Alcohol-induced extracellular ASC specks perpetuate liver inflammation and damage in alcohol-associated hepatitis even after alcohol cessation.

BACKGROUND AIMS: Prolonged systemic inflammation contributes to poor clinical outcomes in severe alcohol-associated hepatitis (AH) even after the cessation of alcohol use. However, mechanisms leading to this persistent inflammation remain to be understood. APPROACH RESULTS: We show that while chronic alcohol induces nucleotide-binding oligomerization domain-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome activation in the liver, alcohol binge results not only in NLRP3 inflammasome activation but also in increased circulating extracellular apoptosis-associated speck-like protein containing a caspase recruitment domain (ex-ASC) specks and hepatic ASC aggregates both in patients with AH and in mouse models of AH. These ex-ASC specks persist in circulation even after the cessation of alcohol use. Administration of alcohol-induced-ex-ASC specks in vivo in alcohol-naive mice results in sustained inflammation in the liver and circulation and causes liver damage. Consistent with the key role of ex-ASC specks in mediating liver injury and inflammation, alcohol binge failed to induce liver damage or IL-1ß release in ASC-deficient mice. Our data show that alcohol induces ex-ASC specks in liver macrophages and hepatocytes, and these ex-ASC specks can trigger IL-1ß release in alcohol-naive monocytes, a process that can be prevented by the NLRP3 inhibitor, MCC950. In vivo administration of MCC950 reduced hepatic and ex-ASC specks, caspase-1 activation, IL-1ß production, and steatohepatitis in a murine model of AH. CONCLUSIONS: Our study demonstrates the central role of NLRP3 and ASC in alcohol-induced liver inflammation and unravels the critical role of ex-ASC specks in the propagation of systemic and liver inflammation in AH. Our data also identify NLRP3 as a potential therapeutic target in AH.

Steatosis, inflammasome upregulation, and fibrosis are attenuated in miR-155 deficient mice in a high fat-cholesterol-sugar diet-induced model of NASH.

Nonalcoholic fatty liver disease (NAFLD) is the most common cause of chronic liver disease globally. miRNAs (miRs) regulate various cellular events that lead to NAFLD. In this study we tested the hypothesis that miR-155 is an important regulator of steatohepatitis and fibrosis pathways. Wild type (WT) or miR-155 deficient (KO) mice received a high fat-high cholesterol-high sugar-diet (HF-HC-HS) for 34 weeks and liver tissues were analyzed. In patients with nonalcoholic steatohepatitis and in the mouse model of HF-HC-HS diet we found increased miR-155 levels in the liver compared to normal livers. Upon HF-HC-HS diet feeding, miR-155 KO mice displayed less liver injury, decreased steatosis, and attenuation in fibrosis compared to WT mice. ALT, triglyceride levels, and genes involved in fatty acid metabolic pathway were increased in WT mice whereas miR-155 KO mice showed attenuation in these parameters. HF-HC-HS diet-induced significant increase in the expression of NLRP3 inflammasome components in the livers of WT mice compared to chow fed diet. Compared to WT mice, miR-155 KO showed attenuated induction in the NLRP3, ASC, and caspase1 inflammasome expression on HF-HC-HS diet. Fibrosis markers such as collagen content and deposition, αSMA, Zeb2, and vimentin were all increased in WT mice and miR-155 KO mice showed attenuated fibrosis marker expression. Overall, our findings highlight a role for miR-155 in HF-HC-HS diet-induced steatosis and liver fibrosis.

Endoplasmic Reticulum Stress-induced Hepatocellular Death Pathways Mediate Liver Injury and Fibrosis via Stimulator of Interferon Genes.

Fibrosis, driven by inflammation, marks the transition from benign to progressive stages of chronic liver diseases. Although inflammation promotes fibrogenesis, it is not known whether other events, such as hepatocyte death, are required for the development of fibrosis. Interferon regulatory factor 3 (IRF3) regulates hepatocyte apoptosis and production of type I IFNs. In the liver, IRF3 is activated via Toll-like receptor 4 (TLR4) signaling or the endoplasmic reticulum (ER) adapter, stimulator of interferon genes (STING). We hypothesized that IRF3-mediated hepatocyte death is an independent determinant of chemically induced liver fibrogenesis. To test this, we performed acute or chronic CCl4 administration to WT and IRF3-, Toll/Interleukin-1R (TIR) domain-containing adapter-inducing interferon-ß (TRIF)-, TRIF-related adaptor molecule (TRAM)-, and STING-deficient mice. We report that acute CCl4 administration to WT mice resulted in early ER stress, activation of IRF3, and type I IFNs, followed by hepatocyte apoptosis and liver injury, accompanied by liver fibrosis upon repeated administration of CCl4 Deficiency of IRF3 or STING prevented hepatocyte death and fibrosis both in acute or chronic CCl4 In contrast, mice deficient in type I IFN receptors or in TLR4 signaling adaptors, TRAM or TRIF, upstream of IRF3, were not protected from hepatocyte death and/or fibrosis, suggesting that the pro-apoptotic role of IRF3 is independent of TLR signaling in fibrosis. Hepatocyte death is required for liver fibrosis with causal involvement of STING and IRF3. Thus, our results identify that IRF3, by its association with STING in the presence of ER stress, couples hepatocyte apoptosis with liver fibrosis and indicate that innate immune signaling regulates outcomes of liver fibrosis via modulation of hepatocyte death in the liver.

Acute Kidney Injury in a Previously Healthy 56-Year-Old Male Following a Direct Visual Internal Urethrotomy of a Bulbar Stricture.

Acute kidney injury (AKI) is a frequent finding in acutely ill and hospitalized patients arising from various etiologies. Anuric AKI, a more pronounced form of AKI in which less than 100 cc of urine is produced per day, is most frequently encountered in hospitalized, septic, and post-surgical patients, often secondary to shock or bilateral urinary tract obstruction. The development of anuric AKI in previously healthy patients after outpatient urological procedures presents a unique challenge to physicians, as many outpatient procedures require the routine perioperative administration of multiple nephrotoxic medications. Further complicating this clinical scenario, some surgical procedures that intrinsically involve iatrogenic injury to the kidney, ureter, bladder, or nearby organ can rarely lead to a phenomenon known as reflex anuria, an anuric state typically associated with AKI. Here, we report an unusual case of a previously healthy 56-year-old male who developed anuric AKI two days after direct visual internal urethrotomy (DVIU) for the treatment of a bulbar stricture. Non-contrast CT revealed no signs of an obstructive process, and laboratory findings supported an intrarenal cause of AKI. Consideration was given to non-steroidal anti-inflammatory drugs (NSAID)-induced nephrotoxicity, gentamicin-associated acute tubular necrosis, and propofol infusion syndrome, in addition to their potential synergistic effects. We also explore this as the first reported case of reflex anuria occurring at the level of the bulbar urethra, as most cases have involved direct injury to the kidney or ureter. Over the course of 10 days, our patient responded well to treatment with supportive measures and dialysis, with his vomiting, electrolyte abnormalities, renal state, and anuria eventually improving.

Pneumatosis Intestinalis, Pneumoperitoneum, Pneumoretroperitoneum, Pneumomediastinum, and Pneumobilia After Pembrolizumab Therapy: A Case Report.

Immune checkpoint inhibitors (ICIs) are becoming increasingly popular in treating cancers resistant to traditional chemotherapy. While ICIs have shown promise in treating cancer, the class of drugs also comes with certain risks, such as the development of pneumatosis intestinalis (PI) in rare cases. Pembrolizumab, an ICI that inhibits programmed cell death protein 1 (PD-1), has, in some rare instances, caused PI. Patients with ICI-induced PI may also present with pneumoperitoneum, pneumoretroperitoneum, pneumomediastinum, and pneumobilia. In the current report, we describe the presentation and management of a 50-year-old female with initial complaints of diffuse abdominal pain, constipation, abdominal distension, nausea, and decreased urine output approximately six months after beginning pembrolizumab and two months after the most recent dose of pembrolizumab. Subsequent CT imaging revealed massive PI with pneumoperitoneum, pneumoretroperitoneum, pneumomediastinum, and pneumobilia suspected to be secondary to pembrolizumab. Here, we discuss the possible mechanisms of ICI-induced PI and evaluate the management of patients presenting with PI and pneumoperitoneum.

Deficiency of miR-208a Exacerbates CCl4-Induced Acute Liver Injury in Mice by Activating Cell Death Pathways.

Acute liver injury (ALI) is associated with multiple cellular events such as necrosis, apoptosis, oxidative stress and inflammation, which can lead to liver failure. In this study, we demonstrate a new role of microRNA (miR)-208a in ALI. ALI was induced in wild-type (WT) and miR-208a knockout (KO) mice by CCl4 administration. Increased alanine aminotransferase and decreased hepatic miR-208a levels were found in WT mice after acute CCl4 treatment. Histopathological evaluations revealed increased necrosis and decreased inflammation in miR-208a KO compared with WT mice after CCl4 treatment. CCl4 treatment induced a higher alanine aminotransferase elevation and increased numbers of circulating extracellular vesicles (exosomes and microvesicles) in miR-208a KO compared with WT mice. We found increased CCl4-induced nuclear factor kappa B activation and tumor necrosis factor-α induction and decreased monocyte chemoattractant protein 1 levels in miR-208a KO compared with WT mice. Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling assay indicated aggravated hepatic apoptosis and necrosis in CCl4 -treated miR-208a KO compared with WT mice. CCl4 treatment induced a greater increase in cleaved caspase-8, p18, and caspase-3 in miR-208a KO compared with WT mice. p53 is involved in various cell death pathways, including necrosis and apoptosis. Our in silico analysis revealed p53 as a predicted miR-208a target, and we found enhanced p53 and cyclophilin D protein expressions in miR-208a KO mice after CCl4 treatment. Increased liver injury in miR-208a KO mice was further associated with increased Bax (B cell lymphoma 2-associated X protein) and p21 expression. Our in vitro results indicated a role of miR-208a in cell death. We found that CCl4-induced cytotoxicity was partially rescued by miR-208a overexpression in RAW macrophages. Altogether, our results revealed a role of miR-208a in ALI in mice and suggest a role for miR-208a in regulating cell death.

FXR and TGR5 Agonists Ameliorate Liver Injury, Steatosis, and Inflammation After Binge or Prolonged Alcohol Feeding in Mice.

Bile acids (BAs) activate various dedicated receptors, including the farnesoid X receptor (FXR) and the Takeda G protein-coupled receptor 5 (TGR5). The FXR agonist obeticholic acid (OCA) is licensed for the treatment of primary biliary cholangitis and has shown promising results in NASH patients, whereas TGR5 agonists target inflammation and metabolism. We hypothesized that FXR and/or TGR5 agonists may be therapeutic in early alcoholic liver disease (ALD) in mice, in which hepatic inflammation plays a major role. OCA, INT-777, and INT-767 are BA derivatives with selective agonist properties for FXR, TGR5, or both, respectively. These compounds were tested in two mouse models (3-day binge model and prolonged Lieber DeCarli diet for 12 days) of early ALD. Serum alanine aminotransferase and liver histology were used to assess liver injury, Oil Red O staining of liver sections to assess steatosis, and real-time polymerase chain reaction to assess changes in gene expression. In the ethanol binge model, treatment with OCA and INT-777 decreased hepatic macrovesicular steatosis and protected from ethanol-induced liver injury. After prolonged ethanol administration, mice treated with OCA, INT-767, or INT-777 showed decreased hepatic steatosis, associated with reduced liver fatty acid synthase protein expression, and protection from liver injury. Treatment with BA receptor agonists in both models of ethanol administration modulated lipogenic gene expression, and decreased liver interleukin-1ß mRNA expression associated with increased ubiquitination of NLRP3 inflammasome through cyclic adenosine monophosphate-induced activation of protein kinase A. Conclusion: OCA, INT-767, or INT-777 administration is effective in reducing acute and chronic ethanol-induced steatosis and inflammation in mice, with varying degrees of efficacy depending on the duration of ethanol administration, indicating that both FXR and TGR5 activation can protect from liver injury in ALD models.