THE HUMAN MILK OLIGOSACCHARIDES 2'-FUCOSYLLACTOSE AND 6'-SIAYLLACTOSE ATTENUATE NEC-ASSOCIATED BRAIN INJURY
Andres J. Gonzalez Salazar*1, Qinjie Zhou1, Mark L. Kovler1, Diego F. Nino2, Maame Efua Sampah1, Yukihiro Yamaguchi1, Peng Lu1, Hongpeng Jia1, William B. Fulton1, Thomas Prindle1, Sanxia Wang1, Rachael H. Buck3, Karen C. Goehring3, Chhinder P. Sodhi1, David Hackam1
1Johns Hopkins University, Baltimore, MD; 2Texas Tech University Health Sciences Center El Paso, El Paso, TX; 3Abbott Nutrition, Abbott Park, IL
Introduction: One of the most debilitating and relevant complications of necrotizing enterocolitis (NEC) is the development of long-lasting neurodevelopmental impairment. We have previously determined that human and mouse NEC leads to activation of toll-like receptor 4 (TLR4) on the hypothalamic microglia, which results in the inflammation, myelin loss, and white matter injury that underlies NEC-associated cognitive impairment. Breast milk prevents NEC and improves long-term cognition through mechanisms that remain unknown. We now hypothesize that human milk oligosaccharides (HMO) - which are the key constituents of breast milk - act as anti-inflammatory molecules to prevent NEC-associated brain injury by inhibiting TLR4 signaling.
Methods: NEC was induced in 7-day-old C57BL/6 mouse pups through four days of gavage-fed formula mixed with NEC bacteria, and periods of brief hypoxia. The brain was examined histologically and by qRT-PCR in the presence of the key HMOs: 2-fucosyllactose (2'-FL) and 6-siallylactose (6'-SL) and was then compared to breastfed (BF) and NEC formula-fed (FF) mice. To further study the association between HMOs and TLR4 signaling on the developing brain, organoids were cultured from embryonic brains (e14.5-16.5) and were examined for injury in the presence of LPS, a potent TLR4 agonist, and LPS with HMOs.
Results: Oral administration of 2'-FL and 6'-SL significantly reduced the extent of brain inflammation in mice in a dose-dependent manner, as revealed by reduced Tnfa expression (BF:0.4'±0.05, FF:1'±0.1, FF+2'-FL:0.5'±0.1, FF+6'-SL:0.5'±0.05, p<0.001) and decreased apoptosis by PUMA expression (BF:44'±3, FF:69'±5, FF+2'-FL:46'±4, FF+6'-SL:44'±4, p<0.001) in the periventricular regions. Strikingly, 2'-FL and 6'-SL reduced TLR4 signaling in embryonic cultured brain organoids, as manifest by the significant reduction of ROS generation by dihydroethidium staining, reduced expression of Il6 (LPS:200'±25, LPS+2'-FL+6'-SL:10'±2, p<0.001) and Tnfa (LPS:1,000'±500, LPS+2'-FL+6'-SL:200'±20, p<0.001), and reduced apoptosis by TUNEL staining, consistent with the cerebral protection seen in vivo.
Conclusions: These findings reveal that the HMOs 2'-FL and 6'-SL can protect the developing brain via inhibition of TLR4 signaling shedding light on the mechanisms behind the benefits of breast milk and paves the way for novel neuroprotective strategies for patients with NEC.
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