Immune Netw.  2014 Feb;14(1):30-37. 10.4110/in.2014.14.1.30.

Co-stimulation of TLR4 and Dectin-1 Induces the Production of Inflammatory Cytokines but not TGF-beta for Th17 Cell Differentiation

• Chang J
• Kim BM
• Chang CH

• KMID: 1508826
• DOI: http://doi.org/10.4110/in.2014.14.1.30

Abstract

Collaboration of TLR and non-TLR pathways in innate immune cells, which acts in concert for the induction of inflammatory cytokines, can mount a specific adaptive immune response tailored to a pathogen. Here, we show that murine DC produced increased IL-23 and IL-6 when they were treated with LPS together with curdlan that activates TLR4 and dectin-1, respectively. We also found that the induction of the inflammatory cytokine production by LPS and curdlan requires activation of IKK. However, the same treatment did not induce DC to produce a sufficient amount of TGF-beta. As a result, the conditioned media from DC treated with LPS and curdlan was not able to direct CD4+ T cells to Th17 cells. Addition of TGF-beta but not IL-6 or IL-1beta was able to promote IL-17 production from CD4+ T cells. Our results showed that although signaling mediated by LPS together with curdlan is a potent stimulator of DC to secrete many pro-inflammatory cytokines, TGF-beta production is a limiting factor for promoting Th17 immunity.

Keyword

TLR; Dectin-1; IL-17; TGF-beta

MeSH Terms

Adaptive Immunity
Cooperative Behavior
Culture Media, Conditioned
Cytokines*
Interleukin-17
Interleukin-23
Interleukin-6
T-Lymphocytes
Th17 Cells*
Transforming Growth Factor beta*
Culture Media, Conditioned
Cytokines
Interleukin-17
Interleukin-23
Interleukin-6
Transforming Growth Factor beta

Figure

• 

  Figure 1 Co-stimulation of DC with LPS and curdlan increased IL-23 production. (A and B) BMDC prepared from C57BL/6 mice were stimulated with LPS (1 µg/ml), curdlan (100 µg/ml), or both. IL-23 was measured by ELISA at 24 h (left panels). mRNA levels of IL-23p19 (middle panels) and IL-12p40 (right panels) were determined at the indicated time by qRT-PCR. (C) IL-10 was measured by ELISA (left panel). IL-23 was measured by ELISA (middle panel). Cells were cotreated with LPS and curdlan in the presence of α-IL-10R (15 µg/ml) or rat IgG1 (15 µg/ml). BMDC prepared from C57BL/6 (Il10+/+) or Il10+/+ mice were treated with LPS, curdlan, or both and IL-23 was measured by ELISA (right panel).

• 

  Figure 2 IKK plays an important role in cytokine expression for both LPS and curdlan treatment. (A) BMDC were pre-treated with indicated inhibitors (10 µM) for 30 min, followed by stimulation with LPS, curdlan, or both for 6 h. IL-6 and IL-10 mRNA levels were determined by qRT-PCR. (B) Cells were treated with LPS (1 µg/ml), curdlan (100 µg/ml), or both for the indicated time. P-TAK1, TAK1, P-IKKα/β, IKKβ, IκBα, P-p38, p38, P-JNK, JNK, P-Erk1/2 and Erk1/2 were detected by immunoblotting.

• 

  Figure 3 Co-treatment of DC with LPS and curdlan was unable to enhance Th17 differentiation. CD4+ T cells from C57BL/6 mice were stimulated with plate-bound anti-CD3 and anti-CD28 in the presence of conditioned media (CM) as described in the Materials and Methods. CM were prepared by treating BMDC with indicated stimulants for 24 h. T cell cultures were then restimulated on day 5 with plate-bound anti-CD3 for 48 h. ELISA was performed to detect IL-17A, IFN-γ and IL-4 production. *UT: CM from untreated DC.

• 

  Figure 4 Exogenous TGF-β but not IL-6 nor IL-1β enhanced IL-17 production in co-stimulated DC. (A) BMDC were stimulated with LPS (1 µg/ml), curdlan (100 µg/ml), or both. Total TGF-β1 and bioactive TGF-β were determined as described in the Materials and Methods. *Media: DC culture media (RPMI+5% serum); UT: CM from untreated DC. (B) CD4+ T cells were stimulated in the presence of CM plus indicated cytokines (IL-6 (40 ng/ml), IL-1β (+: 1 ng/ml, ++: 10 ng/ml), TGF-β (+: 2.5 ng/ml, ++: 5 ng/ml, +++: 25 ng/ml)). IL-17A was measured by ELISA after anti-CD3 restimulation. (C) CD4+ T cells were stimulated in the presence of CM with or without TGF-β (0.5 ng/ml). Cultures were restimulated for 6 h on day 5 with plate-bound anti-CD3 and mRNA levels were assessed by qRT-PCR.

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