Immature neutrophils in cord blood exert increased expression of genes associated with antimicrobial function.

Introduction: The immune systems of both the mother and the newborn face significant challenges during birth. Proper immune regulation after birth is essential for the survival of neonates. Numerous studies have demonstrated that the neonatal immune system is relatively immature, particularly in its adaptive arm, placing the primary responsibility for immune surveillance on innate immunity. Methods: Given the significant role of neutrophils in protecting the neonate after birth, we conducted a study investigating the properties of neutrophils in newborn cord blood using various methodological approaches. Results: Our findings demonstrate the presence of immature low-density neutrophils in the cord blood, which are likely responsible for the observed elevated expression of genes coding for proteins essential to antimicrobial response, including myeloperoxidase, neutrophils elastase, and defensins. Discussion: We propose that these cells function normally and support the protection of newborns early after birth. Furthermore, our results suggest that the mode of delivery might significantly influence the programming of neutrophil function. The presented findings emphasize the importance of distinct neutrophil subpopulations in neonatal immunity and their potential impact on early postnatal health.

Effect of early postnatal supplementation of newborns with probiotic strain E. coli O83:K24:H31 on allergy incidence, dendritic cells, and microbiota.

Introduction: Probiotic administration seems to be a rational approach to promote maturation of the neonatal immune system. Mutual interaction of the microbiota with the host immune system is critical for the setting of appropriate immune responses including a tolerogenic one and thevmaintenance of homeostasis. On the other hand, our knowledge on the modes of actions of probiotics is still scarce. Methods: In our study, probiotic strain Escherichia coli O83:K24:H31 (EcO83) was administered to neonates of allergic mothers (AMs; neonates with increased risk for allergy development) within 48 h after the delivery, and the impact of this early postnatal supplementation on allergy incidence and selected immune markers has been analyzed 10 years after the primary EcO83 administration. Results: We have observed decreased allergy incidence in 10-year-old children supplemented with EcO83 (13 of 52 children were allergic) in comparison with non-supplemented children of AMs (16 of 42 children were allergic). The early postnatal EcO83 supplementation appeared to limit the allergy in the high-risk group (children of AMs) compared to that in the low-risk group (children of healthy mothers). Dendritic cells (DCs) in the peripheral blood of EcO83-supplemented children do not differ significantly in cell surface presence of CD83. The immunomodulatory capacity of EcO83 on DCs was tested in vitro as well. Both directly isolated myeloid and in vitro monocyte-derived DCs from cord blood increased CD83 expression together with interleukin (IL)-10 secretion after EcO83 stimulation. The effect of early postnatal EcO83 supplementation on the microbiota composition of 10-year-old children was characterized by next-generation sequencing, and we have not observed significant changes in the microbiota composition of EcO83-supplemented and non-supplemented children at the age of 10 years. Conclusions: Early postnatal EcO83 supplementation appears to lower allergy incidence in children of AMs. It seems that the beneficial effect of EcO83 is mediated via modulation of DC functional capacities without impacting the microbiota composition. Larger-scale studies will be necessary to confirm these preliminary findings.

Lower Functional and Proportional Characteristics of Cord Blood Treg of Male Newborns Compared with Female Newborns.

Understanding the early events involved in the induction of immune tolerance to harmless environmental antigens and microbiota compounds could reveal potential targets for allergic disease therapy or prevention. Regulatory T cells (Treg), particularly induced Treg (iTreg), are crucial for the induction and maintenance of tolerance against environmental antigens including allergens. A decrease in the number and/or function of Treg or iTreg could represent an early predictor of allergy development. We analyzed proportional and functional properties of Treg in the cord blood of children of allergic mothers (neonates at high risk of allergy development) and healthy mothers (neonates with relatively low risk of allergy development). We observed a higher number of induced Treg in the cord blood of females compared to males, suggesting an impaired capacity of male immunity to set up tolerance to allergens, which could contribute to the higher incidence of allergy observed in male infants. The decreased proportion of iTreg in cord blood compared with maternal peripheral blood documents the general immaturity of the neonatal immune system. We observed a positive correlation in the demethylation of the Treg-specific demethylated region (TSDR) and the proportion of Treg in cord blood. Our data suggest that immaturity of the neonatal immune system is more severe in males, predisposing them to increased risk of allergy development.

Value of cord blood Treg population properties and function-associated characteristics for predicting allergy development in childhood.

Allergic diseases represent some of the most common immunological disorders with high clinical and economic impact. Despite intensive research, there are still few universally accepted and reliable biomarkers capable of predicting their development at an early age. There is therefore a pressing need for identification of potential predictive factors and validation of their prognostic value by correlating them with allergy development. Dysbalance of the branches of immune response, most often excessive Th2 polarization, is the principal cause of allergic diseases. Regulatory T cells (Treg) are a crucial population for the timely establishment of physiological immune polarization and induction and maintenance of tolerance against environmental antigens. This makes them a potentially promising candidate for an early marker predicting allergy development. In our study, we analysed samples of cord blood of children of allergic mothers and children of healthy mothers by flow cytometry and retrospectively correlated the data with clinical allergy status of the children at the age of 6 to 10 years. Studied parameters included cord blood Treg population proportions and functional properties - intracellular presence of IL-10 and TGF-b, MFI of FoxP3. We observed higher percentage of Tregs in cord blood of children who did not develop allergy compared with allergic children. Further, we found higher numbers of IL-10+ Tregs in cord blood of healthy children of healthy mothers than in cord blood of children of allergic mothers and decreased TGF-b+ cord blood Tregs in the group of allergic children of allergic mothers compared to all other groups.

Different immune response of dendritic cells of newborns of allergic and healthy mothers to bacterial stimuli.

Continuous increasing incidence of allergic diseases is calling for identifying early prognostic markers pointing to increased risk of allergy development and establishing protocols for preventive strategies limiting allergy development in predisposed individuals. It is important to better understand the critical events occurring in early postnatal life, especially the interaction of a newborn with microbial compounds important for the maturation of the neonatal immune system and setting immunoregulatory responses as well. Dendritic cells (DC) together with the cytokine microenvironment play an important role in priming of immune responses. The capacity of monocyte-derived DC (moDC) from cord blood of children of healthy and allergic mothers to respond to microbial antigens (Escherichia coli O86 (EcO86) and delipidated Bacillus firmus (DBF)) was tested by flow cytometry and quantitative real-time PCR. Both EcO86 and DBF were able to promote maturation of moDC, but moDC of children of allergic mothers expressed higher levels of activation markers CD80 and CD83. Increased gene expression of IL-6 and lower expression of indol-amine 2,3 dioxygenase were observed in moDC of neonates of allergic mothers, in comparison to healthy ones. A higher gene expression and an increased presence of activation markers on moDC of newborns of allergic mothers indicate a generally higher reactivity of these cells, possibly enabling easier development of inappropriate immune response after an allergen encounter.

Distinct characteristics of Tregs of newborns of healthy and allergic mothers.

Allergic diseases represent a major issue in clinical and experimental immunology due to their high and increasing incidence worldwide. Allergy status of the mother remains the best predictor of an individual's increased risk of allergy development. Dysregulation of the balance between different branches of immune response, chiefly excessive polarization towards Th2, is the underlying cause of allergic diseases. Regulatory T cells (Tregs) play a pivotal role in the timely establishment of physiological immune polarization and are crucial for control of allergy. In our study we used flow cytometry to assess Tregs in cord blood of newborns of healthy (n = 121) and allergic (n = 108) mothers. We observed a higher percentage of Tregs (CD4+CD25+CD127lowFoxP3+) in cord blood of children of allergic mothers. However, the percentage of cells expressing extracellular (PD-1, CTLA-4, GITR) and intracellular (IL-10, TGF-ß) markers of function was lower (significantly for PD-1 and IL-10) within Tregs of these children. Furthermore, Helios- induced Tregs in the cord blood of children of allergic mothers were decreased. These results were supported by a decrease in plasma levels of IL-10 and TGF-ß in cord blood of newborns of allergic mothers, implying lower tolerogenic capacity on the systemic level. Taken together, these findings reflect deficient function of Tregs in the group with higher risk of allergy development. This may be caused by a lower maturation status of the immune system, specifically Tregs, at birth. Such immaturity may represent an important mechanism involved in the increased risk of allergy in children of allergic mothers.

Decreased allergy incidence in children supplemented with E. coli O83:K24:H31 and its possible modes of action.

The growing knowledge of the key role of microbiota in the maturation of neonatal immune system suggests that manipulation of microbiota could be exploited in hampering allergy development. In this study, Escherichia coli O83:K24:H31 (EcO83) was administered to newborns that were followed prospectively. Several immunological characteristics (cytokines, specific IgE, total T regulatory cells (Treg) and subpopulation of natural Treg (nTreg) and induced Treg (iTreg)) were tested in peripheral blood of 8-year-old children. Incidence of allergic disease was decreased in EcO83 supplemented children and significantly elevated levels of IL-10 and IFN-É£ were detected in serum of EcO83 supplemented children. Probiotic supplementation did not influence the numbers of the total Treg population but their functional capacity (intracellular expression of IL-10) was significantly increased in children supplemented with EcO83 in comparison to non-supplemented children. Morover, decreased proportion of iTreg was present in peripheral blood of non-supplemented in comparison to EcO83 supplemented children. Finally, stimulation of cord blood cells with EcO83 promoted both gene expression and secretion of IL-10 and IFN-É£ suggesting that beneficial effect of EcO83 in prevention of allergy development could be mediated by promotion of regulatory responses (by IL-10) and Th1 immune response (by IFN-É£).

Different capacity of in vitro generated myeloid dendritic cells of newborns of healthy and allergic mothers to respond to probiotic strain E. coli O83:K24:H31.

Allergic diseases belong to one of the most common diseases with steadily increasing incidence even among young children. There is an urgent need to identify a prognostic marker pointing to increased risk of allergy development enabling early preventive measures introduction. It has been shown that administration of selected probiotic strains or mixtures could prevent allergy development. In our study, we have tested the capacity of probiotic strain Escherichia coli O83:K24:H31 (E. coli O83) to promote dendritic cell (DC) maturation and polarisation of immune responses. Increased presence of activation marker CD83 was observed on DC stimulated by E. coli O83 and DC of newborns of allergic mothers have significantly more increased cell surface presence of CD83 in comparison to children of healthy mothers. Increased gene expression and secretion of IL-10 was detected in DC stimulated with E. coli O83 being higher in DC of newborns of healthy mothers in comparison to allergic ones. Generally, increased presence of intracellular cytokines (IL-4, IL-13, IFN-gamma, IL-17A, IL-22, IL-10) was detected in CD4+ T cells cocultured with DC of children of allergic mothers in comparison to healthy ones. E. coli O83 primed DC significantly increased IL-10 and IL-17A in CD4 T cells of newborns of healthy mothers in comparison to the levels detected in CD4 T cells cocultured with control non-stimulated DC. We can conclude E. coli O83 induces dendritic cell maturation and IL-10 production in DC. Newborns of allergic mothers have generally increased reactivity of both DC and CD4 T cells which together with decreased capacity of DC of newborns of allergic mothers to produce IL-10 could support inappropriate immune responses development after allergen encounter.