Morphine inhibits murine dendritic cell IL-23 production by modulating Toll-like receptor 2 and Nod2 signaling

Abstract

IL-23, produced by dendritic cells (DCs) and macrophages, plays a critical role in innate immunity against bacterial infection. Our previous studies show that morphine disrupts the IL-23/IL-17 mediated pulmonary mucosal host defense and increases susceptibility to Streptococcus pneumoniae lung infection. To determine the mechanism by which morphine modulates IL-23 production, mouse bone marrow-derived dendritic cells (BMDCs) and macrophages (BMDMs) were treated with morphine, and infected with S. pneumoniae or stimulated with Toll-like receptor (TLR) and Nod2 ligands. We found that a significant increase in IL-23 protein production was observed in S. pneumoniae, TLR2 ligand lipoteichoic acid (LTA), and TLR4 ligand pneumolysin (PLY) stimulated BMDCs and BMDMs. Interestingly, although Nod2 ligand muramyldipeptide (MDP) alone had no effect on IL-23 production, it potentiated LTA induced IL-23 production to the same level as that observed following S. pneumoniae infection, suggesting that S. pneumoniae induced IL-23 production in DCs involves activation of both TLR2 and Nod2 signaling mechanisms. Furthermore, pretreatment of DCs with MyD88 (myeloid differentiation primary response gene 88) and IL-1 receptor-associated kinase (IRAK) 1/4 inhibitors, or TLR2 antibody diminished the S. pneumoniae induced IL-23 and abolished the inhibitory effects of morphine, indicating that S. pneumoniae induced IL-23 production depends on activation of the TLR2-MyD88-IRAK1/4 signaling pathway. Moreover, morphine decreased S. pneumoniae induced phosphorylation of interferon regulatory factor 3 (IRF3) and activating transcription factor 2 in DCs. Taken together, our study shows that morphine impairs S. pneumoniae induced IL-23 production through MyD88-IRAK1/4-dependent TLR2 and Nod2 signaling in DCs.

FIGURE 1.

S. pneumoniae infection and TLR2/4 activation induced IL-23 production in BMDCs and BMDMs. Mouse BMDCs and BMDMs (1 × 10⁶) were infected with S. pneumoniae (m.o.i., 20:1) or stimulated with TLR ligands (5 μg/ml LTA, 1 μg/ml rPLY, 10 μg/ml CpG) or Nod2 ligand (5 μg/ml MDP) for 6 h, and IL-23 concentrations were measured in the supernatant by ELISA. *, p < 0.05; **, p < 0.01, compared with unstimulated controls (U.C.).

FIGURE 2.

Effect of morphine treatment on IL-23 promoter activity, mRNA expression, and protein synthesis in BMDCs infected with S. pneumoniae. A, BMDCs were transfected with 5 μg of the pGL3B-IL-23p19 firefly luciferase reporter plasmid and co-transfected with 0.5 μg of pRL-TK plasmid-driving Renilla luciferase. After 2 h of transfection, cells were treated with vehicle or morphine (1 nm–1 μm) for 24 h prior to infection with S. pneumonia (S. pn). Lysates were harvested after 1 h and assayed for firefly and Renilla luciferase activity using a Dual-Luciferase reporter kit. Data are presented as relative light units after correction for transfection efficiency by normalization with pRL-TK driving Renilla luciferase. B, BMDCs were treated with low and high dose morphine for 24 h and then infected with S. pneumoniae (m.o.i., 20:1) for 2 h. mRNA expression of IL-23 was determined by real-time PCR. C, BMDCs from WT and μ-opioid receptor knock-out (MORKO) mice were treated with low and high dose morphine for 24 h and then infected with S. pneumoniae (m.o.i., 20:1) for 6 h. The protein expression of IL-23 in culture supernatant was measured by enzyme-linked immunosorbent assay. Data shown are the mean ± S.E. of three separate experiments. Statistical differences were determined by a factorial analysis of variance followed by an unpaired t test. *, significance at level p < 0.05; **, significance at level p < 0.01 compared with the control group.

FIGURE 3.

Effect of morphine treatment on IL-23 production by BMDCs in response to Nod2 and various TLR ligands. A, BMDCs were treated with morphine (1 μm) or vehicle for 24 h and then infected with S. pneumoniae (S. pn) or stimulated with rPLY (1 μg/ml), LTA (5 μg/ml), CpG (10 μg/ml), MDP (5 μg/ml), or PBS as a unstimulated control (UC) for 6 h. IL-23 production was determined by ELISA. B, BMDCs were preincubated with anti-TLR2 (10 μg/ml) or isotype control antibody 2 h before S. pneumoniae infection. After 6 h of in vitro cell infection, supernatant of cell culture was collected and used to determine IL-23 production. *, p < 0.05; **, p < 0.01, compared with vehicle controls. Results are representative of three independent experiments.

FIGURE 4.

Effect of morphine treatment and S. pneumoniae infection on TLRs expression in BMDCs. BMDCs were treated with morphine (1 μm) or vehicle for 24 h. Then, cells were infected with S. pneumoniae (m.o.i., 20:1) for 6 h. TLR expression was analyzed by flow cytometry assay. Similar results were obtained in three independent experiments. Gray dashed line, isotype control; gray line, vehicle + unstimulated control; black line, 1 μm morphine; light gray line, S. pneumoniae infection; black dashed line, 1 μm morphine + S. pneumoniae infection.

FIGURE 5.

MyD88-IRAK1/4 signaling pathway participated in the morphine treatment induced inhibitory IL-23 production. A and B, to inhibit MyD88-dependent signaling, cells were pretreated with MyD88 homodimerization inhibitory peptide or control peptide at 24 h before in vitro cell infection with S. pneumoniae (S. pn). C, BMDCs were preincubated with IRAK1/4 inhibitor (50 μm) or control vehicle 2 h before cell infection with S. pneumoniae. IL-23 concentration was measured in the supernatant by ELISA. **, p < 0.01, compared with the vehicle controls, or comparisons indicated by brackets. Results are representative of three independent experiments. ns, not significant.

FIGURE 6.

Effect of morphine treatment on phosphorylation of IRF3 and ATF-2 following infection with S. pneumoniae or stimulation with TLR and Nod2 ligands. Immunofluorescence staining reveals the subcellular distribution of IRF3 and ATF2 after infection with S. pneumoniae and stimulation with TLR or Nod 2 ligands. Of note, IRF3 and ATF2 were stained with green labeled secondary antibody. Original magnification was ×400.

FIGURE 7.

Role of cAMP in the regulation of IL-23 production by morphine in DCs following S. poneumoniae infection. A, morphine treatment resulted in an increase in intracellular cAMP. BMDCs were treated with morphine or vehicle for 24 h and then infected with S. pneumoniae (S. pn) for 30 min. The levels of intracellular cAMP were determined by competitive enzyme immunoassay. B, forskolin inhibited IL-23 synthesis, and the effect of morphine was abolished by pretreatment with forskolin. BMDCs were preincubated with forskolin (100 μm) or vehicle 30 min before cell infection with S. pneumoniae. The data shown are the mean ± S.E. of three experiments. *, p < 0.05; **, p < 0.01 comparisons as indicated by brackets. n.s., not significant.