Altered gut microbiome drives heightened pain sensitivity in a murine model of metastatic triple-negative breast cancer.

The microbiota residing in the gut environment is essential for host homeostasis. Increasing evidence suggests that microbial perturbation (dysbiosis) regulates cancer initiation and progression at local and distant sites. Here, we have identified microbial dysbiosis with the depletion of commensal bacteria as a host-intrinsic factor associated with metastatic dissemination to the bone. Using a mouse model of triple-negative mammary cancer, we demonstrate that a pre-established disruption of microbial homeostasis using an antibiotic cocktail increases tumor growth, enhanced circulating tumor cells, and subsequent dissemination to the bone. We found that the presence of pathogenic bacteria and loss of commensal bacteria in an antibiotic-induced gut environment is associated with sustained inflammation. Increased secretion of G-CSF and MMP-9 in intestinal tissues, followed by increased neutrophil infiltration and severe systemic inflammation in tumor-bearing mice, indicates the direct consequence of a dysbiotic microbiome. Increased neutrophil infiltration to the bone metastatic niche facilitates extravasation and transendothelial migration of tumor cells. It provides a novel, pre-established, and favorable environment to form an immunosuppressive pre-metastatic niche. The presence of tumor cells in immunosuppressive metastatic tumor niche disrupts the balance between osteoblasts and osteoclasts, promotes osteoclast differentiation, and remodels the bone structure. Excessive bone resorption by osteoclasts causes bone degradation and ultimately causes extreme pain in a bone metastatic mouse model. In clinical settings, bone metastasis is associated with intractable severe pain that severely compromises the quality of life in these patients.

Opioid-induced dysbiosis of maternal gut microbiota during gestation alters offspring gut microbiota and pain sensitivity.

There has been a rapid increase in neonates born with a history of prenatal opioid exposure. How prenatal opioid exposure affects pain sensitivity in offspring is of interest, as this may perpetuate the opioid epidemic. While few studies have reported hypersensitivity to thermal pain, potential mechanisms have not been described. This study posits that alterations in the gut microbiome may underly hypersensitivity to pain in prenatally methadone-exposed 3-week-old male offspring, which were generated using a mouse model of prenatal methadone exposure. Fecal samples collected from dams and their offspring were subjected to 16s rRNA sequencing. Thermal and mechanical pain were assessed using the tail flick and Von Frey assays. Transcriptomic changes in whole brain samples of opioid or saline-exposed offspring were investigated using RNA-sequencing, and midbrain sections from these animals were subjected to qPCR profiling of genes related to neuropathic and inflammatory pain pathways. Prenatal methadone exposure increased sensitivity to thermal and mechanical pain and elevated serum levels of IL-17a. Taxonomical analysis revealed that prenatal methadone exposure resulted in significant alterations in fecal gut microbiota composition, including depletion of Lactobacillus, Bifidobacterium, and Lachnospiracea sp and increased relative abundance of Akkermansia, Clostridium sensu stricto 1, and Lachnoclostridium. Supplementation of the probiotic VSL#3 in dams rescued hypersensitivity to thermal and mechanical pain in prenatally methadone-exposed offspring. Similarly, cross-fostering prenatally methadone-exposed offspring to control dams also attenuated hypersensitivity to thermal pain in opioid-exposed offspring. Modulation of the maternal and neonatal gut microbiome with probiotics resulted in transcriptional changes in genes related to neuropathic and immune-related signaling in whole brain and midbrain samples of prenatally methadone-exposed offspring. Together, our work provides compelling evidence of the gut-brain-axis in mediating pain sensitivity in prenatally opioid-exposed offspring.

Sirtuin 2 promotes human cytomegalovirus replication by regulating cell cycle progression.

IMPORTANCE: This study expands the growing understanding that protein acetylation is a highly regulated molecular toggle of protein function in both host anti-viral defense and viral replication. We describe a pro-viral role for the human enzyme SIRT2, showing that its deacetylase activity supports HCMV replication. By integrating quantitative proteomics, flow cytometry cell cycle assays, microscopy, and functional virology assays, we investigate the temporality of SIRT2 functions and substrates. We identify a pro-viral role for the SIRT2 deacetylase activity via regulation of CDK2 K6 acetylation and the G1-S cell cycle transition. These findings highlight a link between viral infection, protein acetylation, and cell cycle progression.

HIV, opioid use, and alterations to the gut microbiome: elucidating independent and synergistic effects.

Background: The microbiome is essential to immune development, defense against pathogens, and modulation of inflammation. Microbial dysbiosis has been reported in various diseases including human immunodeficiency virus (HIV) and opioid use disorder (OUD). Notably, people living with HIV (PLWH) have been reported to both have higher rates of OUD and use opioids at higher rates than the general public. Thus, studying gut microbial alterations in people living with HIV and with OUD could elucidate mechanisms pertaining to how these conditions both shape and are shaped by the microbiome. However, to date few studies have investigated how HIV and OUD in combination impact the microbiome. Aim of review: Here, we review previous studies outlining interactions between HIV, opioid use, and microbial dysbiosis and describe attempts to treat this dysbiosis with fecal microbial transplantation, probiotics, and dietary changes. Key scientific concepts of review: While the limited number of studies prevent overgeneralizations; accumulating data suggest that HIV and opioid use together induce distinct alterations in the gut microbiome. Among the three existing preclinical studies of HIV and opioid use, two studies reported a decrease in Lachnospiraceae and Ruminococcaceae, and one study reported a decrease in Muribaculaceae in the combined HIV and opioid group relative to HIV-alone, opioid-alone, or control groups. These bacteria are known to modulate immune function, decrease colonic inflammation, and maintain gut epithelial barrier integrity in healthy individuals. Accordingly, modulation of the gut microbiome to restore gut homeostasis may be attempted to improve both conditions. While mixed results exist regarding treating dysbiosis with microbial restoration in PLWH or in those with opioid dependency, larger well-defined studies that can improve microbial engraftment in hosts hold much promise and should still be explored.

Opioid-induced microbial dysbiosis disrupts irinotecan (CPT-11) metabolism and increases gastrointestinal toxicity in a murine model.

BACKGROUND AND PURPOSE: Opioids are commonly used for the management of cancer-associated pain and chemotherapy-induced diarrhoea. The chemotherapeutic irinotecan (CPT-11) causes severe gastrointestinal (GI) toxicity due to deconjugation of inactive metabolite SN-38 glucuronide (SN-38G) by bacterial ß-glucuronidases to the active 7-ethyl-10-hydroxycamptothecin (SN-38). Opioids are known to cause gut microbial dysbiosis, this study evaluated whether CPT-11 anti-tumour efficacy and GI toxicity are exacerbated by opioid co-administration. EXPERIMENTAL APPROACH: Eight-week-old C57BL/6 male mice were co-administration with CPT-11 ± opioid. 16S rRNA sequencing was used for gut microbiome analysis. LC-MS analyses of plasma and intestinal extracts were performed to investigate the pharmacokinetic profile of CPT-11. Histological analysis and quantitative real-time polymerase chain reaction were used to determine the severity of intestinal tissue damage. Human liver microsome In vitro assay was performed to confirm the effects of opioids on CPT-11 metabolism. KEY RESULTS: Gut microbiome analysis showed that morphine treatment induced enrichment of ß-glucuronidase-producing bacteria in the intestines of CPT-11-treated mice, resulting in SN-38 accumulation and exacerbation of GI toxicity in the small intestine. Oral administration of both antibiotics and glucuronidase inhibitor protected mice against GI toxicity induced with CPT-11 and morphine co-administration, implicating a microbiome-dependent mechanism. Additionally, morphine and loperamide decreased the plasma concentration of SN-38 and compromised CPT-11 anti-tumour efficacy, this seemed to be microbiome independent. CONCLUSION AND IMPLICATIONS: Gut microbiota play a significant role in opioid and chemotherapeutic agent drug-drug interactions. Inhibition of gut microbial glucuronidase may also prevent adverse GI effects of CPT-11 in patients on opioids.

HIV-Positive Patients on Antiretroviral Therapy Have an Altered Mucosal Intestinal but Not Oral Microbiome.

This study characterized compositional and functional shifts in the intestinal and oral microbiome in HIV-positive patients on antiretroviral therapy compared to HIV-negative individuals. Seventy-nine specimens were collected from 5 HIV-positive and 12 control subjects from five locations (colon brush, colon wash, terminal ileum [TI] brush, TI wash, and saliva) during colonoscopy and at patient visits. Microbiome composition was characterized using 16S rRNA sequencing, and microbiome function was predicted using bioinformatics tools (PICRUSt and BugBase). Our analysis indicated that the ß-diversity of all intestinal samples (colon brush, colon wash, TI brush, and TI wash) from patients with HIV was significantly different from patients without HIV. Specifically, bacteria from genera Prevotella, Fusobacterium, and Megasphaera were more abundant in samples from HIV-positive patients. On the other hand, bacteria from genera Ruminococcus, Blautia, and Clostridium were more abundant in samples from HIV-negative patients. Additionally, HIV-positive patients had higher abundances of biofilm-forming and pathogenic bacteria. Furthermore, pathways related to translation and nucleotide metabolism were elevated in HIV-positive patients, whereas pathways related to lipid and carbohydrate metabolism were positively correlated with samples from HIV-negative patients. Our analyses further showed variations in microbiome composition in HIV-positive and negative patients by sampling site. Samples from colon wash, colon brush, and TI wash were significant between groups, while samples from TI brush and saliva were not significant. Taken together, here, we report altered intestinal microbiome composition and predicted function in patients with HIV compared to uninfected patients, though we found no changes in the oral microbiome. IMPORTANCE Over 37 million people worldwide are living with HIV. Although the availability of antiretroviral therapy has significantly reduced the number of AIDS-related deaths, individuals living with HIV are at increased risk for opportunistic infections. We now know that HIV interacts with the trillions of bacteria, fungi, and viruses in the human body termed the microbiome. Only a limited number of previous studies have compared variations in the oral and gastrointestinal microbiome with HIV infection. Here, we detail how the oral and gastrointestinal microbiome changes with HIV infection, having used 5 different sampling sites to gain a more comprehensive view of these changes by location. Our results show site-specific changes in the intestinal microbiome associated with HIV infection. Additionally, we show that while there were significant changes in the intestinal microbiome, there were no significant changes in the oral microbiome.

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.

Opioids and Sepsis: Elucidating the Role of the Microbiome and microRNA-146.

Sepsis has recently been defined as life-threatening organ dysfunction caused by the dysregulated host response to an ongoing or suspected infection. To date, sepsis continues to be a leading cause of morbidity and mortality amongst hospitalized patients. Many risk factors contribute to development of sepsis, including pain-relieving drugs like opioids, which are frequently prescribed post-operatively. In light of the opioid crisis, understanding the interactions between opioid use and the development of sepsis has become extremely relevant, as opioid use is associated with increased risk of infection. Given that the intestinal tract is a major site of origin of sepsis-causing microbes, there has been an increasing focus on how alterations in the gut microbiome may predispose towards sepsis and mediate immune dysregulation. MicroRNAs, in particular, have emerged as key modulators of the inflammatory response during sepsis by tempering the immune response, thereby mediating the interaction between host and microbiome. In this review, we elucidate contributing roles of microRNA 146 in modulating sepsis pathogenesis and end with a discussion of therapeutic targeting of the gut microbiome in controlling immune dysregulation in sepsis.

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.

Brief Hydromorphone Exposure During Pregnancy Sufficient to Induce Maternal and Neonatal Microbial Dysbiosis.

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.

Prenatal opioid exposure and vulnerability to future substance use disorders in offspring.

The heightened incidence of opioid use during pregnancy has resulted in unprecedented rates of neonates prenatally exposed to opioids. Prenatal opioid exposure (POE) results in significantly adverse medical, developmental, and behavioral outcomes in offspring. Of growing interest is whether POE contributes to future vulnerability to substance use disorders. The effects of POE on brain development is difficult to assess in humans, as the timing, dose, and route of drug exposure together with complex genetic and environmental factors affect susceptibility to addiction. Preclinical models of POE have allowed us to avoid methodological difficulties and confounding factors of POE in humans. Here, we review the effects of maternal opioid exposure on the developing brain with an emphasis on the neurobiological basis of drug addiction and on preclinical models of POE and their limitations. These studies have indicated that POE increases self-administration of drugs, reward-driven behaviors in the conditioned place paradigm, and locomotor sensitization. While addiction is multifaceted and vulnerability to drug addiction is still inconclusive in human studies of prenatally exposed infants, animal studies do provide a noteworthy corroboration of negative behavioral outcomes.

Extracellular LDLR repeats modulate Wnt signaling activity by promoting LRP6 receptor endocytosis mediated by the Itch E3 ubiquitin ligase.

The LOW-density lipoprotein related protein 6 (LRP6) receptor is an important effector of canonical Wnt signaling, a developmental pathway, whose dysregulation has been implicated in various diseases including cancer. The membrane proximal low-density lipoprotein (LDL) receptor repeats in LRP6 exhibit homology to ligand binding repeats in the LDL receptor (LDLR), but lack known function. We generated single amino acid substitutions of LRP6-LDLR repeat residues, which are highly conserved in the human LDLR and mutated in patients with Familial Hypercholesteremia (FH). These substitutions negatively impacted LRP6 internalization and activation of Wnt signaling. By mass spectrometry, we observed that the Itch E3 ubiquitin ligase associated with and ubiquitinated wild type LRP6 but not the LDLR repeat mutants. These findings establish the involvement of LRP6-LDLR repeats in the regulation of canonical Wnt signaling.