Exosomes as Conduits: Facilitating Hepatitis B Virus-Independent Hepatitis D Virus Transmission and Propagation in Hepatocytes.

A number of research studies, including ours, have spotlighted exosomes as critical facilitators of viral dissemination. While hepatitis B virus (HBV) transmission through exosomes has been studied, the focus on its satellite virus, the hepatitis delta virus (HDV), has been unexplored in this context. HDV, although being a defective virus, can replicate its genome autonomously within hepatocytes, independently of HBV. Investigations on Huh7 cells revealed an intriguing phenomenon: the HDV proteins, S-HDAg and L-HDAg, are transmitted between cells without a complete viral structure. Detailed analysis further revealed that the expression of these proteins not only bolstered exosome secretion but also ensured their enrichment within these vesicles. Our experimental approach utilized transfection of various plasmids to examine the role of HDV RNA and proteins in the process. One salient finding was the differential propagation of the HDV proteins S-HDAg and L-HDAg, suggesting intricate molecular mechanisms behind their transmission. Notably, the purity of our exosome preparations was monitored using markers such as TSG101 and CD81. Importantly, these exosomes were found to carry both HDV RNA and proteins, highlighting their role in HDV dissemination. This novel study underscores the role of exosomes in mediating the transmission of HDV components between hepatocytes independent of HBV. These revelations about the exosomal pathway of HDV transmission provide a foundation for the development of innovative therapeutic strategies against HDV infections.

Relationship Between Recreational Cannabis Use and Helicobacter pylori Infection.

Background: Cannabis plant extracts suppress gastric acid secretion and inflammation, and promote gastroduodenal ulcer healing, all of which are triggered by Helicobacter Pylori infection (HPI). Here, we evaluate the association between cannabis use and HPI among a representative community sample. Materials and Methods: We identified respondents who completed cannabis use questions and were tested for HPI (H. pylori IgG antibody seropositivity) from the National Health and Nutrition Examination Survey III dataset (n=4556). Cannabis usage was categorized as ever-use (ever, never), cumulative lifetime use (>10-times, 1-10-times, never), or recent use (>31-days-ago, within-31-days, never). We calculated the crude and adjusted risk (prevalence rate ratio, cPRR and aPRR) of having HPI with cannabis use using generalized Poisson models (SAS 9.4). The models were adjusted for demographics and risk factors for HPI. Results: The prevalence of HPI was lower among ever versus never cannabis users (18.6% vs. 33%, p<0.0001). Cannabis use was associated with a decreased risk of HPI (cPRR: 0.56 confidence interval [95% CI: 0.47-0.67]; p<0.0001), which persisted after adjusting for demographics (aPRR: 0.75 [95% CI: 0.63-0.90]; p=0.0016) and comorbidities (aPRR: 0.79 [95% CI: 0.66-0.95]; p=0.0145). Further, individuals with >10-times lifetime cannabis use had a decreased risk of HPI compared with those with 1-10-times lifetime use (aPRR: 0.70 [95% CI: 0.55-0.89]; p=0.0011) and never-users (aPRR: 0.65 [95% CI: 0.50-0.84]; p=0.0002). Conclusion: Recreational cannabis use is associated with diminished risk of HPI. These observations suggest the need for additional research assessing the effects of medical cannabis formulations on HPI.

Neutrophil extracellular traps contribute to liver damage and increase defective low-density neutrophils in alcohol-associated hepatitis.

BACKGROUND & AIMS: In alcohol-associated hepatitis (AH), inflammation and neutrophil counts correlate with poor clinical outcomes. Here, we investigated how neutrophils contribute to liver damage in AH. METHODS: We isolated blood neutrophils from individuals with AH to examine neutrophil extracellular traps (NETs) and performed RNA sequencing to explore their unique characteristics. RESULTS: We observed a significant increase in NET production in AH. We also observed a unique low-density neutrophil (LDN) population in individuals with AH and alcohol-fed mice that was not present in healthy controls. Transcriptome analysis of peripheral LDNs and high-density neutrophils (HDNs) from individuals with AH revealed that LDNs exhibit a functionally exhausted phenotype, while HDNs are activated. Indeed, AH HDNs exhibited increased resting reactive oxygen species (ROS) production and produced more ROS upon lipopolysaccharide stimulation than control HDNs, whereas AH LDNs failed to respond to lipopolysaccharide. We show that LDNs are generated from HDNs after alcohol-induced NET release in vitro, and this LDN subset has decreased functionality, including reduced phagocytic capacity. Moreover, LDNs showed reduced homing capacity and clearance by macrophage efferocytosis; therefore, dysfunctional neutrophils could remain in the circulation and liver. Depletion of both HDNs and LDNs in vivo prevented alcohol-induced NET production and liver damage in mice. Granulocyte-colony stimulating factor treatment also ameliorated alcohol-induced liver injury in mice. CONCLUSION: Neutrophils contribute to liver damage through increased NET formation which increases defective LDNs in AH. Alcohol induces phenotypic changes in neutrophils; HDNs are activated whereas LDNs are defective. Our findings provide mechanistic insights that could guide the development of therapeutic interventions for AH. IMPACT AND IMPLICATIONS: In this study we discovered heterogeneity of neutrophils in alcohol-associated hepatitis, including high-density and low-density neutrophils that show hyper-activated or exhausted transcriptomic profiles, respectively. We found that alcohol induces neutrophil extracellular trap (NET) formation, which contributes to liver damage. NET release by high-density neutrophils resulted in low-density neutrophils that reside in the liver and escape clean-up by macrophages. Our findings help to understand the opposing neutrophil phenotypes observed in individuals with alcohol-associated hepatitis and provide mechanistic insights that could guide therapeutic strategies targeting neutrophils.

Reticulon-3 modulates the incorporation of replication competent hepatitis C virus molecules for release inside infectious exosomes.

BACKGROUND: Cell released microvesicles specifically, exosomes, play an important role in mediating immunologic escape, treatment resistance, and disease persistence of Hepatitis C virus (HCV) infection. Reports on the molecular compositions of exosomes released by cells under diverse conditions, especially during viral infections, suggest that their cargo contents are not randomly loaded. However, the precise molecular mechanisms directing the selective cargo sorting and loading inside infectious viral exosomes remains elusive. AIM: To decipher the role of Reticulon 3 (RTN3) in the selective molecular cargo sorting and loading inside infectious viral exosomes during HCV infection. METHODS: We used Huh7 cells-JFH1 HCV infection and HCV Full-Length (FL) replicon systems. Additionally, we analyzed human liver and serum exosome samples from healthy and treatment naïve HCV infected individuals. Our experiments made use of molecular biology and immunology techniques. RESULTS: HCV infection (JFH1-Huh7 or HCV-FL replicon cells) was associated with increased RTN3L&S isoforms expression in cells and cell released exosomes. Accordingly, increased expression of RTN3L&S was observed in liver and serum exosome samples of HCV infected individuals compared to healthy controls. RNA-ChIP analysis revealed that RTN3L&S interacted with dsHCV RNA. Lentiviral CRISPR/Cas9 mediated knockdown (KD) of RTN3 and plasmid overexpression (OE) of wild type, C- and N-terminal deletion mutants of RTN3L&S in HCV- infected Huh7 cells differentially impacted the cellular release of infectious viral exosomes. RTN3L&S KD significantly decreased, while RTN3S OE significantly increased the number of Huh7 cell-released infectious exosomes. The C-terminal domain of RTN3 interacted with and modulated the loading of dsHCV RNA inside infectious exosomes. Antiviral treatment of HCV infected Huh7 cells reduced virus-induced RTN3L&S expression and attenuated the release of infectious exosomes. CONCLUSION: RTN3 constitutes a novel regulator and a potential therapeutic target that mediates the specific loading of infectious viral exosomes.

Cannabis use and risk of Clostridioides difficile infection: Analysis of 59,824 hospitalizations.

BACKGROUND: The prevalence of Clostridioides difficile Infection (CDI), the most notorious hospital acquired disease, and of excessive cannabis use (cannabis use disorder (CUD)) have both been steadily rising. Although cannabidiol, an active ingredient of cannabis, maintains gut integrity and suppresses entero-toxins from Clostridioides difficile, the relationship between CUD and CDI has not been studied. METHODS: We selected adult records (age ≥ 18 years) from the Nationwide Inpatient Sample 2014, and identified CUD and other clinical conditions using ICD-9-CM codes. We used propensity scores derived from a multivariate logistic model to match CUD to non-CUD in a 1:1 ratio (29,912:29,912). We estimated the relative risk for CDI using log-binomial regression models with generalized estimating equations [SAS 9.4]. RESULTS: Among the matched hospitalizations (n = 59,824), cannabis usage was associated with a reduced prevalence of CDI (prevalence: 455.5 [95% CI: 385.1-538.8] vs. 636.4 [95% CI: 549.9-736.5] per 100,000 hospitalizations), resulting in a 28% reduced risk of CDI (relative risk: 0.72 [95% CI: 0.58-0.88]; p = 0002). Non-dependent and dependent CUD respectively had 23% and 80% reduced likelihood of CDI when compared to non-cannabis users (0.77 [95% CI: 0.60-0.95] and 0.20 [95% CI: 0.06-0.54]; p < 0.05). Furthermore, dependent users had less risk of CDI compared to non-dependent users (0.26 [95% CI: 0.08-0.88]; p = 0.01). CONCLUSIONS: CUD was associated with a decreased risk of CDI amongst hospitalized patients. Prospective and molecular mechanistic studies are required to elucidate how cannabis and its contents impacts CDI.

Reduced Prevalence of Alcoholic Gastritis in Hospitalized Individuals Who Consume Cannabis.

BACKGROUND: Alcoholic gastritis, a superficial erosive disease of the stomach, is a common manifestation of risky alcohol use. In contrast, cannabis which is frequently co-used with alcohol suppresses gastric acidity and might counteract the deleterious effect of alcohol on the gastric mucosa. However, no clinical study has examined the impact of cannabis use on the development of alcoholic gastritis among risky alcohol users. METHODS: We analyzed hospital discharge records of adults (age ≥ 18 years), from 2014 of the Nationwide Inpatient Sample, with a diagnosis of risky alcohol use (n = 316,916). We used a propensity-based matching algorithm to match cannabis users to nonusers on 1:1 ratio (30,713: 30,713). We then measured the adjusted relative risk (aRR) for having alcoholic gastritis using conditional Poisson regression models with generalized estimating equations. RESULTS: Our study revealed that among risky alcohol users, cannabis co-users have a lower prevalence of alcoholic gastritis compared to noncannabis users (1,289 [1,169 to 1,421] vs. 1,723 [1,583 to 1,875] per 100,000 hospitalizations for risky alcohol use), resulting in a 25% decreased probability of alcoholic gastritis (aRR: 0.75 [0.66 to 0.85]; p-value <0.0001). Furthermore, dependent cannabis usage resulted in a lower prevalence of alcoholic gastritis when compared to both nondependent cannabis users (0.72 [0.52 to 0.99]) and to noncannabis users (0.56 [0.41 to 0.76]). CONCLUSIONS: We reveal that risky alcohol drinking combined with cannabis use is associated with reduced prevalence of alcohol-associated gastritis in patients. Given increased cannabis legislation globally, understanding whether and how the specific ingredients in cannabis plant extract can be used in the treatment of alcoholic gastritis is paramount. In this regard, further molecular mechanistic studies are needed to delineate the mechanisms of our novel findings not only for alcoholic gastritis but also for gastritis from other causes.

Reduced Risk of Alcohol-Induced Pancreatitis With Cannabis Use.

BACKGROUND: Pancreatitis is an increasingly common clinical condition that causes significant morbidity and mortality. Cannabis use causes conflicting effects on pancreatitis development. We conducted a larger and more detailed assessment of the impact of cannabis use on pancreatitis. METHODS: We analyzed data from 2012 to 2014 of the Healthcare Cost and Utilization Project-Nationwide Inpatient Sample discharge records of patients 18 years and older. We used the International Classification of Disease, Ninth Edition codes, to identify 3 populations: those with gallstones (379,125); abusive alcohol drinkers (762,356); and non-alcohol-non-gallstones users (15,255,464). Each study population was matched for cannabis use record by age, race, and gender, to records without cannabis use. The estimation of the adjusted odds ratio (aOR) of having acute and chronic pancreatitis (AP and CP) made use of conditional logistic models. RESULTS: Concomitant cannabis and abusive alcohol use were associated with reduced incidence of AP and CP (aOR: 0.50 [0.48 to 0.53] and 0.77 [0.71 to 0.84]). Strikingly, for individuals with gallstones, additional cannabis use did not impact the incidence of AP or CP. Among non-alcohol-non-gallstones users, cannabis use was associated with increased incidence of CP, but not AP (1.28 [1.14 to 1.44] and 0.93 [0.86 to 1.01]). CONCLUSIONS: Our findings suggest a reduced incidence of only alcohol-associated pancreatitis with cannabis use.

Reduced Incidence and Better Liver Disease Outcomes among Chronic HCV Infected Patients Who Consume Cannabis.

Background and Aim: The effect of cannabis use on chronic liver disease (CLD) from Hepatitis C Virus (HCV) infection, the most common cause of CLD, has been controversial. Here, we investigated the impact of cannabis use on the prevalence of CLD among HCV infected individuals. Methods: We analyzed hospital discharge records of adults (age ≥ 18 years) with a positive HCV diagnosis. We evaluated records from 2007 to 2014 of the Nationwide Inpatient Sample (NIS). We excluded records with other causes of chronic liver diseases (alcohol, hemochromatosis, NAFLD, PBC, HBV, etc.). Of the 188,333 records, we matched cannabis users to nonusers on 1:1 ratio (4,728:4,728), using a propensity-based matching system, with a stringent algorithm. We then used conditional regression models with generalized estimating equations to measure the adjusted prevalence rate ratio (aPRR) for having liver cirrhosis (and its complications), carcinoma, mortality, discharge disposition, and the adjusted mean ratio (aMR) of total hospital cost and length of stay (LOS) [SAS 9.4]. Results: Our study revealed that cannabis users (CUs) had decreased prevalence of liver cirrhosis (aPRR: 0.81[0.72-0.91]), unfavorable discharge disposition (0.87[0.78-0.96]), and lower total health care cost ($39,642[36,220-43,387] versus $45,566[$42,244-$49,150]), compared to noncannabis users (NCUs). However, there was no difference among CUs and NCUs on the incidence of liver carcinoma (0.79[0.55-1.13]), in-hospital mortality (0.84[0.60-1.17]), and LOS (5.58[5.10-6.09] versus 5.66[5.25-6.01]). Among CUs, dependent cannabis use was associated with lower prevalence of liver cirrhosis, compared to nondependent use (0.62[0.41-0.93]). Conclusions: Our findings suggest that cannabis use is associated with decreased incidence of liver cirrhosis, but no change in mortality nor LOS among HCV patients. These novel observations warrant further molecular mechanistic studies.

Abnormal neutrophil traps and impaired efferocytosis contribute to liver injury and sepsis severity after binge alcohol use.

BACKGROUND & AIMS: Neutrophil extracellular traps (NETs) are an important strategy utilized by neutrophils to immobilize and kill invading microorganisms. Herein, we studied NET formation and the process of neutrophil cell death (NETosis), as well as the clearance of NETs by macrophages (MΦ) (efferocytosis) in acute sepsis following binge drinking. METHODS: Healthy volunteers consumed 2 ml of vodka/kg body weight, before blood endotoxin and 16 s rDNA were measured. Peripheral neutrophils were isolated and exposed to alcohol followed by phorbol 12-myristate 13-acetate (PMA) stimulation. Mice were treated with three alcohol binges and intraperitoneal lipopolysaccharide (LPS) to assess the dynamics of NET formation and efferocytosis. In vivo, anti-Ly6G antibody (IA8) was used for neutrophil depletion. RESULTS: Inducers of NETs (endotoxin and bacterial DNA) significantly increased in the circulation after binge alcohol drinking in humans. Ex vivo, alcohol alone increased NET formation, but upon PMA stimulation alcohol attenuated NET formation. Binge alcohol in mice resulted in a biphasic response to LPS. Initially, binge alcohol reduced LPS-induced NET formation and resulted in a diffuse distribution of neutrophils in the liver compared to alcohol-naïve mice. Moreover, indicators of NET formation including citrullinated histone H3, neutrophil elastase, and neutrophil myeloperoxidase were decreased at an early time point after LPS challenge in mice receiving binge alcohol, suggesting decreased NET formation. However, in the efferocytosis phase (15 h after LPS) citrullinated histone-H3 was increased in the liver in alcohol binge mice, suggesting decreased clearance of NETs. In vitro alcohol treatment reduced efferocytosis and phagocytosis of NETotic neutrophils and promoted expression of CD206 on MΦ. Finally, depletion of neutrophils prior to binge alcohol ameliorated LPS-induced systemic inflammation and liver injury in mice. CONCLUSIONS: Dysfunctional NETosis and efferocytosis following binge drinking exacerbate liver injury associated with sepsis. LAY SUMMARY: Disease severity in alcoholic liver disease (ALD) is associated with a significant presence of neutrophils (a type of immune cell) in the liver. It remains unknown how alcohol affects the capacity of neutrophils to control infection, a major hallmark of ALD. We found that binge alcohol drinking impaired important strategies used by neutrophils to contain and resolve infection, resulting in increased liver injury during ALD.

Cannabis use is associated with reduced prevalence of non-alcoholic fatty liver disease: A cross-sectional study.

Cannabis use is associated with reduced prevalence of obesity and diabetes mellitus (DM) in humans and mouse disease models. Obesity and DM are a well-established independent risk factor for non-alcoholic fatty liver disease (NAFLD), the most prevalent liver disease globally. The effects of cannabis use on NAFLD prevalence in humans remains ill-defined. Our objective is to determine the relationship between cannabis use and the prevalence of NAFLD in humans. We conducted a population-based case-control study of 5,950,391 patients using the 2014 Healthcare Cost and Utilization Project (HCUP), Nationwide Inpatient Survey (NIS) discharge records of patients 18 years and older. After identifying patients with NAFLD (1% of all patients), we next identified three exposure groups: non-cannabis users (98.04%), non-dependent cannabis users (1.74%), and dependent cannabis users (0.22%). We adjusted for potential demographics and patient related confounders and used multivariate logistic regression (SAS 9.4) to determine the odds of developing NAFLD with respects to cannabis use. Our findings revealed that cannabis users (dependent and non-dependent) showed significantly lower NAFLD prevalence compared to non-users (AOR: 0.82[0.76-0.88]; p<0.0001). The prevalence of NAFLD was 15% lower in non-dependent users (AOR: 0.85[0.79-0.92]; p<0.0001) and 52% lower in dependent users (AOR: 0.49[0.36-0.65]; p<0.0001). Among cannabis users, dependent patients had 43% significantly lower prevalence of NAFLD compared to non-dependent patients (AOR: 0.57[0.42-0.77]; p<0.0001). Our observations suggest that cannabis use is associated with lower prevalence of NAFLD in patients. These novel findings suggest additional molecular mechanistic studies to explore the potential role of cannabis use in NAFLD development.