Tiny Messengers in the Milky Universe: The Secrets of Human Milk Extracellular Vesicles

Abstract

After birth, the mother-infant dyad switches from aseptic transfer of nutrients through the umbilicus to dependence on milk. During early-life it is crucial that neonate immune system learns to tolerate harmless food and commensal bacteria, while triggering immune responses against harmful microorganisms. This immune education is ensured by an interplay between components of the infant immune system, the digestive tract, the microbiome and milk. Human milk contains maternal immune modulatory components exerting remarkable influence on early-life health and development. In general human milk is described as a complex body fluid composed of water, fat, sugars and proteins and contains milk-specific structures such as milk fat globules and caseins. In addition, milk contains extracellular vesicles (EVs). EVs are lipid bilayer-delimited nanosized particles naturally released and taken up by cells. EVs are composed of biological information derived from their cells of origin and serve as mediators of intercellular communication. As such, human milk EVs can be a means of transfer of information between mother and child. Several studies have indicated that milk EVs are able to attenuate inflammatory responses, support the intestinal epithelium barrier maturation and prevent viral infections. However, most of these studies focused on bovine milk EV composition and their effects on human cells. The molecular make-up of human milk EVs has been studied in relation to proteins and RNAs, however information on the metabolome is lacking. For a more comprehensive understanding of the function of human milk EVs, mapping of the metabolic cargo is of utmost importance. To study the functional effects mediated by human milk EVs and their cargo, separation of milk EVs from other milk components is needed. This is challenging because non-EV particles with overlapping characteristics of EVs are present in milk and can be co-isolated to various degrees. In line with this, the interpretation and reproducibility of published results is often hampered by the lack of appropriate experimental controls to determine which effects can be attributed to EVs and which to other components. Proper, clear and transparent reporting of the sample/data quality is of crucial importance for data reliability and reproducibility. In my thesis I investigated major gaps in the knowledge on human milk EVs by addressing four main research questions: 1. Do milk EVs have the potential to modulate innate and adaptive immune responses? 2. Do milk EVs have a unique composition and is this composition influenced by the immune status of the mother? 3. How do milk EV cargoes relate to functional modulation? 4. How to improve rigor and reproducibility in milk EV research? I believe that the research presented in my thesis contributes to a better understanding of the role of milk EVs in providing a kick-start for life, which may eventually lead to the nutraceutical and clinical application of human milk EVs. Overall, the exploration and comprehension of the enigmatic intricacies hidden within human milk illuminate the profound influence that milk exerts on human development and underscore the importance of unravelling these secrets for the improvement of lifelong well-being.