Prescription opioids induced microbial dysbiosis worsens severity of chronic pancreatitis and drives pain hypersensitivity.

Opioids, such as morphine and oxycodone, are widely used for pain management associated with chronic pancreatitis (CP); however, their impact on the progression and pain sensitivity of CP has never been evaluated. This report investigates the impact of opioid use on the severity of CP, pain sensitivity, and the gut microbiome. C57BL/6 mice were divided into control, CP, CP with morphine/oxycodone, and either morphine or oxycodone alone groups. CP was induced by administration of caerulein (50ug/kg/h, i.p. hourly x7, twice a week for 10 weeks). The mouse-to-pancreas weight ratio, histology, and Sirius red staining were performed to measure CP severity. Tail flick and paw pressure assays were used to measure thermal and mechanical pain. DNA was extracted from the fecal samples and subjected to whole-genome shotgun sequencing. Germ-free mice were used to validate the role of gut microbiome in sensitizing acute pancreatic inflammation. Opioid treatment exacerbates CP by increasing pancreatic necrosis, fibrosis, and immune-cell infiltration. Opioid-treated CP mice exhibited enhanced pain hypersensitivity and showed distinct clustering of the gut microbiome compared to untreated CP mice, with severely compromised gut barrier integrity. Fecal microbiota transplantation (FMT) from opioid-treated CP mice into germ-free mice resulted in pancreatic inflammation in response to a suboptimal caerulein dose. Together, these analyses revealed that opioids worsen the severity of CP and induce significant alterations in pain sensitivity and the gut microbiome in a caerulein CP mouse model. Microbial dysbiosis plays an important role in sensitizing the host to pancreatic inflammation.

Chronic Morphine Treatment and Antiretroviral Therapy Exacerbate HIV-Distal Sensory Peripheral Neuropathy and Induce Distinct Microbial Alterations in the HIV Tg26 Mouse Model.

Distal Sensory Peripheral Neuropathy (DSP) is a common complication in HIV-infected individuals, leading to chronic pain and reduced quality of life. Even with antiretroviral therapy (ART), DSP persists, often prompting the use of opioid analgesics, which can paradoxically worsen symptoms through opioid-induced microbial dysbiosis. This study employs the HIV Tg26 mouse model to investigate HIV-DSP development and assess gut microbiome changes in response to chronic morphine treatment and ART using 16S rRNA sequencing. Our results reveal that chronic morphine and ART exacerbate HIV-DSP in Tg26 mice, primarily through mechanical pain pathways. As the gut microbiome may be involved in chronic pain persistence, microbiome analysis indicated distinct bacterial community changes between WT and Tg26 mice as well as morphine- and ART-induced microbial changes in the Tg26 mice. This study reveals the Tg26 mouse model to be a relevant system that can help elucidate the pathogenic mechanisms of the opioid- and ART-induced exacerbation of HIV-associated pain. Our results shed light on the intricate interplay between HIV infection, ART, opioid use, and the gut microbiome in chronic pain development. They hold implications for understanding the mechanisms underlying HIV-associated pain and microbial dysbiosis, with potential for future research focused on prevention and treatment strategies.

Multi-omics analysis revealing the interplay between gut microbiome and the host following opioid use.

Opioid crisis is an ongoing epidemic since the past several decades in the United States. Opioid use-associated microbial dysbiosis is emerging as a key regulator of intestinal homeostasis and behavioral responses to opioid. However, the mechanistic insight into the role of microbial community in modulating host response is unavailable. To uncover the role of opioid-induced dysbiosis in disrupting intestinal homeostasis we utilized whole genome sequencing, untargeted metabolomics, and mRNA sequencing to identify changes in microbiome, metabolome, and host transcriptome respectively. Morphine treatment resulted in significant expansion of Parasuterella excrementihominis, Burkholderiales bacterium 1_1_47, Enterococcus faecalis, Enterorhabdus caecimuris and depletion of Lactobacillus johnsonii. These changes correlated with alterations in lipid metabolites and flavonoids. Significant alteration in microbial metabolism (metabolism of lipids, amino acids, vitamins and cofactors) and increased expression of virulence factors and biosynthesis of lipopolysaccharides (LPS) and lipoteichoic acid (LTA) were observed in microbiome of morphine-treated animals. In concurrence with changes in microbiome and metabolome extensive changes in innate and adaptive immune response, lipid metabolism, and gut barrier dysfunction were observed in the host transcriptome. Microbiome depleted mice displayed lower levels of inflammation, immune response and tissue destruction compared to mice harboring a dysbiotic microbiome in response to morphine treatment, thus establishing dysbiotic microbiome as mediator of morphine gut pathophysiology. Integrative analysis of multi-omics data highlighted the associations between Parasutterella excrementihominis, Burkholderiales bacterium 1_1_47, Enterococcus faecalis, Enterorhabdus caecimuris and altered levels of riboflavin, flavonoids, and lipid metabolites including phosphocholines, carnitines, bile acids, and ethanolamines with host gene expression changes involved in inflammation and barrier integrity of intestine. Omic analysis also highlighted the role of probiotic bacteria Lactobacillus johnsonii, metabolites flavonoids and riboflavin that were depleted with morphine as important factors for intestinal homeostasis. This study presents for the first time ever an interactive view of morphine-induced changes in microbial metabolism, strain level gut microbiome analysis and comprehensive view of changes in gut transcriptome. We also identified areas of potential therapeutic interventions to limit microbial dysbiosis and present a unique resource to the opioid research community.

Morphine mediated neutrophil infiltration in intestinal tissue play essential role in histological damage and microbial dysbiosis.

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.

Opioid-Use, COVID-19 Infection, and Their Neurological Implications.

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.

Opioid Use, Gut Dysbiosis, Inflammation, and the Nervous System.

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.

Glycogen synthase kinase-3 inhibition rescues sex-dependent contextual fear memory deficit in human immunodeficiency virus-1 transgenic mice.

BACKGROUND AND PURPOSE: A significant number of HIV-1 patients on antiretroviral therapy develop HIV-associated neurocognitive disorders (HAND). Evidence indicate that biological sex may regulate HAND pathogenesis, but the mechanisms remain unknown. We investigated synaptic mechanisms associated with sex differences in HAND, using the HIV-1-transgenic 26 (Tg26) mouse model. EXPERIMENTAL APPROACH: Contextual- and cue-dependent memories of male and female Tg26 mice and littermate wild type mice were assessed in a fear conditioning paradigm. Hippocampal electrophysiology, immunohistochemistry, western blot, qRT-PCR and ELISA techniques were used to investigate cellular, synaptic and molecular impairments. KEY RESULTS: Cue-dependent memory was unaltered in male and female Tg26 mice, when compared to wild type mice. Male, but not female, Tg26 mice showed deficits in contextual fear memory. Consistently, only male Tg26 mice showed depressed hippocampal basal synaptic transmission and impaired LTP induction in area CA1. These deficits in male Tg26 mice were independent of hippocampal neuronal loss and microglial activation but were associated with increased HIV-1 long terminal repeat mRNA expression, reduced hippocampal synapsin-1 protein, reduced BDNF mRNA and protein, reduced AMPA glutamate receptor (GluA1) phosphorylation levels and increased glycogen synthase kinase 3 (GSK3) activity. Importantly, selective GSK3 inhibition using 4-benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione increased levels of synapsin-1, BDNF and phosphorylated-GluA1 proteins, restored hippocampal basal synaptic transmission and LTP, and improved contextual fear memory in male Tg26 mice. CONCLUSION AND IMPLICATIONS: Sex-dependent impairments in contextual fear memory and synaptic plasticity in Tg26 mice are associated with increased GSK3 activity. This implicates GSK3 inhibition as a potential therapeutic strategy to improve cognition in HIV-1 patients.

Morphine tolerance is attenuated in germfree mice and reversed by probiotics, implicating the role of gut microbiome.

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.

Cytoplasmic translocation of polypyrimidine tract-binding protein and its binding to viral RNA during Japanese encephalitis virus infection inhibits virus replication.

Japanese encephalitis virus (JEV) has a single-stranded, positive-sense RNA genome containing a single open reading frame flanked by the 5'- and 3'-non-coding regions (NCRs). The virus genome replicates via a negative-sense RNA intermediate. The NCRs and their complementary sequences in the negative-sense RNA are the sites for assembly of the RNA replicase complex thereby regulating the RNA synthesis and virus replication. In this study, we show that the 55-kDa polypyrimidine tract-binding protein (PTB) interacts in vitro with both the 5'-NCR of the positive-sense genomic RNA--5NCR(+), and its complementary sequence in the negative-sense replication intermediate RNA--3NCR(-). The interaction of viral RNA with PTB was validated in infected cells by JEV RNA co-immunoprecipitation and JEV RNA-PTB colocalization experiments. Interestingly, we observed phosphorylation-coupled translocation of nuclear PTB to cytoplasmic foci that co-localized with JEV RNA early during JEV infection. Our studies employing the PTB silencing and over-expression in cultured cells established an inhibitory role of PTB in JEV replication. Using RNA-protein binding assay we show that PTB competitively inhibits association of JEV 3NCR(-) RNA with viral RNA-dependent RNA polymerase (NS5 protein), an event required for the synthesis of the plus-sense genomic RNA. cAMP is known to promote the Protein kinase A (PKA)-mediated PTB phosphorylation. We show that cells treated with a cAMP analogue had an enhanced level of phosphorylated PTB in the cytoplasm and a significantly suppressed JEV replication. Data presented here show a novel, cAMP-induced, PTB-mediated, innate host response that could effectively suppress JEV replication in mammalian cells.