Human milk affects TLR4 activation and LPS-induced inflammatory cytokine expression in Caco-2 intestinal epithelial cells.

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. 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 LPS/TLR4 signaling. HM enhances LPS-induced TLR4 signaling as well as LPS-induced IEC gene expression of pro-inflammatory cytokines and negative regulators of NF-κB. Human serum albumin (HSA) present in HM contributes to these effects. HSA within HM synergizes with LPS to induce 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.

Biomarkers of Development of Immunity and Allergic Diseases in Farming and Non-farming Lifestyle Infants: Design, Methods and 1 Year Outcomes in the "Zooming in to Old Order Mennonites" Birth Cohort Study.

Traditional farming lifestyle has been shown to be protective against asthma and allergic diseases. The individual factors that appear to be associated with this "farm-life effect" include consumption of unpasteurized farm milk and exposure to farm animals and stables. However, the biomarkers of the protective immunity and those associated with early development of allergic diseases in infancy remain unclear. The "Zooming in to Old Order Mennonites (ZOOM)" study was designed to assess the differences in the lifestyle and the development of the microbiome, systemic and mucosal immunity between infants born to traditional farming lifestyle at low risk for allergic diseases and those born to urban/suburban atopic families with a high risk for allergic diseases in order to identify biomarkers of development of allergic diseases in infancy. 190 mothers and their infants born to Old Order Mennonite population protected from or in Rochester families at high risk for allergic diseases were recruited before birth from the Finger Lakes Region of New York State. Questionnaires and samples are collected from mothers during pregnancy and after delivery and from infants at birth and at 1-2 weeks, 6 weeks, 6, 12, 18, and 24 months, with 3-, 4-, and 5-year follow-up ongoing. Samples collected include maternal blood, stool, saliva, nasal and skin swabs and urine during pregnancy; breast milk postnatally; infant blood, stool, saliva, nasal and skin swabs. Signs and symptoms of allergic diseases are assessed at every visit and serum specific IgE is measured at 1 and 2 years of age. Allergic diseases are diagnosed by clinical history, exam, and sensitization by skin prick test and/or serum specific IgE. By the end of the first year of life, the prevalence of food allergy and atopic dermatitis were higher in ROC infants compared to the rates observed in OOM infants as was the number of infants sensitized to foods. These studies of immune system development in a population protected from and in those at risk for allergic diseases will provide critical new knowledge about the development of the mucosal and systemic immunity and lay the groundwork for future studies of prevention of allergic diseases.

Traditional Farming Lifestyle in Old Older Mennonites Modulates Human Milk Composition.

Background: In addition to farming exposures in childhood, maternal farming exposures provide strong protection against allergic disease in their children; however, the effect of farming lifestyle on human milk (HM) composition is unknown. Objective: This study aims to characterize the maternal immune effects of Old Order Mennonite (OOM) traditional farming lifestyle when compared with Rochester (ROC) families at higher risk for asthma and allergic diseases using HM as a proxy. Methods: HM samples collected at median 2 months of lactation from 52 OOM and 29 ROC mothers were assayed for IgA1 and IgA2 antibodies, cytokines, endotoxin, HM oligosaccharides (HMOs), and targeted fatty acid (FA) metabolites. Development of early childhood atopic diseases in children by 3 years of age was assessed. In addition to group comparisons, systems level network analysis was performed to identify communities of multiple HM factors in ROC and OOM lifestyle. Results: HM contains IgA1 and IgA2 antibodies broadly recognizing food, inhalant, and bacterial antigens. OOM HM has significantly higher levels of IgA to peanut, ovalbumin, dust mites, and Streptococcus equii as well TGF-ß2, and IFN-λ3. A strong correlation occurred between maternal antibiotic use and levels of several HMOs. Path-based analysis of HMOs shows lower activity in the path involving lactoneohexaose (LNH) in the OOM as well as higher levels of lacto-N-neotetraose (LNnT) and two long-chain FAs C-18OH (stearic acid) and C-23OH (tricosanoic acid) compared with Rochester HM. OOM and Rochester milk formed five different clusters, e.g., butyrate production was associated with Prevotellaceae, Veillonellaceae, and Micrococcaceae cluster. Development of atopic disease in early childhood was more common in Rochester and associated with lower levels of total IgA, IgA2 to dust mite, as well as of TSLP. Conclusion: Traditional, agrarian lifestyle, and antibiotic use are strong regulators of maternally derived immune and metabolic factors, which may have downstream implications for postnatal developmental programming of infant's gut microbiome and immune system.

Immunologic components in human milk and allergic diseases with focus on food allergy.

Human milk contains a wide range of immunomodulatory factors, including immunoglobulins, human milk oligosaccharides, cytokines, microbiome, innate factors and food antigens. Maternal diet can influence the content of human milk as it is well-established that dietary antigens can be secreted in human milk after maternal consumption, but whether these dietary antigens promote tolerance or sensitization in the infant is a subject of debate. This review summarizes the current literature on these immunologically active factors in human milk, including the microbiome, innate factors, and maternal diet-derived dietary antigens in the context of development of allergic diseases, with the focus on food allergy.

Determinants of the regulation of Helicobacter pylori adhesins include repeat sequences in both promoter and coding regions as well as the two-component system ArsRS.

PURPOSE: We investigated the transcription of adhesin-encoding genes sabA, hopZ and labA in Helicobacter pylori strain J99. Each possesses a repeating homopolymeric nucleotide tract within their promoter regions, and sabA and hopZ possess repeats within their 5' coding regions. METHODOLOGY: We altered the repeat lengths associated with the adhesin genes and quantified mRNA levels by real-time quantitative PCR. Using adherence to AGS cells and IL-8 assays, we examined the effects of altered transcript levels. We assessed the role of ArsRS in transcription using an arsS null mutant and by examining ArsR binding to promoter regions via electrophoretic mobility shift assays. RESULTS: Extensions or truncations of promoter region repeats in hopZ and labA increased transcript levels, mirroring results shown by our lab and others for mutations in the sabA promoter. Altered lengths of the poly-cytosine thymine tract within the 5' coding region of sabA demonstrated that switching from phase-off to phase-on significantly increased mRNA levels. However, mutations in the poly-thymine tract of sabA, which increased mRNA levels, do not behave synergistically with phase-on mutations. Phase-on mutations of sabA resulted in increased H. pylori adherence to AGS cells, but only a modest effect on IL-8. hopZ and labA, and sabA paralogue sabB, transcript levels were increased in an arsS mutant and ArsR bound the promoter regions for each of these genes in vitro. CONCLUSION: This work highlights the complex nature of adhesin regulation, its impact on H. pylori attachment and the pervasive role of ArsRS in adhesin expression. Such regulation may help facilitate the decades-long persistence of infection.