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

Abstract

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.

Keywords: CD83; Escherichia coli O83:K24:H31; IL-10; allergy; cord blood; dendritic cell; flow cytometry; probiotic.

Figure 1

Cytokine concentration in cord and peripheral blood sera of children until the age of 10 years. Cytokines were determined by ELISA. Typical Th2 cytokines were determined: IL-4 (A), IL-5 (B), IL-6 (C), and IL-13 (D). The concentration of typical Th1 cytokine (IFN-γ) is shown in (E) Dynamics of concentration of immunoregulatory cytokines is presented in (F) for IL-10 and panel (G) for TGF-β. EcO83, Escherichia coli O83:K24:H31; *p ≤ 0.0071 (adjusted p value). IL, interleukin; IFN, interferon; TGF, transforming growth factor.

Figure 2

The proportion and functional characteristics of Treg in the peripheral blood of 10-year-old children were determined by flow cytometry. The percentage of Treg in the peripheral blood in the three basic groups is shown in (A) Percentages of Treg in children divided according to their own as well as maternal allergy status and appropriate probiotic supplementation are presented in (B) The proportion of Treg positive for cytotoxic T-lymphocyte antigen 4 (CTLA-4) in the three basic groups is indicated in (C) CTLA-4-positive Treg in children divided according to their own as well as maternal allergy status and appropriate probiotic supplementation are presented in (D) The presence of programmed death domain 1 (PD-1) on Treg of children divided into the three basic groups (E) and the six subgroups (F). The cell surface presence of glucocorticoid-induced tumor necrosis factor receptor-related gene (GITR) on Treg in children divided into the three basic groups is indicated in (G) and the six subgroups in (H) N HM, non-supplemented children of healthy mothers; N AM, non-supplemented children of allergic mothers; S AM, E coli O83:K24:H31-supplemented children of allergic mothers; H N HM, healthy non-supplemented children of healthy mothers; H N AM, healthy non-supplemented children of allergic mothers; H S AM, healthy E coli O83:K24:H31-supplemented children of allergic mothers; A N HM, allergic non-supplemented children of healthy mothers; A N AM, allergic non-supplemented children of allergic mothers; A S AM, allergic E coli O83:K24:H31-supplemented children of allergic mothers; Treg, regulatory T cells; *p ≤ 0.0125 (adjusted p value); ***p ≤ 0.00025 (adjusted p value).

Figure 3

Proportion of CD83+ myeloid dendritic cells (mDCs) and plasmacytoid dendritic cells (pDCs) in the peripheral blood of 10-year-old children detected by flow cytometry. The presence of CD83 on mDCs of allergic non-supplemented children of healthy mothers (A N HM), allergic non-supplemented children of allergic mothers (A N AM), and allergic E coli O83:K24:H31-supplemented children of allergic mothers (A S AM) is documented in (A) The proportion of CD83+ mDCs in the peripheral blood of children divided according to their allergic status into the six subgroups is shown in (B) Levels of CD83 on mDCs of children divided only according to their allergic status are presented in (C) The proportion of CD83+ pDCs of N HM, N AM, and S AM is documented in (D) Numbers of CD83+ pDCs in the peripheral blood of children divided according to their allergic status into the six subgroups are shown in (E) The percentage of CD83 on mDCs of children divided only according to their allergic status are presented in (F) H N HM, healthy non-supplemented children of healthy mothers; H N AM, healthy non-supplemented children of allergic mothers; H S AM, healthy E coli O83:K24:H31-supplemented children of allergic mothers; A N HM, allergic non-supplemented children of healthy mothers; A N AM, allergic non-supplemented children of allergic mothers; A S AM, allergic E coli O83:K24:H31-supplemented children of allergic mothers; *p ≤ 0.0167 (adjusted p value). H, healthy children regardless of maternal allergy status or Escherichia coli O83:K24:H31 supplementation; A, allergic children regardless of maternal allergy status or Escherichia coli O83:K24:H31 supplementation.

Figure 4

Median fluorescence intensity (MFI) of CD83 on myeloid dendritic cells (mDCs) and plasmacytoid dendritic cells (pDCs). The MFI of CD83 on mDCs of allergic non-supplemented children of healthy mothers (A N HM), allergic non-supplemented children of allergic mothers (A N AM), and allergic E coli O83:K24:H31-supplemented children of allergic mothers (A S AM) is presented in (A) The MFI of CD83+ mDCs in the peripheral blood of children divided according to their allergic status into the six subgroups is documented in (B) Levels of the MFI of CD83 on mDCs of children divided only according to their allergic status are presented in (C) The MFI of CD83+ pDCs of N HM, N AM, and S AM is documented in (D) The MFI of CD83+ pDCs in the peripheral blood of children divided according to their allergic status into the six subgroups is shown in (E) The MFI of CD83 on pDCs of children divided only according to their allergic status is presented in (F) A N HM, allergic non-supplemented children of healthy mothers; A N AM, allergic non-supplemented children of allergic mothers; A S AM, allergic E coli O83:K24:H31-supplemented children of allergic mothers; *p ≤ 0.00167 (adjusted p value); **p ≤ 0.0033 (adjusted p value); ***p ≤ 0.0003 (adjusted p value). H, healthy children regardless of maternal allergy status or Escherichia coli O83:K24:H31 supplementation; A, allergic children regardless of maternal allergy status or Escherichia coli O83:K24:H31 supplementation.

Figure 5

The effect of E coli O83:K24:H31 (EcO83) on the induction of the activation marker CD83 on myeloid dendritic cells (mDCs), monocyte-derived dendritic cells (moDCs), and plasmacytoid dendritic cells (pDCs) obtained from the cord blood of newborns of healthy and allergic mothers. mDCs isolated directly from cord blood cells were stimulated with EcO83, and the cell surface activation marker CD83 was inspected after 24 h (A). The effect of EcO83 stimulation on the maturation of moDCs is presented in (B) The impact of EcO83 stimulation on the induction of CD83 on in vitro-derived pDCs is shown in (C) C H, non-stimulated DCs of newborns of healthy mothers; C A, non-stimulated DCs of newborns of allergic mothers; EcO83 H, DCs of newborns of healthy mothers stimulated with E coli O83:K24:H31; EcO83 A, DCs of newborns of allergic mothers stimulated with E coli O83:K24:H31; LPS H, DCs of newborns of healthy mothers stimulated with lipopolysaccharide; LPS A, DCs of newborns of allergic mothers stimulated with lipopolysaccharide; *p ≤ 0.05; **p ≤ 0.01.

Figure 6

The impact of E. coli O83:K24:H31 (EcO83) stimulation on cytokine secretion by dendritic cells (DCs), as determined by ELISA. The capacity of EcO83 to promote IL-10 secretion by myeloid dendritic cells (mDCs) is shown in (A). The impact of EcO83 on the production of pro-inflammatory cytokines IL-6 and TNF is shown in (B, C), respectively. The capacity of EcO83 to promote IL-10 secretion by monocyte-derived dendritic cells (moDCs) is shown in (D). Release of IL-6 and TNF by moDC is presented in (E, F, respectively. The effect of EcO83 stimulation on the secretion of IL-10 by pDCs is documented in (G). Concentration of pro-inflammatory cytokines IL-6 and TNF in cell culture supernatants of pDC stimulated with EcO83 and LPS is indicated in (H, I). C H, non-stimulated DCs of newborns of healthy mothers; C A, non-stimulated DCs of newborns of allergic mothers; EcO83 H, DCs of newborns of healthy mothers stimulated with E. coli O83:K24:H31; EcO83 A, DCs of newborns of allergic mothers stimulated with E. coli O83:K24:H31; LPS H, DCs of newborns of healthy mothers stimulated with lipopolysaccharide; LPS A, DCs of newborns of allergic mothers stimulated with lipopolysaccharide; *p ≤ 0.0167 (adjusted p value); **p ≤ 0.0033 (adjusted p value); ***p ≤ 0.00033 (adjusted p value). IL, interleukin; TNF, tumor necrosis factor.