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Severe respiratory syncytial virus (RSV) infections are a major reason for young children needing hospitalization, and a global cause of childhood mortality [1, 2]. Frequent infections such as RSV during early childhood also increase the risk of developing chronic lung conditions such as asthma later in life [3]. These early infections occur at a time when the infant gut microbiome is still maturing.

Previous studies have found that disturbances in the development of the gut microbiome are linked to the severity of lower respiratory tract infections. One especially large study in humans found that when a mother’s diet during pregnancy is rich in carbohydrates and deficient in fresh fruits and vegetables, which are key sources of dietary fiber, it can predispose infants to more severe RSV infections [4]. The precise reasons underlying this association are unclear.

In a new study [5], Simon Phipps, an immunologist at the QIMR Berghofer Medical Research Institute in Australia and his colleagues, elucidated the connections between a mouse mother’s diet and her offspring’s susceptibility to severe infections with RSV.

The team began with two groups of mice that were of breeding age. One group was fed a low-fiber diet (LFD) and the other a high-fiber diet (HFD). Pups born to both groups were infected with PVM, a mouse viral equivalent of human RSV, when they were 7 days old. Offspring born to mice fed a low-fiber diet (LFD-reared) suffered more severe respiratory infections. They gained less weight after the infection and showed hallmarks of severe inflammation, including large amounts of mucus, neutrophil activation, higher viral loads, and lower levels of viral proteins in their airways. The researchers also found lower levels of regulatory T cells in the lymph nodes and lungs of the LFD-reared pups compared with HFD-reared pups.

The team then isolated dendritic cells, which help the immune system recognize pathogens, from the HFD-reared pups. They transferred these cells to LFD-reared pups and found that the  viral loads were decreased and the severity of lower respiratory infections was decreased. Similarly, transferring regulatory T cells, another group of immune cells, from HFD-reared pups to LFD-reared pups reduced the severity of the latter group’s infection. The data support “the importance of these two immunoregulatory cell types,” the authors write in their publication.

Higher consumption of dietary fiber can drive the gut microbiome to produce specific metabolites such as short-chain fatty acids, which then modulate immune development and the maturation of dendritic cells and regulatory T cells. The researchers found that one week after birth, HFD-reared pups had higher levels of short-chain fatty acids such as propionate and butyrate in their feces and circulating in their blood.

Both the gut microbiome of pups and their levels of short-chain fatty acids were strongly correlated with maternal dietary fiber. When pups in the LFD-reared group were fostered with mothers fed a high-fiber diet, they were better protected against severe lower respiratory tract infections. But fostering pups from mothers fed a high-fiber diet with those that had consumed a low-fiber diet led to more severe respiratory disease.

To understand how microbes were being transferred from mothers to pups, the researchers performed fecal microbial transplants between the two groups but found that protection from the high-fiber diet was not conferred. Next, they tested the mothers’ milk and found differences in the milk microbiome and short-chain fatty acid composition of the high-fiber and low-fiber groups.

The team enriched and cultured milk microbes from both the high-fiber and low-fiber groups and fed the microbes to LFD-reared pups via a tube to their stomachs. They exposed the animals to PVM. The HFD microbial treatment protected LFD-reared pups from severe infections, altered their gut microbiota, and increased serum and fecal levels of propionate. When the researchers isolated microbial species from milk and repeated the process with individual species, they found that microbes that resulted in greater propionate levels also conferred the most protection against infection. “These findings indicate that the maternal diet affects milk microbiome composition, which, in turn, affects the offspring’s susceptibility to develop [severe lower respiratory infections] sLRI upon [pneumonia virus of mice] PVM infection” , the authors wrote in their paper.

Protection was most closely linked to propionate levels, not other short-chain fatty acids such as butyrate. The authors identified an immunomodulatory cytokine named Flt3L as a key player in the connections between the milk microbiome, propionate, and immunity against severe respiratory infections. Flt3L levels were lower in the stool of LFD-reared pups, and the researchers found Flt3L levels in the gut and serum rose and fell together as HFD-reared pups matured, which “suggests that the gut is the primary source of serum Flt3L in early life,” they wrote in their paper. An anti-Flt3L treatment reversed the effects of propionate treatment in LFD-reared pups, hinting at this cytokine’s key role in helping the gut microbiome protect mouse pups from severe respiratory infections.

Collectively, the study’s data reveal a critical interaction between the milk microbiome and offspring’s immunity in mice. The results reveal how lifestyle factors such as a mother’s diet may influence the health of offspring later in life via her milk.

References

1. Bozzola E, Barni S, Villani A. Respiratory syncytial virus pediatric hospitalization in the COVID-19 era. International Journal of Environmental Research and Public Health. 2022 Nov 22;19(23):15455.

2. Nair H, Simões EA, Rudan I, Gessner BD, Azziz-Baumgartner E, Zhang JS, Feikin DR, Mackenzie GA, Moiïsi JC, Roca A, Baggett HC. Global and regional burden of hospital admissions for severe acute lower respiratory infections in young children in 2010: a systematic analysis. The Lancet. 2013 Apr 20;381(9875):1380-90.

3. Wilkinson TM, Donaldson GC, Johnston SL, Openshaw PJ, Wedzicha JA. Respiratory syncytial virus, airway inflammation, and FEV1 decline in patients with chronic obstructive pulmonary disease. American Journal of Respiratory and Critical Care Medicine. 2006 Apr 15;173(8):871-6.

4. Ferolla FM, Hijano DR, Acosta PL, Rodríguez A, Dueñas K, Sancilio A, Barboza E, Caría A, Gago GF, Almeida RE, Castro L. Macronutrients during pregnancy and life-threatening respiratory syncytial virus infections in children. American Journal of Respiratory and Critical Care Medicine. 2013 May 1;187(9):983-90.

5. Sikder MA, Rashid RB, Ahmed T, Sebina I, Howard DR, Ullah MA, Rahman MM, Lynch JP, Curren B, Werder RB, Simpson J. Maternal diet modulates the infant microbiome and intestinal Flt3L necessary for dendritic cell development and immunity to respiratory infection. Immunity. 2023 May 9;56(5):1098-114.

Researchers probed the gut-healing properties of extracellular vesicles isolated from both cow and human milk [1]. These tiny packages, called milk-derived extracellular vesicles (mEVs), are secreted from mammary cells and carry a cargo of lipids, proteins, and nucleic acids. The new research revealed promising effects of mEVs on the cells lining the gastrointestinal tract, indicating a potential therapy for the “leaky” gut barrier found in many gastrointestinal and metabolic diseases [1].

The GI tract is teeming with partially digested food, microbes, digestive enzymes, and toxins, and one of the main jobs of the intestinal lining is to be a selective barrier between that messy gut environment and the bloodstream. When the gut lining is working properly, it facilitates the absorption of vital nutrients but blocks less savory elements from entering. But when the gut lining is “leaky,” or overly permeable, it can expose the body’s cells to harmful molecules and allow bacteria and toxins to invade the bloodstream. A more permeable gut lining has been associated with a range of diseases, including inflammatory bowel disease, nonalcoholic fatty liver disease, and nonalcoholic steatohepatitis [2].

Because they can be orally administered and have been reported to survive the harsh environment of the digestive tract, mEVs have attracted attention as potential therapeutics. In a recent study published in April 2023 in the journal Science Advances, researchers from National University of Singapore and Ocean University of China set out to investigate how mEVs affect the gut barrier [1], adding to previous promising research by their group and others on both cow and human mEVs [3-4].

The research team reported results from a series of intricate experiments on mEVs. First, they isolated mEVs from both cow and human milk and studied their contents, finding that both types were rich in proteins and small nucleic acids known to be related to gut barrier function and inflammation. They looked at how mEVs affected intestinal cells—both isolated cells and those arranged in a single layer as a model of the intestinal lining—and found that mEVs decreased inflammation and increased the expression of proteins involved in intestinal barrier function. They also tested how well mEVs survived conditions that mimicked a trip through the digestive tract, awash in saliva and stomach, pancreatic, and bile juices. “We confirmed that mEVs remained intact after passage through oral-gastrointestinal digestive conditions,” the researchers wrote, referring to mEVs from both cow and human milk. 

In the next series of experiments, cow mEVs were administered to mice and were found to reach both the small intestine and the colon, where they were absorbed by epithelial cells. Most importantly, in mouse models of colon inflammation and nonalcoholic steatohepatitis, oral mEV administration reduced intestinal inflammation, repaired the gut barrier, and even suppressed inflammation in the liver—indicating that mEVs have effects beyond the digestive tract. 

Because gut inflammation and hyperpermeability are common features of so many diseases, these findings could have exciting implications, wrote Dr. Jiong-Wei Wang, an assistant professor of surgery and physiology at the National University of Singapore and a lead author on the paper, in an email interview with the SPLASH! editorial team. Wang added, “Our findings suggest that mEVs may be used for the treatment of a broad range of diseases instead of one single disease type.” 

The milk we buy at the store contains mEVs, but to mimic the dose given to the mice in the study, you’d need to drink one liter, or about four cups, of milk per day, the researchers estimated in their paper. That’s more than is recommended in the Dietary Guidelines for Americans; plus, lactose is difficult to digest for about two-thirds of the world’s population [5). But the good news is that mEVs themselves, once extracted from milk, have very little lactose, wrote Wang. “They may be an alternative milk product for lactose-intolerant people,” he added.

Though the results are promising, more research is needed before mEVs can be promoted as a therapy for preventing or healing gut disorders. Currently, Wang and his colleagues “are trying to uncover the exact mechanism underlying the therapeutic effects of mEVs.” His team is also preparing to conduct clinical trials in humans, which will be necessary to see if the anti-inflammatory and gut-healing properties of mEVs observed in isolated cells and mice also occur in humans. And even though mEVs are found in cow milk, which has been consumed by humans for thousands of years, concentrated mEV therapeutics will need to be studied to determine appropriate dosing, whether they are safe, and if they cause side effects, Wang wrote in the interview with the SPLASH! editorial team. 

That’s the incremental nature of scientific research, but the current study [1] paves the way for those future explorations of mEVs.

References

1. Tong L, Zhang S, Liu Q, Huang C, Hao H, Tan MS, Yu X, Lou CKL, Huang R, Zhang Z, Liu T, Gong P, Ng CH, Muthiah M, Pastorin G, Wacker MG, Chen X, Storm G, Lee CN, Zhang L, Xi H, Wang JW. Milk-derived extracellular vesicles protect intestinal barrier integrity in the gut-liver axis. Science Advances. 2023 Apr 9:eade5041. 
2. Di Tommaso N, Gasbarrini A, Ponziani FR. Intestinal barrier in human health and disease. International Journal of Environmental Research and Public Health. 2021;18(23):1-23.
3. Tong L, Hao H, Zhang Z, Lv Y, Liang X, Liu Q, Liu T, Gong P, Zhang L, Cao F, Pastorin G, Lee CN, Chen X, Wang JW, Yi H.  Milk-derived extracellular vesicles alleviate ulcerative colitis by regulating the gut immunity and reshaping the gut microbiota. Theranostics. 2021 Jul 11:8570–86.
4. Zonneveld MI, van Herwijnen MJC, Fernandez-Gutierrez MM, Giovanazzi A, de Groot AM, Kleinjan M, van Capel TMM, Sijts AJAM, Taams LS, Garssen J, de Jong EC, Kleerebezem M, Nolte-‘t Hoen ENM, Redegeld FA, Wauben MHM. Human milk extracellular vesicles target nodes in interconnected signalling pathways that enhance oral epithelial barrier function and dampen immune responses. Journal of Extracellular Vesicles. 2021 Mar 10:e12071.
5. Storhaug CL, Fosse SK, Fadnes LT. Country, regional, and global estimates for lactose malabsorption in adults: a systematic review and meta-analysis. Lancet Gastroenterology and Hepatology. 2017 Oct 2:738–46.

Researchers have speculated for decades that one effect of breast milk may be to promote heart maturation in newborns. A recent study has finally confirmed this idea, and has also pinned down the molecular connection. In mice, the study reports, a fatty acid called γ-linolenic acid, or GLA, from maternal milk activates a protein called retinoid X receptor (RXR), which helps heart cells—cardiomyocytes—switch on their pulsing function at birth (1).

“We think the beauty of this study is that it opens up many new avenues of research at the molecular level and also, if relevant in humans, at the clinical level,” says Mercedes Ricote, a molecular biologist at the Spanish National Center for Cardiovascular Research who led the work. 

Ricote and her colleagues did not set out to study milk at all. Their lab was investigating how proteins called transcription factors play a role in cardiomyocyte maturation, and in previous work they identified a protein called RXR as one such factor. In this study, they found that mice in which the gene encoding RXR was knocked out survived until birth, but most neonates did not survive past 24 hours. “If there is no RXR signaling after birth, the hearts of these mice stop beating and they die,” says Ana Paredes, a molecular biologist in Ricote’s laboratory and first author on the study. 

Neonatal mice undergo a significant change in their metabolic profile. In the fetus, cardiomyocytes are powered by glucose oxidation, but after birth that process falls away, and instead these cells begin relying on fatty acid consumption by mitochondria. Paredes and Ricote reasoned that birth also brings two major physiological challenges: breathing, and ingesting maternal milk. Earlier work from the lab showed that RXR must be activated by a ligand to function (2). The identity of that ligand was unknown, although there were hints in the literature that it came from fatty acids. So the researchers hypothesized that maternal milk might carry it. 

To test the idea, Ricote and her colleagues fed dams a specially created diet lacking free fatty acids. Lipidomic studies of the milk these mice produced showed that it lacked omega-6 fatty acids, including GLA. Most neonates suckling from these mothers died within a day or two, just like the pups lacking RXR. The researchers then looked at gene expression in genes regulating fatty acid metabolism and with the help of lipidomics, a method for analyzing multiple fats, found that these genes’ activation depends on the presence of GLA. When they gave GLA to mouse mothers who were fed a fat-free diet, their pups thrived. Conversely, giving GLA directly to pups lacking the RXR gene did not boost their survival—suggesting that GLA activates RXR function. 

“Together, these results reveal a molecular signaling pathway whereby nutrients in the milk of female mice activate a gene-expression program that triggers maturation of cardiomyocytes and prepares them for postnatal function,” write Pingzhu Zhou and William T. Pu at Boston Children’s Hospital, in a review article in Nature accompanying the paper (3).

The researchers note that GLA is not naturally present in the body. “GLA is an essential omega-6 fatty acid—it must be ingested because we cannot produce it,” Paredes says. That may partly explain the significance of mother’s milk in terms of evolution, she adds.  

Ricote speculates that this milk-governed process takes place not only in the heart but in all organs that have a high-energy requirement. The investigators are currently testing whether and how milk plays a role in maintaining energy levels in the brain and the liver. “We think this is a homeostatic process,” she says.  

The work was conducted in mice, and “the relevance of our studies to humans needs further evaluation,” Ricote cautions. Little is known about human variants of the RXR gene, but determining whether premature babies or babies born with cardiac problems have specific variants might provide some hints to whether the mechanism also is present in humans, Paredes says. If so, supplementing such babies’ diets with GLA may help promote their survival, she says. 

Ricote also notes that the lab tested an infant milk formula and found that it contained enough GLA precursors to provide this fatty acid—so babies who eat formula instead of breast milk would still get enough GLA. “That is an important point for women who might who read this study and get scared,” she said. 

References

1. Paredes A, Justo-Méndez R, Jiménez-Blasco D, Núñez V, Calero I, Villalba-Orero M, Alegre-Martí A, Fischer T, Gradillas A, Sant’Anna VAR, Were F, Huang Z, Hernansanz-Agustín P, Contreras C, Martínez F, Camafeita E, Vázquez J, Ruiz-Cabello J, Area-Gómez E, Sánchez-Cabo F, Treuter E, Bolaños JP, Estébanez-Perpiñá E, Rupérez FJ, Barbas C, Enríquez JA, Ricote M. γ-Linolenic acid in maternal milk drives cardiac metabolic maturation. Nature. 2023;618(7964):365-373.

2. Rőszer T, Menéndez-Gutiérrez MP, Cedenilla M, Ricote M. Retinoid X receptors in macrophage biology. Trends Endocrinol Metab. 2013;24(9):460-468.

3. Zhou P, Pu WT. Molecule in mothers’ milk nurses pups’ heart muscle cells to maturity. Nature. 2023:618(7964):242-243.

Being a first-time mother is challenging. Without prior experience, navigating the baby’s sleep and feeding schedules, mastering the swaddle, and knowing when to introduce solid foods can be daunting tasks. But it isn’t just maternal behaviors that benefit from experience—reproductive experience also is associated with improvements in milk production. Across mammals, mothers with more than one pregnancy (multipara) are more effective at transferring milk to their offspring than first-time mothers (primipara) [1].

This difference in lactation performance can be explained, in part, by changes in the mammary gland after the first pregnancy. For example, in mice [2], the mammary gland had a stronger response to hormones produced during second pregnancies compared with the first pregnancy [2]. And in grey seals [3], mammary glands of multiparous mothers had a greater capacity for secreting and storing milk compared with mammary glands of primiparous mothers.

Primiparous mothers can also differ from multiparous mothers in their ability to support the energetic cost of lactation. In rhesus macaque monkeys, mothers often had their first offspring while they were still growing. This meant primiparous macaques had to meet the caloric requirements of body growth at the same time as meeting the energetic costs associated with pregnancy and lactation. And because they were still growing, primiparous macaque mothers had fewer fat stores than multiparous mothers to support those additional costs. With higher energetic costs and fewer stored calories, it isn’t surprising that primiparous macaque mothers were found to produce milk for their daughters that was lower in energy content than milk from multiparous macaque mothers with daughters [4, 5].

Mammal mothers may love all their offspring equally but could be physiologically constrained in their ability to invest equally through milk production. But what this reduced investment might mean for firstborn daughters is not well understood. Nutrition during growth and development is known to affect life history traits such as adult body mass and age at first reproduction. Milk is a very specific type of nutrition, containing not only macro- and micronutrients to support infant growth and development but also hormones and bioactive peptides that play a role in organizing metabolic, reproductive, neuroendocrine, and biobehavioral systems during growth and development [1]. In dairy cows, daughters of primiparous dams produced significantly less milk when they were first-time mothers than did daughters of multiparous dams [6]. Could deficits in milk composition or milk yield experienced by other mammals during a firstborn daughter’s infancy shape her own capacity to synthesize milk throughout her reproductive career?

This intriguing research question was investigated in a new study [1] on a well-studied population of captive rhesus macaques housed at the California National Primate Research Center in Davis, CA. Researchers Pittet and Hinde tested the effect of birth order on milk characteristics, including composition and yield, as well as early life growth rates, age at first reproduction, body mass of offspring, and body mass as a lactating mother, among firstborn (n = 72) and laterborn daughters (n = 202).

Being the daughter of a first-time mother did indeed leave a signature on growth and lactation performance in rhesus macaques. Firstborn daughters grew more quickly during infancy (4 months–1 year of age) than laterborn daughters, but this trend was reversed during the juvenile period (1–2 years of age). By 2 years old, laterborn daughters were approximately 100 grams heavier than firstborn daughters. These differences in body size persisted through adulthood and laterborns were heavier than firstborns throughout their reproductive careers.

Having more body mass meant more energetic resources for milk production. At peak lactation, higher body mass was associated with higher milk yield [1]. As primipara, firstborn daughters produced a similar milk composition to laterborns but made approximately 23% less milk. Milk output improved with subsequent pregnancies, but firstborns still produced 5% less milk at peak lactation, and their milk was on average 0.67% lower in fat concentration compared with laterborns [1].

These small differences in yield and fat content resulted in a large difference in available milk energy (AME), the product of milk energy density (kilocalories/gram) and milk yield (grams). On average, AME from firstborns was 16% lower than AME from laterborns. Importantly, this difference was only partly explained by the larger body mass of laterborns. After controlling for maternal mass in the statistical models, the study found firstborns still produced approximately 10% lower AME than laterborns [1].

Primiparous mothers transferred fewer calories and essential nutrients for somatic maintenance, tissue development, and behavioral activity to firstborn daughters [1]. This “meager milk” not only resulted in a smaller adult body mass for firstborn daughters but could also have influenced their mammary gland morphology, how they used the nutrients they consumed (e.g., are they stored or used immediately?) or even cellular functions (e.g., glucose metabolism). Together, these factors could constrain a firstborn’s ability to match laterborns in AME.

To understand the connection between infant milk intake and adult milk production, the study authors offer a brilliant analogy to the construction of a building [1]. The building itself is the infant’s phenotype—traits influenced by genes and their environment, such as body size, hormone levels, age at first reproduction, and even milk production. The construction materials that help build this house include nutrients from the mother’s milk. But the overall design of the building is also influenced by bioactive signals in milk, such as growth hormones [1]. Maybe the genetic blueprint called for a second story, but the construction materials lacked the necessary building blocks or were too costly to manufacture, so a tiny home was built instead.

It is not yet known how reduced AME from firstborns influenced their daughter’s own milk production (the granddaughters of the current study’s primiparous mothers). But the researchers did find, somewhat surprisingly considering the difference in AME, that infants from firstborns and laterborns were similar in body mass at peak lactation. There’s a long-term cost to cutting corners with the framing and foundation of a building. Pittet and Hinde suggest that infants of firstborns could be devoting a higher proportion of milk energy to growth than infants of laterborns, but they are doing so at a cost. Among this population of rhesus macaques, lower AME is associated with a less confident temperament, suggesting a lack of energy available for play or exploratory activities [1]. Continuing to follow these macaque infants throughout growth and development, including when they become mothers themselves, will help elucidate the intergenerational impacts of constrained milk production among primipara. And ultimately, this type of comparative research can help us understood potential predictors for poor lactation performance among human mothers. Pittet and Hinde rightly emphasize that there is a lack of support for women who experience challenges with breastfeeding, such as low milk supply, while at the same time there is a stigma directed toward mothers who choose to feed their infants formula. Identifying potential intergenerational effects limiting a woman’s capacity for milk production can hopefully limit these dismissive attitudes and pave the way for interventions during infancy to improve lactation outcomes across generations.

References

1. Pittet F, Hinde K. Meager milk: Lasting consequences for adult daughters of primiparous mothers among thesus macaques (Macaca mulatta). Integrative and Comparative Biology. 2023 May 11: icad022.
2. Dos Santos CO, Dolzhenko E, Hodges E, Smith AD, Hannon GJ. An epigenetic memory of pregnancy in the mouse mammary gland. Cell Reports. 2015 May 19;11(7): 1102-1109.
3. Lang SL, Iverson SJ, Bowen WD. The influence of reproductive experience on milk energy output and lactation performance in the grey seal (Halichoerus grypus). PloS one. 2011 May 11;6(5): e19487.
4. Hinde K. First-time macaque mothers bias milk composition in favor of sons. Current Biology. 2007 Nov 20;17(22):R958-9.
5. Hinde K. Richer milk for sons but more milk for daughters: Sex‐biased investment during lactation varies with maternal life history in rhesus macaques. American Journal of Human Biology: The Official Journal of the Human Biology Association. 2009 Jul;21(4): 512-519.
6. Poczynek M, Nogueira LD, Carrari IF, Carneiro JH, Almeida RD. Associations of body condition score at calving, parity, and calving season on the performance of dairy cows and their offspring. Animals. 2023 Feb 8;13(4): 596.