Infection rate among intravenous drug users (IDU) is higher than the general public, and is the major cause of morbidity and hospitalization in the IDU population. Epidemiologic studies provide data on increased prevalence of opportunistic bacterial infections such as TB and pneumonia, and viral infections such as HIV-1 and hepatitis in the IDU population. An important component in the intravenous drug abuse population and in patients receiving medically indicated chronic opioid treatment is opioid withdrawal. Data on bacterial virulence in the context of opioid withdrawal suggest that mice undergoing withdrawal had shortened survival and increased bacterial load in response to Salmonella infection. As the body of evidence in support of opioid dependency and its immunosuppressive effects is growing, it is imperative to understand the mechanisms by which opioids exert these effects and identify the populations at risk that would benefit the most from the interventions to counteract opioid immunosuppressive effects. Thus, it is important to refine the existing animal model to closely match human conditions and to cross-validate these findings through carefully controlled human studies. Better understanding of the mechanisms will facilitate the search for new therapeutic modalities to counteract adverse effects including increased infection rates. This review will summarize the effects of morphine on innate and adaptive immunity, identify the role of the mu opioid receptor in these functions and the signal transduction activated in the process. The role of opioid withdrawal in immunosuppression and the clinical relevance of these findings will also be discussed.

BACKGROUND AND PURPOSEPrevious work in our laboratory showed opioid agents inhibit cytokine expression in astrocytes. Recently, Watkins and colleagues hypothesized that opioid agonists activate toll-like receptor 4 (TLR4) signalling, which leads to neuroinflammation. To test this hypothesis, we characterized LPS and opioid effects on TLR4 signalling in reporter cells. EXPERIMENTAL APPROACHNF-kB reporter cells expressing high levels of TLR4 were used to compare LPS and opioid effects on NF-kB activation, a pathway activated by TLR4 stimulation. KEY RESULTS LPS increased TLR4 signalling in a concentration-dependent manner and was antagonized by LPS antagonist (LPS-RS, from Rhodobacter sphaeroides).A concentration ratio analysis showed that LPS-RS was a competitive antagonist. The opioid agonists, morphine and fentanyl, produced minor activation of TLR4 signalling when given alone. When tested following LPS stimulation, opioid agonists inhibited NF-kB activation but this inhibition was not blocked by the general opioid antagonist, naloxone, nor by the selective m opioid receptor antagonist, b-FNA. Indeed, both naloxone and b-FNA also inhibited NF-kB activation in reporter cells. Further examination of fentanyl and b-FNA effects revealed that both opioid agents inhibited LPS signalling in a non-competitive fashion. CONCLUSIONS AND IMPLICATIONSThese results show that LPS-RS is a competitive antagonist at the TLR4 complex, and that both opioid agonists and antagonists inhibit LPS signalling in a non-competitive fashion through a non-GPCR, opioid site(s) in the TLR4 signalling pathway. If confirmed, existing opioid agents or other drug molecules more selective at this novel site may provide a new therapeutic approach to the treatment of neuroinflammation.

Development of tolerance to endotoxin prevents sustained hyper inflammation during systemic infections. Here we report for the first time that chronic morphine treatment tempers endotoxin tolerance resulting in persistent inflammation, septicemia and septic shock. Morphine was found to down-regulate endotoxin/LPS induced miR-146a and 155 in macrophages. However, only miR-146a over expression, but not miR-155 abrogates morphine mediated hyper-inflammation. Conversely, antagonizing miR-146a (but not miR-155) heightened the severity of morphine-mediated hyper-inflammation. These results suggest that miR-146a acts as a molecular switch controlling hyper-inflammation in clinical and/or recreational use of morphine.

Increased inflammatory signaling in microglia is implicated in the pathogenesis of neurodegenerative diseases, trauma, psychiatric disorders, and anxiety/depression. Understanding inflammatory signaling in microglia is critical for advancing treatment options. Studying rodent-derived microglia has yielded substantial information, yet, much remains to better understand inflammatory signaling in human microglia. Hence, there is great interest in developing immortalized human microglial cell lines. The C20 human microglial cell line was recently developed and our primary objective was to advance our knowledge of inflammatory signaling in these cells. Methods: Expression of the microglia specific marker transmembrane protein 119 (TMEM119) was assessed by western blot analysis. Lipopolysaccharide (LPS)-and interleukin-1β (IL-1β)-induced cytokine/chemokine expression was determined by ELISA. Phosphorylation of inhibitory kappa B alpha (IκBα), nuclear factor (NF)-κB p65, and p38 mitogen-activated protein kinase (p38 MAPK) was measured by western blot analysis. Results: TMEM119 was expressed in unstimulated C20 cells, and to a greater extent in IL-1β-stimulated cells. IL-1β significantly induced IL-6, monocyte chemoattractant protein-1/CCL2, and interferon-γ inducible protein 10/CXCL10 expression. LPS induced CCL2 expression, but not IL-6 or CXCL10 expression. IL-1β induced inflammatory signaling as indicated by increased phosphorylation of IκBα, NF-κB p65 and p38 MAPK. Conclusion: We provide the first evidence that C20 microglia express TMEM119. This is the initial report of IL-1βinduced activation of IκBα, NF-κB p65, and p38 MAPK and subsequent CXCL10, CCL2 and IL-6 secretion in C20 cells. These findings advance our understanding of inflammatory signaling in C20 cells and support the value of this cell line as a research tool.

Opioid-immune crosstalk occurs when opioid drugs alter the activity of the immune system. In this study, the opioid antagonist β-funaltrexamine (β-FNA) decreases the expression and release of an inflammatory chemokine, interferon-γ inducible protein-10 (CXCL10) from normal human astrocytes stimulated by interleukin 1β (IL-1β). β-FNA decreased CXCL10 by an unknown action that did not involve the mu opioid receptor (MOR). As IL-1β acts through its receptor to activate NF-κB/MAPK signaling pathways which leads to CXCL10 expression and release, key steps in the IL-1β signaling pathways were examined following β-FNA treatment. IL-1β-induced activation of p38 mitogen-activated protein kinases (p38 MAPK) was inhibited by β-FNA as shown by decreased p38 MAPK phosphorylation in treated cells. β-FNA also decreased the levels of activated subunits of NF-κB (p50 and p65) in treated astrocytes. The impact of β-FNA was also observed in proteins that act to negatively regulate NF-κB signaling. IL-1β upregulated the expression of A20, a ubiquitin (Ub)-editing enzyme that dampens NF-κB signaling by altering ubiquination patterns on IL-1 receptor second messengers, and the increase in A20 was significantly inhibited by β-FNA treatment. Inhibition of the Ub-activating enzyme E1 by the inhibitor PYR41 also decreased CXCL10 release, like β-FNA, and concurrent treatment with both PYR41 and β-FNA inhibited CXCL10 more than did either agent alone. In mice, lipopolysaccharide-induced CXCL10 expression in the brain was inhibited by treatment with β-FNA. These findings suggest that β-FNA exerts an anti-inflammatory action in vitro and in vivo that is MOR-independent and possibly due to the alkylating ability of β-FNA.