Cholangiopathies caused by biliary epithelial cell (BEC) injury represent a leading cause of liver failure. No effective pharmacologic therapies exist, and the underlying mechanisms remain obscure. We aimed to explore the mechanisms of bile duct repair after targeted BEC injury. Injection of intermedilysin into BEC-specific human CD59 (hCD59) transgenic mice induced acute and specific BEC death, representing a model to study the early signals that drive bile duct repair. Acute BEC injury induced cholestasis followed by CCR2+ monocyte recruitment and BEC proliferation. By using microdissection and next generation RNA sequencing, we identified five genes that were most upregulated in proliferating BECs after acute injury including Mapk8ip2, Cdkn1a, Itgb6, Rgs4, and Ccl2. Immunohistochemistry analyses confirmed robust upregulation of integrin αvβ6 (ITGβ6) expression in this BEC injury model, after bile duct ligation, and in patients with chronic cholangiopathies. Deletion of Itgb6 gene attenuated BEC proliferation post-acute bile duct injury. Macrophage depletion or Ccr2-deficiency impaired ITGβ6 expression and BEC proliferation. In vitro experiments revealed that bile-acid activated monocytes promoted BEC proliferation through ITGβ6. Our data suggest that BEC injury induces cholestasis, monocyte recruitment, and induction of ITGβ6, which work together to promote BEC proliferation, and that therefore represent potential therapeutic targets for cholangiopathies.
Adrien Guillot, Lucia Guerri, Dechun Feng, Seung-Jin Kim, Yeni Ait Ahmed, Janos Paloczi, Yong He, Kornel Schuebel, Shen Dai, Fengming Liu, Pal Pacher, Tatiana Kisseleva, Xuebin Qin, David Goldman, Frank Tacke, Bin Gao
Sepsis is a leading cause of death in critical illness, and its pathophysiology varies depending on preexisting medical conditions. Here we identified nonalcoholic fatty liver disease (NAFLD) as an independent risk factor for sepsis in a large clinical cohort and showed a link between mortality in NAFLD-associated sepsis and hepatic mitochondrial and energetic metabolism dysfunction. Using in vivo and in vitro models of liver lipid overload, we discovered a metabolic coordination between hepatocyte mitochondria and liver macrophages that express triggering receptor expressed on myeloid cells-2 (TREM2). Trem2-deficient macrophages released exosomes that impaired hepatocytic mitochondrial structure and energy supply because of their high content of miR-106b-5p, which blocks Mitofusin 2 (Mfn2). In a mouse model of NAFLD-associated sepsis, TREM2 deficiency accelerated the initial progression of NAFLD and subsequent susceptibility to sepsis. Conversely, overexpression of TREM2 in liver macrophages improved hepatic energy supply and sepsis outcome. This study demonstrates that NAFLD is a risk factor for sepsis, providing a basis for precision treatment, and identifies hepatocyte-macrophage metabolic coordination and TREM2 as potential targets for future clinical trials.
Jinchao Hou, Jue Zhang, Ping Cui, Yingyue Zhou, Can Liu, Xiaoliang Wu, Yun Ji, Sicong Wang, Baoli Cheng, Hui Ye, Liqi Shu, Kai Zhang, Di Wang, Jielin Xu, Qiang Shu, Marco Colonna, Xiangming Fang
Monocyte homing to the liver and adhesion to the liver sinusoidal endothelial cells (LSEC) are key elements in nonalcoholic steatohepatitis (NASH) pathogenesis. We reported previously that vascular cell adhesion molecule 1 (VCAM-1) mediates monocyte adhesion to LSEC. However, the pathogenic role of VCAM-1 in NASH is unclear. Herein, we report that VCAM-1 was a top upregulated adhesion molecule in the NASH mouse liver transcriptome. Open chromatin landscape profiling combined with genome-wide transcriptome analysis showed robust transcriptional upregulation of LSEC-VCAM-1 in murine NASH. Moreover, LSEC-VCAM-1 expression was significantly increased in human NASH. LSEC-VCAM-1 expression was upregulated by palmitate treatment in vitro, and reduced with inhibition of the mitogen-activated protein 3 kinase, mixed lineage kinase 3 (MLK3). Likewise, LSEC-VCAM-1 expression was reduced in the Mlk3-/- mice with diet-induced NASH. Furthermore, VCAM-1 neutralizing antibody or pharmacological inhibition attenuated diet-induced NASH in mice, mainly via reducing the proinflammatory monocyte hepatic population as examined by mass cytometry by time of flight (CyTOF). Moreover, endothelium-specific Vcam1 knockout mice were also protected against NASH. In summary, lipotoxic stress enhances the expression of LSEC-VCAM-1, in part, through MLK3 signaling. Inhibition of VCAM-1 was salutary in murine NASH, and might serve as a potential therapeutic strategy for human NASH.
Kunimaro Furuta, Qianqian Guo, Kevin D. Pavelko, Jeong-Heon Lee, Keith D. Robertson, Yasuhiko Nakao, Jan Melek, Vijay H. Shah, Petra Hirsova, Samar H. Ibrahim
Acute liver failure (ALF) patients display systemic innate immune suppression and increased susceptibility to infections. PD-1 expression by macrophages has been associated with immune suppression during sepsis and cancer. We therefore examined the role of PD-1/PD-L1 pathway in regulating Kupffer cell inflammatory and antimicrobial responses in acetaminophen (APAP) induced acute liver injury. Using intravital imaging and flow cytometry we found impaired Kupffer cell bacterial clearance and systemic bacterial dissemination in mice with liver injury. Increased PD-1 and PD-L1 expression was detected in Kupffer cells and lymphocyte subsets, respectively, during resolution of injury. Gene expression profiling of PD-1+ Kupffer cells revealed an immune-suppressive profile and reduced pathogen responses. Compared to wild-type, PD-1 deficient or anti-PD-1 treated mice with liver injury showed improved Kupffer cell bacterial clearance, reduced tissue bacterial load and protection from sepsis. Blood sample analyses of ALF patients revealed enhanced PD-1 and PD-L1 expression of monocytes and lymphocytes, respectively, and that plasma soluble PD-L1 levels predict patient outcome and sepsis. PD-1 in vitro blockade restored monocyte functionality. Our study describes a role for PD-1/PD-L1 axis in suppressing Kupffer cell and monocyte antimicrobial responses after liver injury and suggests anti-PD-1 immunotherapy as a strategy to reduce infection susceptibility in ALF.
Evangelos Triantafyllou, Cathrin L. C. Gudd, Marie-Anne Mawhin, Hannah C. Husbyn, Francesca M. Trovato, Matthew K. Siggins, Thomas O'Connor, Hiromi Kudo, Sujit K. Mukherjee, Julia A. Wendon, Christine Bernsmeier, Robert D. Goldin, Marina Botto, Wafa Khamri, Mark J.W. McPhail, Lucia A. Possamai, Kevin J. Woollard, Charalambos G. Antoniades, Mark R. Thursz
Neutrophil infiltration around lipotoxic hepatocytes is a hallmark of nonalcoholic steatohepatitis (NASH); however, how these two types of cells communicate remain obscure. We have previously demonstrated that neutrophil-specific microRNA-223 (miR-223) is elevated in hepatocytes to limit NASH progression in obese mice. Here we demonstrated that this elevation of miR-223 in hepatocytes was due to preferential uptake of miR-223-enriched extracellular vesicles (EVs) derived from neutrophils as well other types of cells albeit to a lesser extent. This selective uptake was dependent on the expression of low-density lipoprotein receptor (LDLR) on hepatocytes and apolipoprotein E (APOE) on neutrophil-derived EVs, which was enhanced by free fatty acids. Once internalized by hepatocytes, the EV-derived miR-223 acted to inhibit hepatic inflammatory and fibrogenic gene expression. In the absence of this LDLR-APOE dependent uptake of miR-223-enriched EVs, the progression of steatosis to NASH was accelerated. In contrast, augmentation of this transfer by treatment with an inhibitor of proprotein convertase subtilisin/kexin type 9, a drug used to lower blood cholesterol by upregulating LDLR, ameliorated NASH in mice. This specific role of LDLR and APOE in the selective control of miR-223-enriched EV transfer from neutrophils to hepatocytes may serve as a potential therapeutic target for NASH.
Yong He, Robim M. Rodrigues, Xiaolin Wang, Wonhyo Seo, Jing Ma, Seonghwan Hwang, Yaojie Fu, Eszter Trojnar, Csaba Matyas, Suxian Zhao, Ruixue Ren, Dechun Feng, Pal Pacher, George Kunos, Bin Gao
ABSTRACTIndividuals harboring the loss-of-function (LOF) proprotein convertase subtilising/kexin type 9 Gln152His variation (PCSK9Q152H) have low circulating low-density lipoprotein (LDL) cholesterol levels and are therefore protected against cardiovascular disease (CVD). This uncleavable form of pro-PCSK9, however, is retained in the endoplasmic reticulum (ER) of liver hepatocytes where it would be expected to contribute to ER storage disease (ERSD); a heritable condition known to cause systemic ER stress and liver injury. Here, we examined liver function in members of several French-Canadian families known to carry the PCSK9Q152H variation. We report that PCSK9Q152H carriers exhibited marked hypocholesterolemia and normal liver function despite their lifelong state of ER PCSK9 retention. Mechanistically, hepatic overexpression of PCSK9Q152H using adeno-associated viruses in male mice greatly increased the stability of key ER stress response chaperones in liver hepatocytes and unexpectedly protected against ER stress and liver injury rather than to induce them. Our findings show that ER retention of PCSK9 not only reduced CVD risk in patients but may also protect against ERSD and other ER stress-driven conditions of the liver. In summary, we have uncovered a co-chaperone function for PCSK9Q152H that explains its hepatoprotective effects and generated a translational mouse model for further mechanistic insights into this clinically relevant LOF PCSK9 variant.
Paul F. Lebeau, Hanny Wassef, Jae Hyun Byun, Khrystyna Platko, Brandon Ason, Simon Jackson, Joshua Dobroff, Susan Shetterly, William G. Richards, Ali A. Al-Hashimi, Kevin D. Won, Majambu Mbikay, Annik Prat, An Tang, Guillaume Paré, Renata Pasqualini, Nabil G. Seidah, Wadih Arap, Michel Chretien, Richard C. Austin
Membrane protrusion and adhesion to the extracellular matrix, which involves the extension of actin filaments and formation of adhesion complexes, are the fundamental processes for cell migration, tumor invasion, and metastasis. How cancer cells efficiently coordinate these processes remains unclear. Here, we showed that membrane-targeted CLIC1 spatiotemporally regulates the formation of cell-matrix adhesions and membrane protrusions through the recruitment of PIP5Ks to the plasma membrane. Comparative proteomics identified CLIC1 upregulated in human hepatocellular carcinoma (HCC) and associated with tumor invasiveness, metastasis, and poor prognosis. In response to migration-related stimuli, CLIC1 recruited PIP5K1A and PIP5K1C from the cytoplasm to the leading edge of the plasma membrane, where PIP5Ks generate a PIP2-rich microdomain to induce the formation of integrin-mediated cell-matrix adhesions and the signaling for cytoskeleon extension. CLIC1 silencing inhibited the attachment of tumor cells to culture plates and the adherence and extravasation in the lung alveoli resulting in suppressed lung metastasis in mice. This study reveals an unrecognized mechanism that spatiotemporally coordinates the formation of both lamellipodium/invadopodia and nascent cell-matrix adhesions for directional migration and tumor invasion/metastasis. The unique traits of upregulation and membrane targeting of CLIC1 in cancer cells make it an excellent therapeutic target for tumor metastasis.
Jei-Ming Peng, Sheng-Hsuan Lin, Ming-Chin Yu, Sen-Yung Hsieh
RNA binding protein Apobec1 Complementation Factor (A1CF) regulates posttranscriptional ApoB mRNA editing but the range of RNA targets and long-term impact of altered A1CF expression on liver function are unknown. Here we studied hepatocyte-specific A1cf transgenic (A1cf +/Tg), A1cf+/Tg Apobec1–/– and A1cf –/– mice fed chow or high fat/high fructose diets using RNA-Seq, RNA-CLIP Seq and tissue microarrays from human hepatocellular cancer (HCC). A1cf +/Tg mice exhibited increased hepatic proliferation and steatosis, with increased lipogenic gene expression (Mogat1, Mogat2, Cidea, Cd36) associated with shifts in polysomal RNA distribution. Aged A1cf +/Tg mice developed spontaneous fibrosis, dysplasia and HCC, which was accelerated on a high fat/fructose diet and independent of Apobec1. RNA-Seq revealed increased expression of mRNAs involved in oxidative stress (Gstm3, Gpx3, Cbr3), inflammatory response (Il19, Cxcl14, Tnfα, Ly6c), extracellular matrix organization (Mmp2, Col1a1, Col4a1), proliferation (Kif20a, Mcm2, Mcm4, Mcm6) with a subset of mRNAs (including Sox4, Sox9, Cdh1) identified in RNA CLIP-Seq. Increased A1CF expression in human HCC correlated with advanced fibrosis and with reduced survival in a subset with nonalcoholic fatty liver disease. In conclusion, we show that hepatic A1CF overexpression selectively alters polysomal distribution and mRNA expression, promoting lipogenic, proliferative and inflammatory pathways leading to HCC.
Valerie Blanc, Jesse D. Riordan, Saeed Soleymanjahi, Joseph Nadeau, ILKe Nalbantoglu, Yan Xie, Elizabeth A. Molitor, Blair B. Madison, Elizabeth M. Brunt, Jason C. Mills, Deborah C. Rubin, Irene O.L. Ng, Yeonjung Ha, Lewis R. Roberts, Nicholas O. Davidson
Type 2 diabetes is clinically associated with progressive necroinflammation and fibrosis in nonalcoholic steatohepatitis (NASH). Advanced glycation end-products (AGEs) accumulate during prolonged hyperglycemia, but the mechanistic pathways that lead to accelerated liver fibrosis have not been well defined. In this study, we show that the AGEs clearance receptor AGER1 was downregulated in patients with NASH and diabetes and in our NASH models, whereas the proinflammatory receptor RAGE was induced. These findings were associated with necroinflammatory, fibrogenic, and pro-oxidant activity via the NADPH oxidase 4. Inhibition of AGEs or RAGE deletion in hepatocytes in vivo reversed these effects. We demonstrate that dysregulation of NRF2 by neddylation of cullin 3 was linked to AGER1 downregulation and that induction of NRF2 using an adeno-associated virus–mediated approach in hepatocytes in vivo reversed AGER1 downregulation, lowered the level of AGEs, and improved proinflammatory and fibrogenic responses in mice on a high AGEs diet. In patients with NASH and diabetes or insulin resistance, low AGER1 levels were associated with hepatocyte ballooning degeneration and ductular reaction. Collectively, prolonged exposure to AGEs in the liver promotes an AGER1/RAGE imbalance and consequent redox, inflammatory, and fibrogenic activity in NASH.
Ali Dehnad, Weiguo Fan, Joy X. Jiang, Sarah R. Fish, Yuan Li, Suvarthi Das, Gergely Mozes, Kimberly A. Wong, Kristin A. Olson, Gregory W. Charville, Mohammed Ali, Natalie J. Török
BACKGROUND. Despite an increasing appreciation of the roles that myeloid cells play in tumor progression and therapy, challenges remain in interpreting the tumor-associated myeloid response balance and its translational value. We aimed to construct a simple and reliable myeloid signature for hepatocellular carcinoma (HCC). METHODS. Using in situ immunohistochemistry, we assessed the distribution of major myeloid subtypes in both peri- and intratumoral regions of HCC. A 2-feature-based, myeloid-specific prognostic signature, named the myeloid response score (MRS), was constructed using an L1-penalized Cox regression model based on data from a training subset (n=244) and in a test subset (n=244), an independent internal (n=341), and two external (n= 94; n=254) cohorts. RESULTS. The MRS and the MRS-based nomograms displayed remarkable discriminatory power, accuracy, and clinical usefulness for predicting recurrence and patient survival, superior to current staging algorithms. Moreover, an increase in MRS was associated with a shift in the myeloid response balance from antitumor to protumor activities, accompanied with enhanced CD8+ T cell exhaustion patterns. Additionally, we provide evidence that the MRS was associated with the efficacy of sorafenib treatment for recurrent HCC. CONCLUSION. We identified and validated a simple myeloid signature for HCC which showed remarkable prognostic potential and may serve as a basis for the stratification of HCC immune subtypes. FUNDING. This work was supported by the National Science and Technology Major Project of China, the National Natural Science Foundation of China, the Science and Information Technology of Guangzhou, the Fundamental Research Funds for the Central Universities, and the China Postdoctoral Science Foundation.
Chong Wu, Jie Lin, Yulan Weng, Dan-Ni Zeng, Jing Xu, Shufeng Luo, Li Xu, Mingyu Liu, Qiaomin Hua, Chao-Qun Liu, Jin-Qing Li, Jing Liao, Cheng Sun, Jian Zhou, Min-Shan Chen, Chao Liu, Zhenhong Guo, Shi-Mei Zhuang, Jin-Hua Huang, Limin Zheng