Multiple asynchronous recurrence as a predictive factor for refractoriness against locoregional and surgical therapy in patients with intermediate-stage hepatocellular carcinoma.

Development of subclassification of intermediate-stage hepatocellular carcinoma (HCC) by treatment suitability is in demand. We aimed to identify predictors that define treatment refractoriness against locoregional(transarterial chemoembolization(TACE) or thermal ablation) and surgical therapy. This multicenter retrospective study enrolled 1167 HCC patients between 2015 and 2021. Of those, 209 patients were initially diagnosed with intermediate-stage HCC. Treatment refractoriness was defined as clinical settings that meets the following untreatable progressive conditions by TACE (1) 25% increase of intrahepatic tumor, (2) transient deterioration to Child-Pugh class C, (3) macrovascular invasion or extrahepatic spread, within one year. We then analyzed factors contributing to treatment refractoriness. The Child-Pugh score/class, number of tumors, infiltrative radiological type, and recurrence were significant factors. Focusing on recurrence as a predictor, median time to untreatable progression (TTUP) was 17.2 months in the recurrence subgroup whereas 35.5 months in the initial occurrence subgroup (HR, 2.06; 95% CI, 1.44-2.96; P = 0.001). Median TTUP decreased in cases with more later times of recurrence (3-5 recurrences, 17.3 months; ≥ 6 recurrences, 7.7 months). Recurrence, even more at later times, leads to increased treatment refractoriness. Early introduction of multidisciplinary treatment should be considered against HCC patients after multiple recurrent episodes.

Granulocyte-monocyte/macrophage apheresis for steroid-nonresponsive or steroid-intolerant severe alcohol-associated hepatitis: A pilot study.

BACKGROUND: Patients with severe alcohol-associated hepatitis (SAH) have a high short-term mortality rate. Unmet needs exist in patients who are refractory to corticosteroids (CS) or are ineligible for early liver transplantation. METHODS: This was a prospective, open-label, nonrandomized pilot study conducted at a liver transplant center in Tokyo, Japan, starting in October 2015. Lille model and Model for End-stage Liver Disease (MELD) score-defined CS nonresponsive or CS-intolerant patients with SAH who fulfilled the inclusion criteria (leukocytosis over 10,000/µL, etc.) were considered for enrollment. The median duration from admission to enrollment was 23 days (IQR, 14-31 days), after standard of care. Granulocyte-monocyte/macrophage apheresis (GMA) performed with Adacolumn twice per week, up to 10 times per treatment course, was evaluated. RESULTS: 13 GMA treatments were conducted through December 2021. Maddrey Discriminant Function was 53.217.7 at admission. The overall survival rate was 90.9% at 90 and 180 days. MELD scores significantly improved, from median (IQRs) of 23 (20-25) to 15 (13-21) after GMA (p<0.0001). Estimated mortality risks using the Lille model and MELD scores significantly improved from 20.9%±16.5% to 7.4%±7.3% at 2 months and from 30.4%±21.3% to 11.6%±10.8% at 6 months, respectively (both p<0.01), and were internally validated. The cumulative rate of alcohol relapse was 35.9% per year. No severe adverse events were observed. In exploratory analysis, granulocyte colony-stimulating factor levels were significantly correlated with prognostic systems such as MELD-Sodium scores after GMA (correlation coefficient= -0.9943, p<0.0001) but not before GMA (p=0.62). CONCLUSIONS: Compared to published studies, GMA is associated with a lower-than-expected 90- and 180-day mortality in patients with CS-nonresponsive or CS-intolerant SAH. GMA may meet the needs as a salvage anti-inflammatory therapy for SAH. (Trial registration: UMIN000019351 and jRCTs No.032180221) (274 words).

IL-15-producing splenic B cells play pathogenic roles in the development of autoimmune hepatitis.

Background & Aims: B-cell depletion therapy with an anti-CD20 is an effective treatment strategy for patients with refractory autoimmune hepatitis (AIH). However, the mechanisms underlying B-cell action are unclear. Methods: Herein, we used the adeno-associated virus IL-12 model, in which hepatic IL-12 expression triggers liver injuries characteristic of AIH. We also analysed the clinical samples of patients with AIH. Results: B-cell depletion using anti-CD20 or splenectomy was found to improve liver functions and decrease the cytotoxic CD8+ T-cell (cytotoxic T lymphocyte [CTL]) count in the liver. This improvement was reversed by the adoptive transfer of splenic B cells derived from AAV IL-12-treated mice to splenectomised mice as it caused the hepatic CTL count to increase. RNA-sequencing analysis identified IL-15 as a key factor in pathogenic B cells, which promotes CTL expansion and subsequent migration to the liver via the CXCL9/CXCR3 axis. Indeed, IL-15 neutralisation ameliorated hepatitis by suppressing splenic and hepatic CTLs in vivo. The close distribution of B220+ B cells and CD8+ T cells in the spleen of AIH mice suggested mutual interactions. Mechanistically, IFNγ and CD40L/CD40 signalling were indispensable for the expression of IL-15 in B cells, and in vitro co-culture experiments revealed that splenic CD40L+CD8+ T cells promoted IL-15 production in B cells, which led to CTL expansion. In patients with AIH, high serum IL-15 concentration and IL-15+ B-cell counts, positively correlating with serum alanine aminotransferase levels, support translation and potential therapeutic targeting in human AIH. Conclusions: This investigation elucidated the roles of IL-15-producing splenic B cells that occur in concert with pathogenic CD8+ T cells during the development of AIH. Impact and Implications: IL-15-producing B cells were shown to exacerbate experimental AIH via cytotoxic T lymphocyte expansion. CD40L+CD8+ T cells promoted IL-15 expression in B cells, indicating the mutual interaction of both cells. High serum IL-15 concentrations, IL-15+ B-cell counts, and CD40L+IL-15Rα+CD8+ T-cell counts were confirmed in the blood of patients with AIH.

Successful treatment of refractory enteritis and arthritis with combination of tumour necrosis factor and interleukin-6 inhibition in patients with ulcerative colitis.

An 18 year-old man with autoimmune hepatitis-primary sclerosing cholangitis-overlap syndrome and ulcerative colitis was admitted due to relapsed enteritis and polyarthritis after cessation of infliximab. Colonoscopy and articular ultrasonography revealed large ulcers in the colon with crypt abscess in the specimens and active enthesitis and synovitis, respectively. His intestinitis was improved with golimumab but arthritis was persistent. Golimumab was switched to secukinumab, which was effective for arthritis. However, colitis was flared resulting in total colorectal resection. One month after colectomy, polyarthritis was relapsed. Tocilizumab ameliorated arthritis but enteritis emerged again, and switching tocilizumab to adalimumab improved enteritis but arthritis exacerbated. Finally, we restarted tocilizumab for arthritis with continued adalimumab for enteritis. The dual cytokine blocking strategy, tumour necrosis factor-α and interleukin-6 inhibition, subsided both of his refractory enteritis and arthritis and maintained remission for more than 3 years without any serious adverse event. Our case suggests that enteritis and arthritis in inflammatory bowel disease may be different in pathophysiology and raises the possible usefulness of simultaneous inhibition of two inflammatory cytokines in such cases.

Concurrent de novo Thymoma-associated Paraneoplastic Type 1 Autoimmune Hepatitis and Pure Red Cell Aplasia after Thymectomy: A Case Report and Literature Review.

The precise manipulation of immune tolerance is the holy grail of immunotherapies for both autoimmunity and cancer immunity. Thymomas are well known to be associated with autoimmune diseases. The exact mechanism by which autoreactivity is induced after thymectomy remains to be elucidated. We herein present the case of a 50-year-old lady with concurrent de novo type 1 autoimmune hepatitis (AIH) and pure red cell aplasia (PRCA), 1 month after undergoing a successful total thymectomy for combined squamous cell carcinoma and thymoma (Masaoka stage II). Corticosteroids yielded short-term effects for both AIH and PRCA. Literature on thymoma-associated AIH, an extremely rare immune-related comorbidity, was also reviewed.

Successful Case of Deceased Donor Liver Transplant for Polycystic Liver Disease.

A 58-year-old woman was admitted to hospital for deceased donor liver transplant. Her liver volume, measured by computed tomography, had reached 22,764 cm3 and she was bedridden with performance status 3 because of abdominal distention. The Model for End-Stage Liver Disease score was 24 with exception points. The final weight of the removed liver after cystic fluid puncture was 14 kg. The patient recovered well after transplant and was discharged on postoperative day 43 with a weight of 41 kg and performance status of 1.

Dynamics of type IV collagen 7S fragment on eradication of HCV with direct antiviral agents: Prognostic and metabolomic impacts.

BACKGROUND: Liver fibrosis is one of the cardinal clinical features of chronic hepatitis C (CHC). However, the mechanisms underlying the evolution and reversion of liver fibrosis after hepatitis C virus (HCV) eradication and their relationship with clinical outcomes and metabolic alterations are not fully elucidated. Whether any non-invasive fibrosis marker can predict prognosis is unknown. METHODS: Between October 2014 and September 2019, 418 patients with CHC or compensated cirrhosis with HCV were prospectively recruited in this observational study. 326 patients that were successfully eradicated with interferon-free direct antiviral agents (IFN-free DAAs) were analyzed. Peri-treatment dynamics of serum levels of type IV collagen 7S fragment (4COL7S), a fibrosis marker, and subsequent clinical outcomes, including hepatic decompensation, newly emerged hepatocellular carcinoma (HCC), and all-cause mortality were analyzed. RESULTS: Ten (3.1%) patients died during the observation period. 4COL7S-defined fibrosis progression (n = 97, 29.8%) at SVR was significantly correlated with worse all-cause mortality post-SVR (P = 0.0062) but not with the probability of newly emerged HCC (P = 0.24). Prognostic tendency was more prominent in patients with advanced fibrosis (P< 0.0001). 4COL7S-defined fibrosis progression at SVR and a baseline platelet count less than 10×104/µL were significantly predicted all-cause mortality (P = 0.0051). In exploratory analyses, a decreased 4COL7S at the end of treatment was correlated with a matrix-degrading phenotype that showed higher serum metalloproteinase to tissue inhibitors of metalloproteinase-1 ratios and characteristic metabolic fingerprints such as increased butyrate, some medium-chain fatty acids, anabolic amino acids, and decreased uremia toxins. CONCLUSIONS: Peri-treatment dynamics of serum 4COL7S, a non-invasive fibrosis marker, predict prognosis. Non-invasive fibrosis markers may be useful biomarkers for risk stratification post-SVR.

Th17 cells in the liver: balancing autoimmunity and pathogen defense.

In addition to carcinogenesis, T helper 17 (Th17) cells (a subtype of CD4 + T lymphocytes) are involved in the acute, chronic, and cirrhotic phases of liver diseases; however, their role in the development and progression of liver diseases remains unclear. It is difficult to elucidate the role of Th17 cells in liver diseases due to their dichotomous nature, i.e., plasticity in terms of pathogenic or host protective function depending on environmental and time phase factors. Moreover, insufficient depletion of Th17 cells by inhibiting the cytokines and transcription factors involved in their production causes difficulties in analyzing their specific role in vitro and in vivo murine models, partially due to complex interaction. This review summarizes the recent progress in understanding the plasticity and function of hepatic Th17 cells and type 3 cytokines.

Hepatic Adenosine Triphosphate Reduction Through the Short-Chain Fatty Acids-Peroxisome Proliferator-Activated Receptor γ-Uncoupling Protein 2 Axis Alleviates Immune-Mediated Acute Hepatitis in Inulin-Supplemented Mice.

How liver tolerance is disrupted in immune-mediated liver injury is currently unclear. There is also insufficient information available regarding susceptibility, precipitation, escalation, and perpetuation of autoimmune hepatitis. To explore how dietary fiber influences hepatic damage, we applied the concanavalin A (ConA)-induced acute immune-mediated liver injury model in mice fed a diet supplemented with 6.8% inulin, a water-soluble fermentable fiber. Twelve hours after ConA administration, inulin-supplemented diet-fed mice demonstrated significantly alleviated hepatic damage histologically and serologically, with down-regulation of hepatic interferon-γ and tumor necrosis factor and reduced myeloperoxidase (MPO)-producing neutrophil infiltration. Preconditioning with an inulin-supplemented diet for 2 weeks significantly reduced hepatic adenosine triphosphate (ATP) content; suramin, a purinergic P2 receptor antagonist, abolished the protective effect. Of note, the portal plasma derived from mice fed the inulin-supplemented diet significantly alleviated ConA-induced immune-mediated liver injury. Mechanistically, increased portal short-chain fatty acid (SCFA) levels, such as those of acetate and butyrate, by inulin supplementation leads to up-regulation of hepatic γ-type peroxisome proliferator-activated receptor (Pparg) and uncoupling protein 2 (Ucp2), which uncouples mitochondrial ATP synthesis downstream of PPARγ. Pparg down-regulating small interfering RNA cancelled the protective effect of inulin supplementation against MPO-producing neutrophil infiltration and the subsequent immune-mediated liver injury, suggesting that the SCFA-PPARγ-UCP2 axis plays a key role in the protective effect by inulin supplementation. Moreover, significant changes in the gut microbiota, including increased operational taxonomic units in genera Akkermansia and Allobaculum, also characterized the protective effect of the inulin-supplemented diet. Conclusion: There is a possible unraveled etiopathophysiological link between the maintenance of liver tolerance and dietary fiber. The SCFA-PPARγ-UCP2 axis may provide therapeutic targets for immune-mediated liver injury in the future.

Role of CC chemokine receptor 9 in the progression of murine and human non-alcoholic steatohepatitis.

BACKGROUND & AIMS: The number of patients with non-alcoholic steatohepatitis (NASH) is increasing globally. Recently, specific chemokine receptors have garnered interest as therapeutic targets in NASH. This is the first report to examine the role of the C-C chemokine receptor 9 (CCR9)/C-C chemokine receptor ligand 25 (CCL25) axis, and to reveal its therapeutic potential in NASH. METHODS: Patients with biopsy-proven non-alcoholic liver disease (NAFLD) were recruited and their serum and hepatic chemokine expression was examined. Furthermore, wild-type (WT) and Ccr9-/- mice were fed a high-fat high-cholesterol (HFHC) diet for 24 weeks to establish NASH. RESULTS: Serum CCL25, and hepatic CCR9 and CCL25 expression levels were increased in patients with NASH compared to healthy volunteers. Furthermore, Ccr9-/- mice were protected from HFHC diet-induced NASH progression both serologically and histologically. Flow cytometry and immunohistochemistry analysis showed that CCR9+CD11b+ inflammatory macrophages accumulated in the inflamed livers of HFHC diet-fed mice, while the number was reduced in Ccr9-/- mice. Consistent with human NASH livers, CCR9 was also expressed on hepatic stellate cells (HSCs) in mice with NASH, while CCR9-deficient HSCs showed less fibrogenic potential in vitro. Administration of a CCR9 antagonist hampered further fibrosis progression in mice with NASH, supporting its potential clinical application. Finally, we showed that CCR9 blockade attenuated the development of NAFLD-related hepatocellular carcinoma in HF diet-fed mice injected with diethylnitrosamine. CONCLUSIONS: These results highlight the role of the CCR9/CCL25 axis on macrophage recruitment and fibrosis formation in a murine NASH model, providing new insights into therapeutic strategies for NASH. LAY SUMMARY: Herein, we show that a specific chemokine axis involving a receptor (CCR9) and its ligand (CCL25) contributes to the progression of non-alcoholic steatohepatitis and carcinogenesis in humans and mice. Furthermore, treatment with a CCR9 antagonist ameliorates the development of steatohepatitis and holds promise for the treatment of patients with non-alcoholic steatohepatitis.

Sustained virologic response to direct-acting antiviral therapy in patients with chronic hepatitis C and hepatocellular carcinoma: A systematic review and meta-analysis.

BACKGROUND & AIMS: The effect of hepatocellular carcinoma (HCC) on the response to interferon-free direct-acting antiviral (DAA) therapy in patients with chronic hepatitis C (CHC) infection remains unclear. Using a systematic review and meta-analysis approach, we aimed to investigate the effect of DAA therapy on sustained virologic response (SVR) among patients with CHC and either active, inactive or no HCC. METHODS: PubMed, Embase, Web of Science, and the Cochrane Central Register of Controlled Trials were searched from 1/1/2013 to 9/24/2018. The pooled SVR rates were computed using DerSimonian-Laird random-effects models. RESULTS: We included 49 studies from 15 countries, comprised of 3,341 patients with HCC and 35,701 without HCC. Overall, the pooled SVR was lower in patients with HCC than in those without HCC (89.6%, 95% CI 86.8-92.1%, I2 = 79.1% vs. 93.3%, 95% CI 91.9-94.7%, I2 = 95.0%, p = 0.0012), translating to a 4.8% (95% CI 0.2-7.4%) SVR reduction by meta-regression analysis. The largest SVR reduction (18.8%) occurred in patients with active/residual HCC vs. inactive/ablated HCC (SVR 73.1% vs. 92.6%, p = 0.002). Meanwhile, patients with HCC who received a prior liver transplant had higher SVR rates than those who did not (p <0.001). Regarding specific DAA regimens, patients with HCC treated with ledipasvir/sofosbuvir had lower SVR rates than patients without HCC (92.6%, n = 884 vs. 97.8%, n = 13,141, p = 0.026), but heterogeneity was high (I2 = 84.7%, p <0.001). The SVR rate was similar in patients with/without HCC who were treated with ombitasvir/paritaprevir/ritonavir ±â€¯dasabuvir (n = 101) (97.2% vs. 94.8%, p = 0.79), or daclatasvir/asunaprevir (91.7% vs. 89.8%, p = 0.66). CONCLUSION: Overall, SVR rates were lower in patients with HCC, especially with active HCC, compared to those without HCC, though heterogeneity was high. Continued efforts are needed to aggressively screen, diagnose, and treat HCC to ensure higher CHC cure rates. LAY SUMMARY: There are now medications (direct-acting antivirals or "DAAs") that can "cure" hepatitis C virus, but patients with hepatitis C and liver cancer may be less likely to achieve cure than those without liver cancer. However, patients with liver cancer are also more likely to have advanced liver disease and risk factors that can decrease cure rates, so better controlled studies are needed to confirm these findings.

Gut pathobionts underlie intestinal barrier dysfunction and liver T helper 17 cell immune response in primary sclerosing cholangitis.

Primary sclerosing cholangitis (PSC) is a chronic inflammatory liver disease and its frequent complication with ulcerative colitis highlights the pathogenic role of epithelial barrier dysfunction. Intestinal barrier dysfunction has been implicated in the pathogenesis of PSC, yet its underlying mechanism remains unknown. Here, we identify Klebsiella pneumonia in the microbiota of patients with PSC and demonstrate that K. pneumoniae disrupts the epithelial barrier to initiate bacterial translocation and liver inflammatory responses. Gnotobiotic mice inoculated with PSC-derived microbiota exhibited T helper 17 (TH17) cell responses in the liver and increased susceptibility to hepatobiliary injuries. Bacterial culture of mesenteric lymph nodes in these mice isolated K. pneumoniae, Proteus mirabilis and Enterococcus gallinarum, which were prevalently detected in patients with PSC. A bacterial-organoid co-culture system visualized the epithelial-damaging effect of PSC-derived K. pneumoniae that was associated with bacterial translocation and susceptibility to TH17-mediated hepatobiliary injuries. We also show that antibiotic treatment ameliorated the TH17 immune response induced by PSC-derived microbiota. These results highlight the role of pathobionts in intestinal barrier dysfunction and liver inflammation, providing insights into therapeutic strategies for PSC.

Intestinal barrier regulates immune responses in the liver via IL-10-producing macrophages.

The gut-liver axis is of clinical importance as a potential therapeutic target in a wide range of liver diseases; however, the mechanisms underlying interactions between microbial products and immune responses in the liver remain unknown. In this study, we demonstrated that IL-10-producing macrophages contribute to immune tolerance in the inflamed liver under intestinal barrier disruption in a murine tandem model of dextran sulfate sodium (DSS) colitis and concanavalin A (Con A) hepatitis. Intestinal barrier disruption protected mice from subsequent liver injury, and the severity of colitis directly affected susceptibility to such injury. The protective effect of DSS-Con A was canceled in gut-sterilized mice, suggesting that gut microbiota play a substantial role in this process. Altered gut microbiota and their metabolites, along with a disrupted intestinal barrier, directly gave rise to immunological permissiveness in the inflamed liver. We identified 1-methylnicotinamide (1-MNA) as a candidate metabolite capable of suppressing liver injury with the potential to induce IL-10-producing macrophages. Consistently, expression of nicotinamide N-methyltransferase, which converts nicotinamide to 1-MNA, was upregulated in the liver of DSS-Con A mice, and this effect was abrogated by gut sterilization. Collectively, our results provide a mechanistic insight into the regulation of immunological balance in the liver via the gut-liver axis.

Dual effects of the Nrf2 inhibitor for inhibition of hepatitis C virus and hepatic cancer cells.

BACKGROUND: We previously showed that knockdown of nuclear factor E2-related factor 2 (Nrf2) resulted in suppression of hepatitis C virus (HCV) infection. In this study, whether brusatol, an Nrf2 inhibitor, has dual anti-HCV and anticancer effects was explored. METHODS: The anti-HCV effect of brusatol was investigated by analyzing HCV RNA and proteins in a hepatic cell line persistently-infected with HCV, HPI cells, and by analyzing HCV replication in a replicon-replicating hepatic cell line, OR6 cells. Then, dual anti-HCV and anticancer effects of brusatol and enhancement of the effects by the combination of brusatol with anticancer drugs including sorafenib, which has been reported to have the dual effects, were then investigated. RESULTS: Brusatol suppressed the persistent HCV infection at both the RNA and protein levels in association with a reduction in Nrf2 protein in the HPI cells. Analysis of the OR6 cells treated with brusatol indicated that brusatol inhibited HCV persistence by inhibiting HCV replication. Combination of brusatol with an anticancer drug not only enhanced the anticancer effect but also, in the case of the combination with sorafenib, strongly suppressed HCV infection. CONCLUSIONS: Brusatol has dual anti-HCV and anticancer effects and can enhance the comparable effects of sorafenib. There is therefore the potential for combination therapy of brusatol and sorafenib for HCV-related hepatocellular carcinoma.

On-treatment decrease of NKG2D correlates to early emergence of clinically evident hepatocellular carcinoma after interferon-free therapy for chronic hepatitis C.

BACKGROUND AND AIMS: Interferon (IFN)- free direct antiviral agents (DAAs) with rapid HCV eradication might evoke immunological reconstitutions, and some early recurrences of HCC after IFN-free DAAs have been reported. This study aimed to investigate whether natural killer group 2, member D (NKG2D) predicts early emergence of HCC after IFN-free DAAs. METHODS: We conducted a clinical practice-based observational study of 101 patients infected with genotype 1 HCV who received IFN-free (DAAs), and stratified them into those who did or did not develop early (i.e., during the 6-month surveillance period following treatment.) recurrence or occurrence of clinically evident HCC. We also analyzed the peripheral blood mononuclear cells, both before treatment and at end of treatment (EOT), of 24 of the patients who received IFN-free DAAs, and 16 who received IFN-combined protease inhibitor. RESULTS: We found early emergence of clinically evident HCC after IFN-free DAAs in 12 (12%) patients. Higher pre-treatment NKG2D expression, higher FIB-4 score, previous HCC history and failure to achieve sustained viral response were significant factors correlating to early HCC emergence. After IFN-free DAAs, a rapid decrease of NKG2D at EOT correlated with early HCC emergence in the IFN-free DAA-treated patients, but not in patients treated with the IFN-combined regimen. The decrease of NKG2D until EOT was predictive of early HCC emergence at a cut-off of -52% (AUC = 0.92). CONCLUSIONS: On-treatment decrease of NKG2D may be a useful predictor of early emerging HCC in patients treated with IFN-free DAAs.

Bone marrow-derived macrophages distinct from tissue-resident macrophages play a pivotal role in Concanavalin A-induced murine liver injury via CCR9 axis.

The fundamental mechanism how heterogeneous hepatic macrophage (Mφ) subsets fulfill diverse functions in health and disease has not been elucidated. We recently reported that CCR9+ inflammatory Mφs play a critical role in the course of acute liver injury. To clarify the origin and differentiation of CCR9+Mφs, we used a unique partial bone marrow (BM) chimera model with liver shielding for maintaining hepatic resident Mφs. First, irradiated mice developed less liver injury with less Mφs accumulation by Concanavalin A (Con A) regardless of liver shielding. In mice receiving further BM transplantation, CD11blowF4/80high hepatic-resident Mφs were not replaced by transplanted donors under steady state, while under inflammatory state by Con A, CCR9+Mφs were firmly replaced by donors, indicating that CCR9+Mφs originate from BM, but not from hepatic-resident cells. Regarding the mechanism of differentiation and proliferation, EdU+CCR9+Mφs with a proliferative potential were detected specifically in the inflamed liver, and in vitro study revealed that BM-derived CD11b+ cells co-cultured with hepatic stellate cells (HSCs) or stimulated with retinoic acids could acquire CCR9 with antigen-presenting ability. Collectively, our study demonstrates that inflammatory Mφs originate from BM and became locally differentiated and proliferated by interaction with HSCs via CCR9 axis during acute liver injury.

Prominent steatosis with hypermetabolism of the cell line permissive for years of infection with hepatitis C virus.

Most of experiments for HCV infection have been done using lytic infection systems, in which HCV-infected cells inevitably die. Here, to elucidate metabolic alteration in HCV-infected cells in a more stable condition, we established an HCV-persistently-infected cell line, designated as HPI cells. This cell line has displayed prominent steatosis and supported HCV infection for more than 2 years, which is the longest ever reported. It enabled us to analyze metabolism in the HCV-infected cells integrally combining metabolomics and expression arrays. It revealed that rate-limiting enzymes for biosynthesis of cholesterol and fatty acids were up-regulated with actual increase in cholesterol, desmosterol (cholesterol precursor) and pool of fatty acids. Notably, the pentose phosphate pathway was facilitated with marked up-regulation of glucose-6-phosphate dehydrogenase, a rete-limiting enzyme, with actual increase in NADPH. In its downstream, enzymes for purine synthesis were also up-regulated resulting in increase of purine. Contrary to common cancers, the TCA cycle was preferentially facilitated comparing to glycolysis pathway with a marked increase of most of amino acids. Interestingly, some genes controlled by nuclear factor (erythroid-derived 2)-like 2 (Nrf2), a master regulator of antioxidation and metabolism, were constitutively up-regulated in HPI cells. Knockdown of Nrf2 markedly reduced steatosis and HCV infection, indicating that Nrf2 and its target genes play important roles in metabolic alteration and HCV infection. In conclusion, HPI cell is a bona fide HCV-persistently-infected cell line supporting HCV infection for years. This cell line sustained prominent steatosis in a hypermetabolic status producing various metabolites. Therefore, HPI cell is a potent research tool not only for persistent HCV infection but also for liver metabolism, overcoming drawbacks of the lytic infection systems.

C-C motif chemokine receptor 9 positive macrophages activate hepatic stellate cells and promote liver fibrosis in mice.

UNLABELLED: Chemokine receptors mediate migration of immune cells into the liver, thereby promoting liver inflammation. C-C motif chemokine receptor (CCR) 9(+) macrophages are crucial in the pathogenesis of acute liver inflammation, but the role and underlying mechanisms of this macrophage subset in chronic liver injury and subsequent liver fibrosis are not fully understood. We confirmed that tumor necrosis factor alpha (TNF-α)-producing CCR9(+) macrophages accumulated during the initiation of carbon tetrachloride (CCl4 )-induced liver injury, and CCR9 deficiency attenuated the degree of liver damage. Accumulation of CCR9(+) macrophages persisted prominently during the process of liver fibrosis induced by repetitive CCl4 or thioacetamide (TAA)/leptin administration. Increased CCR9 expression was also found on activated hepatic stellate cells (HSCs). Importantly, experimental liver fibrosis was significantly ameliorated in CCR9(-/-) mice compared with wild-type (WT) mice, assessed by α-smooth muscle actin (α-SMA) immunostain, Sirius red staining, and messenger RNA (mRNA) expression levels of α-SMA, collagen 1α1, transforming growth factor (TGF)-ß1, and tissue inhibitor of metalloproteinase (TIMP)-1. Accumulated CD11b(+) macrophages in CCl4 -treated WT mice showed marked increases in TNF, NO synthase-2, and TGF-ß1 mRNA expression compared with CCR9(-/-) mice, implying proinflammatory and profibrogenic properties. Hepatic CD11b(+) macrophages from CCl4 -treated WT mice (i.e., CCR9(+) macrophages), but not CD8(+) T lymphocytes or non-CD11b(+) cells, significantly activated HSCs in vitro compared with those from CCR9(-/-) mice. TNF-α or TGF-ß1 antagonism attenuated CCR9(+) macrophage-induced HSC activation. Furthermore, C-C motif chemokine ligand (CCL) 25 mediated migration and, to a lesser extent, activation of HSCs in vitro. CONCLUSION: Accumulated CD11b(+) macrophages are critical for activating HSCs through the CCR9/CCL25 axis and therefore promote liver fibrosis. CCR9 antagonism might be a novel therapeutic target for liver fibrosis.

CCR9+ macrophages are required for acute liver inflammation in mouse models of hepatitis.

BACKGROUND & AIMS: Antigen-presenting cells (APCs) are involved in the induction of liver inflammation. We investigated the roles of specific APCs in the pathogenesis of acute liver injury in mice. METHODS: We used concanavalin A (con A) or carbon tetrachloride to induce acute liver inflammation in mice and studied the roles of macrophages that express CCR9. RESULTS: After injection of con A, we detected CCR9(+)CD11b(+)CD11c(-) macrophages that express tumor necrosis factor (TNF)-α in livers of mice, whereas CCR9(+)Siglec-H(+)CD11b(-)CD11c(low) plasmacytoid DCs (pDCs), which are abundant in normal livers, disappeared. The CCR9(+) macrophages were also detected in the livers of RAG-2(-/-) mice, which lack lymphocytes and natural killer T cells, after injection of con A. Under inflammatory conditions, CCR9(+) macrophages induced naive CD4(+) T cells to become interferon gamma-producing Th1 cells in vivo and in vitro. CCR9(-/-) mice injected with con A did not develop hepatitis unless they also received CCR9(+) macrophages from mice that received con A; more CCR9(+) macrophages accumulated in their inflamed livers than CCR9(+) pDCs, CCR9(-) pDCs, or CCR9(-) macrophages isolated from mice that had received injections of con A. Levels of CCL25 messenger RNA increased in livers after injection of con A; neutralizing antibodies against CCL25 reduced the induction of hepatitis by con A by blocking the migration of CCR9(+) macrophages and their production of TNF-α. Peripheral blood samples from patients with acute hepatitis had greater numbers of TNF-α-producing CCR9(+)CD14(+)CD16(high) monocytes than controls. CONCLUSIONS: CCR9(+) macrophages contribute to the induction of acute liver inflammation in mouse models of hepatitis.

Evaluation of liver fibrosis by transient elastography using acoustic radiation force impulse: comparison with Fibroscan(®).

BACKGROUND: Accurate evaluation of liver fibrosis in patients with chronic liver damage is required to determine the appropriate treatment. Various approaches, including laboratory tests and transient elastography, have been used to evaluate liver fibrosis. Recently, transient elastography with acoustic radiation force impulse (ARFI) has been developed and applied with conventional ultrasonography. The aim of this study was to evaluate the clinical utility of transient elastography with ARFI and to compare the results with this method and those of the Fibroscan(®) procedure. METHODS: One hundred and thirty-one patients with liver damage, who underwent liver biopsy at our department, were enrolled prospectively in this study. Elastography with ARFI (applied with ACUSON S2000(®)), and Fibroscan(®) was performed at the same time as liver biopsy. These measurements were compared with histological findings in liver biopsy specimens, and measurement accuracy was evaluated by receiver-operating characteristic analysis. RESULTS: Elastography values with both procedures were significantly correlated with the stages of liver fibrosis and there was little difference in the results obtained using the 2 procedures. The accuracy of differential diagnosis between no fibrosis at F0 and more than F1 stage was insufficient with ARFI, but this procedure was sufficient for diagnosing advanced fibrosis. The accuracy of ARFI was almost equivalent to that of the Fibroscan(®) method. Moreover, both ARFI and Fibroscan(®) values increased in proportion to the severity of hepatic inflammation when fibrosis stage is low, but not in proportion to the severity of steatosis. CONCLUSIONS: Transient elastography with ARFI is simple, non-invasive and useful for diagnosing the stage of fibrosis in chronic liver disease. The utility of ARFI was almost equivalent to that of the Fibroscan(®) method.