IL-4 and IL-13 differentially regulate TLR-induced eosinophil-basophil differentiation of cord blood CD34+ progenitor cells.

Intrauterine environmental exposures have been shown to influence neonatal immunity and subsequent allergic disease development. We have previously shown that fewer lipopolysaccharide (LPS)-stimulated eosinophil-basophil (Eo/B) colonies grow from cord blood (CB) of high-atopic risk infants, compared to low-atopic risk infants. In the present study, we investigated whether a surrogate ex vivo TH2 milieu (i.e., either IL-4 or IL-13) could represent an underlying mechanism to explain our previous findings. CB CD34+ cells from healthy donors were cultured with IL-4 or IL-13 (in combination with LPS) and assessed for Eo/B differentiation using methylcellulose cultures and flow cytometry for related intracellular signalling pathways. Pharmacological inhibitors were added to the methylcellulose cultures to determine the effect of blocking intracellular signalling in CB CD34+ cells in relation to Eo/B colony forming unit (CFU) formation. Stimulation of CD34+ cells with IL-4, but not IL-13, reduced Eo/B CFU formation in the presence of LPS; this was found to be dependent on IL-4Rα and not IL-13Rα1. Additionally, IL-4 reduced the expression of ERK 1/2 after LPS stimulation, which was recovered by inhibition of IL-4Rα. While IL-13 did not have an inhibitory effect on ERK 1/2 expression, inhibition of ERK 1/2 significantly reduced Eo/B CFU formation. Thus, the responsiveness of CB CD34+ progenitor cells to LPS is differentially regulated by the TH2 cytokines, IL-4 and IL-13. This may have implications for in utero interactions between placental-derived pro-allergic cytokines and neonatal progenitor cells influencing Eo/B-mediated inflammatory responses in early life.

Toll-like receptor-mediated eosinophil-basophil differentiation: autocrine signalling by granulocyte-macrophage colony-stimulating factor in cord blood haematopoietic progenitors.

Eosinophils are multi-functional leucocytes that play a role in inflammatory processes including allergy and infection. Although bone marrow (BM) inflammatory cells are the main source of eosinophil-basophil (Eo/B) differentiation-inducing cytokines, a recent role has been demonstrated for cytokine induction through Toll-like receptor (TLR)-mediated signalling in BM progenitors. Having previously demonstrated that cord blood (CB) progenitors induce Eo/B colony-forming units (CFU) after lipopolysaccharide (LPS) stimulation, we sought to investigate the intracellular mechanisms by which LPS induces Eo/B differentiation. Freshly isolated CD34-enriched human CB cells were stimulated with LPS (and/or pharmacological inhibitors) and assessed for alterations in haematopoietic cytokine receptor expression and signalling pathways by flow cytometry, Eo/B CFU in methylcellulose cultures, and cytokine secretion using Luminex assays. The LPS stimulation resulted in a significant increase in granulocyte-macrophage colony-stimulating factor (GM-CSF)-responsive, as opposed to interleukin-5-responsive, Eo/B CFU, which also correlated with significant increases in CD34(+) cell GM-CSFRα expression. Functionally, CB CD34(+) cells secrete abundant amounts of GM-CSF following LPS stimulation, via a p38 mitogen-activated protein kinase (MAPK)-dependent mechanism; this secretion was responsible for Eo/B CFU formation ex vivo, as shown by antibody blockade. We show for the first time that LPS stimulation of CB progenitor cells results in autocrine activation of p38 MAPK-dependent GM-CSF secretion facilitating Eo/B differentiation ex vivo. This work provides evidence that early life exposure to products of bacterial agents can modulate Eo/B differentiation, representing a novel mechanism by which progenitor cells can respond to microbial stimuli and so affect immune and inflammatory responses.

Maternal allergy modulates cord blood hematopoietic progenitor Toll-like receptor expression and function.

BACKGROUND: Little is known regarding the prenatal determinants of innate immune responses in relation to infant allergic risk. Environmental exposures, including microbial stimuli, might predispose susceptible subjects to atopy and asthma in early infancy or even in utero. OBJECTIVE: Because Toll-like receptors (TLRs) recognize microbial products and because cord blood (CB) progenitor alterations have been observed in neonates at risk for atopy, we investigated the expression and function of TLRs on CB hematopoietic progenitors in relation to atopic risk, as defined by maternal allergic sensitization. METHODS: Thirty-two (15 with low and 17 with high atopic risk) infant CB samples were assessed for phenotypic and functional alterations in CD34(+) cells by means of flow cytometry and methylcellulose culture, respectively. CD34(+) hematopoietic progenitors were stained for TLR-2, TLR-4, TLR-9, GM-CSF receptor α, IL-5 receptor α, and IL-3 receptor α or cultured in methylcellulose assays for hematopoietic cytokine-stimulated eosinophil-basophil (Eo/B) colony-forming units (CFUs) with or without LPS. RESULTS: High-atopic-risk infants had significantly lower CB CD34(+) cell TLR-2, TLR-4, and TLR-9 expression (P = .009). High-risk infant progenitors gave rise to significantly more Eo/B CFUs (P = .002) with hematopoietic cytokine (IL-3, IL-5, or GM-CSF) stimulation ex vivo. Although LPS costimulation induced Eo/B CFUs from both low- and high-risk infant CB CD34(+) cells, this response was significantly (P = .020) muted in the high-risk CB progenitors. CONCLUSIONS: Neonatal CB CD34(+) hematopoietic progenitor cell TLR expression and functional responsiveness are altered in CB from atopic at-risk infants. Maternal allergic sensitization might modulate hematopoietic progenitor TLR expression and function in utero; specifically, Eo/B "lineage priming" at birth might be circumvented through engagement of TLR pathways in early life.