠早产的早产斯普拉格-道利幼崽暴露于缺氧/低温环境，并随机分为对照组——由代孕母鼠喂养的大鼠，在出生后48小时腹腔注射生理盐水（载体）；对照组+单克隆抗体——由代孕母鼠喂养的大鼠，在出生后48小时腹腔注射10μg ALT-100；NEC组——经口灌喂、配方奶喂养并注射生理盐水的大鼠；以及NEC+单克隆抗体组——经口灌喂、配方奶喂养并在出生后48小时腹腔注射10μg ALT-100的大鼠。在剖宫产分娩96小时后对远端回肠进行处理，用于组织学、生化、分子和RNA测序研究。与对照组相比（<0.0001），生理盐水处理的NEC幼崽粪便潜血和回肠组织学损伤明显增加，而在ALT-100单克隆抗体处理的NEC幼崽中这些发现显著减少（<0.01）。回肠组织的实时PCR显示，与接受ALT-100单克隆抗体的幼崽相比，NEC幼崽的NAMPT增加（<0.01）。与NEC+单克隆抗体组幼崽相比，NEC组幼崽血清肿瘤坏死因子α（TNFα）、白细胞介素6（IL-6）、白细胞介素-8（IL-8）和NAMPT水平升高（<0.01）。最后，RNA测序证实NEC组幼崽中TGFβ和TLR4信号通路失调，而ALT-100单克隆抗体治疗可使其减弱。这些数据有力地支持了eNAMPT参与NEC病理生物学过程以及中和eNAMPT作为满足NEC治疗未满足需求的一种策略。
