TLR2 and TLR4 stimulation differentially induce cytokine secretion in human neonatal, adult, and murine mononuclear cells

Abstract

Toll-like receptor 2 (TLR2) and TLR4 signaling may induce differential secretion of T helper 1 (Th1) and Th2 cytokines, potentially influencing the development of autoimmune or atopic diseases. To date, the influence of the type of stimulus, timing, and dose of TLR2 and TLR4 ligands on cytokine secretion has not been well established. We tested whether the innate stimuli peptidoglycan (Ppg, TLR2 agonist) and lipid A (LpA, TLR4 agonist) differentially affect the secretion of interleukin-13 (IL-13) (Th2) and interferon-gamma (IFN-gamma) (Th1). Further, we examined the influence of the maturity of the immune system, species, dose, and timing of stimuli in human cord and adult peripheral blood mononuclear cells (PBMC) and murine cells in vitro and in vivo. Stimulation with Ppg induced the secretion of both IL-13 and IFN-gamma, influenced by time and dose in neonates, adults, and mice. In contrast, stimulation with LpA induced primarily time-independent and dose-independent production of IFN-gamma. Pulmonary administration of Ppg in vivo in mice resulted in secretion of IL-13, whereas administration of LpA resulted in secretion of IFN-gamma in bronchoalveolar lavage (BAL). Therefore, TLR2 and TLR4 stimuli differentially influence IL-13 and IFN-gamma secretion in neonates, adults, and mice, supporting a critical role for innate stimuli in the modulation of cytokine responses.

Copyright Mary Ann Liebert, Inc.

Figures

FIG. 1

Lymphocyte proliferation following innate stimulation in WT and TLR2 KO mice. Stimulation with LpA (TLR4) induced lymphocyte proliferation in WT as well as TLR2 KO mice. In contrast, stimulation with Ppg (TLR2) induced lymphocyte proliferation only in WT but not in TLR2 KO mice. Lymphocyte proliferation was determined as stimulation index (SI) following stimulation of murine spleen cells with LpA and Ppg (both 100 μg/ml) for 72 h by 3H-thymidine uptake, as described in Materials and Methods (n = 5 mice per group).

FIG. 2

CBMC IL-13 and IFN-γ secretion following innate stimulation. (A) Secretion of IL-13 by CBMC following stimulation with Ppg was increased compared with secretion following LpA stimulation (p < 0.001). Both stimuli induced higher levels of IL-13 secretion than in unstimulated cells (M) (p = 0.003 and p < 0.001). (B) CBMC stimulated with LpA and Ppg showed higher levels of IFN-γ secretion than in un-stimulated CBMC (p = 0.002 and p = 0.001). (C) Lymphocyte proliferation was intact at 72 h after stimulation with LpA and Ppg. (A and B) Cytokine concentrations from supernatants of CBMC harvested 72 h after stimulation with LpA (0.1 μg/ml) and Ppg (10 μg/ml) were measured with ELISA. (C) Lymphocyte proliferation was determined following stimulation with the indicated doses of LpA and Ppg for 72 h by 3H-thymidine uptake, as described in Materials and Methods (n = 30 for M and Ppg, n = 15 for LpA).

FIG. 3

Comparison of IL-13 and IFN-γ secretion in CBMC and PBMC following administration of different innate stimuli. Secretion of (A) IL-13 and (B) IFN-γ in cord blood (n = 30 for M and Ppg, n = 15 for LpA) and in adults (n = 7) was similar after stimulation with LpA and Ppg. Cytokine concentrations in supernatants of PBMC harvested 72 h after stimulation with LpA (0.1 μg/ml) and Ppg (10 μg/ml) were measured using ELISA.

FIG. 4

Secretion of IL-13 and IFN-γ by isolated murine spleen cells. Secretion of (A) IL-13 and (B) IFN-γ by isolated murine spleen cells was higher after stimulation with Ppg compared with cells stimulated with LpA or LPS and unstimulated cells (M) (p < 0.001). IFN-γ secretion after stimulation with LpA and LPS was increased compared with secretion by un-stimulated cells (M) (p < 0.01). (C) Lymphocyte proliferation was intact following stimulation with Ppg, LpA, and LPS. (A, B, and C) Cytokine concentrations from supernatants of spleen cells harvested at 44 h after stimulation with 10 μg/ml Ppg, 100 μg/ml LpA, and 10 μg/ml LPS were measured using ELISA. Lymphocyte proliferation was determined following stimulation with the indicated doses of Ppg, LpA, and LPS for 44 h by 3H-thymidine uptake, as described in Materials and Methods (n = 6 mice per group).

FIG. 5

Dose-dependent and time-dependent effects on secretion of IL-13 following stimulation with Ppg and LpA in human adult PBMC. (A) IL-13 secretion induced by Ppg stimulation was dose dependent and significantly higher with 10 μg/ml Ppg than in un-stimulated cells (p = 0.03). (B) IL-13 secretion induced by LpA stimulation was independent of dose. (C) IL-13 secretion of un-stimulated cells (M) and following LpA stimulation was independent of time. IL-13 secretion was higher after 72 h of stimulation with 10 μg/ml Ppg than after 24 and 16 h of stimulation (p < 0.01). (A, B, and C) Cytokine concentrations from supernatants of adult PBMCs were harvested at the indicated times after stimulation with the indicated doses (μg/ml) of Ppg and LpA and measured using ELISA (n = 7).

FIG. 6

Dose-dependent and time-dependent effects on secretion of IL-13 and IFN-γ following stimulation with Ppg, LpA, and LPS in murine spleen cells. (A and E) Higher doses of Ppg resulted in higher levels of IL-13 and IFN-γ secretion (p < 0.01). (B, C, F, and G) IL-13 and IFN-γ secretion induced by LpA and LPS was independent of dose. (D and H) IL-13 and IFN-γ secretion increased from 22 to 44 h after stimulation with Ppg (p < 0.01) and was similar after stimulation with LpA and LPS. (A, B, C, D, E, F, G, and H) Cytokine concentrations from supernatants of murine spleen cells harvested at 44 h (A, B, C, E, F, and G) or at 22 h and 44 h after stimulation with the indicated doses (μg/ml) of Ppg, LpA and LPS (D and H) were measured with ELISA (n = 6 mice per group).

FIG. 7

Pulmonary stimulation with LpA and Ppg in vivo. (A and B) Intratracheal (i.t.) stimulation with Ppg induced higher levels of IL-13 in BAL fluid in mice compared with stimulation with LpA and PBS, whereas IFN-γ secretion was higher following stimulation with LpA compared with Ppg and PBS (p < 0.01). Mice underwent i.t. administration of Ppg (75 μg), LpA (75 μg), or PBS (0.2 ml of 0.5× PBS), and BAL was performed 18 or 42 h later, as previously described.(26,27) Cytokine concentrations from supernatants of BAL cells were measured with ELISA as described in Materials and Methods (n = 6 mice per group). (C) AHR (Penh) was increased in OVA-sensitized and challenged mice compared with PBS mice. OVA mice treated with LpA i.t. showed significantly decreased Penh compared with OVA mice. AHR was determined by Penh measured at baseline and after metacholine challenge (100 mg/ml) in a murine model of allergic sensitization and challenged with OVA, as described in Materials and Methods (n = 6 mice per group).