Innate Immune Responses of Human Neonatal Cells to Bacteria from the Normal Gastrointestinal Flora

Abstract

The hygiene hypothesis postulates that the prevalence of allergy has increased due to decreased microbial stimulation early in life, leading to delayed maturation of the immune system. The aim of this study was to examine the cytokine pattern produced from cord blood mononuclear cells relative to adult cells after stimulation with bacterial strains from the normal flora. Mononuclear cells from cord and adult blood samples were stimulated with the following bacteria: Bifidobacterium adolescentis, Enterococcus faecalis, Lactobacillus plantarum, Streptococcus mitis, Corynebacterium minutissimum, Clostridium perfringens, Bacteroides vulgatus, Escherichia coli, Pseudomonas aeruginosa, Veillonella parvula, and Neisseria sicca. The levels of interleukin 12 (IL-12), tumor necrosis factor alpha (TNF-␣), IL-10, and IL-6 were measured by enzyme-linked immunosorbent assay. The TNF-␣ production was also analyzed after blocking CD14, Toll-like receptor 2 (TLR-2), and TLR-4 prior to stimulation with bacteria. The levels of IL-12 and TNF-␣ were similar in cord and adult cells. Gram-positive bacteria induced considerably higher levels of IL-12 and TNF-␣ than gram-negative bacteria in both cord and adult cells. The levels of IL-6 were significantly higher in newborns than in adults, whereas the levels of IL-10 were similar in newborns and adults. Gram-negative and gram-positive bacteria induced similar levels of IL-6 and IL-10 in cord cells. L. plantarum bound or signaled through CD14, TLR-2, and TLR-4, whereas E. coli acted mainly through CD14 and TLR-4. These results indicate that the innate immune response in newborns to commensal bacteria is strong and also suggest that different bacterial strains may have differential effects on the maturation of the immune system of infants.

Pattern of Cytokine Responses to Gram-Positive and Gram-Negative Commensal Bacteria Is Profoundly Changed when Monocytes Differentiate into Dendritic Cells

“…1, bacterial stimulation induced low levels of IL-12p70 from both purified monocytes and DCs in the absence of IFN-␥. This contrasted with the high levels obtained from PBMCs or monocytes enriched by adherence, which might be explained by the finding that T cells and their secreted soluble factors primed the APCs for IL-12 production (20,25). As T cells isolated from the lamina propria of the normal human gut spontaneously secrete IFN-␥, we investigated whether IFN-␥ affected the responses of the APCs (26).…”

“…Only few studies have examined how clinical isolates of intact bacteria affect the immune system through the interaction with DCs and monocytes. It has previously been shown that enriched monocytes and PBMCs generally produce higher levels of IL-12 and TNF in response to a panel of gram-positive bacterial strains than in response to gram-negative strains (20,25). Other workers have shown that murine macrophages elicit higher levels of IL-12 in response to intact gram-negative bacteria than in response to gram-positive organisms (29).…”

“…It has previously been shown that gram-positive and gramnegative commensal bacteria have different capacities to induce cytokine production from blood mononuclear cells in both neonates and adults (20,25) and that they also differ in the ability to trigger prostaglandin secretion (21). This indicates that the composition of the flora might be important for how APCs modulate acquired immunity.…”

“…The triggering of APCs through TLRs initiates a signal transduction cascade that culminates in the activation of transcription factors, such as NF-B, which leads to secretion of cytokines and expression of costimulatory molecules. Furthermore, CD14 plays an essential role in the response to microbial components from both gram-positive and gram-negative bacteria (4,25,36). CD14 lacks an intracellular domain and therefore has no signaling capacity, but it acts as a coreceptor for TLRs (4).…”

“…Concentrations of IL-12p70, tumor necrosis factor (TNF), IL-6, and IL-10 in cell culture supernatants were determined by enzymelinked immunosorbent assays (ELISAs). A standard ELISA procedure was used as described in detail elsewhere (25). All antibodies and standards were purchased from Pharmingen (San Diego, Calif.).…”