Mrigya Babuta

Neuroinflammation and microglial activation are common features in Alzheimer’s disease (AD) and excessive alcohol consumption. Alcohol induces liver damage and inflammation, and altered serum aminotransferases have been reported in patients with AD. Small extracellular vesicles (sEVs) and their cargo are emerging as mediators of intercellular communication in AD and alcohol-induced liver injury. However, the effects of alcohol use on AD progression remain to be elucidated. In this study, we evaluated the effects of repeated binge drinking on the liver and brain in the APP/PS1 mouse model of AD. Alcohol-exposed APP/PS1 mice showed elevated serum alanine aminotransferase levels, indicating liver injury absent in controls, along with increased pro-inflammatory cytokine expression in both liver and brain. Alcohol increased the biogenesis and release of sEVs in AD mice and in mouse hepatocytes. These sEVs were enriched in mature miR-122-5p that is predominant in hepatocytes. In vivo, miR-122-5p was detected in the brain of alcohol-exposed AD mice suggesting EV-mediated miR-122-5p transfer to the brain. Microglial uptake of hepatocyte-derived sEVs was confirmed in vitro. sEVs from alcohol-treated hepatocytes sensitized BV-2 microglia to LPS stimulation, significantly enhancing CCL2, CXCL2, CCL3, and TNF-α production. Transfection of microglia with a miR-122-5p mimic partially recapitulated the pro-inflammatory sensitization by sEVs from alcohol-exposed hepatocytes, which was reversed by miR-122-5p inhibitor delivery. ADAM10, a miR-122-5p target, was reduced in both wild-type and AD brains after alcohol exposure, with cell-specific effects. These results suggest that alcohol-induced hepatocyte-derived sEVs may modulate liver-to-brain signaling via miR-122-5p and contribute to neuroinflammation in AD.

Background: Cyclic GMP–AMP synthase (cGAS) catalyzes the production of cGAMP, which activates the STING–IRF3 signaling pathway. Previous investigations indicated a role for STING–IRF3 in early alcohol-associated liver disease (ALD). In this study, we examined the role of cGAS in liver damage and inflammation in early ALD. Methods: Wild-type (WT) or cGAS knockout (cGAS-KO) mice received a single dose of alcohol (5 g/kg), and in WT mice with or without a cGAS inhibitor, RU.521 (5 mg/kg) or 2′–3′ cGAMP or control. Liver and serum were evaluated after 9 hours of alcohol administration. Results: Alcohol gavage in cGAS knockout (cGAS-KO) mice led to increased liver damage compared with WT mice. Inhibition of cGAS with the small molecule, RU.521, also resulted in increased serum alanine and aspartate aminotransferases. cGAS deficiency or inhibition made the liver susceptible to alcohol-induced apoptosis. Alcohol-fed cGAS-KO mice and WT mice treated with the cGAS inhibitor showed enhanced unfolded protein response in the liver. This was associated with elevated levels of the proinflammatory cytokines, macrophage migration inhibitory factor, CD68 ⁺ cells, Ly6G ⁺ cells, and chemokines like lipocalin-2 and neutrophil elastase. Primary hepatocytes isolated from cGAS-KO mice displayed increased levels of cleaved caspase-3, XBP1s, and CHOP after alcohol administration as compared with WT control hepatocytes. Interestingly, restoration of cGAMP levels after cGAS inhibition improved liver damage and autophagic flux. Conclusions: Our findings highlight that cGAS deficiency or cGAS inhibition predisposes the liver to increased damage, apoptosis, and unfolded protein response in early ALD. An increase in the proinflammatory cytokine MIF and chemokines involved in neutrophil recruitment might contribute to the increased liver damage. In summary, our findings indicate a protective role for cGAS activation in early ALD.

Venous thromboembolism is frequent in ovarian cancer especially after surgery. Elaboration of tissue factor bearing extracellular vesicles (TF+EV) has been linked to thrombosis in cancer and hypercoagulability of surgery. Rosuvastatin can reduce the generation of EV and decrease VTE risk in non-cancer populations. The use of rosuvastatin to mitigate post-surgical thrombosis by lowering TF+EV following ovarian cancer surgery has not been investigated. In a pilot phase 2 trial in women undergoing ovarian cancer, women were randomized to enoxaparin 40 mg daily for 30 days with or without rosuvastatin 40 mg daily (administered from days 15- 60). Women who elected to forgo randomization received enoxaparin 40 mg daily for 30 days per standard of care. Total EV and TF+EV were assessed at baseline, day 30, and day 60. Bilateral lower extremity ultrasound was performed on day 30 and 60. A total of 24 women enrolled in the trial, 15 underwent randomization and 7 women were randomized to enoxaparin with rosuvastatin (17 received enoxaparin alone). There were no statistical differences observed in circulating TF+EV with the addition of rosuvastatin to enoxaparin at day 30 or day 60. Similarly, there were no differences in C-reactive protein or D-dimer between groups. There were no lower extremity deep vein thrombosis identified on screening ultrasounds, although portal vein thrombosis was diagnosed in enoxaparin-only arm. No major hemorrhages were observed. The addition of rosuvastatin to enoxaparin following ovarian cancer surgery does not appear to impact number of circulating TF+EV nor alter markers of thromboinflammation.

Severe alcohol-associated hepatitis (AH) is a life-threatening form of alcohol-associated liver disease. Liver neutrophil infiltration is a hallmark of AH, yet the effects of alcohol on neutrophil functions remain elusive. Identifying therapeutic targets to reduce neutrophil-mediated liver damage is essential. Bruton’s tyrosine kinase (BTK) plays an important role in neutrophil development and function; however, the role of BTK in AH is unknown. Using RNA sequencing of circulating neutrophils, we found an increase in Btk expression ( P = 0.05) and phosphorylated BTK (pBTK) in patients with AH compared with healthy controls. In vitro, physiologically relevant doses of alcohol resulted in a rapid, TLR4-mediated induction of pBTK in neutrophils. In a preclinical model of AH, administration of a small-molecule BTK inhibitor (evobrutinib) or myeloid-specific Btk knockout decreased proinflammatory cytokines and attenuated neutrophil-mediated liver damage. We found that pBTK was essential for alcohol-induced bone marrow granulopoiesis and liver neutrophil infiltration. In vivo, BTK inhibition or myeloid-specific Btk knockout reduced granulopoiesis, circulating neutrophils, liver neutrophil infiltration, and liver damage in a mouse model of AH. Mechanistically, using liquid chromatography–tandem mass spectrometry, we identified CD84 as a kinase target of BTK, which is involved in granulopoiesis. In vitro, CD84 promoted alcohol-induced interleukin-1β and tumor necrosis factor–α in primary human neutrophils, which was inhibited by CD84-blocking antibody treatment. Our findings define the role of BTK and CD84 in regulating neutrophil inflammation and granulopoiesis, with potential therapeutic implications in AH.

Background: The recent increase in the incidence of alcohol-associated hepatitis (AH) coincides with the obesity epidemic in the United States. However, current mouse models do not fully replicate the combined insults of obesity, metabolic dysfunction–associated steatohepatitis, and alcohol. The aim of this study was to develop a new mouse model that recapitulates the robust inflammatory and fibrotic phenotype characteristic of human MetALD. Methods: Eight- to 10-week-old male C57BL/6 mice were fed chow or high fat-cholesterol-sugar diet (metabolic dysfunction–associated steatohepatitis diet) and in each group, some received alcohol in drinking water (ad libitum) and weekly alcohol binges (EtOH) for 3 months. The liver was assessed for features of AH. Results: MetALD mice displayed increased liver damage indicated by highly elevated ALT and bilirubin levels compared to all other groups. Liver steatosis was significantly greater in the MetALD mice compared to all other experimental groups. The inflammatory phenotype of MetALD was also recapitulated, including increased IL-6 and IL-1β protein levels as well as increased CD68+ macrophages and Ly6G+ neutrophils in the liver. Sirius red staining and expression of collagen 1, alpha-smooth muscle actin indicated advanced fibrosis in the livers of MetALD mice. In addition, indicators of epithelial-to-mesenchymal transition markers were increased in MetALD mice compared to all other groups. Furthermore, we found increased ductular reaction, dysregulated hedgehog signaling, and decreased liver synthetic functions, consistent with severe AH. Conclusions: Alcohol administration in mice combined with metabolic dysfunction–associated steatohepatitis diet recapitulates key characteristics of human AH including liver damage, steatosis, robust systemic inflammation, and liver immune cell infiltration. This model results in advanced liver fibrosis, ductular reaction, decreased synthetic function, and hepatocyte dedifferentiation, suggesting a robust model of MetALD in mice. Export

Binge drinking in obese patients positively correlates with accelerated liver damage and liver-related death. However, the underlying mechanism and the effect of alcohol use on the progression of metabolic-dysfunction-associated steatotic liver disease (MASLD) remain unexplored. Here, we show that short-term feeding of a metabolic-dysfunction-associated steatohepatitis (MASH) diet plus daily acute alcohol binges for three days induce liver injury and activation of the NLRP3 inflammasome. We identify that a MASH diet plus acute alcohol binges promote liver inflammation via increased infiltration of monocyte-derived macrophages, neutrophil recruitment, and NET release in the liver. Our results suggest that both monocyte-derived macrophages and neutrophils are activated via NLRP3, while the administration of MCC950, an NLRP3 inhibitor, dampens these effects.In this study, we reveal important intercellular communication between hepatocytes and neutrophils. We discover that the MASH diet plus alcohol induces IL-1β via NLRP3 activation and that IL-1β acts on hepatocytes and promotes the production of CXCL1 and LCN2. In turn, the increase in these neutrophils recruits chemokines and causes further infiltration and activation of neutrophils in the liver. In vivo administration of the NLRP3 inhibitor, MCC950, improves the early phase of MetALD by preventing liver damage, steatosis, inflammation, and immune cells recruitment.

The prolyl hydroxylation of hypoxia-inducible factor 1α (HIF-1α) mediated by the EGLN–pVHL pathway represents a classic signalling mechanism that mediates cellular adaptation under hypoxia. Here we identify RIPK1, a known regulator of cell death mediated by tumour necrosis factor receptor 1 (TNFR1), as a target of EGLN1–pVHL. Prolyl hydroxylation of RIPK1 mediated by EGLN1 promotes the binding of RIPK1 with pVHL to suppress its activation under normoxic conditions. Prolonged hypoxia promotes the activation of RIPK1 kinase by modulating its proline hydroxylation, independent of the TNFα–TNFR1 pathway. As such, inhibiting proline hydroxylation of RIPK1 promotes RIPK1 activation to trigger cell death and inflammation. Hepatocyte-specific Vhl deficiency promoted RIPK1-dependent apoptosis to mediate liver pathology. Our findings illustrate a key role of the EGLN–pVHL pathway in suppressing RIPK1 activation under normoxic conditions to promote cell survival and a model by which hypoxia promotes RIPK1 activation through modulating its proline hydroxylation to mediate cell death and inflammation in human diseases, independent of TNFR1.

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-1β 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-1β 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-1β 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.

The actin cytoskeleton, a dynamic network of actin filaments and associated F-actin–binding proteins, is fundamentally important in eukaryotes. α-Actinins are major F-actin bundlers that are inhibited by Ca²⁺ in nonmuscle cells. Here we report the mechanism of Ca²⁺-mediated regulation of Entamoeba histolytica α-actinin-2 (EhActn2) with features expected for the common ancestor of Entamoeba and higher eukaryotic α-actinins. Crystal structures of Ca²⁺-free and Ca²⁺-bound EhActn2 reveal a calmodulin-like domain (CaMD) uniquely inserted within the rod domain. Integrative studies reveal an exceptionally high affinity of the EhActn2 CaMD for Ca²⁺, binding of which can only be regulated in the presence of physiological concentrations of Mg²⁺. Ca²⁺ binding triggers an increase in protein multidomain rigidity, reducing conformational flexibility of F-actin–binding domains via interdomain cross-talk and consequently inhibiting F-actin bundling. In vivo studies uncover that EhActn2 plays an important role in phagocytic cup formation and might constitute a new drug target for amoebic dysentery.

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.