Richard Charboneau

Morphine Impairs Host Innate Immune Response and Increases Susceptibility to Streptococcus pneumoniae Lung Infection

Chronic morphine use impairs host innate immune response and increases susceptibility to bacteria and virus. In this study a novel mouse model of chronic morphine treatment, followed by intranasal inoculation with Streptococcus pneumoniae, was used to investigate microbial events and host innate immune response. Our results show that chronic morphine treatment markedly delayed neutrophil recruitment and increased bacterial burden in the lung, spleen, and blood with a subsequent increase in mortality. In morphine-treated animals, before neutrophil recruitment, a significant decrease in TNF-α, IL-1, IL-6, MIP-2, and KC was observed both in bronchoalveolar lavage fluids and in lung tissue. In the early phase of infection, we found that accumulation of galectin-3 in the alveolar space of streptococcus-infected lungs was decreased after morphine treatment. The transcription factor NF-κB in lung resident cells was also inhibited after morphine treatment. Taken together, these results suggest that chronic morphine treatment in an S. pneumoniae infection model suppresses NF-κB gene transcription in lung resident cells, which, in turn, modulates the transcriptional regulation of MIP-2 and inflammatory cytokines. The decreased synthesis of MIP-2 and inflammatory cytokines coupled with the decreased release of galectin-3 result in reduced migration of neutrophils to the site of infection, thereby increasing susceptibility to S. pneumoniae infection after morphine treatment.

Opioid Drug Abuse and Modulation of Immune Function: Consequences in the Susceptibility to Opportunistic Infections

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.

The immunosuppressive effects of chronic morphine treatment are partially dependent on corticosterone and mediated by the μ-opioid receptor

Wild‐type and μ‐opioid receptor knockout (MORKO) mice were used to investigate the role of corticosterone (CORT) and the μ‐opioid receptor (MOR) in chronic morphine‐mediated immunosuppression. We found that although plasma CORT concentrations in CORT infusion (10 mg/kg/day) and morphine‐pellet implantation (75 mg) mice were similar (400–450 ng/ml), chronic morphine treatment resulted in a significantly higher (two‐ to threefold) inhibition of thymic, splenic, and lymph node cellularity; inhibition of thymic‐lymphocyte proliferation; inhibition of IL‐2 synthesis; and activation of macrophage nitric oxide (NO) production when compared with CORT infusion. In addition, results show that the inhibition of IFN‐γ synthesis and splenic‐ and lymph node‐lymphocyte proliferation and activation of macrophage TNF‐α and IL‐1β synthesis occurred only with chronic morphine treatment but not with CORT infusion. These morphine effects were abolished in MORKO mice. The role of the sympathetic nervous system on morphine‐mediated effects was investigated by using the ganglionic blocker chlorisondamine. Our results show that chlorisondamine was able to only partially reverse morphines inhibitory effects. The results clearly show that morphine‐induced immunosuppression is mediated by the MOR and that although some functions are amplified in the presence of CORT or sympathetic activation, the inhibition of IFN‐γ synthesis and activation of macrophage‐cytokine synthesis is CORT‐independent and only partially dependent on sympathetic activation.

Morphine Negatively Regulates Interferon-γ Promoter Activity in Activated Murine T Cells through Two Distinct Cyclic AMP-dependent Pathways

To explore the mechanism by which morphine promotes the incidence of HIV infection, we evaluated the regulatory role of morphine on the interferon-γ (IFN-γ) promoter in activated T cells from wild type and μ-opioid receptor knockout mice. Our results show that morphine inhibited anti-CD3/CD28-stimulated IFN-γ promoter activity in a dose-dependent manner. Chronic morphine treatment of T cells increased intracellular cAMP. To evaluate the role of cAMP in morphines modulatory function, the effects of dibutyryl cyclic AMP and forskolin were investigated. Both dibutyryl cyclic AMP and forskolin treatment inhibited IFN-γ promoter activity. Treatment with pertussis toxin, but not with a protein kinase A inhibitor, antagonized morphines inhibitory effects. Morphine inhibited phosphorylation of ERK1/2 and p38 MAPK; in addition, morphine treatment in the presence of either ERK1/2 or p38 MAPK inhibitor (PD98059 or SB203580) resulted in an additive inhibition of IFN-γ promoter activity. The transcription factor activator protein-1, NF-κB, and nuclear factor of activated T cells (NFAT) were negatively regulated by morphine. Overexpression of NF-κB p65 rescued the inhibitory effect of morphine on IFN-γ promoter activity. However, only when NFATc1 was co-overexpressed with c-fos was the inhibitory effect of morphine on IFN-γ promoter counteracted. The inhibitory effects of morphine were not observed in T cells obtained from μ-opioid receptor knockout mice, suggesting that morphine modulation of IFN-γ promoter activity is mediated through the μ-opioid receptor. In summary, our data indicate that morphine modulation of IFN-γ promoter activity is mediated through two distinct cAMP-dependent pathways, the NF-κB signaling pathway and the ERK1/2, p38 MAPK, AP-1/NFAT pathway.

Chronic morphine treatment differentiates T helper cells to Th2 effector cells by modulating transcription factors GATA 3 and T-bet.

Chronic morphine treatment in animal models has been shown to alter a number of immune parameters including suppression of cellular immunity. T helper cell differentiation into Th2 effector cell may be a major contributing factor to impaired cellular immunity following chronic drug abuse. We had previously shown that chronic morphine treatment in vivo and in vitro decreases IL-2 and IFNgamma (Th1) protein levels and increases IL-4 and IL-5 (Th2) protein levels in a time-dependent manner. In addition in this paper, we show that chronic morphine treatment resulted in a decrease in IFNgamma and IL-2 mRNA and an increase in IL-4 and IL-5 mRNA accumulation in murine splenocytes. Furthermore, chronic morphine treatment inhibited IFNgamma promoter activity and increased IL-4 promoter activity in respective promoter transfected primary T cells. In addition, we also demonstrate that chronic morphine treatment resulted in an increase in GATA 3 binding to DNA consensus elements in electromobility shift assays and an increase in GATA 3 protein and mRNA levels. In contrast, chronic morphine treatment resulted in a decrease in T-bet mRNA levels. From these data, we conclude that chronic morphine treatment differentiates T helper cell to Th2 effector cells by modulating key master switches that results in committing T helper cell to a Th2 phenotype.

μ-Opioid Receptor Mediates Chronic Restraint Stress-Induced Lymphocyte Apoptosis

Psychological stress is associated with immunosuppression in both humans and animals. Although it was well established that psychological stressors stimulate the hypothalamic-pituitary-adrenal axis and the sympathetic nervous system, resulting in the release of various hormones and neurotransmitters, the mechanisms underlying these phenomena are poorly understood. In this study, μ-opioid receptor knockout (MORKO) mice were used to investigate whether the μ-opioid receptor mediates the immunosuppression induced by restraint stress. Our results showed that wild-type (WT) mice subjected to chronic 12-h daily restraint stress for 2 days exhibited a significant decrease in splenocyte number with a substantial increase in apoptosis and CD95 (Fas/APO-1) expression of splenocytes. The effects are essentially abolished in MORKO mice. Furthermore, inhibition of splenic lymphocyte proliferation, IL-2, and IFN-γ production induced by restraint stress in WT mice was also significantly abolished in MORKO mice. Interestingly, both stressed WT and MORKO mice showed a significant elevation in plasma corticosterone and pituitary proopiomelanocortin mRNA expression, although the increase was significantly lower in MORKO mice. Adrenalectomy did not reverse restraint stress-induced immunosuppression in WT mice. These data clearly established that the μ-opioid receptor is involved in restraint stress-induced immune alterations via a mechanism of apoptotic cell death, and that the effect is not mediated exclusively through the glucocorticoid pathway.

MORPHINE SYNERGIZES WITH LIPOPOLYSACCHARIDE IN A CHRONIC ENDOTOXEMIA MODEL

Emergent or elective surgical procedures may be complicated by sepsis, resulting in critical illness that can lead to organ failure and death. The opioid drug, morphine is widely used to alleviate pain in post-surgical patients; however, it is well documented that chronic treatment of mice with morphine affects the proliferation, differentiation and function of immune cells. Thus, morphine might be expected to exacerbate the effects of sepsis, which also compromises the immune system. To test this notion, we investigated the effect on several immune functions of a clinical dose of morphine (4 mg/kg) superimposed upon a lipopolysaccharide (LPS)-induced infection model. Our results show that this relatively low dose of morphine, though generally having no effects on immune parameters by itself, significantly augmented LPS responses. A clinical dose of morphine (4 mg/kg body weight) superimposed upon an animal model of sepsis resulted in a significant increase in mortality at 48 h. In the absence of the drug, most septic animals died after 96 h. Phenotypic responses such as, decreased thymic cellularity, compromised mitogenic response and inhibition of IL-2 synthesis that are evident at 48-72 h after LPS injection appear as early as 24 h in animals that receive morphine in addition to LPS. In addition, our results show that in T cells there is a shift from TH1 type cytokine elaboration to a TH2 type cytokine elaboration in animals that receive both LPS and morphine.

Chronic Morphine Administration Delays Wound Healing by Inhibiting Immune Cell Recruitment to the Wound Site

Patients prescribed morphine for the management of chronic pain, and chronic heroin abusers, often present with complications such as increased susceptibility to opportunistic infections and inadequate healing of wounds. We investigated the effect of morphine on wound-healing events in the presence of an infection in an in vivo murine model that mimics the clinical manifestations seen in opioid user and abuser populations. We show for the first time that in the presence of an inflammatory inducer, lipopolysaccharide, chronic morphine treatment results in a marked decrease in wound closure, compromised wound integrity, and increased bacterial sepsis. Morphine treatment resulted in a significant delay and reduction in both neutrophil and macrophage recruitment to the wound site. The delay and reduction in neutrophil reduction was attributed to altered early expression of keratinocyte derived cytokine and was independent of macrophage inflammatory protein 2 expression, whereas suppression of macrophage infiltration was attributed to suppressed levels of the potent macrophage chemoattractant monocyte chemotactic protein-1. When the effects of chronic morphine on later wound healing events were investigated, a significant suppression in angiogenesis and myofibroblast recruitment were observed in animals that received chronic morphine administration. Taken together, our findings indicate that morphine treatment results in a delay in the recruitment of cellular events following wounding, resulting in a lack of bacterial clearance and delayed wound closure.

Morphine Induces Defects in Early Response of Alveolar Macrophages to Streptococcus pneumoniae by Modulating TLR9-NF-κB Signaling

Resident alveolar macrophages and respiratory epithelium constitutes the first line of defense against invading lung pneumococci. Results from our study showed that increased mortality and bacterial outgrowth and dissemination seen in morphine-treated mice were further exaggerated following depletion of alveolar macrophages with liposomal clodronate. Using an in vitro alveolar macrophages and lung epithelial cells infection model, we show significant release of MIP-2 from alveolar macrophages, but not from lung epithelial cells, following 4 h of exposure of cells to pneumococci infection. Morphine treatment reduced MIP-2 release in pneumococci stimulated alveolar macrophages. Furthermore, morphine treatment inhibited Streptococcus pneumoniae-induced NF-κB-dependent gene transcription in alveolar macrophages following 2 h of in vitro infection. S. pneumoniae infection resulted in a significant induction of NF-κB activity only in TLR9 stably transfected HEK 293 cells, but not in TLR2 and TLR4 transfected HEK 293 cells, and morphine treatment inhibited S. pneumoniae-induced NF-κB activity in these cells. Moreover, morphine treatment also decreased bacterial uptake and killing in alveolar macrophages. Taken together, these results suggest that morphine treatment impairs TLR9-NF-κB signaling and diminishes bacterial clearance following S. pneumoniae infection in resident macrophages during the early stages of infection, leading to a compromised innate immune response.

Morphine Induces CD4+ T Cell IL-4 Expression through an Adenylyl Cyclase Mechanism Independent of the Protein Kinase A Pathway

Impaired host defense mechanisms after major operative procedures and trauma are recognized as important factors in the development of infectious complication. Trauma is associated with impaired cellular immunity and CD4+ T cell Th2 differentiation. We have previously implicated morphine treatment as a possible mechanism for Th2 differentiation after injury. In this investigation we first establish that morphine treatment in vivo results in Th2 differentiation and that this effect is mediated through a naltrexone-sensitive opioid receptor. We investigated the intracellular mechanism by which morphine controls CD4+ T cell differentiation and demonstrate that morphine treatment in vitro 1) increases anti CD3/CD28 Ab-induced CD4+ T cell IL-4 protein synthesis, IL-4 mRNA, and GATA-3 mRNA accumulation through a pertussis toxin-sensitive receptor; 2) results in a dose-dependent increase in anti-CD3/CD28 Ab-induced CD4+ T cell cytoplasmic cAMP concentration; and 3) increases the forskolin-stimulated cytoplasmic cAMP level through a pertussis toxin-sensitive receptor. We also demonstrate that chronic morphine treatment increases anti-CD3/CD28 Ab-induced IL-4 promoter activity and IL-4 immunoprotein expression through a p38 MAPK-dependent, but protein kinase A- and Erk1/Erk2-independent, mechanism.