Human Breast Milk-Derived Exosomes Attenuate Lipopolysaccharide-Induced Activation in Microglia

Microglia mediate the immune response to many insults in the central nervous system, including lipopolysaccharide (LPS), a bacterial endotoxin that initiates an exaggerated neuroinflammatory response in neonates, especially preterm infants. Exosomes, nano-sized vesicles (30–200 nm) involved in intercellular communication, are implicated in many pathophysiological processes through the effects of their highly regulated cargo. Human breast milk, which is rich in exosomes, plays a vital role in neonatal immune system maturation and adaptation. Activated microglia may cause brain-associated injuries or disorders, especially in the hyperinflammatory response that commonly occurs in the immature neonatal immune system. Therefore, safe and effective options are vital and urgently needed for this susceptible population. We hypothesize that human breast milk-derived exosomes (hBME) attenuate LPS-induced activation of microglia by decreasing p38 mitogen-activated protein kinase (MAPK) and nuclear factor-kappa-light-chain-enhancer of activated B cells (NF-κB) p50/p65 phosphorylation downstream of toll-like receptor 4 (TLR4) in microglia. We isolated hBME and characterized them using nanoparticle tracking analysis, transmission electron microscopy, fluorescence-activated cell sorting, and western blot (WB) analysis. Biochemical analysis of microglia exposed to LPS and hBME indicated that hBME modulated the expression of signaling molecules in the canonical NF-κB pathway and their products, cluster of differentiation 40 (CD40), NOD-, LRR-, and pyrin domain-containing protein 3 (NLRP3), and cytokines interleukin (IL)-1β and IL-10. WB analysis shows an increase in nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor alpha (IκBα) and IL-10, and a decrease in p38 MAPK, NF-κB p65, CD40, NLRP3, and IL-1β. Enzyme-linked immunosorbent assay (ELISA) analysis showed a decrease in expression of IL-1β and an increase in IL-10. Immunocytochemistry showed morphological changes consistent with microglial activation were inhibited, and the expression of pro-inflammatory marker, ionized calcium-binding adaptor molecule 1 (Iba1), was downregulated after treatment with hBME. Our findings indicate that hBME have an anti-inflammatory effect on microglia. Here, we present hBME as a novel, biologically safe, easily accessible, and reproducible therapeutic option for attenuating the microglial response to LPS in neonates. This work was supported by the National Institutes of Health (1R15DA045564-01) and # 5R25MH080661-13/2004445114. This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.