Morphine disrupts interleukin-23 (IL-23)/IL-17-mediated pulmonary mucosal host defense against Streptococcus pneumoniae infection

Abstract

Streptococcus pneumoniae is a pathogen that causes serious respiratory disease and meningitis in the immunocompromised drug abuse population. However, the precise mechanisms by which drug abuse compromises the host immune defense to pulmonary S. pneumoniae infection is not fully understood. Using a well-established murine model of opiate abuse and S. pneumoniae lung infection, we explored the influence of morphine treatment on the interleukin-23 (IL-23)/IL-17 axis and related innate immunity. Impairment of early IL-23/IL-17 production caused by morphine treatment was associated with delayed neutrophil migration and decreased pneumococcal clearance. Furthermore, morphine treatment impaired MyD88-dependent IL-23 production in alveolar macrophages and dendritic cells in response to in vitro S. pneumoniae cell infection. Moreover, morphine treatment significantly inhibited the S. pneumoniae-induced phosphorylation of interferon response factor 3 (IRF3), ATF2, and NF-kappaBp65. T-cell receptor delta (TCRdelta)-deficient mice showed a decrease in IL-17 production and a severely weakened capacity to clear lung S. pneumoniae infection. Finally, morphine treatment resulted in diminished secretion of antimicrobial proteins S100A9 and S100A8/A9 during early stages of S. pneumoniae infection. In conclusion, morphine treatment causes a dysfunction in IL-23-producing dendritic cells and macrophages and IL-17-producing gammadeltaT lymphocytes in response to S. pneumoniae lung infection. This leads to diminished release of antimicrobial S100A8/A9 proteins, compromised neutrophil recruitment, and more-severe infection.

FIG. 1.

Protein production and mRNA expression of IL-23 and IL-17 in the lungs, BAL fluids, and cells after S. pneumoniae infection. Mice were intranasally infected (10⁷ CFU per mouse) with S. pneumoniae (serotype 3). At various time points postinfection, lung tissue and BAL fluid samples were collected. The concentration of IL-23 (A) and IL-17 (B) in lung homogenates and BAL fluids was measured by ELISA. The mRNA expression of IL-23 (C) and IL-17 (D) in the lung tissues and BAL cells was determined by real-time RT-PCR. Data represent the mean ± SEM of the results for three independent experiments (n ≥ 6 mice per group).

FIG. 2.

Levels of IL-23 (A) and IL-17 (B), recruitment of neutrophils (C), and bacterial burden (D) in the lung tissues and BAL fluids in morphine-treated and placebo-treated control mice. Data are expressed as the mean ± SEM of the results for three independent experiments. *, P < 0.05; **, P < 0.01 (compared with the placebo-treated control group; n ≥ 6).

FIG. 3.

Administration of rIL-17 improves bacterial clearance in morphine-treated mice. Mice were infected with 10⁷ CFU of S. pneumoniae followed by intranasal administration of 15 ng rIL-17 (or vehicle) 2 h later. Animals were then sacrificed 24 h postinfection. Error bars represent mean ± SEM. **, P < 0.01.

FIG. 4.

Effect of morphine treatment on pneumococcus-induced MyD88-dependent IL-23 production and phosphorylation of IFR3, ATF2, and NF-κBp65. (A) Mouse BMDCs and AMs (1 × 10⁶) were treated with morphine (1 μM) or vehicle for 24 h and then infected with S. pneumoniae for 6 h, and IL-23 concentration was measured in the supernatant by ELISA. **, P < 0.01, compared with the vehicle controls. Results are representative of three independent experiments. (B) BMDCs were pretreated with naltrexone (10 μM) or vehicle for 1 h before morphine treatment (10 nM to 1 μM) and then infected with S. pneumoniae (MOI, 10:1) for 6 h. IL-23 concentration was measured in the supernatant by ELISA. **, P < 0.01, compared with the vehicle controls. Results are representative of three independent experiments. (C) BMDCs were pretreated with MyD88 inhibitory or control peptide as described in Materials and Methods and then treated with either morphine (1 μM) or vehicle for 24 h and infected with S. pneumoniae for 6 h. The data are presented as mean concentration ± SEM. **, P < 0.05 compared with the vehicle control group; n ≥ 6. (D) Levels of p-IRF3 were compared by naïve PAGE and immunoblotting. Phosphorylation of ATF2 and NF-κBp65 was compared by Western blot analysis. To verify equality of loading, blots were reprobed with anti-β-actin or anti-TATA binding protein (TBP). Shown are representative results from one of three independent experiments.

FIG. 5.

Effect of morphine treatment on IL-23/IL-17 production and bacterial clearance in TCRδ^−/− and WT mice. IL-23 and IL-17 concentrations are shown at 4 h after intranasal infection with S. pneumoniae. Bacterial burden is shown at 24 h following S. pneumoniae infection. Each value represents the mean ± SEM. Mean values from three independent experiments are shown. n.s., not significant; *, P < 0.05; **, P < 0.01, compared with the placebo controls.

FIG. 6.

Effect of morphine treatment on the secretion of S100A9 and S100A8/A9 during the early stages of S. pneumoniae infection. At various times postinfection, the lungs and BAL fluid samples were collected. The concentrations of S100A9 and S100A8/A9 were measured by ELISA in the lung homogenates and BAL fluids. Data represent the mean ± SEM of the results for ≥6 mice per group. *, P < 0.05; **, P < 0.01, compared with the placebo treatment group.