Sabita Roy

Hypoxia-induced microRNA-424 expression in human endothelial cells regulates HIF-α isoforms and promotes angiogenesis

Adaptive changes to oxygen availability are critical for cell survival and tissue homeostasis. Prolonged oxygen deprivation due to reduced blood flow to cardiac or peripheral tissues can lead to myocardial infarction and peripheral vascular disease, respectively. Mammalian cells respond to hypoxia by modulating oxygen-sensing transducers that stabilize the transcription factor hypoxia-inducible factor 1α (HIF-1α), which transactivates genes governing angiogenesis and metabolic pathways. Oxygen-dependent changes in HIF-1α levels are regulated by proline hydroxylation and proteasomal degradation. Here we provide evidence for what we believe is a novel mechanism regulating HIF-1α levels in isolated human ECs during hypoxia. Hypoxia differentially increased microRNA-424 (miR-424) levels in ECs. miR-424 targeted cullin 2 (CUL2), a scaffolding protein critical to the assembly of the ubiquitin ligase system, thereby stabilizing HIF-α isoforms. Hypoxia-induced miR-424 was regulated by PU.1-dependent transactivation. PU.1 levels were increased in hypoxic endothelium by RUNX-1 and C/EBPα. Furthermore, miR-424 promoted angiogenesis in vitro and in mice, which was blocked by a specific morpholino. The rodent homolog of human miR-424, mu-miR-322, was significantly upregulated in parallel with HIF-1α in experimental models of ischemia. These results suggest that miR-322/424 plays an important physiological role in post-ischemic vascular remodeling and angiogenesis.

Evidence for opioid receptors on cells involved in host defense and the immune system

Although the role of opiates and opioids in the physiological and pathological function of the immune system is only beginning to be unraveled, converging lines of evidence indicate that the opioid receptors expressed by immune cells are often the same or similar to the neuronal subtypes, particularly delta and kappa. Recent studies also point to the existence of novel opioid receptors and/or binding sites on immune cells that are selective for morphine. Opioids and their receptors, particularly those with high affinity for delta agonists, appear to function in an autocrine/paracrine manner. Thus, opioid peptides generated from immune-derived proenkephalin A act as cytokines, capable of regulating myriad functions of both granulocytes and mononuclear cells. Further identification and characterization of receptors and signal transduction pathways that account for some of the unique properties of opiate binding and immunomodulation (e.g., dose-dependent effects of morphine that occur at exceptionally low concentrations relative to the Kds of the neuronal mu receptor or the morphine binding site reported on activated human T-cells) represents one of the major research challenges ahead. Elucidating mechanisms, such as these, may provide unique therapeutic opportunities through the application of opioid immunopharmacology to disorders involving immune responses in peripheral organs and the central nervous system.

Effects of opioids on the immune system

Both therapeutic and chronic uses of opioids compromise the optimal functioning of the immune system. Overwhelming evidence suggests that opioid use affects both innate immunity and adaptive immunity. Chronic administration of opioids decreases the proliferative capacity of macrophage progenitor cells and lymphocytes. Additionally, the differentiated function of immune cells is significantly affected by opioids. These effects are mediated by either a direct action of opioids on the target cells or by indirect centrally mediated pathways. Molecular biological and biochemical characterization suggest that immune cells differentially express classical opioid receptors. Interestingly, these studies also reveal the presence of a novel class of opioid receptors in immune cells. We believe that this low affinity morphine binding site mediates the antiproliferative effects of morphine.

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.

MU-opioid receptor-knockout mice: role of μ-opioid receptor in morphine mediated immune functions

The role of the mu-opioid receptor in immune function was investigated using mu-opioid receptor knockout mice (MOR-KO). Morphine modulation of several immune functions, including macrophage phagocytosis and macrophage secretion of TNF-alpha, was not observed in the MOR-KO animals, suggesting that these functions are mediated by the classical mu-opioid receptor. In contrast, morphine reduction of splenic and thymic cell number and mitogen-induced proliferation were unaffected in MOR-KO mice, as was morphine inhibition of IL-1 and IL-6 secretion by macrophages. These latter results are consistent with morphine action on a naloxone insensitive morphine receptor, a conclusion supported by previous studies characterizing a nonopioid morphine binding site on immune cells. Alternatively, morphine may act either directly or indirectly on these cells, by a mechanism mediated by either delta or kappa opioid receptors.

Modulation of Immune Function by Morphine: Implications for Susceptibility to Infection

The outcome of microbial infection in an organism is a dynamic process that depends on factors derived from both the microorganism and the host. In chronic human infections, the kind of immune response made in response to pathogens may be of vital importance to host defense. An inappropriate immune response may not only result in lack of protection, but even contribute to disease severity. Chronic morphine use and abuse has been documented to result in severe immune consequence (Roy and Loh 1996; Dinda et al. 2005; Friedman and Eisenstein 2004) and thus may pose a significant risk factor to opportunistic infection. It is therefore not surprising that epidemiological studies show increased prevalence of opportunistic infections such as tuberculosis, HIV infection, and pneumonia in opioid abusers (Quaglio et al. 2002; Nath et al. 2002; Georges et al. 1999). Besides the sharing of unsterilized, contaminated needles, the occurrence of infections in these patients has been attributed to the immune modulatory effect of morphine. In animal studies where confounding variables such as nutritional status, environmental influences, history of drug use, and genetic variability can be controlled, morphine treatment resulted in significant immune deficits. Defense against microbes is mediated by the early reactions of innate immunity and the later response of adaptive immunity. Chronic morphine has been shown to affect both these arms of immune defense (Vallejo et al. 2004). This review on the immunological effects of morphine summarizes the effects of this drug on innate and adaptive immunity, identifies the role of the mu opioid receptor in these functions, and finally discusses how changes in these parameters increase the risk for opportunistic infection.

μ-Opioid receptor-knockout mice: the role of μ-opioid receptor in gastrointestinal transit

Abstract The role of μ-opioid receptor in gastrointestinal transit was investigated using μ-opioid receptor knockout mice (MOR-KO). Our result establishes unequivocally that inhibition of GI transit by morphine is a μ-opioid receptor mediated function. In addition, we show that neither δ nor κ receptor agonist given supraspinally or peripherally are able to inhibit GI transit in MOR-KO animals. It was interesting to observe that basal GI motility was lower in MOR-KO (−/−) compared to heterozygous (±) and wild type (+/+) animals.

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.