The gut-liver axis in hepatobiliary diseases.

Recent advances in the analysis of intestinal bacteria have led to reports of variations in intestinal bacterial levels among hepatobiliary diseases. The mechanisms behind the changes in intestinal bacteria in various hepatobiliary diseases include the abnormal composition of intestinal bacteria, weakening of the intestinal barrier, and bacterial translocation outside the intestinal tract, along with their metabolites, but many aspects remain unresolved. Further research employing clinical studies and animal models is expected to clarify the direct relationship between intestinal bacteria and hepatobiliary diseases and to validate the utility of intestinal bacteria as a diagnostic biomarker and potential therapeutic target. This review summarizes the involvement of the microbiota in the pathogenesis of hepatobiliary diseases via the gut-liver axis.

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).

Bacteriophage therapy against pathological Klebsiella pneumoniae ameliorates the course of primary sclerosing cholangitis.

Primary sclerosing cholangitis (PSC) is characterized by progressive biliary inflammation and fibrosis. Although gut commensals are associated with PSC, their causative roles and therapeutic strategies remain elusive. Here we detect abundant Klebsiella pneumoniae (Kp) and Enterococcus gallinarum in fecal samples from 45 PSC patients, regardless of intestinal complications. Carriers of both pathogens exhibit high disease activity and poor clinical outcomes. Colonization of PSC-derived Kp in specific pathogen-free (SPF) hepatobiliary injury-prone mice enhances hepatic Th17 cell responses and exacerbates liver injury through bacterial translocation to mesenteric lymph nodes. We developed a lytic phage cocktail that targets PSC-derived Kp with a sustained suppressive effect in vitro. Oral administration of the phage cocktail lowers Kp levels in Kp-colonized germ-free mice and SPF mice, without off-target dysbiosis. Furthermore, we demonstrate that oral and intravenous phage administration successfully suppresses Kp levels and attenuates liver inflammation and disease severity in hepatobiliary injury-prone SPF mice. These results collectively suggest that using a lytic phage cocktail shows promise for targeting Kp in PSC.

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.

Myeloid TLR4 signaling promotes post-injury withdrawal resolution of murine liver fibrosis.

The fate of resolution of liver fibrosis after withdrawal of liver injury is still incompletely elucidated. Toll-like receptor 4 (TLR4) in tissue fibroblasts is pro-fibrogenic. After withdrawal of liver injury, we unexpectedly observed a significant delay of fibrosis resolution as TLR4 signaling was pharmacologically inhibited in vivo in two murine models. Single-cell transcriptome analysis of hepatic CD11b+ cells, main producers of matrix metalloproteinases (MMPs), revealed a prominent cluster of restorative Tlr4-expressing Ly6c2-low myeloid cells. Delayed resolution after gut sterilization suggested its microbiome-dependent nature. Enrichment of a metabolic pathway linking to a significant increase of bile salt hydrolase-possessing family Erysipelotrichaceae during resolution. Farnesoid X receptor-stimulating secondary bile acids including 7-oxo-lithocholic acids upregulated MMP12 and TLR4 in myeloid cells in vitro. Fecal material transplant in germ-free mice confirmed phenotypical correlations in vivo. These findings highlight a pro-fibrolytic role of myeloid TLR4 signaling after injury withdrawal and may provide targets for anti-fibrotic therapy.

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.

Enrichment of type I interferon signaling in colonic group 2 innate lymphoid cells in experimental colitis.

Group 2 innate lymphoid cells (ILC2s) serve as frontline defenses against parasites. However, excluding helminth infections, it is poorly understood how ILC2s function in intestinal inflammation, including inflammatory bowel disease. Here, we analyzed the global gene expression of ILC2s in healthy and colitic conditions and revealed that type I interferon (T1IFN)-stimulated genes were up-regulated in ILC2s in dextran sodium sulfate (DSS)-induced colitis. The enhancement of T1IFN signaling in ILC2s in DSS-induced colitis was correlated with the downregulation of cytokine production by ILC2s, such as interleukin-5. Blocking T1IFN signaling during colitis resulted in exaggeration of colitis in both wild-type and Rag2-deficient mice. The exacerbation of colitis induced by neutralization of T1IFN signaling was accompanied by reduction of amphiregulin (AREG) in ILC2s and was partially rescued by exogenous AREG treatment. Collectively, these findings show the potential roles of T1IFN in ILC2s that contribute to colitis manifestation.

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.

CCR9 axis inhibition enhances hepatic migration of plasmacytoid DCs and protects against liver injury.

Plasmacytoid dendritic cells (pDCs) perform dual proinflammatory and immunosuppressive roles. We recently reported the potential of pDC therapy for treatment of intractable acute liver failure. However, establishment of efficient methods to deliver pDCs to the liver is essential for future clinical therapeutic applications. The present study demonstrates a higher abundance of liver and peripheral blood pDCs in mice lacking C-C motif chemokine receptor 9 (CCR9), a pDC gut-homing receptor, than in WT mice. Adoptive transfer of Ccr9-/- pDCs resulted in a higher efficiency of migration to the liver than WT pDCs did, while WT pDCs migrated efficiently to the original target organ, the small intestine. Further, Ccr9-/- pDCs consistently migrated efficiently to livers with concanavalin A-induced inflammation, and exerted a more effective immunosuppressive effect, resulting in better protection against acute liver inflammation than that demonstrated by WT pDCs. These findings highlight the therapeutic potential of the manipulation of the CCR9 axis as an approach to improve migration of immunosuppressive pDCs to the liver in order to exploit their beneficial effects in acute liver disease.

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.

Vagus nerve-mediated intestinal immune regulation: therapeutic implications of inflammatory bowel diseases.

The pathophysiology of inflammatory bowel diseases (IBDs) involves immunological, genetic and environmental factors. Through its ability to sense environmental stimuli, the autonomic nervous system plays a key role in the development and persistence of IBDs. The vagus nerve (VN), which contains sensory and motor neurons, travels throughout the body to innervate the gut and other visceral organs in the thoracic and abdominopelvic cavities. Recent studies show that the VN has anti-inflammatory effects via the release of acetylcholine, in what is known as the cholinergic anti-inflammatory pathway (CAIP). In the gut immune system, the CAIP is proposed to be activated directly by signals from the gut and indirectly by signals from the liver, which receives gut-derived bioactive substances via the portal vein and senses the status of the gut. The gut-brain axis and liver-brain-gut reflex arc regulate a wide variety of peripheral immune cells to maintain homeostasis in the gut. Therefore, targeting the neural reflex by methods such as VN stimulation is now under investigation for suppressing intestinal inflammation associated with IBDs. In this review, we describe the role of the VN in the regulation of intestinal immunity, and we discuss novel therapeutic approaches for IBDs that target neuroimmune interactions.

Early Dynamics of MELD Scores Predict Corticosteroid Responsiveness to Severe Acute-Onset Autoimmune Hepatitis.

Timely diagnosis and management of severe acute-onset autoimmune hepatitis (SA-AIH), a potential cause of acute liver failure (ALF), are challenging. An initial trial of corticosteroids (CS) followed by an assessment of clinical responses over 1-2 weeks is advocated by the latest international practice guidelines1,2 and expert reviews.3,4 Consideration of a second-line drug while evaluating for liver transplantation (LT) is also recommended.2 Established predictors of "CS responsiveness" to guide decision-making are nonexistent. Herein, we determined the diagnostic abilities of early dynamics to define CS responsiveness in SA-AIH using the model for end-stage liver disease (MELD) scores.

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.

Utility of the SARC-F Questionnaire for Sarcopenia Screening in Patients with Chronic Liver Disease: A Multicenter Cross-Sectional Study in Japan.

Diagnosing sarcopenia is challenging. This multicenter cross-sectional study aimed to evaluate the utility of the SARC-F score system for identifying sarcopenia in patients with chronic liver disease (CLD). We enrolled 717 patients from five participating centers who completed the SARC-F between November 2019 and March 2021. Sarcopenia was diagnosed based on the Japan Society of Hepatology Working Group on Sarcopenia in Liver Disease Consensus. Muscle strength was estimated using a grip dynamometer, and muscle mass was assessed using computed tomography or bioelectrical impedance analysis. The association between SARC-F and sarcopenia was analyzed using a logistic regression model. The optimal SARC-F cutoff value for identifying sarcopenia was determined using receiver operating characteristic (ROC) curve analysis. Of the 676 eligible patients, 15% were diagnosed with sarcopenia. The SARC-F distribution was 0 points in 63% of patients, 1 point in 17%, 2 points in 7%, 3 points in 4%, and ≥4 points in 8%. The SARC-F items of "Strength" (odds ratio (OR), 1.98; 95% confidence interval (CI), 1.03-3.80) and "Falls" (OR, 2.44; 95% CI, 1.48-4.03) were significantly associated with sarcopenia. The SARC-F value of 1 point showed a higher discriminative ability for identifying sarcopenia than the 4 points that are conventionally used (p < 0.001), with an area under the ROC curve of 0.68, sensitivity of 0.65, specificity of 0.68, positive predictive value of 0.27, and negative predictive value of 0.92. SARC-F is useful for identifying patients with CLD who are at risk of sarcopenia.

CD8+ tissue-resident memory T cells promote liver fibrosis resolution by inducing apoptosis of hepatic stellate cells.

Non-alcoholic steatohepatitis (NASH) is a leading cause of chronic liver disease that can progress to liver fibrosis. Recent clinical advance suggests a reversibility of liver fibrosis, but the cellular and molecular mechanisms underlying NASH resolution remain unclarified. Here, using a murine diet-induced NASH and the subsequent resolution model, we demonstrate direct roles of CD8+ tissue-resident memory CD8+ T (CD8+ Trm) cells in resolving liver fibrosis. Single-cell transcriptome analysis and FACS analysis revealed CD69+CD103-CD8+ Trm cell enrichment in NASH resolution livers. The reduction of liver CD8+ Trm cells, maintained by tissue IL-15, significantly delayed fibrosis resolution, while adoptive transfer of these cells protected mice from fibrosis progression. During resolution, CD8+ Trm cells attracted hepatic stellate cells (HSCs) in a CCR5-dependent manner, and predisposed activated HSCs to FasL-Fas-mediated apoptosis. Histological assessment of patients with NASH revealed CD69+CD8+ Trm abundance in fibrotic areas, further supporting their roles in humans. These results highlight the undefined role of liver CD8+ Trm in fibrosis resolution.

Acetaldehyde exposure underlies functional defects in monocytes induced by excessive alcohol consumption.

Increased intestinal permeability and hepatic macrophage activation by endotoxins are involved in alcohol-induced liver injury pathogenesis. Long-term alcohol exposure conversely induces endotoxin immune tolerance; however, the precise mechanism and reversibility are unclear. Seventy-two alcohol-dependent patients with alcohol dehydrogenase-1B (ADH1B, rs1229984) and aldehyde dehydrogenase-2 (ALDH2, rs671) gene polymorphisms admitted for alcohol abstinence were enrolled. Blood and fecal samples were collected on admission and 4 weeks after alcohol cessation and were sequentially analyzed. Wild-type and ALDH2*2 transgenic mice were used to examine the effect of acetaldehyde exposure on liver immune responses. The productivity of inflammatory cytokines of peripheral CD14+ monocytes in response to LPS stimulation was significantly suppressed in alcohol dependent patients on admission relative to that in healthy controls, which was partially restored by alcohol abstinence with little impact on the gut microbiota composition. Notably, immune suppression was associated with ALDH2/ADH1B gene polymorphisms, and patients with a combination of ALDH2*1/*2 and ADH1B*2 genotypes, the most acetaldehyde-exposed group, demonstrated a deeply suppressed phenotype, suggesting a direct role of acetaldehyde. In vitro LPS and malondialdehyde-acetaldehyde adducted protein stimulation induced direct cytotoxicity on monocytes derived from healthy controls, and a second LPS stimulation suppressed the inflammatory cytokines production. Consistently, hepatic macrophages of ethanol-administered ALDH2*2 transgenic mice exhibited suppressed inflammatory cytokines production in response to LPS compared to that in wild-type mice, reinforcing the contribution of acetaldehyde to liver macrophage function. These results collectively provide new perspectives on the systemic influence of excessive alcohol consumption based on alcohol-metabolizing enzyme genetic polymorphisms.

Vulnerability to recurrent episodes of acute decompensation/acute-on-chronic liver failure characterizes those triggered by indeterminate precipitants in patients with liver cirrhosis.

BACKGROUND: Acute decompensation (AD) of liver cirrhosis (LC) and subsequent acute-on-chronic liver failure (ACLF) are fatal and impair quality of life. Insufficient knowledge of the highly heterogeneous natural history of LC, including decompensation, re-compensation, and possible recurrent decompensation, hinders the development and application of novel therapeutics. Approximately 10%-50% of AD/ACLF is reported to be precipitated by any indeterminate (unidentifiable, cryptogenic, or unknown) acute insults; however, its clinical characteristics are unclear. METHODS: We conducted a single-center observational study of 2165 consecutively admitted patients with LC from January 2012 to December 2019. A total of 466 episodes of AD/ACLF in 285 patients, including their 285 first indexed AD/ACLF, were extracted for analysis. Stratified analyses of different acute precipitants, classified as indeterminate (AD/ACLFIND), bacterial infection (AD/ACLFBAC), gastrointestinal bleeding, active alcoholism, and miscellaneous, were performed. RESULTS: AD/ACLFIND was the leading acute precipitant (28%), followed by AD/ACLFBAC (23%). AD/ACLFIND showed better survival outcomes than AD/ACLFBAC (P = 0.03); however, hyperbilirubinemia, hyponatremia, or leukocytosis significantly and uniquely characterized subgroups of AD/ACLFIND with comparable or even worse survival outcomes than those of AD/ACLFBAC. Patients with subsequent AD/ACLF significantly tended to suffer from AD/ACLF with any organ failure in AD/ACLFIND but not in AD/ACLFBAC (P = 0.004, for trend). In competing risk analysis, patients with AD/ACLFIND were significantly more vulnerable to suffer from recurrent episodes of AD/ACLF within 180 days, compared to those triggered by other precipitants (P = 0.04). CONCLUSIONS: AD/ACLFIND, the leading acute precipitant, also plays a role in subsequent AD/ACLF. An abruptly exacerbating, remitting, and relapsing nature of systemic inflammation underlying AD/ACLF may also be useful for risk estimation.

C-C motif chemokine receptor 9 regulates obesity-induced insulin resistance via inflammation of the small intestine in mice.

AIMS/HYPOTHESIS: Accumulation of adipose tissue macrophages is considered pivotal in the development of obesity-associated inflammation and insulin resistance. In addition, recent studies suggest an involvement of the intestine as the primary organ in inducing hyperglycaemia and insulin resistance. We have reported that the C-C motif chemokine receptor (CCR) CCR9 is associated with intestinal immunity and has a pathogenic role in various liver diseases. However, its contribution to type 2 diabetes is unknown. In the current study, we aimed to clarify the involvement of CCR9 in the pathology of type 2 diabetes and the potential underlying mechanisms. METHODS: To elucidate how CCR9 affects the development of metabolic phenotypes, we examined the impact of CCR9 deficiency on the pathogenesis of type 2 diabetes using male C57BL/6J (wild-type [WT]) and CCR9-deficient (CCR9 knockout [KO]) mice fed a 60% high-fat diet (HFD) for 12 weeks. RESULTS: WT and Ccr9KO mice fed an HFD exhibited a comparable weight gain; however, glucose tolerance and insulin resistance were significantly improved in Ccr9KO mice. Moreover, visceral adipose tissue (VAT) and the liver of Ccr9KO mice presented with less inflammation and increased expression of glucose metabolism-related genes than WT mice. Ccr9 and Ccl25 expression were specifically higher in the small intestine but was not altered by HFD feeding and type 2 diabetes development. Accumulation of IFN-γ-producing CD4+ T lymphocytes and increased intestinal permeability in the small intestine was observed in WT mice following HFD feeding, but these changes were suppressed in HFD-fed Ccr9KO mice. Adoptive transfer of gut-tropic CCR9-expressing T lymphocytes partially reversed the favourable glucose tolerance found in Ccr9KO mice via exacerbated inflammation in the small intestine and VAT. CONCLUSIONS/INTERPRETATION: CCR9 plays a central role in the pathogenesis of type 2 diabetes by inducing an inflammatory shift in the small intestine. Our findings support CCR9 as a new therapeutic target for type 2 diabetes via the gut-VAT-liver axis.

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.