Background:Sepsis, a life-threatening condition, involves dysregulated host responses to infection that frequently lead to multi-organ failure. Despite advancements in supportive care, current therapies for this disease remain limited, necessitating novel treatment approaches. Interleukin-39 (IL-39), a recently identified member of the IL-12 family, has emerged as a key mediator of inflammatory diseases; however, its functional role in the pathogenesis of sepsis remains largely unknown. Methods:Serum levels of IL-39 were analyzed in septic patients and healthy individuals. A cecal ligation and puncture (CLP)-induced sepsis murine model was employed. Recombinant human IL-39 (rhIL-39) or an IL-39-neutralizing antibody was administered to CLP mice to investigate both the causative role of IL-39 as well as the anti-IL-39 therapeutic potential. The survival rates, the severity of pulmonary injuries, the levels of pro-inflammatory cytokines, and the infiltration of neutrophils in septic mice were assessed. Bulk RNA sequencing was performed to explore the mechanisms underlying IL-39 promotion of sepsis. Results:Septic patients had significantly elevated serum levels of IL-39 compared to healthy individuals. The levels of IL-39 were strongly correlated with clinical severity and 90-day mortality in the septic patients. In CLP-induced septic mice, rhIL-39 administration enhanced pulmonary damage, increased systemic inflammation and worsened overall survival. Conversely, IL-39 neutralizing antibodies reversed these effects and improved the survival outcome. Mechanistically, RNA sequencing analysis revealed IL-39 treatment markedly increased several chemokines and pro-inflammatory cytokines, and enriched "cytokine-cytokine receptor interaction" pathway. Finally, rhIL-39 enhanced neutrophil recruitment to lung tissues, a process that was proven to be linked to the CXCL1-CXCR2 pathway activation by in vitro analyses. Conclusions:This study demonstrates that IL-39 is a critical exacerbating factor in sepsis progression, driving neutrophil-mediated tissue injury via CXCL1-CXCR2 signaling. These findings identify IL-39 as both a prognostic biomarker and a promising potential therapeutic target for sepsis management.

Hepatocellular carcinoma (HCC) is an increasingly prevalent and deadly disease that is initiated by different etiological factors, such as alcohol-associated liver disease (ALD), metabolic dysfunction-associated steatohepatitis (MASH), viral hepatitis, and other hepatotoxic and hepatocarcinogenic agents. The tumor microenvironment (TME) of HCC is characterized by several different fibroblastic and immune cell types, all of which affect the initiation, progression and metastasis of this malignant cancer. This complex immune TME can be divided into an innate component that includes macrophages, neutrophils, dendritic cells, myeloid-derived suppressor cells, mucosal-associated invariant T cells, natural killer cells, natural killer T cells, and innate lymphoid cells, as well as an adaptive component that includes CD4+ T cells, CD8+ T cells, regulatory T cells, and B cells. In this review, we discuss the latest findings shedding light on the direct or indirect roles of these immune cells (and fibroblastic-like cells such as hepatic stellate cells) in the pathogenesis of HCC. Henceforth, further characterization of this heterogeneous TME is highly important for studying the progression of HCC and developing novel immunotherapeutic treatment options. In line with this, we also review novel groundbreaking experimental techniques and animal models aimed at specifically elucidating this complex TME and discuss emerging immune-based therapeutic strategies intended to treat HCC and predict the efficacy of these immunotherapies.

Background & Aims: Reactive oxygen species (ROS) act as modulators triggering cellular dysfunctions and organ damage including liver fibrosis in which hepatic stellate cell (HSC) activation plays a key role. Previous studies suggest that microRNA-144 (miR-144) acts as a pro-oxidant molecule; however, whether and how miR-144 affects HSC activation and liver fibrosis remain unknown. Methods: Carbon tetrachloride (CCl4) and bile duct ligation (BDL)-induced experimental liver fibrosis models were used. Hepatic miR-144 expression was analyzed by miRNA in situ hybridization with RNAscope probe. The in vivo effects of silencing or overexpressing miR-144 were examined with an adeno-associated virus 6 (AAV6) carrying miR-144 inhibitor or mimics in fibrotic mouse experimental models. Results: In this study, we demonstrated that ROS treatment significantly upregulated miR-144 in HSCs, which further promoted HSC activation in vitro. Interestingly, miR-144 was preferentially elevated in HSCs of experimental liver fibrosis in mice and in human liver fibrotic tissues. Furthermore, in vivo loss or gain-of-function experiments via AAV6 carrying miR-144 antagomir or agomir revealed that blockade of miR-144 in HSCs mitigated, while overexpression of miR-144 in HSCs accelerated the development of experimental liver fibrosis. Mechanistically, SIN3 transcription regulator family member A (SIN3A), a transcriptional repressor, was identified to be the target of miR-144 in HSCs. MiR-144 downregulated Sin3A, and in line with this result, specific knockdown of Sin3a in HSCs remarkedly activated p38 MAPK signaling pathway to promote HSC activation, eventually exacerbating liver fibrosis. Conclusions: Oxidative stress-driven miR-144 fuels HSC activation and liver fibrogenesis by limiting the SIN3A-p38 axis. Thus, a specific inhibition of miR-144 in HSCs could be a novel therapeutic strategy for the treatment of liver fibrosis.

Primary liver cancers ranked as the sixth most commonly diagnosed cancers and the third-leading cause of cancer-related death in 2020. Despite encouraging findings on diagnosis and treatments, liver cancer remains a life-threatening disease with a still increasing incidence. Therefore, it is of interest to better characterise and understand the mechanistic process occurring at early steps of carcinogenesis. Inflammatory responses in liver diseases participate in the activation of liver progenitor cells (LPCs) facultative compartment but also to their transformation into cancer stem cells (CSCs) and give rise to primary liver cancer including hepatocellular carcinoma and cholangiocarcinoma. Higher intratumoural heterogeneity has been associated with poorer prognosis and linked to tumour escape from the immune surveillance and to resistance to chemotherapy. A better understanding of the malignant transformation of LPC as tumour initiating cells (ie, CSC) should also provide a potential new therapeutic target for anticancer therapy. In this review, we summarise the recent reports identifying underlying mechanisms by which chronic liver inflammatory responses could trigger the early steps in liver carcinogenesis, notably through the transformation of LPCs into tumour initiating cells.

Supplementary Table 5: Difference in gene expression between Immune High (IH) and Immune Low (IL) subgroups from the validation dataset (n=20, Coulouarn et al, Carcinogenesis, 2012) (33).

Alcohol-associated liver disease (ALD) represents a major public health issue worldwide and is a leading etiology of liver cirrhosis. Alcohol-related liver injuries include a range of manifestations including alcoholic hepatitis (AH), simple steatosis, steatohepatitis, hepatic fibrosis, cirrhosis and liver cancer. Liver disease occurs from several pathological disturbances such as the metabolism of ethanol, which generates reactive oxygen species (ROS) in hepatocytes, alterations in the gut microbiota, and the immune response to these changes. A common hallmark of these liver affections is the establishment of an inflammatory environment, and some (broad) anti-inflammatory approaches are used to treat AH (eg, corticosteroids). Macrophages, which represent the main innate immune cells in the liver, respond to a wide variety of (pathogenic) stimuli and adopt a large spectrum of phenotypes. This translates to a diversity of functions including pathogen and debris clearance, recruitment of other immune cells, activation of fibroblasts, or tissue repair. Thus, macrophage populations play a crucial role in the course of ALD, but the underlying mechanisms driving macrophage polarization and their functionality in ALD are complex. In this review, we explore the various populations of hepatic macrophages in alcohol-associated liver disease and the underlying mechanisms driving their polarization. Additionally, we summarize the crosstalk between hepatic macrophages and other hepatic cell types in ALD, in order to support the exploration of targeted therapeutics by modulating macrophage polarization.

Primary liver cancers rank among the deadliest cancers worldwide and often develop in patients with chronic liver diseases in an inflammatory context. This review highlights recent reports on the mechanisms of inflammatory-mediated hepatic cell transformation that trigger the tumorigenic process (initiation steps) and the impact of the immune response favoring tumor cell expansion (progression steps). Several cytokines, namely interleukin (IL)-6, IL-17, IL-1beta, and tumor necrosis factor-alpha, have been described to play a prominent role in the initiation of liver cancers. Additionally, inflammation contributes to cancer progression by favoring tumor escape from anti-tumor immune response, angiogenesis, and metastasis through tumor growth factor-beta and matrix metalloprotease upregulation. These recent studies allowed the development of novel therapeutic strategies aiming at regulating liver inflammation. These strategies are based on the use of anti-inflammatory agents, antibodies targeting immune checkpoint molecules such as programmed death ligand 1 and molecules targeting angiogenic factors, metastasis key factors, and microRNAs involved in tumor development. This review aims at summarizing the recent studies reporting different mechanisms by which the liver inflammatory responses could contribute to liver cancer development.

AbstractPurpose:Combined hepatocellular-cholangiocarcinoma (cHCC-CCA) is a rare malignancy associated with an overall poor prognosis. We aimed to investigate the immune profile of cHCC-CCA and determine its impact on disease outcome.Experimental Design:We performed a multicenter study of 96 patients with cHCC-CCA. Gene expression profile was analyzed using nCounter PanCancer IO 360 Panel. Densities of main immune cells subsets were quantified from digital slides of IHC stainings. Genetic alterations were investigated using targeted next-generation sequencing.Results:Two main immune subtypes of cHCC-CCA were identified by clustering analysis: an “immune-high” (IH) subtype (57% of the cases) and an “immune-low” (IL) subtype (43% of the cases). Tumors classified as IH showed overexpression of genes related to immune cells recruitment, adaptive and innate immunity, antigen presentation, cytotoxicity, immune suppression, and inflammation (P < 0.0001). IH cHCC-CCAs also displayed activation of gene signatures recently shown to be associated with response to immunotherapy in patients with HCC. Quantification of immunostainings confirmed that IH tumors were also characterized by higher densities of immune cells. Immune subtypes were not associated with any genetic alterations. Finally, multivariate analysis showed that the IH subtype was an independent predictor of improved overall survival.Conclusions:We have identified a subgroup of cHCC-CCA that displays features of an ongoing intratumor immune response, along with an activation of gene signatures predictive of response to immunotherapy in HCC. This tumor subclass is associated with an improved clinical outcome. These findings suggest that a subset of patients with cHCC-CCA may benefit from immunomodulating therapeutic approaches.

Chronic inflammation is a key component in the development of virtually all types of primary liver cancers. However, how chronic inflammation potentiates or even may initiate liver parenchymal cell transformation remains unclear. Cancer stem cells (CSCs) represent an exciting target for novel anticancer therapeutic strategies in several types of cancers and were also described in primary liver cancers as tumor initiating cells. Recently, we reported a key role of Interleukin (IL)-17 in Liver Progenitor Cell (LPC) accumulation in preneoplastic cirrhotic livers. In this study, we evidenced in vitro, that long-term stimulation of LPCs with IL-17 led to their transformation into CSCs. Indeed, they acquired CSC-marker expression, and self-renewal properties, showed by their increased capacity to form spheroids. The miRNome analysis revealed that long-term IL-17 treatment of LPCs led to a 90% decrease in miR-122 expression. In a model using immunodeficient mice, ectopic engraftment of LPCs in an IL-17-enriched environment led to tumor occurrence with an aggressive phenotype. Contrastingly, in a murine model of hepatocellular carcinoma induced by a unique injection of diethyl-nitrosamine associated with chronic administration of carbon tetrachloride, IL-17-deficiency or anti-IL-17 therapy protected mice from liver tumor growth. In conclusion, we showed that a chronic exposure of LPCs to IL-17 cytokine promotes their transformation into CSCs. In addition, we demonstrated that IL-17-neutralizing strategies limit CSC occurrence and liver tumor progression through miR-122 restored-expression.

Kupffer cells (KCs), which are liver-resident macrophages, originate from the fetal yolk sac and represent one of the largest macrophage populations in the body. However, the current data on the origin of the cells that restore macrophages during liver injury and regeneration remain controversial. Here, we address the question of whether liver macrophage restoration results from circulating monocyte infiltration or local KC proliferation in regenerating livers after partial hepatectomy (PHx) and uncover the underlying mechanisms. By using several strains of genetically modified mice and performing immunohistochemical analyses, we demonstrated that local KC proliferation mainly contributed to the restoration of liver macrophages after PHx. Peak KC proliferation was impaired in Il6-knockout (KO) mice and restored after the administration of IL-6 protein, whereas KC proliferation was not affected in Il4-KO or Csf2-KO mice. The source of IL-6 was identified using hepatocyte- and myeloid-specific Il6-KO mice and the results revealed that both hepatocytes and myeloid cells contribute to IL-6 production after PHx. Moreover, peak KC proliferation was also impaired in myeloid-specific Il6 receptor-KO mice after PHx, suggesting that IL-6 signaling directly promotes KC proliferation. Studies using several inhibitors to block the IL-6 signaling pathway revealed that sirtuin 1 (SIRT1) contributed to IL-6-mediated KC proliferation in vitro. Genetic deletion of the Sirt1 gene in myeloid cells, including KCs, impaired KC proliferation after PHx. In conclusion, our data suggest that KC repopulation after PHx is mainly driven by local KC proliferation, which is dependent on IL-6 and SIRT1 activation in KCs.

To assess the performance of 405 nm–induced autofluorescence for the characterization of primary liver nodules on ex vivo resected specimens. Forty resected liver specimens bearing 53 primary liver nodules were included in this IRB-approved prospective study. Intratissular spectroscopic measurements were performed using a 25-G fibered-needle on all ex vivo specimens: 5 autofluorescence measurements were performed in both nodules and adjacent parenchyma. The spectra derivatives of the 635 and 670 nm autofluorescence peaks observed in nodules and in adjacent liver parenchyma were compared (Kruskal–Wallis and Mann–Whitney when appropriate). A total of 42 potentially evolutive primary liver nodules—34 hepatocellular carcinomas, 4 intrahepatic cholangiocarcinomas, 4 hepatocellular adenomas—and 11 benign nodules—5 focal nodular hyperplasias, 6 regenerative nodules—were included. Both 635 and 670 nm Δderivatives were significantly higher in benign as compared to potentially evolutive (PEV) nodules (respectively 32.9 ± 4.5 vs 15.3 ± 1.4; p < 0.0001 and 5.7 ± 0.6 vs 2.5 ± 0.1; p < 0.0001) with respective sensitivity and specificity of 78% and 91% for distinguishing PEV from benign nodules. 405 nm–induced autofluorescence enables the discrimination of benign from PEV primary liver nodules, suggesting that autofluorescence imaging could be used to optimize US targeted liver biopsies. • 405 nm–induced autofluorescence can distinguish liver tumors from the adjacent liver parenchyma. • The analysis of autofluorescence imaging observed within primary liver tumors can discriminate benign tumors from those requiring follow-up or targeted liver biopsy. • In current practice, autofluorescence imaging could be embedded within biopsy needle, to enable, in addition to ultrasound guidance, optimal targeting of liver nodules which could optimize tissue sampling.

PURPOSE:The impact of tertiary lymphoid structures (TLS) in hepatocellular carcinoma (HCC) progression is being extensively investigated. However, their presence during the early steps of human liver carcinogenesis remains unknown. We thus aimed to determine whether TLS are induced in preneoplastic/early hepatic lesions (EHL), and whether they are associated with a particular immune profile.EXPERIMENTAL DESIGN:A series of 127 EHLs (low/high-grade dysplastic nodules, early HCC, and small and progressed HCC) was included in the study. TLSs were investigated by pathologic reviewing. Densities of immune cells were assessed using IHC. A subset of lesions was microdissected and gene expression profiling was performed with a custom NanoString panel.RESULTS:Compared with surrounding cirrhotic nodules, EHL of all stages displayed increased densities of T cells, B cells, and dendritic cells. Immature TLSs were identified in 24% of EHL. Gene expression profiling identified a subset of EHL with elevated mRNA levels of various cytokines involved in immune cells' recruitment and TLS induction. This subgroup of EHL also showed overexpression of genes related to T- and B-cells' activation and antigen presentation, as well as those related to immunosuppression and immune exhaustion.CONCLUSIONS:Local immune activation occurs in the very early steps of liver carcinogenesis; however, it may not be fully efficient and paradoxically favor immune evasion and progression to full-blown HCC. These results have implications for the development of anti-HCC chemopreventive strategies in cirrhotic patients.

Chronic liver injury with any etiology can progress to fibrosis and the end-stage diseases cirrhosis and hepatocellular carcinoma. The progression of liver disease is controlled by a variety of factors, including liver injury, inflammatory cells, inflammatory mediators, cytokines, and the gut microbiome. In the current review, we discuss recent data on a large number of cytokines that play important roles in regulating liver injury, inflammation, fibrosis, and regeneration, with a focus on interferons and T helper (Th) 1, Th2, Th9, Th17, interleukin (IL)-1 family, IL-6 family, and IL-20 family cytokines. Hepatocytes can also produce certain cytokines (such as IL-7, IL-11, and IL-33), and the functions of these cytokines in the liver are briefly summarized. Several cytokines have great therapeutic potential, and some are currently being tested as therapeutic targets in clinical trials for the treatment of liver diseases, which are also described.

Potential conflict of interest: Nothing to report. To the Editor: We read this article by Behnke et al. recently published in Hepatology with great interest.1 This study demonstrated the role of B cells in favoring liver regeneration, notably by maintaining splenic CD169+ macrophages and hepatic IL‐6 expression. This is of great importance to us, because we believe there is still a lot to investigate to unravel the full scope of the immune mechanisms implicated in the tremendous liver regenerative abilities. Behnke et al. showed that splenectomy or genetic B‐cell depletion resulted in reduced numbers of splenic CD169+ macrophages. In another experiment, Behnke et al. used CD169‐DTR transgenic mice in which CD169+ cells are depleted following diphtheria toxin injection. After partial hepatectomy, diphtheria injected CD169‐DTR mice had a lower survival rate, decreased liver/body weight ratio, and Ki67 staining reflecting a diminished regenerative response, as well as lower hepatic IL‐6 expression, as compared to control mice. However, we came across some imprecisions while reading the article. Although, as stated by the authors, spleen‐resident macrophages are CD169+, other macrophage populations express CD169 (including liver‐tissue–resident Kupffer cells and bone‐marrow–derived monocytes).2 Interestingly, Gupta et al. showed that tissue‐resident macrophages are effectively ablated in the spleen and liver from diphtheria toxin‐treated CD169‐DTR mice. It is also well known that macrophage depletion dramatically alters liver regeneration after partial hepatectomy.3 The confusion about CD169+ macrophage tissue distribution remains in the rest of the study, as the authors have not characterized hepatic macrophage populations in their models. This analysis would certainly strengthen the authors’ conclusions. Overall, we are enthusiastic about the authors’ findings on innate and adaptive immune responses in liver regeneration. Thus, we are looking forward to getting greater precision regarding the fate of liver macrophages throughout tissue regeneration in these models.

Background & aims The recent approval of direct acting anti-virals (DAA) has dramatically changed the landscape of hepatitis C virus (HCV) therapy. Whether viral clearance could promote liver carcinogenesis is debated. It has been hypothesized that changes in intrahepatic immune surveillance following viral cure could favour tumour growth. This study aimed at characterizing the intrahepatic immune changes induced by HCV cure following DAA therapy. Methods Patients with compensated cirrhosis who underwent surgical resection for hepatocellular carcinoma (HCC) after sustained virological response (SVR) to DAA therapy were included. A control group of untreated HCV-infected patients with compensated cirrhosis was selected. RNA was extracted from tumoral and non-tumoral tissues and analysed using the Nanostring Immuno-Oncology-360 panel. Immune cells were quantified by immunohistochemistry. Results Twenty patients were included: 10 patients with a DAA-induced SVR and 10 untreated controls. All of them had a de novo BCLC 0/A HCC. Non-tumoral tissue profiling showed down-regulation of interferon-related genes (including MX1, ISG15 and IFIT1) after DAA therapy. No other differences in immune profiles/immune cell densities were identified between the two groups. The intra-tumoral immune profiles of HCCs that occurred after DAA therapy were not qualitatively or quantitatively different from those of tumours occurring in untreated patients. Conclusion In conclusion, removal of HCV infection after DAA-based therapy results only in a down-regulation of interferon-stimulated genes in non-tumoral tissues from patients with cirrhosis who develop HCC. These minor changes in the liver immune microenvironment are unlikely to favour HCC occurrence or recurrence after DAA-induced SVR.

BACKGROUND & AIMS: Hepatic ischemia/reperfusion injury is a complication of liver surgery that involves mitochondrial dysfunction resulting from mitochondrial permeability transition pore (mPTP) opening. Cyclophilin D (PPIF or CypD) is a peptidyl-prolyl cis-trans isomerase that regulates mPTP opening in the inner mitochondrial membrane. We investigated whether and how recently created small-molecule inhibitors of CypD prevent opening of the mPTP in hepatocytes and the resulting effects in cell models and livers of mice undergoing ischemia/reperfusion injury. METHODS: We measured the activity of 9 small-molecule inhibitors of cyclophilins in an assay of CypD activity. The effects of the small-molecule CypD inhibitors or vehicle on mPTP opening were assessed by measuring mitochondrial swelling and calcium retention in isolated liver mitochondria from C57BL/6J (wild-type) and Ppif-/- (CypD knockout) mice and in primary mouse and human hepatocytes by fluorescence microscopy. We induced ischemia/reperfusion injury in livers of mice given a small-molecule CypD inhibitor or vehicle before and during reperfusion and collected samples of blood and liver for histologic analysis. RESULTS: The compounds inhibited peptidyl-prolyl isomerase activity (half maximal inhibitory concentration values, 0.2-16.2 mu mol/L) and, as a result, calcium-induced mitochondrial swelling, by preventing mPTP opening (half maximal inhibitory concentration values, 1.4-132 mu mol/L) in a concentration-dependent manner. The most potent inhibitor (C31) bound CypD with high affinity and inhibited swelling in mitochondria from livers of wild-type and Ppif(-/-) mice (indicating an additional, CypD-independent effect on mPTP opening) and in primary human and mouse hepatocytes. Administration of C31 in mice with ischemia/reperfusion injury before and during reperfusion restored hepatic calcium retention capacity and oxidative phosphorylation parameters and reduced liver damage compared with vehicle. CONCLUSIONS: Recently created small-molecule inhibitors of CypD reduced calcium-induced swelling in mitochondria from mouse and human liver tissues. Administration of these compounds to mice during ischemia/reperfusion restored hepatic calcium retention capacity and oxidative phosphorylation parameters and reduced liver damage. These compounds might be developed to protect patients from ischemia/reperfusion injury after liver surgery or for other hepatic or nonhepatic disorders related to abnormal mPTP opening.