TLR8-mediated activation of human monocytes inhibits TL1A expression

Abstract

TLR play important roles in inflammation and innate immune response to pathogens. TLR8 recognizes ssRNA and induces NF-kappaB via MyD88 signaling. TL1A is a member of the TNF superfamily that markedly enhances IFN-gamma production by IL-12/IL-18-stimulated peripheral and mucosal CD4(+) T cells. TL1A expression is increased in the mucosa of patients with inflammatory bowel disease and is considered a key mediator of Crohn's disease (CD). We have previously shown that TL1A is strongly induced by immune complexes (IC) but not TLR ligands in antigen-presenting cells. However, a potential interaction between these pro-inflammatory signaling pathways has not been investigated. IC-induced TL1A expression of monocytes was potently inhibited by a TLR8 or TLR7/8 ligand (R848) in a dose-dependent manner. Furthermore, when co-cultured with CD4(+) T cells, TLR8 ligands inhibited TL1A production, resulting in almost complete inhibition of IFN-gamma production by the CD4(+) T cells. Furthermore, we demonstrate that IFN-alpha is not required for this suppressive effect by TLR8 signaling. Our data demonstrate for the first time a direct interaction between TLR and TL1A signaling pathways. TLR8 activation may be an important, novel pathway for targeted treatment of Th1-mediated diseases, such as CD.

FIGURE 1. The TLR8 ligand R848 is an efficient inhibitor of IC-induced TL1A expression

CD14+ monocytes were incubated with LPS, Pam₃, Flagellin, or R848 in the presence or absence of IC for 16 h. The control (Co) represents incubation in medium alone. (A) TL1A mRNA was analyzed by real-time PCR. Data are expressed as % of β-Actin mRNA expression. (B) Supernatants were harvested and TL1A protein levels analyzed by ELISA. Means ± SD are shown (n=2). Shown is one representative experiment out of three independent experiments.

FIGURE 2. TLR8 expression and induction of cytokines in IC-stimulated monocytes

(A) Monocytes were isolated from the peripheral blood of healthy donors (n=6) and incubated for 16 h. RT-PCR for TLR8 is shown. The positive control (pos Co) was purchased from InvivoGen. M represents molecular weight markers (B–D) Differential production of cytokines by monocytes isolated from individual donors and stimulated with 3M-002 (TLR8 agonist) or R848 (TLR7/8 agonist) in the presence or absence of IC. The control (Co) represents incubation in medium alone. (B) IL-6 (n=8), (C) TNF-α (n=8), and (D) IL-10 (n=5) levels in supernatants were measured by ELISA. Means are shown as black bars. *, p<0.05; **, p<0.001; ***, p<0.0001 (Student’s t-test).

FIGURE 3

TLR8 ligands inhibit IC-induced TL1A expression. TL1A mRNA, TL1A secretion, and TL1A membrane expression were determined following stimulation of monocytes with 10 μM 3M-002 (TLR8 agonist) or 10 μM R848 (TLR7/8 agonist) in the absence or presence of IC. The control (Co) represents incubation in medium alone. (A) TL1A transcript levels were quantified by real-time RT-PCR and expressed as percent β-actin transcript levels (n=16). (B) TL1A secretion in supernatants was measured by ELISA (n=30). (C) TL1A expression on the surface of monocytes was evaluated by flow cytometry (n=20)and the results presented as % monocytes expressing TL1A. Means are shown as black bars. *, p<0.05; **, p<0.001; ***, p<0.0001 (Student’s t-test).

FIGURE 4

TLR8 agonist and R848 abrogate IC-stimulated monocyte enhancement of IFN-γ production by autologous CD4⁺ T cells. CD4⁺ T cells were isolated and incubated overnight with IL-12 and IL-18. The next day, autologous T cells were either cultured alone or co-cultured in the presence or absence of 3M-002 (TLR8 agonist) or R848 (TLR7/8 agonist) with monocytes that had been preincubated with or without IC for 16 h. The control (Co) represents incubation in medium alone. After 48 h, supernatants were harvested and analyzed for (A) TL1A and (B) IFN-γ production by ELISA. Representative data of two experiments with similar results is shown (n=3).

FIGURE 5. IFN-α is not required for TLR8 inhibition of IC-induced TL1A production

(A) IFN-α and (B) TL1A production in monocytes stimulated with or without anti-IFN-α antibodyor control IgG1 in the presence or absence of IC and 3M-002 (TLR8 agonist) or R848 (TLR7/8 agonist) were measured by ELISA (n=3 per treatment group). (C) IFN-α and (D) TL1Aproduction in monocytes stimulated with or without recombinant IFN-α in the presence or absence of IC and 3M-002 (TLR8 agonist) were measured by ELISA. One representative experiment is shown (n=3). (E) TL1A production in monocytes stimulated with or without anti-IFN-α receptor antibody or control IgG1 in the presence or absence of IC and 3M-002 (TLR8 agonist) or R848 (TLR7/8 agonist) was measured by ELISA. One representative experiment out of two independent experiments is shown (n=3).