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Opioid use disorder (OUD) is defined as the chronic use or misuse of prescribed or illicitly obtained opioids and is characterized by clinically significant impairment. The etiology of OUD is multifactorial as it is influenced by genetics, environmental factors, stress response and behavior. Given the profound role of the gut microbiome in health and disease states, in recent years there has been a growing interest to explore interactions between the gut microbiome and the central nervous system as a causal link and potential therapeutic source for OUD. This review describes the role of the gut microbiome and opioid-induced immunopathological disturbances at the gut epithelial surface, which collectively contribute to OUD and perpetuate the vicious cycle of addiction and relapse., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Prenatal opioid exposure is associated with significantly adverse medical, developmental, and behavioral outcomes in offspring, though the underlying mechanisms driving these impairments are still unclear. Accumulating evidence implicates gut microbial dysbiosis as a potential modulator of these adverse effects. However, how opioid exposure during pregnancy alters the maternal and neonatal microbiome remain to be elucidated. Here, we utilize a murine model of brief hydromorphone exposure during pregnancy (gestation day 11-13; i.p.; 10 mg/kg) to examine its impact on the maternal and neonatal microbiome. Fecal samples were collected at various timepoints in dams (4 days post hydromorphone exposure, birth, and weaning) and offspring (2, 3, and 5 weeks) to interrogate longitudinal changes in the microbiome. Stomach contents at 2 weeks were also collected as a surrogate for breastmilk and microbial analysis was performed using 16S rRNA sequencing. Alongside alterations in the maternal gut microbial composition, offspring gut microbiota exhibited distinct communities at 2 and 3 weeks. Furthermore, functional profiling of microbial communities revealed significant differences in microbial community-level phenotypes gram-negative, gram-positive, and potentially pathogenic in maternal and/or neonatal hydromorphone exposed groups compared with controls. We also observed differences in stomach microbiota in opioid-exposed vs non-exposed offspring, which suggests breast milk may also play a role in shaping the development of the neonatal gut microbiota. Together, we provide evidence of maternal and neonatal microbial dysbiosis provoked even with brief hydromorphone exposure during pregnancy., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an imminent threat to human health and public safety. ACE2 and transmembrane serine protease 2 proteins on host cells provide the viral entry point to SARS-CoV-2. Although SARS-CoV-2 mainly infects the respiratory system, there have been reports of viral neurotropism and central nervous system injury as indicated by plasma biomarkers, including neurofilament light chain protein and glial fibrillary acidic protein. Even with a small proportion of infections leading to neurological manifestation, the overall number remains high. Common neurological manifestations of SARS-CoV-2 infection include anosmia, ageusia, encephalopathy, and stroke, which are not restricted to only the most severe infection cases. Opioids and opioid antagonists bind to the ACE2 receptor and thereby have been hypothesized to have therapeutic potential in treating COVID-19. However, in the case of other neurotropic viral infections such as human immunodeficiency virus (HIV), opioid use has been established to exacerbate HIV-mediated central nervous system pathogenesis. An analysis of electronic health record data from more than 73 million patients shows that people with Substance Use Disorders are at higher risk of contracting COVID-19 and suffer worse consequences then non-users. Our in-vivo and in-vitro unpublished studies show that morphine treatment causes increased expression of ACE2 in murine lung and brain tissue as early as 24 h post treatment. At the same time, we also observed morphine and lipopolysaccharides treatment lead to a synergistic increase in ACE2 expression in the microglial cell line, SIM-A9. This data suggests that opioid treatment may potentially increase neurotropism of SARS-CoV-2 infection. We have previously shown that opioids induce gut microbial dysbiosis. Similarly, gut microbiome alterations have been reported with SARS-CoV-2 infection and may play a role in predicting COVID-19 disease severity. However, there are no studies thus far linking opioid-mediated dysbiosis with the severity of neuron-specific COVID-19 infection., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Jalodia, Antoine, Braniff, Dutta, Ramakrishnan and Roy.)

Introduction: During the first year of the COVID-19 pandemic, over 93,000 Americans lost their lives to a preventable overdose. Medications for opioid use disorder (OUD) have been shown to decrease mortality in OUD but are underutilized. Through this case-based learning exercise, first-year medical students applied physiologic and pharmacologic principles to the diagnosis and treatment of OUD., Methods: Faculty facilitated a case discussion over a 1-hour large-group case-based learning (CBL) session. Facilitators utilized PowerPoint slides to illustrate graphs and figures while discussing the case. To evaluate students on the CBL learning objectives, three pharmacology exam questions were administered; students also evaluated the CBL's effectiveness in meeting educational objectives on three Likert-scale questions and via open-ended feedback., Results: First-year medical students ( n = 200) completed the CBL. The mean score on the exam questions was 91%. Students agreed or strongly agreed that the CBL was an effective way to learn pharmacology principles (69%), that it reinforced pharmacologic fundamentals (70%), and that it showed how pharmacology fundamentals were important in the real world of clinical medicine (86%). Qualitative feedback on the CBL was generally positive, including satisfaction with the small-group setting and practical applications of pharmacology to clinical practice., Discussion: This CBL exercise contains content critical for preparing students to combat the modern opioid epidemic. The exercise provides an opportunity for learners to review fundamental pharmacodynamic and pharmacokinetic principles so as to ready them for clinical clerkships and beyond., (© 2022 Taldone et al.)

Substance use disorder (SUD) is a prevalent disease that has caused hundreds of thousands of deaths and affected the lives of even more. Despite its global impact, there is still no known cure for SUD, or the psychological symptoms associated with drug use. Many of the behavioral consequences of drug use prevent people from breaking the cycle of addiction or cause them to relapse back into the cycle due to the physical and psychological consequences of withdrawal. Current research is aimed at understanding the cause of these drug related behaviors and therapeutically targeting them as a mechanism to break the addiction cycle. Research on opioids suggests that the changes in the microbiome during drug use modulated drug related behaviors and preventing these microbial changes could attenuate behavioral symptoms. This review aims to highlight the relationship between the changes in the microbiome and behavior during opioid treatment, as well as highlight the additional research needed to understand the mechanism in which the microbiome modulates behavior to determine the best therapeutic course of action., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Herlihy and Roy.)

Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

COVID-19 is the most consequential pandemic of the 21 st century. Since the earliest stage of the 2019-2020 epidemic, animal models have been useful in understanding the etiopathogenesis of SARS-CoV-2 infection and rapid development of vaccines/drugs to prevent, treat or eradicate SARS-CoV-2 infection. Early SARS-CoV-1 research using immortalized in-vitro cell lines have aided in understanding different cells and receptors needed for SARS-CoV-2 infection and, due to their ability to be easily manipulated, continue to broaden our understanding of COVID-19 disease in in-vivo models. The scientific community determined animal models as the most useful models which could demonstrate viral infection, replication, transmission, and spectrum of illness as seen in human populations. Until now, there have not been well-described animal models of SARS-CoV-2 infection although transgenic mouse models (i.e. mice with humanized ACE2 receptors with humanized receptors) have been proposed. Additionally, there are only limited facilities (Biosafety level 3 laboratories) available to contribute research to aid in eventually exterminating SARS-CoV-2 infection around the world. This review summarizes the most successful animal models of SARS-CoV-2 infection including studies in Non-Human Primates (NHPs) which were found to be susceptible to infection and transmitted the virus similarly to humans (e.g., Rhesus macaques, Cynomolgus, and African Green Monkeys), and animal models that do not require Biosafety level 3 laboratories (e.g., Mouse Hepatitis Virus models of COVID-19, Ferret model, Syrian Hamster model). Balancing safety, mimicking human COVID-19 and robustness of the animal model, the Murine Hepatitis Virus-1 Murine model currently represents the most optimal model for SARS-CoV-2/COVID19 research. Exploring future animal models will aid researchers/scientists in discovering the mechanisms of SARS-CoV-2 infection and in identifying therapies to prevent or treat COVID-19., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Caldera-Crespo, Paidas, Roy, Schulman, Kenyon, Daunert and Jayakumar.)

The gut microbial ecosystem exhibits a complex bidirectional communication with the host and is one of the key contributing factors in determining mucosal immune homeostasis or an inflammatory state. Opioid use has been established to induce gut microbial dysbiosis consistent with increased intestinal tissue inflammation. In this study, we investigated the role of infiltrated immune cells in morphine-induced intestinal tissue damage and gut microbial dysbiosis in mice. Results reveal a significant increase in chemokine expression in intestinal tissues followed by increased neutrophil infiltration post morphine treatment which is direct consequence of a dysbiotic microbiome since the effect is attenuated in antibiotics treated animals and in germ-free mice. Neutrophil neutralization using anti-Ly6G monoclonal antibody showed a significant decrease in tissue damage and an increase in tight junction protein organization. 16S rRNA sequencing on intestinal samples highlighted the role of infiltrated neutrophils in modulating microbial community structure by providing a growth benefit for pathogenic bacteria, such as Enterococcus , and simultaneously causing a significant depletion of commensal bacteria, such as Lactobacillus . Taken together, we provide the first direct evidence that neutrophil infiltration contributes to morphine-induced intestinal tissue damage and gut microbial dysbiosis. Our findings implicate that inhibition of neutrophil infiltration may provide therapeutic benefits against gastrointestinal dysfunctions associated with opioid use.

Vaccines offer a promising prophylactic and therapeutic intervention to counteract opioid use disorders (OUD) and fatal overdoses. Vaccines generate opioid-specific antibodies that bind the target opioid, reducing drug distribution to the brain and preventing drug-induced behavioral and pharmacological effects. Due to their selectivity, anti-opioid vaccines can be administered in combination with FDA-approved medications. Because patients with OUD or other substance use disorders may be affected by other multifactorial co-morbidities, such as infection or depression, it is important to test whether vaccine efficacy is modified by factors that may impact individual innate or adaptive immunity. To that end, this study tested whether housing conditions would affect the efficacy of two lead vaccine formulations targeting oxycodone and fentanyl in male mice and rats, and further analyzed whether differences in the gastrointestinal (GI) microbiome would be correlated with either vaccine efficacy or housing conditions. Results showed that housing mice and rats in either conventional (non-controlled) or specific pathogen-free (SPF, sterile barrier maintained) environment did not affect vaccine-induced antibody responses against oxycodone and fentanyl, nor their efficacy against oxycodone- and fentanyl-induced antinociception, respiratory depression, and bradycardia. Differences in the GI microbiome detected via 16S rRNA gene sequencing were related to the housing environment. This study supports use of anti-opioid vaccines in clinical populations that may display deficits in microbiome function.

Growing evidence from animal and human studies show that opioids have a major impact on the composition and function of gut microbiota. This leads to disruption in gut permeability and altered microbial metabolites, driving both systemic and neuroinflammation, which in turn impacts central nervous system (CNS) homeostasis. Tolerance and dependence are the major comorbidities associated with prolonged opioid use. Inflammatory mediators and signaling pathways have been implicated in both opioid tolerance and dependence. We provide evidence that targeting the gut microbiome during opioid use through prebiotics, probiotics, antibiotics, and fecal microbial transplantation holds the greatest promise for novel treatments for opioid abuse. Basic research and clinical trials are required to examine what is more efficacious to yield new insights into the role of the gut-brain axis in opioid abuse., (Copyright © 2021 Cold Spring Harbor Laboratory Press; all rights reserved.)

HIV trans-activator of transcription (Tat), one of the cytotoxic proteins secreted from HIV-infected cells, is also known to facilitate chemokine-mediated transmigration of monocytes into the brain leading, in turn, to neuroinflammation and thereby contributing to the development of HIV-associated neurocognitive disorders (HAND). The mechanism(s) underlying HIV Tat-mediated enhancement of monocyte transmigration, however, remain largely unknown. CXC chemokine receptor 3 (CXCR3) that is expressed by the peripheral monocytes is known to play a role in the monocyte influx and accumulation. In the present study, we demonstrate for the first time that exposure of human monocytes to HIV Tat protein resulted in upregulated expression of CXCR3 leading, in turn, to increased monocyte transmigration across the blood-brain barrier (BBB) both in the in vitro and in vivo model systems. This process involved activation of toll-like receptor 4 (TLR4), with downstream phosphorylation and activation of TANK-binding kinase 1 (TBK1), and subsequent phosphorylation and nuclear translocation of interferon regulatory factor 3 (IRF3), ultimately leading to enhanced expression of CXCR3 in human monocytes. These findings imply a novel molecular mechanism underlying HIV Tat-mediated increase of monocyte transmigration across the BBB, while also implicating a novel role of CXCR3-dependent monocyte transmigration in HIV Tat-mediated neuroinflammation., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Niu, Liao, Hu, Moidunny, Roy and Buch.)

HPV-induced cervical cancer is one of the prevalent gynecological cancers world-wide. In the present study, we determined the efficacy of Minnelide, a prodrug which is converted to its active form (Triptolide) in vivo against cervical cancer cells. Our studies show that Triptolide inhibited HPV-16 and HPV-18 positive cells at nanomolar concentrations. Tumor cells treated with Triptolide failed to grow in 3-D cultures in a concentration-dependent manner. Triptolide markedly reduced E6 and E7 transcript levels. Further studies revealed that exposure to Triptolide increased the levels of p53 and pRb. As a consequence, Caspase-3/7 activation and apoptosis was induced in cervical cancer cells by Triptolide. Subsequently, we evaluated the efficacy of Minnelide in xenotransplantation models of cervical cancer. Minnelide at very low doses effectively inhibited the growth of established cervical cancers in all the three animal models tested. Furthermore, Minnelide treatment was more effective when combined with platinum-based chemotherapy. These studies show that Minnelide can be used to inhibit the growth of cervical cancer., Competing Interests: The University of Minnesota has a patent for Minnelide, which has been licensed to Minneamrita Therapeutics, LLC. Ashok Saluja is the co-founder and the Chief Scientific Officer of this company and this relationship is managed by the University of Miami as per the University regulations., (AJCR Copyright © 2021.)

Intestinal barrier dysfunction and subsequent microbial translocation play crucial roles in persistent immune activation leading to HIV disease progression. Opioid use is associated with worse outcome in HIV-infected patients. The exacerbated disease progression by opioids is mainly driven by excessive intestinal inflammation and increased gut permeability. The objective of this study is to investigate how opioids potentiate HIV disease progression by compromising intestinal barrier function and impairing intestinal epithelial self-repair mechanism. In the present study, abnormal intestinal morphology and reduced epithelial proliferation were observed in bone marrow-liver-thymus humanized mice and in HIV-infected patients who were exposed to opioids. In bone marrow-liver-thymus mice, HIV, and morphine independently, and additively induced gut dysbiosis, especially depletion of Lachnospiraceae, Ruminococcaceae, and Muribaculaceae. We also observed that the abundance of Lachnospiraceae, Ruminococcaceae, and Muribaculaceae negatively correlated with apoptosis of epithelial cells, and intestinal IL-6 levels. Previous studies have shown that these bacterial families play crucial roles in maintaining intestinal homeostasis because they include most short-chain fatty acid-producing members. Short-chain fatty acids have been shown to maintain stem cell populations and suppress inflammation in the gut by inhibiting histone deacetylases (HDAC). In addition, we demonstrate that morphine exposure inhibited growth of intestinal organoids derived from HIV transgenic mice by suppressing Notch signaling in an HDAC-dependent manner. These studies implicate an important role for HDAC in intestinal homeostasis and supports HDAC modulation as a therapeutic intervention in improving care of HIV patients, especially in opioid-abusing population., (Copyright © 2020 Meng, Banerjee, Zhang, Sindberg, Moidunny, Li, Robbins, Girotra, Segura, Ramakrishnan and Roy.)

Prolonged exposure to opioids results in analgesic tolerance, drug overdose, and death. The mechanism underlying morphine analgesic tolerance still remains unresolved. We show that morphine analgesic tolerance was significantly attenuated in germfree (GF) and in pan-antibiotic-treated mice. Reconstitution of GF mice with naïve fecal microbiota reinstated morphine analgesic tolerance. We further demonstrated that tolerance was associated with microbial dysbiosis with selective depletion in Bifidobacteria and Lactobacillaeae. Probiotics, enriched with these bacterial communities, attenuated analgesic tolerance in morphine-treated mice. These results suggest that probiotic therapy during morphine administration may be a promising, safe, and inexpensive treatment to prolong morphine's efficacy and attenuate analgesic tolerance. We hypothesize a vicious cycle of chronic morphine tolerance: morphine-induced gut dysbiosis leads to gut barrier disruption and bacterial translocation, initiating local gut inflammation through TLR2/4 activation, resulting in the activation of proinflammatory cytokines, which drives morphine tolerance., Competing Interests: The authors declare no conflict of interest.

Human Immunodeficiency Virus (HIV) pathogenesis has been closely linked with microbial translocation, which is believed to drive inflammation and HIV replication. Opioid drugs have been shown to worsen this symptom, leading to a faster progression of HIV infection to Acquired Immunodeficiency Syndrome (AIDS). The interaction of HIV and opioid drugs has not been studied at early stages of HIV, particularly in the gut microbiome where changes may precede translocation events. This study modeled early HIV infection by examining Simian Immunodeficiency Virus (SIV)-infected primates at 21 days or less both independently and in the context of opioid use. Fecal samples were analyzed both for 16S analysis of microbial populations as well as metabolite profiles via mass spectrometry. Our results indicate that changes are minor in SIV treated animals in the time points examined, however animals treated with morphine and SIV had significant changes in their microbial communities and metabolic profiles. This occurred in a time-independent fashion with morphine regardless of how long the animal had morphine in its system. Globally, the observed changes support that microbial dysbiosis is occurring in these animals at an early time, which likely contributes to the translocation events observed later in SIV/HIV pathogenesis. Additionally, metabolic changes were predictive of specific treatment groups, which could be further developed as a diagnostic tool or future intervention target to overcome and slow the progression of HIV infection to AIDS.

Objective: Microbial translocation and monocyte activation predict mortality in treated HIV. We examined whether substance use independently contributes to these pathophysiologic processes., Design: Cross-sectional study at baseline for a randomized controlled trial., Methods: HIV-positive, methamphetamine-using MSM with undetectable HIV viral load (less than 40 copies/ml) were enrolled. We examined if plasma biomarkers of monocyte activation and intestinal barrier integrity were associated with the following: reactive urine toxicology results (Tox+) for stimulants (i.e., methamphetamine or cocaine) and substance use severity measured by the Addiction Severity Index. Multiple linear regression models adjusted for age, antiretroviral therapy regimen, CD4 T-cell count, interleukin-6, and alcohol use severity., Results: The sample of 84 virally suppressed MSM had a median CD4 T-cell count of 645 cells/μl. Those who were Tox+ for stimulants displayed higher soluble CD14 (sCD14) levels (2087 versus 1801 ng/ml; P = 0.009), and this difference remained significant after adjusting for covariates (standardized beta = 0.23, P = 0.026). Greater substance use severity was also independently associated with higher sCD14 after adjusting for covariates (standardized beta = 0.29, P = 0.013). Being Tox+ for stimulants and substance use severity were not associated with soluble CD163 (sCD163) or intestinal fatty acid binding protein (iFABP) levels (P > 0.05)., Conclusions: Monocyte activation is one plausible mechanism by which stimulant use may increase clinical HIV progression.

Allogeneic hematopoietic stem cell transplantation is hampered by chronic graft-versus-host disease (cGVHD), resulting in multiorgan fibrosis and diminished function. Fibrosis in lung and skin leads to progressive bronchiolitis obliterans (BO) and scleroderma, respectively, for which new treatments are needed. We evaluated pirfenidone, a Food and Drug Administration (FDA)-approved drug for idiopathic pulmonary fibrosis, for its therapeutic effect in cGVHD mouse models with distinct pathophysiology. In a full major histocompatibility complex (MHC)-mismatched, multiorgan system model with BO, donor T-cell responses that support pathogenic antibody production are required for cGVHD development. Pirfenidone treatment beginning one month post-transplant restored pulmonary function and reversed lung fibrosis, which was associated with reduced macrophage infiltration and transforming growth factor-β production. Pirfenidone dampened splenic germinal center B-cell and T-follicular helper cell frequencies that collaborate to produce antibody. In both a minor histocompatibility antigen-mismatched as well as a MHC-haploidentical model of sclerodermatous cGVHD, pirfenidone significantly reduced macrophages in the skin, although clinical improvement of scleroderma was only seen in one model. In vitro chemotaxis assays demonstrated that pirfenidone impaired macrophage migration to monocyte chemoattractant protein-1 (MCP-1) as well as IL-17A, which has been linked to cGVHD generation. Taken together, our data suggest that pirfenidone is a potential therapeutic agent to ameliorate fibrosis in cGVHD., (© 2017 by The American Society of Hematology.)

NF-κB has an essential role in the initiation and progression of pancreatic cancer and specifically mediates the induction of epithelial-mesenchymal transition and invasiveness. In this study, we demonstrate the importance of activated NF-κB signaling in EMT induction, lymphovascular metastasis, and neural invasion. Modulation of NF-κB activity was accomplished through the specific NF-κB inhibitor (BAY 11-7085), triptolide, and Minnelide treatment, as well as overexpression of IKBα repressor and IKK activator plasmids. In the classical lymphovascular metastatic cascade, inhibition of NF-κB decreased the expression of several EMT transcription factors (SNAI1, SNAI2, and ZEB1) and mesenchymal markers (VIM and CDH2) and decreased in vitro invasion, which was rescued by IKK activation. This was further demonstrated in vivo via BAY 11-7085 treatment in a orthotopic model of pancreatic cancer. In vivo NF-κB inhibition decreased tumor volume; decreased tumor EMT gene expression, while restoring cell-cell junctions; and decreasing overall metastasis. Furthermore, we demonstrate the importance of active NF-κB signaling in neural invasion. Triptolide treatment inhibits Nerve Growth Factor (NGF) mediated, neural-tumor co-culture in vitro invasion, and dorsal root ganglia (DRG) neural outgrowth through a disruption in tumor-neural cross talk. In vivo, Minnelide treatment decreased neurotrophin expression, nerve density, and sciatic nerve invasion. Taken together, this study demonstrates the importance of NF-κB signaling in the progression of pancreatic cancer through the modulation of EMT induction, lymphovascular invasion, and neural invasion., Competing Interests: disclosure statement: University of Minnesota has a patent for Minnelide, which has been licensed to Minneamrita Therapeutics, LLC. AKS is the co-founder and the Chief Scientific Officer of this company. Dr. Banerjee is a consultant with Minneamrita Therapeutics LLC and this relationship is managed by University of Miami. The other authors have nothing to disclose.

Aim: Opioids are the most prescribed analgesics for moderate and severe pain management; however, chronic use impairs host innate immune response and increases susceptibility to infection. Recently, autophagy has been shown to be an innate defence mechanism against bacterial infection. The effect of autophagy-induced bacterial clearance following morphine treatment has not been previously investigated., Methods: Autophagosomes were visualized by confocal microscopy following GFP-LC3 transfection and also by transmission electron microscopy. The relative protein levels were analysed by Western blot. Macrophages were transfected with GFP-mcherry-LC3 simultaneously to monitor autolysosome formation and subsequent events that lead to degradation., Results: Morphine treatment potentiated LPS-induced vesicular translocation of GFP-LC3 with a concurrent increase in LC3-II levels. In addition, morphine upregulated LPS-induced Beclin1 level, but downregulated Bcl-2 level. We further show that p38 MAP kinase signalling is required for autophagy activation. In contrast, morphine inhibited LPS-induced autophagosome maturation and autophagolysosomal fusion as indicated by the failure to recruit LAMP1 into autophagosome and reduced degradation of SQSTM1/p62 protein level. Morphine modulation of LPS-induced autophagosome maturation visualized using co-localization of GFP-mcherry-LC3 was TLR4 independent, but mediated through μ opioid receptor signalling. Correspondingly, morphine and LPS co-treatment significantly increased Streptococcus pneumoniae load, when compared with LPS treatment alone., Conclusion: These observations imply that although morphine treatment facilitates LPS-induced autophagy, it inhibits autophagolysosomal fusion leading to decreased bacterial clearance and increased bacterial load. These observations support the increased susceptibility to infection and the prevalence of persistent infection in the drug abuse population., (© 2015 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd.)

Opioids are known to exacerbate HIV pathogenesis, however current studies have been limited by models of HIV infection. Given that HIV causes many systemic effects via direct infection of host cells as well as indirect bystander effects, it is important to establish a systemic infection model in a small animal so that genetic tools can be utilized to elucidate the mechanisms of action. In this study, the systemic effects of EcoHIV infection, a modified HIV which can infect mouse cells, are examined in conjunction with morphine. EcoHIV infection with opioid treatment induced bacterial translocation from the lumen of the gut into systemic compartments such as liver, which is similar to observations in human patients with LPS. Bacterial translocation corresponds with alterations in gut morphology, disorganization of the tight junction protein occludin, and a concurrent increase in systemic inflammation in both IL-6 and TNFα. Long term infection also had increased expression of inflammatory cytokines in the CNS when co-treated with morphine. Overall, this study shows that EcoHIV is an appropriate model to study the effects of opioids on HIV pathogenesis, including the HIV-induced pathology at early stages of pathogenesis in the gut.

The first line of defense against invading bacteria is provided by the innate immune system. Morphine and other opiates can immediately disrupt the body's first line of defense against harmful external bacteria. Opiate, for example morphine, abuse degrades physical and physiologic barriers, and modulates phagocytic cells (macrophages, neutrophils) and, nonspecific cytotoxic T cells (gammadelta T), natural killer cells, and dendritic cells, that are functionally important for carrying out a rapid immune reaction to invading pathogens. In vitro studies with innate immune cells from experimental animals and humans and in vivo studies with animal models have shown that opiate abuse impairs innate immunity and is responsible for increased susceptibility to bacterial infection. However, to better understand the complex interactions between opiates, innate immunity, and bacterial infection and develop novel approaches to treat and even prevent bacterial infection in the opiate-abuse population, there is an urgent need to fill the numerous gaps in our understanding of the cellular and molecular mechanisms by which opiate abuse increases susceptibility to bacterial infection.

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-kappaB-dependent gene transcription in alveolar macrophages following 2 h of in vitro infection. S. pneumoniae infection resulted in a significant induction of NF-kappaB 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-kappaB 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-kappaB 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.

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

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, mu-opioid receptor knockout (MORKO) mice were used to investigate whether the mu-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-gamma 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 mu-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.

Wild-type and mu-opioid receptor knockout (MORKO) mice were used to investigate the role of corticosterone (CORT) and the mu-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-gamma synthesis and splenic- and lymph node-lymphocyte proliferation and activation of macrophage TNF-alpha and IL-1beta 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 morphine's 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-gamma synthesis and activation of macrophage-cytokine synthesis is CORT-independent and only partially dependent on sympathetic activation.