Interleukin-18 signaling promotes activation of hepatic stellate cells in mouse liver fibrosis.

BACKGROUND AND AIMS: Nucleotide-binding oligomerization domain-like receptor-family pyrin domain-containing 3 (NLRP3) inflammasome activation has been shown to result in liver fibrosis. Mechanisms and downstream signaling remain incompletely understood. Here, we studied the role of IL-18 in hepatic stellate cells (HSCs), and its impact on liver fibrosis. APPROACH AND RESULTS: We observed significantly increased serum levels of IL-18 (128.4 pg/ml vs. 74.9 pg/ml) and IL-18 binding protein (BP; 46.50 ng/ml vs. 15.35 ng/ml) in patients with liver cirrhosis compared with healthy controls. Single cell RNA sequencing data showed that an immunoregulatory subset of murine HSCs highly expresses Il18 and Il18r1 . Treatment of cultured primary murine HSC with recombinant mouse IL-18 accelerated their transdifferentiation into myofibroblasts. In vivo , IL-18 receptor-deficient mice had reduced liver fibrosis in a model of fibrosis induced by HSC-specific NLRP3 overactivation. Whole liver RNA sequencing analysis from a murine model of severe NASH-induced fibrosis by feeding a choline-deficient, L-amino acid-defined, high fat diet showed that genes related to IL-18 and its downstream signaling were significantly upregulated, and Il18-/- mice receiving this diet for 10 weeks showed protection from fibrotic changes with decreased number of alpha smooth muscle actin-positive cells and collagen deposition. HSC activation triggered by NLRP3 inflammasome activation was abrogated when IL-18 signaling was blocked by its naturally occurring antagonist IL-18BP. Accordingly, we observed that the severe inflammatory phenotype associated with myeloid cell-specific NLRP3 gain-of-function was rescued by IL-18BP. CONCLUSIONS: Our study highlights the role of IL-18 in the development of liver fibrosis by its direct effect on HSC activation identifying IL-18 as a target to treat liver fibrosis.

NLR Family Pyrin Domain-Containing 3 Inflammasome Activation in Hepatic Stellate Cells Induces Liver Fibrosis in Mice.

The NLR family pyrin domain-containing 3 (NLRP3) inflammasome plays an important role in liver fibrosis (LF) development. However, the mechanisms involved in NLRP3-induced fibrosis are unclear. Our aim was to test the hypothesis that the NLRP3 inflammasome in hepatic stellate cells (HSCs) can directly regulate their activation and contribute to LF. Primary HSCs isolated from wild-type (WT), Nlrp3-/- , or Nlrp3L351PneoR knock-in crossed to inducible (estrogen receptor Cre-CreT) mice were incubated with lipopolysaccharide (LPS) and adenosine triphosphate (ATP), or 4OH-tamoxifen, respectively. HSC-specific Nlrp3L351P knock-in mice were generated by crossing transgenic mice expressing lecithin retinol acyltransferase (Lrat)-driven Cre and maintained on standard rodent chow for 6 months. Mice were then sacrificed; liver tissue and serum were harvested. Nlrp3 inflammasome activation along with HSC phenotype and fibrosis were assessed by RT-PCR, western blotting, fluorescence-activated cell sorting (FACS), enzyme-linked immunosorbent assay, immunofluorescence (IF), and immunohistochemistry (IHC). Stimulated WT HSCs displayed increased levels of NLRP3 inflammasome-induced reactive oxygen species (ROS) production and cathepsin B activity, accompanied by an up-regulation of mRNA and protein levels of fibrotic makers, an effect abrogated in Nlrp3-/- HSCs. Nlrp3L351P CreT HSCs also showed elevated mRNA and protein expression of fibrotic markers 24 hours after inflammasome activation induced with 4-hydroxytamoxifen (4OHT). Protein and mRNA expression levels of fibrotic markers were also found to be increased in isolated HSCs and whole liver tissue from Nlrp3L351P Lrat Cre mice compared to WT. Liver sections from 24-week-old NlrpL351P Lrat Cre mice showed fibrotic changes with increased alpha smooth muscle actin (αSMA) and desmin-positive cells and collagen deposition, independent of inflammatory infiltrates; these changes were also observed after LPS challenge in 8-week-old NlrpL351P Lrat Cre mice. Conclusion: Our results highlight a direct role for the NLRP3 inflammasome in the activation of HSCs directly triggering LF.

Novel Drivers of the Inflammatory Response in Liver Injury and Fibrosis.

Hepatocyte demise as well as signals released by stressed hepatocytes have been now recognized as important triggers of liver inflammation. While traditional concepts classically viewed hepatocyte cell death to occur by either a nonlytic, noninflammatory form (apoptosis), or lytic, proinflammatory nonregulated cell death (necrosis), recent studies have provided evidence for additional mechanisms that can contribute to both acute and chronic liver damage. Two novel forms of cell death, pyroptosis and necroptosis, are of particular importance as they are highly regulated and intrinsically proinflammatory. Additionally, stressed hepatocytes may also release signals to attract and activate monocytes into proinflammatory macrophages. In this review, the authors discuss recent developments supporting the role of novel triggers of liver inflammation in various forms of liver injury and their potential translational implications.

Oxidized linoleic acid metabolites induce liver mitochondrial dysfunction, apoptosis, and NLRP3 activation in mice.

Circulating oxidized linoleic acid (LA) metabolites (OXLAMs) are increased in patients with nonalcoholic steatohepatitis (NASH) and their levels correlate with disease severity. However, the mechanisms by which OXLAMs contribute to NASH development are incompletely understood. We tested the hypothesis that LA or OXLAMs provided directly through the diet are involved in the development of hepatic injury. C57BL/6 mice were fed an isocaloric high-fat diet containing low LA, high LA, or OXLAMs for 8 weeks. The livers of OXLAM-fed mice showed lower triglyceride concentrations, but higher FA oxidation and lipid peroxidation in association with increased oxidative stress. OXLAM-induced mitochondrial dysfunction was associated with reduced Complex I protein and hepatic ATP levels, as well as increased mitochondrial biogenesis and cytoplasmic mitochondrial DNA. Oxidative stress increased thioredoxin-interacting protein (TXNIP) in the liver and stimulated the activation of mitochondrial apoptosis signal-regulating kinase 1 (ASK1) leading to apoptosis. We also found increased levels of NOD-like receptor protein 3 (NLRP3) inflammasome components and Caspase-1 activation in the livers of OXLAM-fed mice. In vitro, OXLAMs induced hepatocyte cell death, which was partly dependent on Caspase-1 activation. This study identified key mechanisms by which dietary OXLAMs contribute to NASH development, including mitochondrial dysfunction, hepatocyte cell death, and NLRP3 inflammasome activation.

Shear wave elastography-based liver fibrosis assessment in patients with chronic hepatitis E displays elevated liver stiffness regardless of previous antiviral therapy.

Background: Hepatitis E virus (HEV) infection especially in immunocompromised individuals can lead to chronic hepatitis. Aggressive courses of chronic hepatitis E leading to liver cirrhosis in a short period of time have been described, but evidence on the degree of liver involvement in chronic hepatitis E is rare. We therefore aimed to quantify liver fibrosis in patients with chronic active hepatitis E compared to patients with sustained virological response after ribavirin (RBV) treatment using 2D-shear wave elastography (2D-SWE) to measure liver stiffness. Methods: Patients with chronic hepatitis E underwent 2D-SWE, B-mode and Doppler ultrasound and laboratory testing in order to assess severity of liver involvement. Results: In this cross-sectional study, we included 14 patients of whom 8 had ongoing chronic hepatitis E and 6 patients had been successfully treated for chronic hepatitis E. The most frequent cause for immunosuppression was prior kidney transplantation (n=12), one patient was a multivisceral transplant recipient, one had been treated for lymphoma. Five patients cleared HEV after RBV therapy, one patient reached viral clearance after reduction of his immunosuppressive medication. Using 2D-SWE measurement, 71.4% displayed increased stiffness indicative of liver fibrosis: 57.1% classified as significant fibrosis and 14.3% as severe fibrosis. Liver stiffness did not differ between patients with active chronic hepatitis E and in patients who had cleared HEV (1.59 and 1.54 m/s respectively). Compared with a control group of kidney transplant recipients without hepatitis E (1.44 m/s), the patients with a history of hepatitis E displayed a significantly higher liver stiffness (P=0.04). Conclusions: In our cohort of chronic hepatitis E patients, elevated liver stiffness indicating liver fibrosis was common and significantly higher than in controls. This is consistent with prior sparse reports of the presence of liver fibrosis or cirrhosis in chronic hepatitis E and emphasizes the need for HEV testing, therapy and research on new therapeutic options. As elevated liver stiffness was also present in patients after HEV treatment, continuous liver surveillance including elastography and ultrasound should be considered.

Shear Wave Elastography and Shear Wave Dispersion Imaging in the Assessment of Liver Disease in Alpha1-Antitrypsin Deficiency.

Liver affection of Alpha1-antitrypsin deficiency (AATD) can lead to cirrhosis and hepatocellular carcinoma (HCC). A noninvasive severity assessment of liver disease in AATD is urgently needed since laboratory parameters may not accurately reflect the extent of liver involvement. Preliminary data exist on two-dimensional shear wave elastography (2D-SWE) being a suitable method for liver fibrosis measurement in AATD. AATD patients without HCC were examined using 2D-SWE, shear wave dispersion imaging (SWD) and transient elastography (TE). Furthermore, liver steatosis was assessed using the controlled attenuation parameter (CAP) and compared to the new method of attenuation imaging (ATI). 29 AATD patients were enrolled, of which 18 had the PiZZ genotype, eight had PiMZ, two had PiSZ and one had a PiZP-Lowell genotype. 2D-SWE (median 1.42 m/S, range 1.14-1.83 m/S) and TE (median 4.8 kPa, range 2.8-24.6 kPa) values displayed a significant correlation (R = 0.475, p < 0.05). 2D-SWE, ATI (median 0.56 dB/cm/MHz, range 0.43-0.96 dB/cm/MHz) and CAP (median 249.5 dB/m, range 156-347 dB/m) values were higher in PiZZ when compared to other AATD genotypes. This study provides evidence that 2D-SWE is a suitable method for the assessment of liver disease in AATD. The newer methods of SWD and ATI require further evaluation in the context of AATD.

Bile Acids Activate NLRP3 Inflammasome, Promoting Murine Liver Inflammation or Fibrosis in a Cell Type-Specific Manner.

Bile acids (BA) as important signaling molecules are considered crucial in development of cholestatic liver injury, but there is limited understanding on the involved cell types and signaling pathways. The aim of this study was to evaluate the inflammatory and fibrotic potential of key BA and the role of distinct liver cell subsets focusing on the NLRP3 inflammasome. C57BL/6 wild-type (WT) and Nlrp3-/- mice were fed with a diet supplemented with cholic (CA), deoxycholic (DCA) or lithocholic acid (LCA) for 7 days. Additionally, primary hepatocytes, Kupffer cells (KC) and hepatic stellate cells (HSC) from WT and Nlrp3-/- mice were stimulated with aforementioned BA ex vivo. LCA feeding led to strong liver damage and activation of NLRP3 inflammasome. Ex vivo KC were the most affected cells by LCA, resulting in a pro-inflammatory phenotype. Liver damage and primary KC activation was both ameliorated in Nlrp3-deficient mice or cells. DCA feeding induced fibrotic alterations. Primary HSC upregulated the NLRP3 inflammasome and early fibrotic markers when stimulated with DCA, but not LCA. Pro-fibrogenic signals in liver and primary HSC were attenuated in Nlrp3-/- mice or cells. The data shows that distinct BA induce NLRP3 inflammasome activation in HSC or KC, promoting fibrosis or inflammation.