Differential induction of interleukin-10 and interleukin-12 in dendritic cells by microbial toll-like receptor activators and skewing of T-cell cytokine profiles

Abstract

Dendritic cells (DCs) discriminate different microbial pathogens and induce T-cell responses of appropriate effector phenotypes accordingly. Microbial recognition and differentiation are mediated in part by pattern recognition receptors such as Toll-like receptors (TLRs), whereas the development of T-cell effector functions is critically dependent on DC-derived cytokines such as interleukin-12 (IL-12) and IL-10. However, it is not entirely clear to what extent various microbial TLR activators could induce different functional states of DCs that favor different T-cell effector phenotypes. Toward a better understanding of this issue, we examined IL-10 and IL-12 production and T-cell-polarizing potentials of murine bone marrow-derived DCs after stimulation by three microbial TLR activators, namely, lipopolysaccharide (LPS), peptidoglycan (PGN), and zymosan. We found that the three stimuli induced drastically different profiles of IL-10 and IL-12 production in DCs. Further, these stimuli differentially conditioned CD40-dependent IL-10 and IL-12 production by DCs. Finally, LPS-, PGN-, and zymosan-stimulated DCs primed distinct T-cell cytokine profiles. Our results support the notion that microbe-specific information sensed through different TLRs by DCs is linked to differential Th priming through DC-derived cytokines.

Figures

FIG. 1.

Cytokine production by LPS-, PGN-, and zymosan-stimulated DCs. BM-derived BALB/c DCs were stimulated with indicated concentrations of LPS, PGN, or zymosan, and 24-h supernatants were harvested to measure IL-10, IL-12p70, and TNF-α by enzyme-linked immunosorbent assay. None of these cytokines was measurable (

FIG. 2.

IL-10 and IL-12p40 mRNA levels in LPS-, PGN-, and zymosan-stimulated DCs. DCs were stimulated for 12 h with 1 μg of LPS, 10 μg of PGN, or 5 μg of zymosan/ml, and then total RNA was isolated and subjected to RNase protection assay with the RiboQuant multiprobe template set mCK2b. The bands corresponding to IL-12p40, IL-10, and GAPDH (glyceraldehyde-3-phosphate dehydrogenase) are shown. The data represent two independent experiments.

FIG. 3.

IL-10 and IL-12p70 production in microbe-exposed DCs in response to CD40 ligation. (A and B) DCs were stimulated for 24 h with 1 μg of LPS, 10 μg of PGN, or 5 μg of zymosan/ml, together with an anti-CD40 antibody (▨) or control rat immunoglobulin G (□). (C and D) DCs were stimulated with microbial stimuli for 12 h, washed, and then further cultured for 24 h in the presence of the anti-CD40 antibody (▨) or control antibody (□) At the end of these culture periods, supernatants were harvested to measure IL-12p70 (A and C) and IL-10 (B and D). N.D., not detectable.

FIG. 4.

In vitro priming of distinct Th effector phenotypes by LPS-, PGN-, and zymosan-stimulated DCs. BALB/c DCs were left untreated (A) or were stimulated with 1 μg of LPS, 10 μg of PGN, or 5 μg of zymosan/ml for 12 h (B) and then cocultured with 2 × 105 C57BL/6 CD4+ CD45RBhigh T cells (DC/T ratio = 1:10). T cells were harvested 10 days later to enumerate cytokine-producing cells after brief restimulation. (A) T cells primed by untreated DCs were double stained for IFN-γ and IL-4 or for IFN-γ and IL-10. (B) T cells primed by microbe-exposed DCs were stained for individual cytokines separately. Gates were drawn based on isolate controls for individual specific antibodies. The y axis is the fluorescent intensity in log10 scale, whereas the x axis is the linear-scale forward scatter. The data represent five independent experiments.