Jean-Philippe Pradere

Fate tracing reveals hepatic stellate cells as dominant contributors to liver fibrosis independent of its aetiology

Although organ fibrosis causes significant morbidity and mortality in chronic diseases, the lack of detailed knowledge about specific cellular contributors mediating fibrogenesis hampers the design of effective anti-fibrotic therapies. Different cellular sources including tissue-resident and bone marrow-derived fibroblasts, pericytes and epithelial cells have been suggested to give rise to myofibroblasts, but their relative contributions remain controversial, with profound differences between organs and different diseases. Here we employ a novel Cre-transgenic mouse that marks 99% of hepatic stellate cells (HSCs), a liver-specific pericyte population, to demonstrate that HSCs give rise to 82-96% of myofibroblasts in models of toxic, cholestatic and fatty liver disease. Moreover, we exclude that HSCs function as facultative epithelial progenitor cells in the injured liver. On the basis of these findings, HSCs should be considered the primary cellular target for anti-fibrotic therapies across all types of liver disease.

The HMGB1/RAGE axis triggers neutrophil-mediated injury amplification following necrosis

In contrast to microbially triggered inflammation, mechanisms promoting sterile inflammation remain poorly understood. Damage-associated molecular patterns (DAMPs) are considered key inducers of sterile inflammation following cell death, but the relative contribution of specific DAMPs, including high-mobility group box 1 (HMGB1), is ill defined. Due to the postnatal lethality of Hmgb1-knockout mice, the role of HMGB1 in sterile inflammation and disease processes in vivo remains controversial. Here, using conditional ablation strategies, we have demonstrated that epithelial, but not bone marrow-derived, HMGB1 is required for sterile inflammation following injury. Epithelial HMGB1, through its receptor RAGE, triggered recruitment of neutrophils, but not macrophages, toward necrosis. In clinically relevant models of necrosis, HMGB1/RAGE-induced neutrophil recruitment mediated subsequent amplification of injury, depending on the presence of neutrophil elastase. Notably, hepatocyte-specific HMGB1 ablation resulted in 100% survival following lethal acetaminophen intoxication. In contrast to necrosis, HMGB1 ablation did not alter inflammation or mortality in response to TNF- or FAS-mediated apoptosis. In LPS-induced shock, in which HMGB1 was considered a key mediator, HMGB1 ablation did not ameliorate inflammation or lethality, despite efficient reduction of HMGB1 serum levels. Our study establishes HMGB1 as a bona fide and targetable DAMP that selectively triggers a neutrophil-mediated injury amplification loop in the setting of necrosis.

The Yin and Yang of Toll-like Receptors in Cancer

Recognition of non-self molecular patterns by pattern recognition receptors is a cornerstone of innate immunity. Toll-like receptors (TLRs) exert a key role in recognizing pathogen-associated molecular patterns (PAMPs) but have also been implicated in the recognition of damage-associated molecular patterns (DAMPs). As such, TLRs regulate a wide range of biological responses including inflammatory and immune responses during carcinogenesis. The high expression of TLRs by antigen-presenting cells, including dendritic cells, and their ability to induce antitumor mediators such as type I interferon has led to efforts to utilize TLR agonists in tumor therapy in order to convert the often tolerant immune response toward antitumor responses. However, TLRs are also increasingly recognized as regulators of tumor-promoting inflammation and promoters of tumor survival signals. Here, we will review in detail the dichotomous role of TLRs in tumor biology, focusing on relevant TLR-dependent pro- and antitumor pathways, and discuss clinical applications of TLR-targeted therapies for tumor prevention and treatment.

Toll-like receptor 4 and hepatic fibrogenesis

Inflammation is strongly associated with chronic hepatic injury and the ensuing wound-healing process. Recent evidence from mouse models and human studies implicates Toll-like receptors (TLRs) as important regulators of the inflammatory response and a functional link between inflammation and fibrosis in the chronically injured liver. Here, we review mechanisms by which TLR4 and TLR4 ligands from the intestinal microbiota contribute to hepatic injury, inflammation, hepatic stellate cell activation, and fibrosis.

Absence of hepatic stellate cell retinoid lipid droplets does not enhance hepatic fibrosis but decreases hepatic carcinogenesis

Objective Hepatic stellate cells (HSCs) contain a number of bioactive metabolites or their precursors including retinoids in their characteristic lipid droplets. The loss of lipid droplets and retinoids is a hallmark of HSC activation, but it remains unclear whether this loss promotes HSC activation, liver fibrogenesis or carcinogenesis. Design Spontaneous and experimental fibrogenesis as well as a diethylnitrosamine-induced hepatocarcinogenesis were investigated in lecithin-retinol acyltransferase (LRAT)-deficient mice which lack retinoid-containing lipids droplets in their HSCs. Results Following HSC activation, LRAT expression was rapidly lost, emphasising its importance in lipid droplet biology in HSCs. Surprisingly, there was no difference in fibrosis induced by bile duct ligation (BDL) or by eight injections of carbon tetrachloride (CCl4) between wild-type and LRAT-deficient mice. To exclude the possibility that the effects on fibrogenesis were missed due to the rapid downregulation of LRAT following HSC activation, acute as well as spontaneous liver fibrosis was investigated. However, there was no increased fibrosis in 3-, 8- and 12-month-old LRAT-deficient mice and in LRAT-deficient mice after a single injection of CCl4 compared with wild-type mice. To determine whether the absence of retinoids in HSCs affects hepatocarcinogenesis, wild-type and LRAT-deficient mice were injected with diethylnitrosamine. LRAT deficiency decreased diethylnitrosamine-induced injury and tumour load and increased the expression of the retinoic acid responsive genes Cyp26a1, RARb and p21, suggesting that the lower tumour load of LRAT-deficient mice was a result of increased retinoid signalling and subsequent p21-mediated inhibition of proliferation. Conclusions The absence of retinoid-containing HSC lipid droplets does not promote HSC activation but reduces hepatocarcinogenesis.

Lysophosphatidic acid-1-receptor targeting agents for fibrosis.

Introduction: The presence of fibrosis is associated with alterations in organ architecture and is responsible for the morbidity of diseases including pneumopathies, systemic sclerosis, liver cirrhosis, chronic cardiovascular diseases, progressive kidney diseases and diabetes. Although a growing number of pro-fibrotic molecules, mediators and other pathways have been reported, there are currently very few antifibrotic molecules being evaluated in clinical trials. Areas covered: Current knowledge about the contribution of lysophosphatidic acid (LPA), a bioactive mediator acting via specific G-protein coupled receptors (LPAR), in the etiology of fibrosis. In a number of organs, fibrosis is associated with increased LPA production as well as with increased expression of some LPAR subtypes (mainly LPA1R). LPAR–/– knockout mice and treatment of animal models with specific antagonists clearly demonstrate the contribution of LPA1R subtype to the development of kidney, lung, vascular and dermal fibrosis. The involvement of LPA in liver fibrosis is also strongly suspected but still unproven. Expert opinion: Experiments in animal models clearly demonstrate that LPA1R antagonists have interesting anti-fibrotic potencies. This reveals promising perspectives for the design of new therapeutic approaches to prevent fibrosis-associated diseases. Nevertheless, the number of efficient LPA1R antagonists currently available is still low, and none of them has been used in clinical trials so far.

Epithelial Transforming Growth Factor-β Signaling Does Not Contribute to Liver Fibrosis but Protects Mice From Cholangiocarcinoma

BACKGROUND & AIMS Transforming growth factor-β (TGFβ) exerts key functions in fibrogenic cells, promoting fibrosis development in the liver and other organs. In contrast, the functions of TGFβ in liver epithelial cells are not well understood, despite their high level of responsiveness to TGFβ. We sought to determine the contribution of epithelial TGFβ signaling to hepatic fibrogenesis and carcinogenesis. METHODS TGFβ signaling in liver epithelial cells was inhibited by albumin-Cre-, K19-CreERT-, Prom1-CreERT2-, or AAV8-TBG-Cre-mediated deletion of the floxed TGFβ receptor II gene (Tgfbr2). Liver fibrosis was induced by carbon tetrachloride, bile duct ligation, or disruption of the multidrug-resistance transporter 2 gene (Mdr2). Hepatocarcinogenesis was induced by diethylnitrosamine or hepatic deletion of PTEN. RESULTS Deletion of Tgfbr2 from liver epithelial cells did not alter liver injury, toxin-induced or biliary fibrosis, or diethylnitrosamine-induced hepatocarcinogenesis. In contrast, epithelial deletion of Tgfbr2 promoted tumorigenesis and reduced survival of mice with concomitant hepatic deletion of Pten, accompanied by an increase in tumor number and a shift from hepatocellular carcinoma to cholangiocarcinoma. Surprisingly, both hepatocyte- and cholangiocyte-specific deletion of Pten and Tgfbr2 promoted the development of cholangiocarcinoma, but with different latencies. The prolonged latency and the presence of hepatocyte-derived cholangiocytes after AAV8-TBG-Cre-mediated deletion of Tgfbr2 and Pten indicated that cholangiocarcinoma might arise from hepatocyte-derived cholangiocytes in this model. Pten deletion resulted in up-regulation of Tgfbr2, and deletion of Tgfbr2 increased cholangiocyte but not hepatocyte proliferation, indicating that the main function of epithelial TGFBR2 is to restrict cholangiocyte proliferation. CONCLUSIONS Epithelial TGFβ signaling does not contribute to the development of liver fibrosis or formation of hepatocellular carcinomas in mice, but restricts cholangiocyte proliferation to prevent cholangiocarcinoma development, regardless of its cellular origin.

Negative regulation of NF-κB p65 activity by serine 536 phosphorylation

The kinase IKK fine-tunes inflammation by both activating and inhibiting signaling by the p65 subunit of NF-κB. IKK is the start and stop signal By phosphorylating inhibitor of κB (IκB), the kinase IKK targets it for destruction, thus enabling the dimeric transcription factor nuclear factor κB (NF-κB) to translocate to the nucleus and alter target gene expression. Pradère et al. generated knock-in mice expressing a mutant p65 subunit of NF-κB that could not be phosphorylated by IKK at Ser534 (the mouse homolog of human p65 Ser536). The mutated p65 translocated to the nucleus properly, but the protein exhibited enhanced stability, which resulted in exacerbated responses to inflammatory stimuli in vivo. Experiments with human cells indicated that this regulatory mechanism was conserved. Thus, in addition to stimulating NF-κB signaling by phosphorylating IκB, IKK limits inflammation by targeting this regulatory site in mouse and human p65. Nuclear factor κB (NF-κB) is a master regulator of inflammation and cell death. Whereas most of the activity of NF-κB is regulated through the inhibitor of κB (IκB) kinase (IKK)–dependent degradation of IκB, IKK also phosphorylates subunits of NF-κB. We investigated the contribution of the phosphorylation of the NF-κB subunit p65 at the IKK phosphorylation site serine 536 (Ser536) in humans, which is thought to be required for the activation and nuclear translocation of NF-κB. Through experiments with knock-in mice (S534A mice) expressing a mutant p65 with an alanine-to-serine substitution at position 534 (the murine homolog of human Ser536), we observed increased expression of NF-κB–dependent genes after injection of mice with the inflammatory stimulus lipopolysaccharide (LPS) or exposure to gamma irradiation, and the enhanced gene expression was most pronounced at late time points. Compared to wild-type mice, S534A mice displayed increased mortality after injection with LPS. Increased NF-κB signaling in the S534A mice was at least in part explained by the increased stability of the S534A p65 protein compared to that of the Ser534-phosphorylated wild-type protein. Together, our results suggest that Ser534 phosphorylation of p65 in mice (and, by extension, Ser536 phosphorylation of human p65) is not required for its nuclear translocation, but instead inhibits NF-κB signaling to prevent deleterious inflammation.

HMGB1 links chronic liver injury to progenitor responses and hepatocarcinogenesis

Cell death is a key driver of disease progression and carcinogenesis in chronic liver disease (CLD), highlighted by the well-established clinical correlation between hepatocellular death and risk for the development of cirrhosis and hepatocellular carcinoma (HCC). Moreover, hepatocellular death is sufficient to trigger fibrosis and HCC in mice. However, the pathways through which cell death drives CLD progression remain elusive. Here, we tested the hypothesis that high-mobility group box 1 (HMGB1), a damage-associated molecular pattern (DAMP) with key roles in acute liver injury, may link cell death to injury responses and hepatocarcinogenesis in CLD. While liver-specific HMGB1 deficiency did not significantly affect chronic injury responses such as fibrosis, regeneration, and inflammation, it inhibited ductular/progenitor cell expansion and hepatocyte metaplasia. HMGB1 promoted ductular expansion independently of active secretion in a nonautonomous fashion, consistent with its role as a DAMP. Liver-specific HMGB1 deficiency reduced HCC development in 3 mouse models of chronic injury but not in a model lacking chronic liver injury. As with CLD, HMGB1 ablation reduced the expression of progenitor and oncofetal markers, a key determinant of HCC aggressiveness, in tumors. In summary, HMGB1 links hepatocyte death to ductular reaction, progenitor signature, and hepatocarcinogenesis in CLD.

Abstract 2299: Gremlin 1 labels a mesenchymal progenitor in the gastrointestinal tract, bone and tumor microenvironment.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Introduction: The cellular origin and contribution of mesenchymal stem cells (MSCs) in the gastrointestinal tract, bone marrow and tumor microenvironment are unknown. More specific markers of MSCs are needed. We hypothesized that Grem1 expression would be a specific marker of MSCs. Methods: We developed Grem1-BAC-CreERT, αSMA-BAC-CreERT, Grem1-BAC-EGFP-peptide 2A-DTR-peptide 2A-CreERT and Vimentin-BAC-CreERT transgenic lines to characterize the origin, function and kinetics of gastrointestinal, bone marrow and tumor-associated mesenchyme. These mice were crossed to reporters, Grem1fl/fl and diphtheria toxin associated lines. The cellular fate and function of the Grem1, αSMA and Vimentin expressing cells, was analyzed by direct fluorescence microscopy and immunostaining at several time points (up to 12 months) and in several cancer models. Results: In the bone marrow, Grem1 recombination was identified in a very rare population of CD45/Ter119/CD31 triple negative cells (0.004% of all cells from compact bone extraction). In the bone marrow, Grem1-EGFP identified a discrete population of perisinusoidal cells. Grem1-recombined cells dramatically expand within MSC cultures forming large colony forming units, with a CFU-F efficiency of 3%. Grem1-recombined cells could be differentiated in vitro into adipocytes, osteoblasts, chondrocytes and αSMA(+) myofibroblasts. Furthermore, these cells are long-lived, recoverable from the bone at least 9 months after tamoxifen induction and can be transplanted. In the small intestine, 24 hours after tamoxifen induction, there were single Grem1 recombined cells found immediately subjacent to the basement membrane. These initially rare αSMA(-) recombined cells expanded over the next 12 months to completely trace the periepithelial fibroblast sheath. In both the gut and bone marrow, perinatal tamoxifen induction led to dramatically accelerated lineage tracing. Syngeneic tumorigenicity and carcinogenesis studies (including hepatic metastatic model (splenic injection of MC38, C57Bl/6 colorectal cancer cell line), MNU/Hfelis and AOM/DSS gastrointestinal cancer models) in our Grem1-BAC-specific lines revealed that Grem1 marked a cellular origin of cancer-associated mesenchyme, although many cancer-associated fibroblasts also arise from a specific αSMA+ stromal population. The exact lineage relationship between these Grem1 and αSMA+ cells is being tested. Conclusions: Grem1-expression labels a specific mesenchymal progenitor in the bone and gut that can give rise to multiple mesenchymal lineages during development and in the peritumoral stroma. We are currently examining the functional relevance of these cells and Grem1 production in supporting the normal and pathological stem cell niche in the bone marrow, gut and tumor microenvironment. Citation Format: Daniel L. Worthley, Yiling Si, Samuel Asfaha, Benedikt Westphalen, Yagnesh Tailor, Michael Churchill, Jean-Philippe Pradere, Robert Schwabe, Siddhartha Mukherjee, Timothy Wang. Gremlin 1 labels a mesenchymal progenitor in the gastrointestinal tract, bone and tumor microenvironment. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2299. doi:10.1158/1538-7445.AM2013-2299