Effects Of Opiate Abuse Modulation On Innate Immunity In Streptococcus Pneumoniae Infection Model And The Mechanisms

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. Interleukin-23 (IL-23) has been recently identified as a cytokine closely related to IL-12. IL-23 is secreted by activated macrophages and dendritic cells (DCs) and induces memory T-cell proliferation and is the critical factor required for T-cell IL-17expression in response to bacterial challenge. IL-23 release leads to the production of IL-17. Furthermore, IL-17 promotes neutrophilic inflammation by upregulating CXC chemokines and hematopoietic growth factors. However, whether the IL-23/IL-17 axis contributes to modulating the innate immunity in response to S. pneumoniae lung infection in opiate abuser has not been addressed. Resident lung phagocytic cells, primarily dendritic cells and alveolar macrophages, are likely the first immune cells exposed to S. pneumoniae upon inhalation of the organism into the lungs. Dendritic cells and alveolar macrophages express Toll-like receptors (TLRs) and nucleotide-binding oligomerization domain (Nod) receptors. However, how morphine modulates S. pneumoniae induced IL-23 production through TLRs and Nod receptors dependent signaling pathways remain to be elucidated.In this study, we used our laboratory's well-established murine model of opiate abuse and S. pneumoniae lung infection and in vitro cell S. pneumoniae infection model, we explored the influence of morphine treatment on the interleukin-23 (IL-23)/IL-17 axis in vivo study and how morphine treatment modulates S. pneumoniae infection induced IL-23 production in vitro study.Chapter1: Morphine impairs innate immune functions related the IL-23/IL-17 axis following S. pneumoniae lung infection(1)IL-23 and IL-17 expression during S. pneumoniae lung infection IL-23/ IL-17 protein and mRNA levels following S. pneumoniae infection in the lung tissues, BAL fluids and cells were quantified. IL-23 was rapidly released as early as 2 h following S. pneumoniae infection in both the lung tissues and BAL fluids. In the lung tissues, IL-17 was detected within 2 h after S. pneumonia infection. In the BAL fluids, IL-17 was not detectable during the early stages of infection. Furthermore, mRNA levels of IL-23 and IL-17 in the lung tissues were markedly up-regulated by S. pneumoniae infection as early as 2 h.mRNA levels of IL-23 and IL-17 in the BAL cells are similar with protein levels, separately.(2)Morphine treatment disrupts pulmonary IL-23 and IL-17 expression in the early stages of S. pneumoniae lung infectionMorphine treatment caused a decrease in both IL-23 and IL-17 synthesis during the early stages of infection. This decrease in IL-23 and IL-17 production was associated with delayed and decreased neutrophil recruitment into BAL and lung tissues following S.pneumoniae infection, and an increased bacterial burden within the lungs, and the initiation of systemic disease.(3)Effect of rIL-17 on clearance of S. pneumoniae infection .Administration of recombinant murine IL-17 (15ng/mouse) significantly improved lung antibacterial host defense and reduced bacterial dissemination to the blood.(4)Morphine treatment impairs AMs and DCs IL-23 production in response to in vitro cell infectionS.pneumoniae infection stimulated IL-23 production by DCs were markedly higher than that by macrophages. Morphine treatment inhibited this response in BMDC.The morphine action on S. pneumoniae induced IL-23 was abolished by naltrexone.(5)Morphine inhibits S. pneumoniae-induced IL-23 production through the MyD88-dependent IRF-3, ATF-2, and NF-κB signalingMyD88 inhibitor treatment resulted in decreased IL-23 production in a dose dependent manner in BMDCs.Morphine treatment caused a significant decreased in S. pneumoniae induced IL-23 production in BMDCs when pretreated with MyD88 control peptide. However, no difference in IL-23 production was observed between morphine and vehicle treated BMDCs when pretreated with MyD88 inhibitory peptide .Morphine inhibited S.pneumoniae induced IRF-3, ATF-2 and NF-κBp65 phosphorylation.(6)γδT cells contribute to IL-17 expression in response to S. pneumoniae lung infection S.pneumoniae infection induced IL-23 production in the BAL fluids and lung tissues of both WT and TCRδ-/- mice. Morphine treatment diminished IL-23 production in the early stages of S. pneumoniae infection. However, significant levels of IL-17 production, induced by S. pneumoniae infection, were only detected in the lungs derived from WT mice, but not in the lungs of TCRδ-/- mice. Morphine significantly impaired IL-17 production in S. pneumoniae infected WT mice. The number of live bacteria in the lungs was significantly higher (**p<0.01) in TCRδ-/- mice than in WT mice 24 h postinfection. Morphine treatment in the WT mice resulted in a greater bacterial burden in the BAL and lungs compared to placebo treated WT mice. Interestingly, no difference in bacterial burden was observed between morphine and placebo treatment of TCRδ-/- mice.(7) Morphine inhibits the secretion of antimicrobial proteins S100A9 and S100A8/A9Morphine treatment markedly decreased S100A9 and S100A8/A9 production in the early stages of S. pneumoniae infection.Chapter2: Morphine modulates dendritic cell interleukin 23 production through TR2 and Nod-2 synergistic signaling(1)Dendritic cells are main source of IL-23 in response to S. pneumoniae infectionThe levels of IL-23 in the culture supernatants of BMDCs and BMDMs were significantly increased following infection with S. pneumoniae or stimulation with LTA or PLY. CpG or MDP didn't induce a significant IL-23 production in DCs. The levels of IL-23 were about 6-fold higher in culture supernatants of BMDCs compared to those of BMDMs.(2)Morphine modulates the promoter activity, mRNA transcription and protein production of IL-23 induced by S. pneumoniae in DCs through theμ-opioid receptorsMorphine treatment resulted in an approximately 65 % reduction in IL-23 promoter activity and mRNA levels and more than 50% of IL-23 production when compared with vehicle control in DCs derived from WT mice. Moreover, this morphine effect on IL-23 protein production was completely abolished in DCs derived from MORKO mice.(3)Morphine impairs IL-23 production following S. pneumoniae infection through the TLR2 in synergy with Nod2 signaling pathway DCs were stimulated with single TLR or Nod2 agonist, or combination of TLRs and Nod2 agonists. The combination of MDP and LTA stimulated a significant IL-23 `production, which was at same level of IL-23 production as S. pneumoniae infection. Morphine treatment significantly inhibited these effects.Pretreatment of DCs with anti-TLR2 partially attenuated S. pneumoniae induced IL-23 production. Furthermore, pretreatment of DCs with anti-TLR2 abolished inhibitory effect of morphine on IL-23 production following the infection.(4)Effect of S. pneumoniae infection and morphine treatment on the expressions of TLRsFACS shows that S. pneumoniae infection induced the TLR2 and 4 expressions in DCs. However, morphine treatment did not significantly alter the expressions of TLRs in DCs.(5)Morphine treatment impairs IL-23 production in a MyD88-IRAK1/4 dependent mannerMyD88 inhibitory peptide significantly inhibited S. pneumoniae induced IL-23 production in a dose dependent manner, Meanwhile MyD88 control peptide did not show a significant effect on IL-23 production. When DCs were pretreated with MyD88 control peptide, morphine treatment resulted in a decrease in S. pneumoniae, LTA and PLY induced IL-23 production. However, when DCs were pretreated with MyD88 inhibitor, no difference in IL-23 production was observed between morphine and vehicle treated DCs following infection with S. pneumoniae or stimulated with LTA or PLY.The IRAK1/4 inhibitor caused a clear reduction in IL-23 production following the infection. Pretreatment of DCs with IRAK1/4 inhibitor totally attenuated the inhibitory actions of morphine on IL-23 production following S. pneumoniae(6)Morphine treatment modulates the phosphoralytion of ATF-2 and IRF-3 induced by infection with S. pneumoniae or stimulated with TLR2 plus Nod2 ligandsDCs were infected with S. pneumoniae or stimulated with LTA, MDP or LTA plus MDP for 45 min, then fixed and stained for the subcellular distribution of IRF-3 and ATF-2 using immune-fluorescence staining. We only detected a nuclear translocation of IRF3 and ATF2 in response to infection with S. pneumoniae, and stimulation with LTA plus MDP, or LTA alone. Morphine treatment significantly decreased the phosphorylation of IRF-3 and ATF-2. (7)Cyclic AMP is involved in the inhibitory effects of morphine on S. pneumoniae induced IL-23 productionWhen compared with non-infected DCs, S. pneumoniae infection didn't cause an alteration in levels of intracellular cAMP determined by immunoassay. Chronic morphine treatment resulted in a significant increase in intracellular cAMP in DCs. Forskolin treatment significantly inhibited IL-23 production in S. pneumoniae infected DCs, and abolished morphine's inhibitory effect.ConclusionMorphine treatment causes a dysfunction in IL-23-producing dendritic cells and macrophages and IL-17-producingγδT 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. Morphine impairs S. pneumoniae induced IL-23 production through MyD88-IRAK1/4-IRF3 and MyD88-IRAK1/4- ATF2 dependent TLR2 and Nod-2 synergistic signaling in DCs. Morphine-induced increase intracellular cAMP throughμ-opioid receptor may be a potential mechanism by which morphine treatment inhibits S. pneumonia infection induced IL-23 production.