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Negative effect of human milk, but not infant milk formulations, on cell stimulation via TLR2 and TLR3. A and B , Mono Mac-6 monocytes (2 ϫ 10 5 cells) or DCs (1 ϫ 10 4 cells) were stimulated for 16 h with the indicated concentrations of the TLR2-specific ligands (MM6) Pam 3 -Cys-Ser-(Lys) 4 (Pam3), peptidoglycan (PGN), and heat-killed L. monocytogenes (HKLM; 10:1 bacteria:cell) or the TLR3-specific ligand (DCs) and viral dsRNA mimic synthetic poly(I:C) in the absence or presence of 0.125% human breast milk (HM) or 125 ng/ml purified milk-derived sCD14 (TLR2 stimulation). IL-8 levels in the culture supernatants were determined by ELISA. C , Fluorescence profiles of CD86 expression in DCs (1 ϫ 10 4 cells) stimulated or not with 80 g/ml poly(I:C) for 16 h in the absence or presence of 0.125% human milk. The shaded profile corresponds to the staining with the PE-conjugated isotype-matched control Ab. D , IL-8 levels in the culture supernatants of Mono Mac-6 cells stimulated as in A with 5 g/ml PGN in the absence or presence of 0.125% human milk, 0.125% infant milk formulations ( n ϭ 3), or 125 ng/ml purified milk-derived sCD14. Results shown in A to D are from one experiment ( Ϯ SD in A , B , and D ) representative of four ( A–C ) or three ( D ) performed with milk collected at day 1, 2 (shown), 3, and 5 postpartum. The sCD14 concentration in the 0.125% human milk samples ( n ϭ 3) used in the experiments shown in A–D was 122 Ϯ 5 ng/ml (day 2). The differences in IL-8 release between human milk and purified sCD14 supplemented cultures or cells (poly(I:C) stimulation) were significant: ءء , p Ͻ 0.001; ءءء , p Ͻ 0.0001.
Source publication
The mechanisms controlling innate microbial recognition in the neonatal gut are still to be fully understood. We have sought specific regulatory mechanisms operating in human breast milk relating to TLR-mediated microbial recognition. In this study, we report a specific and differential modulatory effect of early samples (days 1-5) of breast milk o...
Contexts in source publication
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... enhancing effect of milk posed the question of whether milk exerts only a positive regulation on TLR-mediated cell stimulation. In contrast to the enhancing effect on TLR4 and TLR5, in the presence of milk cell stimulation induced via TLR2 by a number of TLR2-specific ligands was found significantly reduced ( Fig. 3A), as compared with that in the presence of the coreceptor sCD14 (used at the same concentration as in the milk sample) or to stimulation in the absence of any supplement (cells alone) at high ligand concentration. A similarly negative effect of milk was observed when DCs were stimulated via TLR3 with the viral dsRNA mimic TLR3 ligand ...
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... 3A), as compared with that in the presence of the coreceptor sCD14 (used at the same concentration as in the milk sample) or to stimulation in the absence of any supplement (cells alone) at high ligand concentration. A similarly negative effect of milk was observed when DCs were stimulated via TLR3 with the viral dsRNA mimic TLR3 ligand poly(I:C) (Fig. 3B). Interestingly, poly(I:C)-induced CD86 expression in DCs was not affected by milk (Fig. 3C). This was also in contrast to the positive effect observed when TLR4 was tested (Fig. 2B). The inhibitory effect was not reproduced by using infant milk formulations (Fig. 3D), like the enhancing effect on TLR4 (Fig. ...
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... as in the milk sample) or to stimulation in the absence of any supplement (cells alone) at high ligand concentration. A similarly negative effect of milk was observed when DCs were stimulated via TLR3 with the viral dsRNA mimic TLR3 ligand poly(I:C) (Fig. 3B). Interestingly, poly(I:C)-induced CD86 expression in DCs was not affected by milk (Fig. 3C). This was also in contrast to the positive effect observed when TLR4 was tested (Fig. 2B). The inhibitory effect was not reproduced by using infant milk formulations (Fig. 3D), like the enhancing effect on TLR4 (Fig. ...
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... DCs were stimulated via TLR3 with the viral dsRNA mimic TLR3 ligand poly(I:C) (Fig. 3B). Interestingly, poly(I:C)-induced CD86 expression in DCs was not affected by milk (Fig. 3C). This was also in contrast to the positive effect observed when TLR4 was tested (Fig. 2B). The inhibitory effect was not reproduced by using infant milk formulations (Fig. 3D), like the enhancing effect on TLR4 (Fig. ...
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... of milk on TLR4 signaling. Similarly, the primary events responsible for the positive effect on TLR5 and negative effects on TLR2 and TLR3 remain to be elucidated. With regard to the negative effect, milk markedly inhibited the TLR3-mediated poly(I:C)-induced cy- tokine production by DCs without affecting costimulatory mole- cule expression (Fig. 3, B and C), suggesting that the MyD88- dependent (cytokine production), but not the independent pathway, which controls costimulatory molecule expression and is the main signaling pathway for TLR3 (31), was affected. This was in marked contrast to the effect of milk on TLR4, because here both cytokine production and costimulatory molecule ...
Citations
... Bifidobacterium are predominant in the gut microbiota of infants and they are considered to be important for infant health and development [8,52,53]. Bifidobacterium in breast milk has been reported to activate immunoglobulin A (IgA)-producing plasma cells in the neonatal gut [54] and could control inflammation through mucosal host-microbe crosstalk [55]. The present study showed a lower relative abundance of Bifidobacterium in IG breast milk at baseline but a higher abundance after the relaxation intervention, suggesting the intervention may have increased the relative abundance of Bifidobacterium in IG breast milk. ...
Background: This secondary analysis of data from a randomized controlled trial (RCT) investigated how the maternal gut, breast milk, and infant gut microbiomes may contribute to the effects of a relaxation intervention, which reduced maternal stress and promoted infant weight gain. Methods: An RCT was undertaken in healthy Chinese primiparous mother–infant pairs (340/7–376/7gestation weeks). Mothers were randomly allocated to either the intervention group (IG, listening to relaxation meditation) or the control group (CG). Outcomes were the differences in microbiome composition and the diversity in the maternal gut, breast milk, and infant gut at 1 (baseline) and 8 weeks (post-intervention) between IG and CG, assessed using 16S rRNA gene amplicon sequencing of fecal and breastmilk samples. Results: In total, 38 mother–infant pairs were included in this analysis (IG = 19, CG = 19). The overall microbiome community structure in the maternal gut was significantly different between the IG and CG at 1 week, with the difference being more significant at 8 weeks (Bray–Curtis distance R² = 0.04 vs. R² = 0.13). Post-intervention, a significantly lower α-diversity was observed in IG breast milk (observed features: CG = 295 vs. IG = 255, p = 0.032); the Bifidobacterium genera presented a higher relative abundance. A significantly higher α-diversity was observed in IG infant gut (observed features: CG = 73 vs. IG = 113, p < 0.001). Conclusions: The findings were consistent with the hypothesis that the microbiome might mediate observed relaxation intervention effects via gut–brain axis and entero-mammary pathways; but confirmation is required.
... Bifidobacterium are predominant in the gut microbiota of infants, and they are considered to be important for infant health and development [8,33,34]. Bifidobacterium in breast milk has been reported to activate immunoglobulin A (IgA)-producing plasma cells in the neonatal gut [35] and could control inflammation through mucosal host-microbe crosstalk [36]. The present study showed a lower relative abundance of Bifidobacterium in IG breast milk at baseline but higher abundance after the relaxation intervention, suggesting the intervention may have increased the relative abundance of Bifidobacterium in IG breast milk. ...
Background: This study explored maternal gut, breast milk and infant gut microbiomes as possi-ble mediators of the observed effects of a relaxation intervention which reduced maternal stress and promoted infant weight gain. Methods: A randomized controlled trial was conducted in healthy Chinese primiparous mother-infant pairs. Mothers were randomly assigned to interven-tion group (IG, listening to relaxation meditation) or control group (CG). Outcomes were differ-ences in microbiome composition and diversity in maternal gut, breast milk and infant gut at 1- and 8-weeks between IG and CG, assessed by 16S rRNA gene amplicon sequencing of fecal and breastmilk samples. Results: 38 mother-infant pairs were included in this analysis (IG=19, CG=19). Overall microbiome community structure in the maternal gut was significantly different between IG and CG at 1-week and the difference was more significant at 8 weeks (Bray-Curtis distance R2=0.04 vs. R2=0.13). Post-intervention, the α-diversity was significantly lower in IG breast milk (observed features: CG=295 vs. IG=255, p=0.032); the Bifidobacterium genera presented higher relative abundance. In parallel, significantly higher α-diversity was observed in IG infant gut (observed features: CG=73 vs. IG=113, p
... Interestingly, it has been observed that colostrum can augment the response of epithelial cells to LPS, possibly due to its high concentration of sCD14 (23). Several in vitro and in vivo studies have examined the effects of the specific components present in human milk. ...
... In our in vitro model of gut epithelial cells and macrophages, we successfully replicated the role of raw milk in augmenting the inflammatory response to bacterial stimuli. In a study by LeBouder et al., it was observed that the response of HT-29 cells to LPS was reversed by employing a blocking antibody against sCD14 (23). CD14 exists in both the cell membrane-bound form (mCD14) and soluble form (sCD14), both of which can modulate the TLR4 signaling pathway (56,57). ...
Introduction
The gastrointestinal and immune systems of premature infants are not fully developed, rendering them more vulnerable to severe complications like necrotizing enterocolitis. Human milk offers a rich array of bioactive factors that collectively contribute to reducing the incidence of gut infections and inflammatory conditions. When a mother's milk is unavailable, preterm infants are often provided with donor human milk processed in Human Milk Banks. However, it remains uncertain whether pasteurized milk confers the same level of risk reduction as unprocessed milk. This uncertainty may stem from the well-documented adverse effects of heat treatment on milk composition. Yet, our understanding of the comprehensive impact on protective mechanisms is limited.
Methods
In this study, we conducted a comparative analysis of the effects of raw versus pasteurized milk and colostrum versus mature milk on cellular functions associated with the gut epithelial barrier and responses to inflammatory stimuli. We utilized THP-1 and HT-29 cell lines, representing monocyte/macrophages and gut epithelial cells, respectively.
Results
Our observations revealed that all milk types stimulated epithelial cell proliferation. However, only raw colostrum increased cell migration and interfered with the interaction between E. coli and epithelial cells. Furthermore, the response of epithelial and macrophage cells to lipopolysaccharide (LPS) was enhanced solely by raw colostrum, with a milder effect observed with mature milk. In contrast, both raw and pasteurized milk diminished the LPS induced response in monocytes. Lastly, we examined how milk affected the differentiation of monocytes into macrophages, finding that milk reduced the subsequent inflammatory response of macrophages to LPS.
Discussion
Our study sheds light on the impact of human milk on certain mechanisms that potentially account for its protective effects against necrotizing enterocolitis, highlighting the detrimental influence of pasteurization on some of these mechanisms. Our findings emphasize the urgency of developing alternative pasteurization methods to better preserve milk properties. Moreover, identifying the key components critically affected by these protective mechanisms could enable their inclusion in donor milk or formula, thereby enhancing immunological benefits for vulnerable newborns.
... Many of these factors possess anti-in ammatory properties by in uencing the TLR4 pathway, suggesting that HM has the capacity to in uence innate immune function and establishment of immune homeostasis (1). Most studies on the immunomodulatory function of HM have focused on the effect of HM on professional immune cells, but only a handful of studies have examined the effect of HM on intestinal epithelial cells (IECs) (4,5). ...
Background: Human milk (HM) components affect immune cell toll-like receptor 4 (TLR4) signaling. However, studies examining the immunomodulatory impacts of HM on TLR4 signaling in intestinal epithelial cells (IECs) are limited.
Methods: This study utilized both a TLR4 reporter cell line and a Caco-2 IEC model to examine the effects of HM on lipopolysaccharide (LPS)-induced TLR4 activation and cytokine responses, respectively. Additionally, we performed fast protein liquid chromatography and mass spectrometry to identify a HM component that contributes to the effect of HM on TLR4 signaling.
Results: HM enhances LPS-induced TLR4 signaling as well as IEC gene expression of pro-inflammatory cytokines and negative regulators of NF-κB. Human serum albumin (HSA) present in HM contributes to this effect.
Conclusion: HSA within HM synergizes with LPS to induce TLR4 signaling as well as IEC gene expression of pro-inflammatory cytokines and negative regulators of NF-κB. Altogether, this study provides mechanistic evidence behind the immunomodulatory function of HM on IECs, which may contribute to an enhanced immune response in breast-fed neonates.
... Both isoforms were over -expressed in the mastitic samples (FC = 36.05, Novel mRNA isoform Gene_926_6 is predicted to be the gene Pellino and this gene has been found to be expressed in various studies looking at immune response in humans [41] and mastitis in the goat mammary gland [42]. However, in neither of these studies was it the main focus so limited information could be found on its functionality in relation to mastitis or the immune system. ...
Background
Mastitis is a very common disease in the dairy industry that producers encounter daily. Transcriptomics, using RNA-Sequencing (RNA - Seq) technology, can be used to study the functional aspect of mastitis resistance to identify animals that have a better immune response to mastitis. When the cow has mastitis, not only genes but also specific mRNA isoforms generated via alternative splicing (AS) could be differentially expressed (DE), leading to the phenotypic variation observed. Therefore, the objective of this study was to use large gap read mapping to identify mRNA isoforms DE between healthy and mastitic milk somatic cell samples (N = 12). These mRNA isoforms were then categorized based on being 1) annotated mRNA isoforms for gene name and length, 2) annotated mRNA isoforms with different transcript length and 3) novel mRNA isoforms of non - annotated genes.
Results
Analysis identified 333 DE transcripts (with at least 2 mRNA isoforms annotated, with at least one being DE) between healthy and mastitic samples corresponding to 303 unique genes. Of these 333 DE transcripts between healthy and mastitic samples, 68 mRNA isoforms are annotated in the bovine genome reference (ARS.UCD.1.2), 249 mRNA isoforms had novel transcript lengths of known genes and 16 were novel transcript lengths of non - annotated genes in the bovine genome reference (ARS.UCD.1.2). Functional analysis including gene ontology, gene network and metabolic pathway analysis was performed on the list of 288 annotated and unique DE mRNA isoforms. In total, 67 significant metabolic pathways were identified including positive regulation of cytokine secretion and immune response. Additionally, numerous DE novel mRNA isoforms showed potential involvement with the immune system or mastitis. Lastly, QTL annotation analysis was performed on coding regions of the DE mRNA isoforms, identifying overlapping QTLs associated with clinical mastitis and somatic cell score.
Conclusion
This study identified novel mRNA isoforms generated via AS that could lead to differences in the immune response of Holstein dairy cows and be potentially implemented in future breeding programs.
... Human milk components can selectively recognize pathogens and differentiate them from commensals. TLR signaling is important for such differential recognition [47,48]. In human milk there are specific TLR responses on different cellular components based on the TLR activated; such responses are not found in infant formulas [48]. ...
... TLR signaling is important for such differential recognition [47,48]. In human milk there are specific TLR responses on different cellular components based on the TLR activated; such responses are not found in infant formulas [48]. TLR2, TLR3, TLR5 and soluble cluster of differentiation (sCD)14, and human β-defensin-1 (hBD-1), function as pattern recognition receptors for innate immune recognition [18]. ...
... TLR2, TLR3, TLR5 and soluble cluster of differentiation (sCD)14, and human β-defensin-1 (hBD-1), function as pattern recognition receptors for innate immune recognition [18]. PRRs in human milk and other bioactive substances in the intestine of the breastfed infant create an anti-inflammatory environment, while TLR responses can be modified by soluble tolllike receptors (sTLRs) and sCD14 [18,48,49]. ...
Background: Immunological protection against novel mucosal pathogens is crucial to us as our immunity is unable to effectively defend against specific pathogens without previous immune encounters, as experienced in the SARS-CoV-2 pandemic. However, a nursing neonate is protected from many novel infections by exclusive human milk feeding despite having a naïve immune system without much previous pathogen exposure. It is observed that SARS-CoV-2 is not transmitted to the nursing infant through human milk and that natural maternal infections produce specific antibody responses in human milk. Furthermore, maternal vaccination against SARS-CoV-2 may modify some of these responses compared to natural infections. In this setting, it was felt necessary to also explore if early, innate immunity in human milk can protect against SARS-CoV-2.To explore this hypothesis, I reviewed the pathogenic mechanisms of COVID-19 focusing on the methods of viral entry through the human mucosae, infection establishment, immune dysregulation, and disease causation, and integrated these with the early actions by human milk feeding on mucosal infections. I then extrapolated the relevant pathways of human milk immune protection as potentials to protect against SARS-CoV-2. Methods: This was divided into three steps which firstly included a literature search, secondly a stepwise analysis and synthesis of data, and thirdly, an integration of data to form a hypothesis. The first step searched articles in two areas. In the first area, articles included were on the infection and pathogenesis of SARS-CoV-2, and in the second area, articles included were on innate immunity in human milk. In the second step, I analyzed the immunological actions in human milk against mucosal infections, on the whole, and synthesized some of these relevant actions against the pathogenesis of SARS-CoV-2 infections. In the third step, I integrated human milk immune pathways that could interfere with the establishment of SARS-CoV-2 infection, viral invasion, immune dysregulation, and the progression of the disease. Results: Infection by SARS-CoV-2 can theoretically be reduced or mitigated by the effect of early immune constituents in human milk. Human milk feeding may confer protection against all stages of the disease including the establishment of SARS-CoV-2 infection, invasion, and immune dysregulation and these actions may benefit both the individual and the community. Limitations: The multifunctional and dynamic nature in which human milk constituents function in a nursing infant cannot be fully reproduced by studying isolated components under experimental conditions. Even when such factors can theoretically offer protection against the virus, this concept has to be further researched in large cohorts of nursing infants. Conclusion: The role of human milk in preventing infection by SARS-CoV-2 must be explored further and if true, exclusive human milk feeding must be considered another reason for the smaller number of infections observed in children compared to adults in the pandemic. The additional counseling of human milk feeding for protection against novel pathogens, besides its established role in reducing neonatal mortality, would enhance rates of exclusive human milk feeding. General health can be developed and promoted through the potential immunotherapy provided by it.
... Breast milk has been championed in the last 20 years for its extensive benefits in disease prevention. Studies by LeBouder illustrate the bioactive components of breastmilk on immune modulation in the first few days after birth (118,250). Others have provided evidence of enhanced microbiota diversity and richness in the gut interacting with TLRs and is protective of conditions such as NEC. A major avenue of research at the moment is unpacking the bioactive cargo within exosomes present in colostrum including coding RNA (mRNA) and noncoding RNA (ncRNA), such as miRNA and long noncoding RNA (lncRNA) (251). ...
Toll-like receptors (TLRs) are crucial transmembrane receptors that form part of the innate immune response. They play a role in the recognition of various microorganisms and their elimination from the host. TLRs have been proposed as vital immunomodulators in the regulation of multiple neonatal stressors that extend beyond infection such as oxidative stress and pain. The immune system is immature at birth and takes some time to become fully established. As such, babies are especially vulnerable to sepsis at this early stage of life. Findings suggest a gestational age-dependent increase in TLR expression. TLRs engage with accessory and adaptor proteins to facilitate recognition of pathogens and their activation of the receptor. TLRs are generally upregulated during infection and promote the transcription and release of proinflammatory cytokines. Several studies report that TLRs are epigenetically modulated by chromatin changes and promoter methylation upon bacterial infection which have long-term influences on immune responses. TLR activation is reported to modulate cardiorespiratory responses during infection and may play a key role in driving homeostatic instability observed during sepsis. Although complex, TLR signalling and downstream pathways are potential therapeutic targets in the treatment of neonatal diseases. By reviewing the expression and function of key toll-like receptors, we aim to provide an important framework to understand the functional role of these receptors in response to stress and infection in premature infants.
... Milk-derived sCD14 promotes LPS recognition by TLR4 leading to the secretion of pro-inflammatory cytokines [132]. Finally, human breast milk (especially the breast milk produced during early lactation called colostrum) contains additional unidentified proteins that enhance innate immune recognition by neonatal cells [133]. ...
... Interestingly, miR-146a induction and neonatal epithelial TLR4 tolerization depend on microbial signals (Fig. 3), since TLR4 deficient mice fail to develop epithelial innate immune tolerance [170]. This may explain why milk proteins have been shown to enhance TLR4 recognition ensuring miR-146a upregulation and TLR4 tolerization [133]. On the other hand, caesarian section-born mice maintain epithelial IRAK1 protein expression promoting epithelial stimulation and cell death [170]. ...
Education of our intestinal immune system early in life strongly influences adult health. This education strongly relies on series of events that must occur in well‐defined time windows. From initial colonization by maternal‐derived microbiota during delivery to dietary changes from mother's milk to solid foods at weaning, these early‐life events have indeed long‐standing consequences on our immunity, facilitating tolerance to environmental exposures or, on the contrary, increasing the risk of developing noncommunicable diseases such as allergies, asthma, obesity, and inflammatory bowel diseases. In this review, we provide an outline of the recent advances in our understanding of these events and how they are mechanistically related to intestinal immunity development and education. First, we review the susceptibility of neonates to infections and inflammatory diseases, related to their immune system and microbiota changes. Then, we highlight the maternal factors involved in protection and education of the mucosal immune system of the offspring, the role of the microbiota, and the nature of neonatal immune system until weaning. We also present how the development of some immune responses is intertwined in temporal and spatial windows of opportunity. Finally, we discuss pending questions regarding the neonate particular immune status and the activation of the intestinal immune system at weaning.
... Breast milk provided by healthy and well-nourished mothers is generally regarded as the optimal form of nourishment for full-term infants up to 6 months，which is the source of personalized nutrition and bioactive compounds for proper growth and development of infants and also confers additional beneficial effects such as immunological protection or lesser risk of later allergies. 1 Studies have shown that early life is vulnerable to microbial attacks on the gut. For example, bifidobacteriaceae, a family commonly associated with the gut of breast-fed babies, occurs at high prevalence early in life. 2 Newborn piglets under 2 weeks of age are also susceptible to microbes in breast milk. ...
BACKGROUND
The nutrients in human milk, particularly amino acids and minerals, are important for infant growth and development. Since there are few reports of amino acids and minerals in Chinese breast milk, we conducted this study to establish a representative preliminary database of breast milk nutrients in Chinese breast milk. In this study, we collected breast milk from healthy mothers in seven cities in western, southern and central China. The composition, content and proportion of total amino acids and ten elements (potassium, sodium, calcium, magnesium, iron, zinc, manganese, copper, selenium and phosphorus) in human milk in different lactation stages were investigated.
RESULTS
In this study, it was found that the content of total essential amino acids (671.47 mg 100 mL⁻¹) in Chinese breast milk was higher compared with the European Society for Paediatric Gastroenterology Hepatology and Nutrition (ESPGHAN) (574 mg 100 mL⁻¹), but the content of leucine (LEU) (129.01 mg 100 mL⁻¹) and cysteine (CYS) (20.31 mg 100 mL⁻¹) was much lower than that recommended by ESPGHAN. Moreover, it was found that the content of most of these ten elements decreased during lactation, and the content of calcium in Chinese breast milk was lower compared with ESPGHAN. In addition, the content of selenium (7.23–20.55 mg 1000 mL⁻¹) in breast milk from the three cities Nanchang, Shanghai and Guangzhou in China was much higher than that recommended by ESPGHAN.
CONCLUSIONS
In a word, amino acids and minerals in Chinese human milk showed a significant difference from other countries. Human milk meal or infant food should be regulated to meet the requirements of the infant and to maintain the balance of the amino acids and minerals. © 2020 Society of Chemical Industry
... 61 It has been hypothesized that Bifidobacterium control inflammation through mucosal host-microbe crosstalk. 78 An association was shown between low levels of intestinal Bifidobacterium microbiota during infancy and an increased risk of atopy later in life. [79][80][81] The original source of breast milk microbiota is unclear. ...
The early‐life microbiome is gaining appreciation as a major influencer in human development and long‐term health. Multiple factors are known to influence the initial colonization, development, and function of the neonatal gut microbiome. In addition, alterations in early‐life gut microbial composition is associated with several chronic health conditions such as obesity, asthma, and allergies. In this review, we focus on both maternal and infant factors known to influence early‐life gut colonization. Also reviewed is the important role of infant feeding, including evidence‐based strategies for maternal and infant supplementation with the goal to protect and/or restore the infant gut microbiome.
