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. 2013 Mar;20(3):427-32.
doi: 10.1128/CVI.00703-12. Epub 2013 Jan 23.

Blueprints of signaling interactions between pattern recognition receptors: implications for the design of vaccine adjuvants

Kim Timmermans  1 , Theo S Plantinga, Matthijs Kox, Michiel Vaneker, Gert Jan Scheffer, Gosse J Adema, Leo A B Joosten, Mihai G Netea
Affiliations

Blueprints of signaling interactions between pattern recognition receptors: implications for the design of vaccine adjuvants

Kim Timmermans et al. Clin Vaccine Immunol. 2013 Mar.

Abstract

Innate immunity activation largely depends on recognition of microorganism structures by Pattern Recognition Receptors (PRRs). PRR downstream signaling results in production of pro- and anti-inflammatory cytokines and other mediators. Moreover, PRR engagement in antigen-presenting cells initiates the activation of adaptive immunity. Recent reports suggest that for the activation of innate immune responses and initiation of adaptive immunity, synergistic effects between two or more PRRs are necessary. No systematic analysis of the interaction between the major PRR pathways were performed to date. In this study, a systematical analysis of the interactions between PRR signaling pathways was performed. PBMCs derived from 10 healthy volunteers were stimulated with either a single PRR ligand or a combination of two PRR ligands. Known ligands for the major PRR families were used: Toll-like receptors (TLRs), C-type lectin receptors (CLRs), NOD-like receptors (NLRs), and RigI-helicases. After 24 h of incubation, production of tumor necrosis factor alpha (TNF-α), interleukin-1 beta (IL-1β), IL-6, and IL-10 was measured in supernatants by enzyme-linked immunosorbent assay (ELISA). The consistency of the PRR interactions (both inhibitory and synergistic) between the various individuals was assessed. A number of PRR-dependent signaling interactions were found to be consistent, both between individuals and with regard to multiple cytokines. The combinations of TLR2 and NOD2, TLR5 and NOD2, TLR5 and TLR3, and TLR5 and TLR9 acted as synergistic combinations. Surprisingly, inhibitory interactions between TLR4 and TLR2, TLR4 and Dectin-1, and TLR2 and TLR9 as well as TLR3 and TLR2 were observed. These consistent signaling interactions between PRR combinations may represent promising targets for immunomodulation and vaccine adjuvant development.

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Figures

Fig 1
Fig 1
Interaction classifications. On the y axis, production of a cytokine is depicted. On the x axis, the ligands (A and B) used to stimulate the PBMCs are depicted; PBMCs were stimulated with either a single ligand or a combination of two PRR ligands. To identify a synergistic effect, the cytokine production after stimulation with both ligands combined should be higher than the combined values of the two single effects. An inhibitory effect was defined as a cytokine response to a combination of ligands that was maximally 0.75-fold of the sum of the cytokine responses to either of the individual ligands and lower than the cytokine response to a single ligand or to both of the ligands. A cytokine production of the combination of stimuli higher than production of each of the single stimulations, but not higher than the sum of the two, is considered additive and is thus classified as “no effect/additive effect.”
Fig 2
Fig 2
Overview of interaction PRRs. Color indicates type of interaction in at least 7 of 10 of the healthy volunteers. Data represent results of an ELISA on culture supernatants after 24 h of stimulation at 37°C with combinations of ligands as specified in Table 1. *, P < 0.05 (Wilcoxon matched-pair test, comparison of cytokine production upon stimulation with both ligands with sum of cytokine production values upon stimulation with each of the ligands separately). Red, synergistic effect; green, no effect/additive effect; blue, inhibitory effect; white, variable effect; black, experiment not performed.
Fig 3
Fig 3
Flagellin interactions. PBMCs were stimulated for 24 h with the indicated ligand or combinations of ligands, and cytokines were measured in the supernatants by ELISA (n = 10 volunteers). The bars indicate a synergistic interaction of flagellin with CpG or poly(I·C) for the production of IL-1β. Data are expressed as medians with interquartile ranges. *, significantly different (P < 0.05) from stimulation with single ligands and the sum of both single ligands; #, significantly different (P < 0.05) from no-ligand (RPMI) results (Wilcoxon matched-pair test).
Fig 4
Fig 4
Dose response. LPS plus Pam3Cys PBMCs were stimulated for 24 h with the indicated ligand or combinations of ligands, and cytokines were measured in the supernatants by ELISA (n = 2 volunteers). The bars indicate different concentrations of Pam3Cys, while increasing concentrations of LPS are depicted on the x axis. Increasing concentrations of LPS and Pam3Cys resulted in increased IL-1β and TNF-α production for single ligands, but no synergistic production of either IL-1β or TNF-α was observed when LPS and Pam3Cys were combined. Data are depicted as medians.
Fig 5
Fig 5
Interindividual variation. Data represent TNF-α production after 24 h of PBMC stimulation with MDP, LPS, or MDP and LPS (n = 10 volunteers). Five individuals exhibited no interaction, three demonstrated an inhibitory effect, and two produced TNF-α in a synergistic manner after combined stimulation.
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