Interactions Between Endogenous Opioids and the Immune System.

The endogenous opioid system, which consists of opioid receptors and their ligands, is widely expressed in the nervous system and also found in the immune system. As a part of the body's defense machinery, the immune system is heavily regulated by endogenous opioid peptides. Many types of immune cells, including macrophages, dendritic cells, neutrophils, and lymphocytes are influenced by endogenous opioids, which affect cell activation, differentiation, proliferation, apoptosis, phagocytosis, and cytokine production. Additionally, immune cells also synthesize and secrete endogenous opioid peptides and participate peripheral analgesia. This chapter is structured into two sections. Part one focuses on immunoregulatory functions of central endogenous opioids; and part two describes how opioid peptide-containing immune cells participate in local analgesia.

Current overview of opioids in progression of inflammatory bowel disease; pharmacological and clinical considerations.

Inflammatory bowel diseases (IBD) belong to a subgroup of persistent, long-term, progressive, and relapsing inflammatory conditions. IBD may spontaneously develop in the colon, resulting in tumor lesions in inflamed regions of the intestine, such as invasive carcinoma. The benefit of opioids for IBD treatment is still questionable, thereby we investigated databases to provide an overview in this context. This review demonstrates the controversial role of opioids in IBD therapy, their physiological and pharmacological functions in attenuating the IBD symptoms, and in improving inflammatory, oxidative stress, and the quality of life factors in IBD subjects. Data were extracted from clinical, in vitro, and in vivo studies in English, between 1995 and 2019, from PubMed, Google Scholar, Scopus, and Cochrane library. Based on recent reports, there are promising opportunities to target the opioid system and control the IBD symptoms. This study suggests a novel approach for future treatment of functional and inflammatory disorders such as IBD.

Multiple potential targets of opioids in the treatment of acute respiratory distress syndrome from COVID-19.

COVID-19 can present with a variety of clinical features, ranging from asymptomatic or mild respiratory symptoms to fulminant acute respiratory distress syndrome (ARDS) depending on the host's immune responses and the extent of the associated pathologies. This implies that several measures need to be taken to limit severely impairing symptoms caused by viral-induced pathology in vital organs. Opioids are most exploited for their analgesic effects but their usage in the palliation of dyspnoea, immunomodulation and lysosomotropism may represent potential usages of opioids in COVID-19. Here, we describe the mechanisms involved in each of these potential usages, highlighting the benefits of using opioids in the treatment of ARDS from SARS-CoV-2 infection.

Immunohistochemical Analysis of Opioid Receptors in Peripheral Tissues.

Immunohistochemical staining is widely used to identify opioid receptors in specific cell types throughout the nervous system. Opioid receptors are not restricted to the central nervous system, but are also present in peripheral sensory neurons, where their activation exerts analgesic effects without inducing centrally mediated side effects. Here, we describe immunohistochemical analysis of µ-opioid receptors in the peripheral sensory neuron cell bodies, along the axons and their peripheral endings in the hind paw skin, as well as in the spinal cord, under naïve and sciatic nerve damage conditions in mice. Importantly, we consider the ongoing debate on the specificity of antibodies.

Real-Time Quantitative Reverse Transcription PCR for Detection of Opioid Receptors in Immune Cells.

Real-time quantitative reverse transcription-PCR (qRT-PCR ) is a highly sensitive molecular biology method based on the amplification of the cDNA of mRNA to detect and quantify the levels of mRNA of interest. In this chapter, we describe real-time qRT-PCR to detect and quantify mRNA of opioid receptors in immune cells. Specifically, we analyze mouse immune cells isolated from the blood and sciatic nerves exposed to a chronic constriction injury, which represents a model of neuropathic pain. We describe in detail the requirements and techniques to induce the chronic constriction injury, to isolate immune cells from the blood and injured nerves, to isolate the total RNA from immune cells, to perform a cDNA reverse transcription from the total RNA, and to perform real-time qRT-PCR for µ-, δ-, and κ-opioid receptor mRNAs.

Quantitative Analysis of MOR-1 Internalization in Spinal Cord of Morphine-Tolerant Mice.

The biological process of opioid analgesic tolerance remains nowadays elusive. In particular the mechanism by which opioid receptor desensitization occurs has not been completely elucidated to date. One possible hypothesis involves the internalization of MOR. Here, we describe a simple in vitro protocol to investigate the localization of MOR-1 after repeated morphine administration in the spinal cord of morphine-tolerant mice, using western blotting and immunofluorescence techniques.

Evaluation of Murine Macrophage Cytokine Production After In Vivo Morphine Treatment.

The discovery of opioid receptor expression on immune cells has originated a large research activity on the possible modulation by opioid drugs of immune system responses. In this chapter we describe an easy methodology useful to obtain information about the potential immunomodulatory activity of opioid drugs. An in vivo treatment schedule is used, and macrophages are studied for their ability to release different cytokines.

Measurement of Macrophage Toll-Like Receptor 4 Expression After Morphine Treatment.

The immune system is a complex and finely orchestrated system, and many soluble molecules and receptors contribute to its regulation.Recent studies have suggested that many of the modulatory effects induced by morphine on innate immunity, and in particular the effects on macrophage activation and function, can be due to the modulation of an important macrophage surface receptor, the toll-like receptor (TLR), that is primarily involved in early regulatory steps. In this chapter we describe a RT-real-time PCR method for assessing TLR expression in macrophage after in vivo morphine treatment.

Chronic Methadone Use Alters the CD8+ T Cell Phenotype In Vivo and Modulates Its Responsiveness Ex Vivo to Opioid Receptor and TCR Stimuli.

Endogenous opioid peptides are released at sites of injury, and their cognate G protein-coupled opioid receptors (ORs) are expressed on immune cells. Although drugs of misuse appropriate ORs, conflicting reports indicate immunostimulatory and immunosuppressive activity, in that opioid users have elevated infection risk, opioids activate innate immune cells, and opioids attenuate inflammation in murine T cell-mediated autoimmunity models. The i.v. use of drugs transmits bloodborne pathogens, particularly viruses, making the study of CD8+ T cells timely. From a cohort of nonuser controls and methadone users, we demonstrate, via t-Stochastic Neighbor Embedding and k-means cluster analysis of surface marker expression, that chronic opioid use alters human CD8+ T cell subset balance, with notable decreases in T effector memory RA+ cells. Studying global CD8+ T cell populations, there were no differences in expression of OR and several markers of functionality, demonstrating the need for finer analysis. Purified CD8+ T cells from controls respond to opioids ex vivo by increasing cytoplasmic calcium, a novel finding for OR signal transduction, likely because of cell lineage. CD8+ T cells from controls exposed to µ-OR agonists ex vivo decrease expression of activation markers CD69 and CD25, although the same markers are elevated in µ-OR-treated cells from methadone users. In contrast to control cells, T cell subsets from methadone users show decreased expression of CD69 and CD25 in response to TCR stimulus. Overall, these results indicate a direct, selective role for opioids in CD8+ T cell immune regulation via their ability to modulate cell responses through the opioid receptors and TCRs.

Agonist-selective NOP receptor phosphorylation correlates in vitro and in vivo and reveals differential post-activation signaling by chemically diverse agonists.

Agonists of the nociceptin/orphanin FQ opioid peptide (NOP) receptor, a member of the opioid receptor family, are under active investigation as novel analgesics, but their modes of signaling are less well characterized than those of other members of the opioid receptor family. Therefore, we investigated whether different NOP receptor ligands showed differential signaling or functional selectivity at the NOP receptor. Using newly developed phosphosite-specific antibodies to the NOP receptor, we found that agonist-induced NOP receptor phosphorylation occurred primarily at four carboxyl-terminal serine (Ser) and threonine (Thr) residues, namely, Ser346, Ser351, Thr362, and Ser363, and proceeded with a temporal hierarchy, with Ser346 as the first site of phosphorylation. G protein-coupled receptor kinases 2 and 3 (GRK2/3) cooperated during agonist-induced phosphorylation, which, in turn, facilitated NOP receptor desensitization and internalization. A comparison of structurally distinct NOP receptor agonists revealed dissociation in functional efficacies between G protein-dependent signaling and receptor phosphorylation. Furthermore, in NOP-eGFP and NOP-eYFP mice, NOP receptor agonists induced multisite phosphorylation and internalization in a dose-dependent and agonist-selective manner that could be blocked by specific antagonists. Our study provides new tools to study ligand-activated NOP receptor signaling in vitro and in vivo. Differential agonist-selective NOP receptor phosphorylation by chemically diverse NOP receptor agonists suggests that differential signaling by NOP receptor agonists may play a role in NOP receptor ligand pharmacology.

Intermittent blockade of OGFr and treatment of autoimmune disorders.

IMPACT STATEMENT: This mini-review presents information on the intermittent blockade of the opioid growth factor (OGF)-OGF receptor (OGFr) axis by low-dose naltrexone (LDN), and the role of enkephalin (i.e. OGF) in autoimmune disorders, specifically multiple sclerosis, Crohn's, and fibromyalgia. Clinical reports on subjects taking LDN have documented reduced fatigue, few side-effects, and improved overall health. Preclinical studies on mice with experimental autoimmune encephalomyelitis (EAE), the animal model of multiple sclerosis, revealed that immunization for EAE reduces serum OGF. Intermittent OGFr blockade with LDN restores serum enkephalin levels that correlate with reduced behavioral and pathological signs of EAE; LDN also increases enkephalin levels in naïve mice. The interplay between LDN, and the onset and treatment of autoimmune diseases, chronic pain, and other addictive behaviors requires further investigation, but highlights a central role for enkephalins and intermittent blockade of the OGF-OGFr pathway in pathogenesis and treatment of these disorders.

Generation and Characterization of Antibodies against Opioid Receptors from Zebrafish.

The opioid system is well conserved among species and plays a critical role in pain and addiction systems. The use of zebrafish as an experimental model to study development and genetics is extraordinary and has been proven to be relevant for the study of different diseases. The main drawback to its use for the analysis of different pathologies is the lack of protein tools. Antibodies that work in other models are not suitable for zebrafish due to the low degree of homology that exists among the opioid receptor protein sequences in different species. Here we report the successful generation and characterization of antibodies against the mu, delta 1 and delta 2 opioid receptors in zebrafish. The antibodies obtained, which are specific for each receptor due to the use of the C-terminus as antigens, work for Western blotting and immunohistochemistry. In addition, the antibodies against mu and delta 1 opioid receptors, but not those against delta 2, are able to immunoprecipitate the corresponding receptor from zebrafish lysates. The development of opioid receptor antibodies is an asset to the further study of the endogenous opioid system in zebrafish.

Opioid and Opiate Immunoregulatory Processes.

The discovery of the ability of the nervous system to communicate through "public" circuits with other systems of the body is attributed to Ernst and Berta Scharrer, who described the neurosecretory process in 1928. Indeed, the immune system has been identified as another important neuroendocrine target tissue. Opioid peptides are involved in this communication (i.e., neuroimmune) and with that of autoimmunoregulation (communication between immunocytes). The significance of opioid neuropeptide involvement with the immune system is ascertained from the presence of novel δ, µ., and κ receptors on inflammatory cells that result in modulation of cellular activity after activation, as well as the presence of specific enzymatic degradation and regulation processes. In contrast to the relatively uniform antinociceptive action of opiate and opioid signal molecules in neural tissues, the presence of naturally occurring morphine in plasma and a novel µ3 opiate-specific receptor on inflammatory cells adds to the growing knowledge that opioid and opiate signal molecules may have antagonistic actions in select tissues. In examining various disorders (e.g., human immunodeficiency virus, substance abuse, parasitism, and the diffuse inflammatory response associated with surgery) evidence has also been found for the involvement of opiate/opioid signaling in prominent mechanisms. In addition, the presence of similar mechanisms in man and organisms 500 million years divergent in evolution bespeaks the importance of this family of signal molecules. The present review provides an overview of recent advances in the field of opiate and opioid immunoregulatory processes and speculates as to their significance in diverse biological systems.

Nociceptin/orphanin FQ (N/OFQ) modulates immunopathology and airway hyperresponsiveness representing a novel target for the treatment of asthma.

BACKGROUND AND PURPOSE: There is evidence supporting a role for the nociceptin/orphanin FQ (N/OFQ; NOP) receptor and its endogenous ligand N/OFQ in the modulation of neurogenic inflammation, airway tone and calibre. We hypothesized that NOP receptor activation has beneficial effects upon asthma immunopathology and airway hyperresponsiveness. Therefore, the expression and function of N/OFQ and the NOP receptor were examined in healthy and asthmatic human airway tissues. The concept was further addressed in an animal model of allergic asthma. EXPERIMENTAL APPROACH: NOP receptor expression was investigated by quantitative real-time PCR. Sputum N/OFQ was determined by RIA. N/OFQ function was tested using several assays including proliferation, migration, collagen gel contraction and wound healing. The effects of N/OFQ administration in vivo were studied in ovalbumin (OVA)-sensitized and challenged mice. KEY RESULTS: NOP receptors were expressed on a wide range of human and mouse immune and airway cells. Eosinophils expressed N/OFQ-precursor mRNA and their number correlated with N/OFQ concentration. N/OFQ was found in human sputum and increased in asthma. Additionally, in asthmatic human lungs N/OFQ immunoreactivity was elevated. NOP receptor activation inhibited migration of immunocytes and increased wound healing in airway structural cells. Furthermore, N/OFQ relaxed spasmogen-stimulated gel contraction. Remarkably, these findings were mirrored in OVA-mice where N/OFQ treatment before or during sensitization substantially reduced airway constriction and immunocyte trafficking to the lung, in particular eosinophils. N/OFQ also reduced inflammatory mediators and mucin production. CONCLUSIONS AND IMPLICATIONS: We demonstrated a novel dual airway immunomodulator/bronchodilator role for N/OFQ and suggest targeting this system as an innovative treatment for asthma.

Evaluation of murine macrophage cytokine production after in vivo morphine treatment.

The discovery of opioid receptors expression on immune cells has originated a large research activity on the possible modulation by opioid drugs of immune system responses. In the present chapter we describe an easy methodology useful to obtain information about the potential immunomodulatory activity of opioid drugs. An in vivo treatment schedule is used, and macrophages are studied for their ability to release different cytokines.

Endogenous opiates and behavior: 2013.

This paper is the thirty-sixth consecutive installment of the annual review of research concerning the endogenous opioid system. It summarizes papers published during 2013 that studied the behavioral effects of molecular, pharmacological and genetic manipulation of opioid peptides, opioid receptors, opioid agonists and opioid antagonists. The particular topics that continue to be covered include the molecular-biochemical effects and neurochemical localization studies of endogenous opioids and their receptors related to behavior, and the roles of these opioid peptides and receptors in pain and analgesia; stress and social status; tolerance and dependence; learning and memory; eating and drinking; alcohol and drugs of abuse; sexual activity and hormones, pregnancy, development and endocrinology; mental illness and mood; seizures and neurologic disorders; electrical-related activity and neurophysiology; general activity and locomotion; gastrointestinal, renal and hepatic functions; cardiovascular responses; respiration and thermoregulation; and immunological responses.

Peripheral sensitization increases opioid receptor expression and activation by crotalphine in rats.

Inflammation enhances the peripheral analgesic efficacy of opioid drugs, but the mechanisms involved in this phenomenon have not been fully elucidated. Crotalphine (CRP), a peptide that was first isolated from South American rattlesnake C.d. terrificus venom, induces a potent and long-lasting anti-nociceptive effect that is mediated by the activation of peripheral opioid receptors. Because the high efficacy of CRP is only observed in the presence of inflammation, we aimed to elucidate the mechanisms involved in the CRP anti-nociceptive effect induced by inflammation. Using real-time RT-PCR, western blot analysis and ELISA assays, we demonstrate that the intraplantar injection of prostaglandin E2 (PGE2) increases the mRNA and protein levels of the µ- and κ-opioid receptors in the dorsal root ganglia (DRG) and paw tissue of rats within 3 h of the injection. Using conformation state-sensitive antibodies that recognize activated opioid receptors, we show that PGE2, alone does not increase the activation of these opioid receptors but that in the presence of PGE2, the activation of specific opioid receptors by CRP and selective µ- and κ-opioid receptor agonists (positive controls) increases. Furthermore, PGE2 down-regulated the expression and activation of the δ-opioid receptor. CRP increased the level of activated mitogen-activated protein kinases in cultured DRG neurons, and this increase was dependent on the activation of protein kinase Cζ. This CRP effect was much more prominent when the cells were pretreated with PGE2. These results indicate that the expression and activation of peripheral opioid receptors by opioid-like drugs can be up- or down-regulated in the presence of an acute injury and that acute tissue injury enhances the efficacy of peripheral opioids.

Immunomodulatory effects of endogenous and synthetic peptides activating opioid receptors.

The main role of endogenous opioid peptides is the modulation of pain. Opioid peptides exert their analgesic activity by binding to the opioid receptors distributed widely in the central nervous system (CNS). However, opioid receptors are also found on tissues and organs outside the CNS, including the cells of the immune system, indicating that opioids are capable of exerting additional effects in periphery. Morphine, which is a gold standard in the treatment of chronic pain, is well-known for its immunosuppressive effects. Much less is known about the immunomodulatory effects exerted by endogenous (enkephalins, endorphins, dynorphins and endomorphins) and synthetic peptides activating opioid receptors. In this review we tried to summarize opioid peptide-mediated modulation of immune cell functions which can be stimulatory as well as inhibitory.

Opioids and immune modulation: more questions than answers.

Opioid addicts are more likely to present with infections suggesting opioids are immune modulators. The potential sites/mechanism(s) for this modulation are controversial and on close inspection not well supported by the current literature. It has long been assumed that opioid-induced immune modulation occurs via a combination of direct actions on the immune cell itself, via the hypothalamic-pituitary-adrenal (HPA) axis, or both. Opioid receptors are classified as MOP (µ, mu), DOP (δ, delta), and KOP (κ, kappa)--classical naloxone sensitive receptors--or NOP (the receptor for nociceptin/orphanin FQ), which is naloxone insensitive. Opioids currently used in clinical practice predominantly target the MOP receptor. There do not appear to be classical opioid receptors present on immune cells. The evidence for HPA activation is also poor and shows some species dependence. Most opioids used clinically or as drugs of abuse do not target the NOP receptor. Other possible target sites for immune modulation include the sympathetic nervous system and central sites. We are currently unable to accurately define the cellular target for immune modulation and suggest further investigation is required. Based on the differences observed when comparing studies in laboratory animals and those performed in humans we suggest that further studies in the clinical setting are needed.

Nociceptin / orphanin FQ (NOP) receptors as novel potential target in the treatment of gastrointestinal diseases.

The nociceptin system, which consists of endogenous nociceptin/orphanin FQ and NOP receptors, is present in the central nervous system (CNS), as well as respiratory, cardiovascular, urogenital and gastrointestinal (GI) tissues. It is critically involved in nociception and pain signaling, as well as modulation of hormone and neurotransmitter release, stress responses and reversal of stress-induced analgesia. In the GI tract, the nociceptin system participates in the maintenance of homeostasis by affecting secretion and motility. Here we give an overview on the nociceptin system in the GI tract. The nociceptin system is an attractive target for novel drugs, which may be effective in the treatment of inflammatory or functional GI disorders, such as inflammatory bowel diseases (IBD) and irritable bowel syndrome (IBS).