Newborns are at increased risk of overwhelming infection, yet the mechanisms underlying this susceptibility are incompletely defined. In this study we report a striking 1- to 3-log decrease in sensitivity of monocytes in human neonatal cord blood, compared with monocytes in adult peripheral blood, to the TNF-α-inducing effect of multiple TLR ligands, including bacterial lipopeptides (BLPs), LPS, and the imidazoquinoline compound, imiquimod. In marked contrast, TNF-α release in response to R-848, a TLR ligand that is a congener of imiquimod, was equivalent in newborn and adult blood. Differences in ligand-induced TNF-α release correlated with divergent ligand-induced changes in monocyte TNF-α mRNA levels. Newborn and adult monocytes did not differ in basal mRNA or protein expression of TLRs or mRNA expression of functionally related molecules. Newborn monocytes demonstrated diminished LPS-induced, but equivalent R-848-induced, phosphorylation of p38 mitogen-activated protein kinase and altered BLP- and LPS-induced acute modulation of cognate receptors, suggesting that the mechanism accounting for the observed differences may be localized proximal to ligand recognition by surface TLRs. Remarkably, newborn plasma conferred substantially reduced BLP-, LPS-, and imiquimod-induced TNF-α release on adult monocytes without any effect on R-848-induced TNF-α release, reflecting differences in a plasma factor(s) distinct from soluble CD14. Impaired response to multiple TLR ligands may significantly contribute to immature neonatal immunity. Conversely, relative preservation of responses to R-848 may present unique opportunities for augmenting innate and acquired immunity in the human newborn.

Newborns suffer a higher frequency and severity of microbial infection than older children and healthy middle-aged adults (1). Invasive neonatal infections are associated with high morbidity and mortality, necessitating a conservative diagnostic and therapeutic approach toward newborns presenting with fever or other signs of infection. The relatively poor response of neonates to most vaccines further compounds the challenge of addressing infections in this population. Although immaturity of the acquired immune system at birth has been well described (2), less is known of the relative function of innate immunity in the newborn (3).

Over the past decade, there has been rapid progress in defining the molecular mechanisms by which the human host’s innate immune system recognizes and responds to a variety of microbe-associated molecules (4). These microbial products activate host cells via TLRs (5). In addition to microbial products, the synthetic imidazoquinolines (6), imiquimod and its congener, resiquimod (R-848), activate murine cells via TLR7 (7), whereas in human cells, R-848 also activates via TLR8 (8). Both imiquimod, which has been approved as a topical immunomodulatory therapy for human papilloma virus infection, and R-848 enhance the release of Th1-type cytokines, including TNF-α (9, 10).

Innate immune recognition of microbial products at normally sterile sites such as blood begins with fluid-phase recognition of microbial products by host factors that can greatly enhance or inhibit ligand-induced cellular signaling. For example, by efficiently delivering LPS monomers to the endotoxin receptor complex composed of membrane CD14, TLR4, and myeloid differentiation protein-2, the LPS-binding protein greatly enhances LPS-induced inflammatory responses, accounting for the ability of human plasma/serum to greatly amplify LPS-induced inflammatory activity (11). At higher concentrations, however, LPS-binding protein serves to shuttle LPS to plasma lipoproteins and thereby detoxify it (12). Soluble CD14 (sCD14)3 is also a constituent of human plasma that modulates the activity of LPS on host cells (13). Less is known about plasma factors that may modulate signaling by other TLR ligands.

Engagement of TLRs activates cytosolic signaling via a family of adapter molecules, including MyD88 and Toll-IL receptor domain-containing adaptor protein (TIRAP) (14). After TLR activation, these adapter molecules recruit IL-1R-associated kinase-4, activation of which initiates a cascade leading to phosphorylation of MAPKs, translocation of NF-κB, and consequent transcription of multiple genes, including that encoding TNF-α (15).

Despite substantial progress in understanding TLR-activated signaling at the molecular level, very little is known about the expression and function of these pathways at birth. The newborn immune system has been generally considered functionally immature, and some studies of neonatal and adult leukocytes with respect to release of cytokines upon stimulation in vitro have suggested that newborn responses are impaired (16, 17). However, these studies were largely conducted before the recognition of the crucial role of TLRs in this process and before the definition of the various TLR ligands, including the bacterial lipopeptides and imidazoquinoline compounds (15). Even in the case of LPS, where several studies have explored the relative responses of newborn and adult cells (17), much of the published work was performed before the realization that commercial preparations of LPS are often contaminated with TLR2 ligands (18). A recent study described a correlation between reduced responsiveness of newborn mononuclear cells to LPS and reduced MyD88 expression (19). However, to our knowledge, there are no published reports of the activity of a range of well-defined TLR ligands and expression of their cognate receptors and signaling intermediates in newborn monocytes.

Given the importance of the TLR system in host defense and the paucity of information about its function in human newborns, we evaluated the responses of newborn monocytes to TLR ligands. Whole blood is an attractive, minimally perturbed, model system to assess innate immune recognition in a biologic fluid containing soluble (i.e., plasma components) and cellular (e.g., monocyte) factors. We therefore compared the abilities of newborn and adult blood monocytes to release TNF-α in response to an array of TLR ligands and discovered a substantial and selective impairment in neonatal response to triacylated BLP (tBLP; TLR1/2), mycoplasma-associated lipopeptide (TLR2/6), LPS (TLR4), and imiquimod (TLR7). Remarkably, the response to the imidazoquinoline compound, R-848 (TLR7/8), was fully intact. Mechanistic studies have indicated that the differences between newborns and adults lie early in the TLR pathway, revealing that noncellular components in newborn cord plasma confer greatly reduced ligand-induced TNF-α release to the compounds tested, with the notable exception of R-848.

Peripheral blood was collected from healthy adult volunteers (n = 26 individual volunteers; mean age, 27 years; 45% male and 55% female) and newborn cord blood (n = 63; mean gestational age, 39 wk; 43% male and 57% female) collected immediately after cesarean section delivery (epidural anesthesia) of the placenta or from the umbilical cord immediately after vaginal birth, but before delivery of the placenta. Births at which antibiotics were administered during labor and/or delivery and births to HIV-positive mothers were excluded. Human experimentation guidelines of the U.S. Department of Health and Human Services and Brigham and Women’s Hospital were observed, following protocols approved by the local institutional review board. Blood was anticoagulated with 129 mM sodium citrate (BD Biosciences, Franklin Lakes, NJ). Hemocytes were collected by centrifugation of blood, followed by washing three times with HBSS buffer without magnesium or calcium (Invitrogen Life Technologies, Grand Island, NY) and then resuspension in either autologous or heterologous 100% plasma.

TLR ligands included the synthetic tBLP, Pam3-CSSNA (Bachem Bioscience, King of Prussia, PA) corresponding to the N terminus of a BLP from Escherichia coli B/r (20), the synthetic diacylated BLP macrophage-activating lipopeptide-2 (MALP; S-(2,3-bis-acyloxypropyl)-cysteine-GNNDESNISFKEK; Alexis Biochemicals, Lausen, Switzerland) from Mycoplasma fermentans (21), ultrapure Re 595 LPS from Salmonella minnesota (List Biologicals, Campbell, CA), imiquimod (3M Pharmaceuticals, Northridge, CA), and R-848 (InvivoGen, San Diego, CA). Specificity of individual TLR ligands for their cognate receptors was confirmed using either NF-κB luciferase reporter and TLR cotransfected human embryonic kidney 293 cells or a neutralizing mAb to TLR2 (22), as previously described.

Heparinized blood was layered onto Ficoll-Hypaque gradients, and the PBMC layer was collected and subjected to hypotonic lysis to remove RBC. Monocytes were isolated from PBMC by positive selection using magnetic microbeads coupled to an anti-CD14 mAb according to the manufacturer’s instructions (Miltenyi Biotec, Auburn, CA) and stimulated in the presence of 100% autologous serum.

After incubation of TLR ligands in blood or monocyte suspensions for 5 h at 37°C with end-over-end rotation, samples were diluted with 5 vol of ice-cold RPMI 1640 medium (Invitrogen Life Technologies) and centrifuged at 1000 × g at 4°C for 5 min. The supernatant was recovered and stored at −20°C until assay of TNF-α by ELISA (R&D Systems, Minneapolis, MN).

Both purified recombinant human sCD14 and sCD14 ELISA for measurement of concentrations in citrated newborn and adult plasma or serum were obtained from R&D Systems. For experiments in which sCD14 was replenished in newborn cord blood, either 500 or 1000 ng of pure sCD14 was added/ml whole blood.

Total RNA was isolated using a silica gel-based membrane (RNeasy, Qiagen, Valencia, CA) and treated with DNase (Qiagen) to avoid contamination with genomic DNA. Random-primed cDNA was prepared using an RT kit according to the manufacturer’s instructions (BD Clontech, Palo Alto, CA). TaqMan PCR was performed to measure the relative mRNA levels of TLR or TLR-related molecules as previously described (23), except for TIRAP primers: forward, 5′-CCTGAGCTCCGATTCATGT-3′; probe, FAM-5′-CCCTGATGGTGGCTTTCGTCAA-3′-TAMRA; and reverse, 5′-CGCATGACAGCTTCTTTGA-3′. Bonferroni’s method of statistical analysis for multiple comparisons was used to compare relative mRNA expression in newborn and adult monocytes. Human TNF-α mRNA was measured using specific PreDeveloped Assay Reagents (Applied Biosystems, Foster City, CA).

The total cellular TLR2 content of purified monocytes or control THP-1 cells was measured using a TLR2 ELISA as follows. Maxisorp plates were coated with 0.25 μg/well mAb 2420 in PBS overnight at 4°C. After a brief wash with PBS, plates were incubated with shaking at room temperature in blocking buffer (150 mM NaCl, 10 mM HEPES (pH 7.2), 0.25% BSA, 0.05% Tween 20, 1 mM EDTA, and 0.05% NaN3). Cell lysates were prepared in 1% Triton X-100, 150 mM NaCl, 10% glycerol, 2 mM EDTA, and 25 mM HEPES, pH 7.2, supplemented with a standard protease inhibitor mixture. One hundred microliters of fresh blocking buffer was added to each well, followed by up to 100 μl of sample (balance block solution), and the mixture was incubated at 4°C with shaking overnight. After washing three times with PBS, each well was incubated with 200 μl of mAb 2392:HRP conjugate for 1 h, then washed three times with PBS/0.05% Tween 20 and once with PBS, developed with 100 μl of ABTS solution (Calbiochem, San Diego, CA), stopped with 1 M H2SO4 (100 μl), and measured at 405 nm. The TLR2 ELISA specificity was confirmed by testing lysates prepared from human embryonic kidney 293 cells transiently transfected with plasmids encoding tagged versions of all human TLRs (1, 2, 3, 4, 5, 6, 7, 8, 9, 10), with only TLR2-expressing cells producing a measurable signal.

TLR ligands were added to citrated blood at a final concentration of 100 ng/ml (LPS) or 10 μg/ml (tBLP). In some experiments 10 μg/ml brefeldin A (Sigma-Aldrich, St. Louis, MO) was added to the blood before the TLR ligand to inhibit TNF-α secretion and enhance detection of intracellular TNF-α. Quantitative surface expression of TLRs and CD14 was measured using PE-conjugated mAbs (eBiosciences, San Diego, CA) incubated at room temperature for 30 min. To identify monocytes, samples stained for TLRs with PE-conjugated mAbs were costained for CD14 using an FITC-conjugated CD14 mAb (eBiosciences). After RBC lysis using 1× FACSLyse solution and permeabilization using 1× FACSPerm2 Solution (BD Biosciences), samples were washed with 1× PBS/0.5% human serum albumin. To determine which blood leukocytes synthesize TNF-α in response to TLR ligands, cells were stained for intracellular TNF-α according to the manufacturer’s protocol (BD Biosciences). TNF-α was stained with a PE-conjugated TNF-α mAb using murine IgG1 as control and monocytes were identified using FITC-conjugated CD14 mAb. Phosphorylated p38 MAPK was stained in permeabilized cells using a PE-conjugated phospho-specific (pT180/pY182) p38 mouse IgG1 mAb (clone 36; BD Biosciences). Flow cytometry was performed using a MoFlo cytometer (DakoCytomation, Fort Collins, CO) with a 488-nm laser. Data were analyzed with Summit version 7.19 software (DakoCytomation). To compare intracellular TNF-α production by monocytes in newborn and adult blood, a TNF-α production index was calculated based on the mean fluorescence intensity (MFI): (% of total leukocytes that are monocytes) × (% of monocytes that are TNF-α-positive) × (MFI of TNF-α positive monocytes/MFI of monocytes stained with an isotype control Ab).

To assess the relative abilities of newborn cord blood and adult peripheral blood to mount an innate immune response to microbial products, we tested TLR ligand-induced TNF-α release in whole human blood ex vivo. Remarkably, TNF-α release in response to tBLP, MALP, LPS, and imiquimod was ∼10- to 1000-fold greater in adult blood than in newborn cord blood relative to the ligand dose-response curve (Fig. 1, A–D). In contrast, newborn blood released equivalent amounts of TNF-α in response to R-848 (TLR7/8), which at 1 μg/ml induced ∼30,000 pg/ml in both newborn and adult blood (Fig. 1,E). Because the method of birth (i.e., vaginal vs cesarean section) can alter the production of some cytokines by cord blood mononuclear cells (24), we also examined TLR ligand-induced TNF-α release in cord blood derived from vaginal delivery (Fig. 2). TLR ligand-induced TNF-α release from cord blood derived from vaginal delivery was indistinguishable from that derived from cesarean section delivery, with low tBLP-induced (Fig. 2,A) and low LPS-induced (Fig. 2,B) TNF-α release relative to adult peripheral blood, but preserved R-848-induced TNF-α release (Fig. 2,C). Time-course experiments demonstrated similar kinetics of ligand-induced TNF-α release in newborn and adult blood (Fig. 3), indicating that the differences observed were not due to delayed kinetics of TNF-α release in the newborn. A similar pattern of differences, with relative preservation of R-848-induced TNF-α release, was observed using heparin instead of citrate as anticoagulant (not shown).

FIGURE 1.

Impaired ligand-induced TNF-α release in newborn cord blood in response to BLPs, LPS, and imiquimod, but preserved response to R-848. TNF-α release from newborn cord blood and adult peripheral blood was measured after a 5-h incubation with tBLP (TLR1/2; A), MALP (TLR2/6; B), LPS (TLR4; C), imiquimod (TLR7; D), and R-848 (TLR7/8; E). Ligand structures are indicated above each panel: ▪, N-acyl-S-diacylglycerylcysteine of BLP; open and filled hexagons, Kdo and GlcN sugars of Re595 LPS, respectively. The number of independent determinations (N) is indicated. ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001; ∗∗∗∗, p < 0.0001.

FIGURE 1.

Impaired ligand-induced TNF-α release in newborn cord blood in response to BLPs, LPS, and imiquimod, but preserved response to R-848. TNF-α release from newborn cord blood and adult peripheral blood was measured after a 5-h incubation with tBLP (TLR1/2; A), MALP (TLR2/6; B), LPS (TLR4; C), imiquimod (TLR7; D), and R-848 (TLR7/8; E). Ligand structures are indicated above each panel: ▪, N-acyl-S-diacylglycerylcysteine of BLP; open and filled hexagons, Kdo and GlcN sugars of Re595 LPS, respectively. The number of independent determinations (N) is indicated. ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001; ∗∗∗∗, p < 0.0001.

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FIGURE 2.

Cord blood derived from both cesarean section and vaginal deliveries demonstrates impaired tBLP- and LPS-induced, but preserved R-848-induced, TNF-α release. Blood was incubated with the indicated concentrations of tBLP, LPS, or R-848 for 5 h, then assayed for TNF-α by ELISA. Adult controls are shown for comparison. There were three to five study subjects in each category.

FIGURE 2.

Cord blood derived from both cesarean section and vaginal deliveries demonstrates impaired tBLP- and LPS-induced, but preserved R-848-induced, TNF-α release. Blood was incubated with the indicated concentrations of tBLP, LPS, or R-848 for 5 h, then assayed for TNF-α by ELISA. Adult controls are shown for comparison. There were three to five study subjects in each category.

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FIGURE 3.

Lower magnitude, but similar kinetics, of tBLP-, LPS-, and R-848-induced TNF-α release in newborn cord vs adult peripheral blood. Results are representative of one of three similar experiments.

FIGURE 3.

Lower magnitude, but similar kinetics, of tBLP-, LPS-, and R-848-induced TNF-α release in newborn cord vs adult peripheral blood. Results are representative of one of three similar experiments.

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To determine which cells produced TNF-α in whole blood, flow cytometry for intracellular TNF-α was used. Of the leukocyte populations present in whole blood, monocytes accounted for the vast majority of TNF-α-positive cells in both adult peripheral and newborn cord blood (not shown). Consistent with relative extracellular TNF-α release (Fig. 1), tBLP- and LPS-induced synthesis of intracellular TNF-α in newborn monocytes was relatively low, whereas R-848 induced TNF-α synthesis was at least as great as that in adult monocytes (Fig. 4 A).

FIGURE 4.

Ligand-induced monocyte TNF-α synthesis. A, Relative ligand-induced intracellular TNF-α production by monocytes in blood, as measured by flow cytometry. Whole blood was incubated with tBLP (10 μg/ml), LPS (10 ng/ml), or R-848 (1 μg/ml) for 4 h, then monocytes were stained with a PE-conjugated anti-TNF-α. Intracellular TNF-α production was calculated as described in Materials and Methods (n = 3–4). B, LPS- and R-848-induced TNF-α release from isolated newborn and adult monocytes tested in autologous serum (n = 3). C, Monocyte TNF-α mRNA synthesis in response to LPS and R-848. Whole blood was incubated with buffer, LPS (100 ng/ml), or R-848 (10 μg/ml) for 6 h, and monocyte total RNA was subjected to TNF-α RT-PCR as described in Materials and Methods (n = 3). ∗, p < 0.05.

FIGURE 4.

Ligand-induced monocyte TNF-α synthesis. A, Relative ligand-induced intracellular TNF-α production by monocytes in blood, as measured by flow cytometry. Whole blood was incubated with tBLP (10 μg/ml), LPS (10 ng/ml), or R-848 (1 μg/ml) for 4 h, then monocytes were stained with a PE-conjugated anti-TNF-α. Intracellular TNF-α production was calculated as described in Materials and Methods (n = 3–4). B, LPS- and R-848-induced TNF-α release from isolated newborn and adult monocytes tested in autologous serum (n = 3). C, Monocyte TNF-α mRNA synthesis in response to LPS and R-848. Whole blood was incubated with buffer, LPS (100 ng/ml), or R-848 (10 μg/ml) for 6 h, and monocyte total RNA was subjected to TNF-α RT-PCR as described in Materials and Methods (n = 3). ∗, p < 0.05.

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To further characterize differences in the release of TNF-α from newborn and adult monocytes, we isolated these cells from blood and resuspended them in autologous serum to equivalent concentrations (106 monocytes/ml). Adult monocytes demonstrated greater LPS-induced TNF-α release than newborn monocytes, whereas R-848-induced TNF-α from isolated newborn and adult monocytes was equivalent (Fig. 4 B).

To confirm the observed differences at the level of TNF-α gene expression, we measured TNF-α mRNA in monocytes derived from blood stimulated with LPS (10 ng/ml) or R-848 (1 μg/ml). RT-PCR analysis revealed impaired LPS-induced increases in newborn monocyte TNF-α mRNA, whereas R-848-induced TNF-α mRNA was indistinguishable between newborns and adults (Fig. 4 C). These results suggest that the mechanism for the observed pattern of differences is upstream of TNF-α gene transcription.

To determine whether differences in responsiveness of newborns to TLR ligands correlated with activation of intracellular signaling cascades, we measured phosphorylation of monocyte p38 MAPK in whole blood stimulated with LPS or R-848. Stimulation of blood monocytes with LPS induced rapid phosphorylation of p38 within 10 min in both newborn and adult monocytes (Fig. 5). However, the peak level of LPS-induced p38 phosphorylation was substantially higher in adults than in newborns (Fig. 5,A). In marked contrast, p38 phosphorylation in response to R-848 was similar for newborns and adults (Fig. 5 B), suggesting that the mechanism for the pattern of differences is localized upstream of p38 MAPK phosphorylation.

FIGURE 5.

Phosphorylation of monocyte p38 MAPK upon stimulation of newborn or adult blood with TLR ligands. Newborn or adult whole blood was stimulated with LPS (10 ng/ml; A) or R-848 (1 μg/ml; B) for the indicated times. Intracellular phospho-p38 was detected with a PE-conjugated mAb. Data represent the difference in p38 MFI between stimulated and unstimulated monocytes at each time point. Results are representative of three similar experiments (n = 3).

FIGURE 5.

Phosphorylation of monocyte p38 MAPK upon stimulation of newborn or adult blood with TLR ligands. Newborn or adult whole blood was stimulated with LPS (10 ng/ml; A) or R-848 (1 μg/ml; B) for the indicated times. Intracellular phospho-p38 was detected with a PE-conjugated mAb. Data represent the difference in p38 MFI between stimulated and unstimulated monocytes at each time point. Results are representative of three similar experiments (n = 3).

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One possible explanation for the reduced response of neonatal monocytes to TLR ligands could be the reduced expression of TLRs or TLR-related signaling intermediates. To test this possibility, we prepared total RNA from monocytes derived from unstimulated blood and measured basal mRNA expression of TLRs1–10, myeloid differentiation protein-2, MyD88, TIRAP, IL-1R-associated kinase-4, and CD14 by RT-PCR. To facilitate comparison, results were expressed as a ratio of basal mRNA expression in neonatal monocytes to that in adult monocytes (Fig. 6 A). Relative mRNA levels were close to 1.0 (i.e., no difference) for all the TLRs and TLR-related molecules measured. Relative basal expression of mRNAs encoding TLRs and TLR-related molecules in newborn monocytes was not significantly different from that in adult monocytes (by both individual comparisons and ANOVA).

FIGURE 6.

Basal expression of TLRs and TLR-related molecules in newborn and adult monocytes. A, Basal monocyte mRNA expression by RT-PCR analysis (n = 7–11). B, Basal total TLR2 protein expression by ELISA (n = 3). C, Basal monocyte surface expression of TLR2, TLR4, and CD14 by flow cytometry of whole blood (n = 8–15).

FIGURE 6.

Basal expression of TLRs and TLR-related molecules in newborn and adult monocytes. A, Basal monocyte mRNA expression by RT-PCR analysis (n = 7–11). B, Basal total TLR2 protein expression by ELISA (n = 3). C, Basal monocyte surface expression of TLR2, TLR4, and CD14 by flow cytometry of whole blood (n = 8–15).

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To confirm that the observed mRNA levels correlated to protein levels, we devised an ELISA to measure total TLR2 protein in newborn and adult monocyte lysates. Similar to TLR2 mRNA levels, total TLR2 protein content per 106 monocytes was indistinguishable between newborn and adult cells, both similar to control monocyte-like THP-1 cells (Fig. 6,B). To further assess the basal monocyte surface expression of TLRs and CD14 in newborns and adults, we compared surface staining of monocytes in newborn cord blood or adult peripheral blood by flow cytometry (Fig. 6 C). Basal surface expression of CD14, TLR2, and TLR4 was indistinguishable between newborn and adult monocytes.

Overall, these studies of basal expression suggest that the differences in TLR ligand-induced TNF-α release between newborn and adult blood monocytes are not due to differences in basal expression of TLRs or the TLR-associated molecules measured.

To determine whether adult and newborn monocytes differed with respect to modulation of surface receptor expression upon stimulation, we measured tBLP- and LPS-induced acute changes in monocyte surface expression of cognate receptors in blood. Of note, addition of tBLP to blood was associated with a rise in monocyte surface TLR1 expression at 5 min in adult, but not newborn, blood (Fig. 7,A). Addition of LPS to blood resulted in a sustained reduction in monocyte surface CD14 in newborns, but not adults (Fig. 7 B). These results raise the possibility that adult and newborn responses to these TLR ligands diverge early in the TLR pathway, at or before interaction with the cell surface.

FIGURE 7.

Modulation of TLR and CD14 surface expression upon stimulation of newborn and adult monocytes. A, Percent change in surface expression of monocyte CD14, TLR1, and TLR2 5 min after the addition of tBLP (10 μg/ml) to whole blood (n = 6). ∗. p < 0.05. B, Monocyte surface expression of CD14 after stimulation of whole blood with LPS (100 ng/ml) for the indicated times (n = 3–5). p < 0.05, by ANOVA.

FIGURE 7.

Modulation of TLR and CD14 surface expression upon stimulation of newborn and adult monocytes. A, Percent change in surface expression of monocyte CD14, TLR1, and TLR2 5 min after the addition of tBLP (10 μg/ml) to whole blood (n = 6). ∗. p < 0.05. B, Monocyte surface expression of CD14 after stimulation of whole blood with LPS (100 ng/ml) for the indicated times (n = 3–5). p < 0.05, by ANOVA.

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To determine whether differences in TLR ligand-induced monocyte TNF-α release correspond to the cellular or soluble (i.e., plasma) fraction of blood, we examined TNF-α release from hemocytes in heterologous plasma (e.g., neonatal cells in adult plasma). Washed newborn or adult hemocytes were resuspended in autologous or heterologous plasma. For purposes of comparison, the effect of heterologous plasma (e.g., neonatal cells in adult plasma) on the activity of the TLR ligands tested was expressed as a modulation index with respect to the ligand dose-response curve in the autologous condition (e.g., neonatal cells resuspended in neonatal plasma), as demonstrated in the inset of Fig. 8. Remarkably, newborn plasma conferred diminished release of TNF-α by adult hemocytes in response to tBLP, MALP, LPS, and imiquimod (Fig. 8, □). Conversely, adult plasma greatly enhanced the release of TNF-α from newborn hemocytes in response to each of these ligands (Fig. 8, ▪). The magnitude of modulation of TNF-α release by plasma ranged from 1–3 logs, suggesting that it could account for the differences observed in whole blood (Fig. 1). In marked contrast, R-848-induced TNF-α release was independent of the source of plasma, such that TNF-α release by newborn hemocytes in adult plasma and that by adult hemocytes in newborn plasma were indistinguishable from that by newborn or adult hemocytes in autologous plasma. These results reveal that the differential responses observed in whole blood are due to differences in the soluble components of newborn and adult plasma.

FIGURE 8.

Differences in the ability of newborn and adult plasma to modulate ligand-induced TNF-α release. Newborn or adult hemocytes were washed and resuspended in autologous or heterologous plasma before addition of TLR ligands and measurement of TNF-α release. For the purposes of comparison, the effects of heterologous plasma on ligand-induced TNF-α release were expressed as a modulation index, as shown in the example provided in the inset (method of data analysis). In this example, the presence of adult plasma in the heterologous condition (N cells/A plasma) resulted in amplification of the ligand-TNF-α dose-response curve such that 0.1 μg/ml ligand yielded as much TNF-α release as 10 μg/ml did under the autologous condition (N cells/N plasma), indicating a modulation index of 100 (i.e., 100-fold increased activity in the presence of adult plasma). Such analysis was performed for each of the TLR ligands tested: tBLP, MALP, LPS, imiquimod, and R-848. For all TLR ligands except R-848, adult plasma increased TNF-α release from newborn hemocytes, whereas newborn plasma reduced TNF-α release from adult hemocytes (n = 3–4; p < 0.05, by Mann-Whitney U test for all comparisons except R-848).

FIGURE 8.

Differences in the ability of newborn and adult plasma to modulate ligand-induced TNF-α release. Newborn or adult hemocytes were washed and resuspended in autologous or heterologous plasma before addition of TLR ligands and measurement of TNF-α release. For the purposes of comparison, the effects of heterologous plasma on ligand-induced TNF-α release were expressed as a modulation index, as shown in the example provided in the inset (method of data analysis). In this example, the presence of adult plasma in the heterologous condition (N cells/A plasma) resulted in amplification of the ligand-TNF-α dose-response curve such that 0.1 μg/ml ligand yielded as much TNF-α release as 10 μg/ml did under the autologous condition (N cells/N plasma), indicating a modulation index of 100 (i.e., 100-fold increased activity in the presence of adult plasma). Such analysis was performed for each of the TLR ligands tested: tBLP, MALP, LPS, imiquimod, and R-848. For all TLR ligands except R-848, adult plasma increased TNF-α release from newborn hemocytes, whereas newborn plasma reduced TNF-α release from adult hemocytes (n = 3–4; p < 0.05, by Mann-Whitney U test for all comparisons except R-848).

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Soluble CD14 can modulate the activity of LPS and additional TLR ligands (13, 25) and has been reported to be expressed at lower levels at birth (26), raising the possibility that differences in the sCD14 content of newborn and adult plasma may contribute to the plasma modulating effects observed (i.e., Fig. 8). We therefore measured sCD14 levels in a subset of our study samples and examined whether differences could account for the observed disparities in plasma modulation of ligand-induced TNF-α release. The concentration of sCD14 in newborn cord blood was ∼40% of that in adult peripheral blood plasma (439 ± 59 vs 1109 ± 30 ng/ml; p = 0.003; Fig. 9,A). However, replenishing newborn cord blood by addition of exogenous sCD14 did not enhance neonatal tBLP- or LPS-induced TNF-α release (Fig. 9 B), indicating that the soluble plasma factor(s) accounting for the differences in the effects of newborn and adult plasma on TLR ligand-induced TNF-α release from mononuclear cells is distinct from sCD14.

FIGURE 9.

Differences in sCD14 concentrations between newborn and adult plasma do not account for discrepancies in tBLP- or LPS-induced TNF-α release. A, The concentration of sCD14 is lower in newborn than adult plasma (439 ± 59 vs 1109 ± 30 ng/ml). B, However, addition of either 500 or 1000 ng of purified sCD14/ml newborn blood (i.e., final sCD14 concentration approximating or exceeding that in adults) did not restore tBLP- or LPS-induced TNF-α release. ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001 (by Student’s t test for adult compared with newborn).

FIGURE 9.

Differences in sCD14 concentrations between newborn and adult plasma do not account for discrepancies in tBLP- or LPS-induced TNF-α release. A, The concentration of sCD14 is lower in newborn than adult plasma (439 ± 59 vs 1109 ± 30 ng/ml). B, However, addition of either 500 or 1000 ng of purified sCD14/ml newborn blood (i.e., final sCD14 concentration approximating or exceeding that in adults) did not restore tBLP- or LPS-induced TNF-α release. ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001 (by Student’s t test for adult compared with newborn).

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Despite substantial progress in defining the means by which microbial products activate cytokine release via TLRs, little is known about the expression and function of the TLR system in human newborns, a population at relatively high risk of microbial infection. We directly compared the responses of newborn and adult monocytes to an array of well-defined TLR ligands under physiologic conditions (i.e., whole human blood) and assessed the relative expressions of TLRs and TLR-related signaling molecules. We have discovered marked impairment in TNF-α release from neonatal monocytes in response to ligands for TLR1/2, TLR2/6, TLR4, and TLR7 (Fig. 1). Responses of newborn blood to these TLR ligands are 10- to 1000-fold lower than those of adult blood, a discrepancy that is independent of the mode of delivery (i.e., vaginal vs cesarean section; Fig. 2) and not due to any differences in the kinetics of ligand-induced TNF-α release (Fig. 3). Of note, these differences are evident despite the higher concentration of monocytes in newborn cord blood (i.e., corrected per monocyte, the observed differences are even greater). In marked contrast, responses to the imidazoquinoline compound, R-848, which is known to activate host cells via TLR7/8, were equivalent in newborn and adult blood (Fig. 1 E), suggesting that the pathway by which R-848 induces monocyte TNF-α release is fully intact in human newborns.

The strength of the whole blood assay system, namely the presence of multiple cellular and soluble modulators of TLR ligands, also presents an important challenge: dissecting the mechanism for the observed differences. We have established that 1) TLR ligand-induced TNF-α in whole blood is largely synthesized by monocytes, and differences in release correlate with differences in intracellular TNF-α synthesis; 2) TLR ligand-induced TNF-α mRNA levels correlate with TNF-α protein synthesis and release, indicating a mechanism upstream of TNF-α mRNA synthesis; 3) the divergence of LPS- and R-848-induced phosphorylation of p38 MAPK suggests that the mechanism for the differences is located upstream of MAPK phosphorylation; 4) the similarity in the measurements of basal expression suggest that the differences observed are not due to differences in basal expression of TLRs or the TLR-related molecules measured; 5) differences in sBLP- and LPS-induced modulation of cognate receptors suggest the divergence of newborn and adult TLR pathways upstream of cell surface recognition events; and 6) the pattern of differences in TLR ligand-induced TNF-α release is the result of differences between newborn and adult blood plasma.

Adult plasma increased TLR ligand-induced TNF-α release from newborn hemocytes, whereas newborn plasma diminished TNF-α release from adult hemocytes, suggesting either that newborn plasma is deficient in a factor that enhances ligand-induced TLR activation or that it contains an inhibitor. Although levels of sCD14 in newborn plasma are only ∼40% of those in adults (Fig. 9,A), repletion experiments demonstrate that these differences in sCD14 concentration do not account for the divergent effects of newborn and adult plasma on TLR ligand-induced TNF-α release (Fig. 9 B). Addition of sCD14, which can neutralize LPS by shuttling it to plasma lipoproteins (13), to newborn blood actually reduced LPS-induced TNF-α release.

Remarkably, newborn and adult plasmas were indistinguishable with respect to their effects on R-848-induced TNF-α release. The preservation of responses to R-848 in newborn plasma is particularly striking in light of its close chemical similarity to its congener imiquimod (see structures in Fig. 1, D and E). R-848 possesses ethanol and ethoxymethyl groups that render it substantially more soluble in aqueous solvents than imiquimod, possibly accounting for its higher potency in animal models of viral infection (10). Because the mechanism(s) by which human plasma modulates signaling by BLPs and imidazoquinolines has yet to be defined, our study sets the stage for the challenge of determining the plasma components responsible for the observed differences.

Yan et al. (19) have recently studied LPS-induced TNF-α production in neonatal mononuclear cells. Several aspects of their study are consistent with ours, including 1) diminished LPS-induced TNF-α release from neonatal mononuclear cells, 2) similar basal expression of TLR4 in neonatal and adult mononuclear cells, and 3) upon LPS stimulation, relatively lower surface CD14 in neonatal monocytes. The authors also noted reduced expression of MyD88 (∼60% of adult levels) and suggested that this reduced expression may contribute to the reduced neonatal response to LPS. Our study does not exclude a contribution of diminished MyD88 protein expression to impaired neonatal responses to TLR ligands, but demonstrates that the differences observed in whole blood (Fig. 1) are largely accounted for by dramatic differences (∼1–3 logs) in the ability of newborn vs adult plasma to modulate TLR ligand-induced TNF-α release (Fig. 8).

What is the biological reason for the presence in newborns of blood plasma conferring reduced monocyte TNF-α responses to multiple TLR ligands? Because inflammation is known to be an important trigger of premature delivery and its attendant complications (27), we speculate that there may be a selective advantage to having muted neonatal TLR-mediated responses in utero to reduce the risk of inflammatory reactions to maternal and microbial molecules. Such plasma-mediated quenching of signaling via TLRs may protect against inflammation, but may also leave the fetus and newborn at relatively high risk of infection.

The susceptibility of newborns heightens the need to prevent infection in this population, but, unfortunately, poor neonatal memory responses have frustrated efforts to achieve effective early vaccination. TLR-based recognition is an important means by which adjuvants activate APCs to initiate adaptive immune responses. Accordingly, TLR ligands such as MALP have been used as vaccine adjuvants (28, 29, 30). Our data raise the possibility that the failure of immunization at birth may relate in part to impaired TLR-based responses to Ags, and that adjuvants chemically similar to BLPs, LPS, or imiquimod may function suboptimally in the newborn, resulting in poor lymphocyte responses to Ags.

By revealing impairment of neonatal monocyte TNF-α release in response to an array of TLR ligands and localizing this lesion to the properties of newborn plasma, our study represents a substantial advance in our understanding of innate immune function at birth. In addition, we identify R-848 as an imidazoquinoline immunomodulator that may be uniquely efficacious as adjunctive therapy of acute infection and/or as a vaccine adjuvant in human newborns. Because imidazoquinolines have been recently approved for the topical treatment of human papilloma virus infection (6), have potential activity as topical or systemic agents for additional viral and parasitic infections (10), and possess vaccine adjuvant activity (31), further study of R-848’s immunostimulatory activity on neonatal cells in vitro and on neonatal animals in vivo is indicated.

We thank Drs. Richard Malley and Dennis Kasper for advice and support, and Dr. Steve Carroll for critical review of the manuscript. Rochelle Jean-Jacques, Jennifer Christianson, Dimitri Sigounas, Mandana Farhadi, and Melissa Coughlin provided expert technical assistance.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1

This work was supported in part by National Institutes of Health Grant KO8AI50583 (to O.L.), National Institutes of Health Contract N01AI25495, and unrestricted grants from Baxter Pharmaceuticals and XOMA (U.S.) LLC (to O.L.).

3

Abbreviations used in this paper: sCD14, soluble CD14; BLP, bacterial lipopeptide; MALP, macrophage-activating lipopeptide; MFI, mean fluorescence intensity; tBLP, triacylated bacterial lipopeptide; TIRAP, Toll-IL receptor domain-containing adaptor protein.

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