Combined TLR2 and TLR4 ligation in the context of bacterial or helminth extracts in human monocyte derived dendritic cells: molecular correlates for Th1/Th2 polarization

Abstract

Background: Recognition of pathogens by dendritic cells (DCs) through interaction with pattern recognition receptors, including Toll like receptors (TLR), is crucial for the initiation of appropriate polarized T helper (Th) cell responses. Yet, the characteristics and differences in molecular profiles of DCs with different T cell polarizing capacities are still poorly defined. To address this issue, the molecular profile of human monocyte derived DCs was characterized after exposure to TLR4 ligand LPS in combination with the Th1 promoting bacterial extracts from Listeria monocytogenes and Escherichia coli or the Th2 promoting helminth derived phospholipids from Schistosoma mansoni and Ascaris lumbricoides, all with TLR2 activating capacity.

Results: With regard to the signalling pathways activated upon exposure to LPS and the TLR2 activating compounds, we find that the ratio of activated Mitogen Activated Protein Kinases (MAPK) p-ERK/p-p38 is lower in DCs stimulated with the bacterial products compared to DCs stimulated with the helminth products, which correlates with the Th1 and Th2 polarizing capacity of these compounds. Furthermore, analysis of the mRNA expression levels of a set of 25 carefully selected genes potentially involved in modulation of T cell polarization revealed that the mRNA expression of notch ligand delta-4 and transcription factor c-fos are differentially regulated and show a strong correlation with Th1 and Th2 polarization, respectively.

Conclusion: This study shows that combined TLR2 and TLR4 activation in the context of different antigen sources can induce very distinct molecular profiles in DCs and suggests that the Th1/Th2 polarizing capacity of compounds can be predicted with the molecular signature they induce in DCs.

Figure 1

TLR activation and T-cell polarization by the different compounds. Mass spectrometry analysis of schPS (A) and ascPS (B). Samples were analysed by LC/MSMS in the negative mode. Neutral loss scans of 87 amu, corresponding to the loss of serine from the phospholipid were obtained. The relative intensity is shown of the detected phosphatidylserine species (indicated by their distinct m/z ratios). C. Activation of TLR2 and TLR4 transfected HEK293 cells. HEK cells were stimulated and IL-8 production in response to activation is shown. CD14 transfected HEK cells were used as negative controls (not shown). One representative experiment out of at least two independent experiments is shown, based on triplicate wells. # > 40.000 pg/ml. 1 a.u. is referring to lipids derived from 2 worm pairs/ml or 12 mg of worm/ml for SchPS and AscPS, respectively. D. T cell polarization was determined by measuring the percentages of cells with intracellular IFN-γ and IL-4 production by FACS analysis. T-cell polarization after LPS stimulation alone (4,6 ± 3,5% IL-4 and 33,9 ± 15,1% IFN-γ producing T cells, respectively) was set to 100% (indicated by the bold lines). Relative amounts of IFN-γ and IL-4 positive T cells induced by the stimuli in the presence of LPS are given. Dark gray (left); IL-4, Light grey (right); IFN-γ. Error bars represent SD of the mean of at least 4 independent experiments where cytokines produced in T cells of single wells of cocultures were measured. Significant differences in IL-4/IFN-γ ratio for the different conditions relative to the LPS control are depicted on the right side of the graph. * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 2

MAPK activation in DCs. Representative histograms of (A) ERK and (B) p38 phosphorylation in DCs 20 minutes after stimulation. C-E. Ratios of p-ERK/p-p38 (C), phosphorylation of ERK (D) and p38 (E) 20 minutes after stimulation in the presence of LPS. Expression induced by LPS (MFI of 49 ± 12 for p-ERK and 16 ± 9 for p-p38) was set to 100% (dashed line). Relative expression levels or ratios are shown. * P < 0.05 compared to LPS stimulation. F and G. Correlation of p-ERK/p-p38 ratio and IL-4 (F) or IFN-γ (G) production by T-cells. All data are relative to stimulation with LPS only and combined results from 4 independent experiments are shown.

Figure 3

Distinct mRNA expression levels after stimulation of immature DCs with Th1 and Th2 inducing compounds. A. mRNA expression levels of the different genes compared to LPS (representing a value of 1). Green and red colours represent expression levels higher and lower, respectively, relative to LPS pulsed DCs. Expression was determined by real-time PCR with TAF-1 as housekeeping gene. Stimuli were clustered hierarchically according to expression profiles (top of figure). B and C. Amount of IL-12 p70 and IL-23 present in the supernatant of DCs 48 hours after stimulation, relative to the amount produced in the presence of LPS only (2518 ± 1733 pg/ml IL-12 & 230 ± 195 pg/ml IL-23). ND: not detectable, * p < 0.05, ** p < 0.01 compared to LPS stimulation only. D and E. Association of delta-4 (D) and c-fos (E) mRNA expression levels with T cell polarization for TLR2 activating stimuli. Diamonds represent HKLM or E. coli, circles the helminth derived lipids. Relative expression levels to LPS control condition (set to 1) from at least 3 independent experiments are shown.