Peter Hasselblatt

Autosomal dominant immune dysregulation syndrome in humans with CTLA4 mutations

The protein cytotoxic T lymphocyte antigen-4 (CTLA-4) is an essential negative regulator of immune responses, and its loss causes fatal autoimmunity in mice. We studied a large family in which five individuals presented with a complex, autosomal dominant immune dysregulation syndrome characterized by hypogammaglobulinemia, recurrent infections and multiple autoimmune clinical features. We identified a heterozygous nonsense mutation in exon 1 of CTLA4. Screening of 71 unrelated patients with comparable clinical phenotypes identified five additional families (nine individuals) with previously undescribed splice site and missense mutations in CTLA4. Clinical penetrance was incomplete (eight adults of a total of 19 genetically proven CTLA4 mutation carriers were considered unaffected). However, CTLA-4 protein expression was decreased in regulatory T cells (Treg cells) in both patients and carriers with CTLA4 mutations. Whereas Treg cells were generally present at elevated numbers in these individuals, their suppressive function, CTLA-4 ligand binding and transendocytosis of CD80 were impaired. Mutations in CTLA4 were also associated with decreased circulating B cell numbers. Taken together, mutations in CTLA4 resulting in CTLA-4 haploinsufficiency or impaired ligand binding result in disrupted T and B cell homeostasis and a complex immune dysregulation syndrome.

Development of pulmonary fibrosis through a pathway involving the transcription factor Fra-2/AP-1

Studies using genetically modified mice have revealed fundamental functions of the transcription factor Fos/AP-1 in bone biology, inflammation, and cancer. However, the biological role of the Fos-related protein Fra-2 is not well defined in vivo. Here we report an unexpected profibrogenic function of Fra-2 in transgenic mice, in which ectopic expression of Fra-2 in various organs resulted in generalized fibrosis with predominant manifestation in the lung. The pulmonary phenotype was characterized by vascular remodeling and obliteration of pulmonary arteries, which coincided with expression of osteopontin, an AP-1 target gene involved in vascular remodeling and fibrogenesis. These alterations were followed by inflammation; release of profibrogenic factors, such as IL-4, insulin-like growth factor 1, and CXCL5; progressive fibrosis; and premature mortality. Genetic experiments and bone marrow reconstitutions suggested that fibrosis developed independently of B and T cells and was not mediated by autoimmunity despite the marked inflammation observed in transgenic lungs. Importantly, strong expression of Fra-2 was also observed in human samples of idiopathic and autoimmune-mediated pulmonary fibrosis. These findings indicate that Fra-2 expression is sufficient to cause pulmonary fibrosis in mice, possibly by linking vascular remodeling and fibrogenesis, and suggest that Fra-2 has to be considered a contributing pathogenic factor of pulmonary fibrosis in humans.

Hepatocyte survival in acute hepatitis is due to c-Jun/AP-1-dependent expression of inducible nitric oxide synthase

Analysis of the molecular factors determining hepatocyte survival or death in response to inflammatory stimuli is essential for understanding the pathogenesis of inflammatory liver disease and for identifying novel therapeutic approaches. c-Jun N-terminal kinase (JNK) is a major mediator of cytokine-induced cell death during hepatitis, but the signaling pathways downstream of JNK remain less well defined. Here we show that the transcription factor c-Jun/AP-1, a prototypic target of JNK, is strongly expressed in the liver of patients with acute liver injury. The molecular function of c-Jun in inflammatory liver disease was analyzed in mice by using the Con A model of T cell-mediated hepatitis. Mice lacking c-Jun in hepatocytes display increased liver cell death and mortality upon Con A injection. This phenotype is caused by impaired expression of inducible nitric oxide synthase (nos2), a direct transcriptional target of c-Jun, and reduced production of hepatoprotective nitric oxide (NO). Moreover, increased hepatotoxicity in mutant mice is likely caused by hypoxia and oxidative stress and can be rescued pharmacologically by liver-specific NO delivery. These findings demonstrate that c-Jun/AP-1 is hepatoprotective during acute hepatitis by regulating nos2/NO expression and thus functionally antagonizes the cell death-promoting functions of JNK.

Impairment of hepatic growth hormone and glucocorticoid receptor signaling causes steatosis and hepatocellular carcinoma in mice

Growth hormone (GH)‐activated signal transducer and activator of transcription 5 (STAT5) and the glucocorticoid (GC)‐responsive glucocorticoid receptor (GR) are important signal integrators in the liver during metabolic and physiologic stress. Their deregulation has been implicated in the development of metabolic liver diseases, such as steatosis and progression to fibrosis. Using liver‐specific STAT5 and GR knockout mice, we addressed their role in metabolism and liver cancer onset. STAT5 single and STAT5/GR double mutants developed steatosis, but only double‐mutant mice progressed to liver cancer. Mechanistically, STAT5 deficiency led to the up‐regulation of prolipogenic sterol regulatory element binding protein 1 (SREBP‐1) and peroxisome proliferator activated receptor gamma (PPAR‐γ) signaling. Combined loss of STAT5/GR resulted in GH resistance and hypercortisolism. The combination of both induced expression of adipose tissue lipases, adipose tissue lipid mobilization, and lipid flux to the liver, thereby aggravating STAT5‐dependent steatosis. The metabolic dysfunctions in STAT5/GR compound knockout animals led to the development of hepatic dysplasia at 9 months of age. At 12 months, 35% of STAT5/GR‐deficient livers harbored dysplastic nodules and ∼60% hepatocellular carcinomas (HCCs). HCC development was associated with GH and insulin resistance, enhanced tumor necrosis factor alpha (TNF‐α) expression, high reactive oxygen species levels, and augmented liver and DNA damage parameters. Moreover, activation of the c‐Jun N‐terminal kinase 1 (JNK1) and STAT3 was prominent. Conclusion: Hepatic STAT5/GR signaling is crucial for the maintenance of systemic lipid homeostasis. Impairment of both signaling cascades causes severe metabolic liver disease and promotes spontaneous hepatic tumorigenesis. (HEPATOLOGY 2011;54:1398–1409)

Purinergic P2Y2 Receptors Promote Neutrophil Infiltration and Hepatocyte Death in Mice With Acute Liver Injury

BACKGROUND & AIMS During progression of liver disease, inflammation affects survival of hepatocytes. Endogenous release of adenosine triphosphate (ATP) in the liver activates purinergic P2 receptors (P2R), which regulate inflammatory responses, but little is known about the roles of these processes in the development of acute hepatitis. METHODS We induced acute hepatitis in C57BL/6 mice by intravenous injection of concanavalin A and then analyzed liver concentrations of ATP and expression of P2R. We assessed P2Y(2)R(-/-) mice and C57BL/6 wild-type mice injected with suramin, a pharmacologic inhibitor of P2YR. Toxic liver failure was induced in mice by intraperitoneal injection of acetaminophen. Hepatocyte-specific functions of P2R signaling were analyzed in primary mouse hepatocytes. RESULTS Induction of acute hepatitis in wild-type C57BL/6 mice released large amounts of ATP from livers and induced expression of P2Y(2)R. Liver damage and necrosis were greatly reduced in P2Y(2)R(-/-) mice and C57BL/6 mice given injections of suramin. Acetaminophen-induced liver damage was reduced in P2Y(2)R(-/-) mice. Analysis of liver-infiltrating immune cells during acute hepatitis revealed that expression of P2Y(2)R in bone marrow-derived cells was required for liver infiltration by neutrophils and subsequent liver damage. Hepatic expression of P2Y(2)R interfered with expression of genes that regulate cell survival, and promoted tumor necrosis factor-α-mediated cell death, in a cell-autonomous manner. CONCLUSIONS Extracellular ATP and P2Y(2)R have cell-type specific, but synergistic functions during liver damage that regulate cellular immune responses and promote hepatocyte death. Reagents designed to target P2Y(2)R might be developed to treat inflammatory liver disease.

Comprehensive Intestinal T Helper Cell Profiling Reveals Specific Accumulation of IFN-γ+IL-17+Coproducing CD4+ T Cells in Active Inflammatory Bowel Disease

Objective:Skewed T helper (TH) cell responses and specific functions of TH1, TH2, TH17, and Treg cells have been implicated in the pathogenesis of inflammatory bowel disease (IBD) that led to the establishment of the pathogenic TH1/TH2 and TH17/Treg cell imbalance paradigms. However, the relevant TH cell population driving mucosal inflammation is still unknown. Methods:We performed a comprehensive TH cell profiling of circulating and intestinal lymphocytes isolated from patients with Crohn’s disease (CD; n = 69) and ulcerative colitis (UC; n = 41) undergoing endoscopy or surgical resection and compared them with healthy controls (n = 45). Mucosal inflammation was assessed endoscopically and histologically. TH cells were analyzed by flow cytometric evaluation of cytokine production and differentiation marker expression. Results:Specialized TH cell populations were enriched in the intestinal mucosa compared with peripheral blood. Specifically, we observed a concomitant upregulation of TH17 cells and Tregs in active inflammatory lesions in patients with both CD and UC compared with quiescent/mildly inflamed lesions and healthy tissue. Of note, interferon &ggr;+ interleukin (IL)-17+coproducing CD4+ T cells with high expression of T-bet, CD26, and IL-22 resembling recently described pathogenic TH17 cells were specifically enriched in the inflamed mucosal tissue. Conclusions:Our results argue against the controversial TH1/TH2 or TH17/Treg paradigms. In contrast, they suggest that a subpopulation of TH17 cells sharing a TH1 signature may be specifically involved in intestinal inflammation in CD and UC. These findings provide a better understanding of IBD pathogenesis and may help explain the efficacy of anti-IL-12p40/IL-23 and failure of anti-IL-17A therapies despite the enrichment of TH17 cells.

MiR-146a regulates the TRAF6/TNF-axis in donor T cells during GVHD.

Acute graft-versus-host disease (GVHD) limits the success of allogeneic hematopoietic cell transplantation (allo-HCT); therefore, a better understanding of its biology may improve therapeutic options. We observed miR-146a up-regulation in T cells of mice developing acute GVHD compared with untreated mice. Transplanting miR-146a(-/-) T cells caused increased GVHD severity, elevated tumor necrosis factor (TNF) serum levels, and reduced survival. TNF receptor-associated factor 6 (TRAF6), a verified target of miR-146a, was up-regulated in miR-146a(-/-) T cells following alloantigen stimulation. Higher TRAF6 levels translated into increased nuclear factor-κB activity and TNF production in miR-146a(-/-) T cells. Conversely, the detrimental effect of miR-146a deficiency in T cells was antagonized by TNF blockade, whereas phytochemical induction of miR-146a or its overexpression using a miR-146a mimic reduced GVHD severity. In humans, the minor genotype of the single nucleotide polymorphism rs2910164 in HCT donors, which reduces expression of miR-146a, was associated with severe acute GVHD (grade III/IV). We show that miR-146a functions as a negative regulator of donor T cells in GVHD by targeting TRAF6, leading to reduced TNF transcription. Because miR-146a expression can be exogenously enhanced, our results provide a novel targeted molecular approach to mitigate GVHD.

The transcription factor c‐Jun protects against sustained hepatic endoplasmic reticulum stress thereby promoting hepatocyte survival

Endoplasmic reticulum (ER) stress due to accumulation of hepatoviral or misfolded proteins is increasingly recognized as an important step in the pathogenesis of inflammatory, toxic, and metabolic liver diseases. ER stress results in the activation of several intracellular signaling pathways including Jun N‐terminal kinase (JNK). The AP‐1 (activating protein 1) transcription factor c‐Jun is a prototypic JNK target and important regulator of hepatocyte survival, proliferation, and liver tumorigenesis. Because the functions of c‐Jun during the ER stress response are poorly understood, we addressed this issue in primary hepatocytes and livers of hepatocyte‐specific c‐Jun knockout mice. ER stress was induced pharmacologically in vitro and in vivo and resulted in a rapid and robust induction of c‐Jun protein expression. Interestingly, ER‐stressed hepatocytes lacking c‐Jun displayed massive cytoplasmic vacuolization due to ER distension. This phenotype correlated with exacerbated and sustained activation of canonical ER stress signaling pathways. Moreover, sustained ER stress in hepatocytes lacking c‐Jun resulted in increased cell damage and apoptosis. ER stress is also a strong inducer of macroautophagy, a cell‐protective mechanism of self‐degradation of cytoplasmic components and organelles. Interestingly, autophagosome numbers in response to ER stress were reduced in hepatocytes lacking c‐Jun. To further validate these findings, macroautophagy was inhibited chemically in ER‐stressed wildtype hepatocytes, which resulted in cytoplasmic vacuolization and increased cell damage closely resembling the phenotypes observed in c‐Jun‐deficient cells. Conclusion: Our findings indicate that c‐Jun protects hepatocytes against excessive activation of the ER stress response and subsequent cell death and provide evidence that c‐Jun functionally links ER stress responses and macroautophagy. (HEPATOLOGY 2012)

The role of the transcription factor AP-1 in colitis-associated and beta-catenin-dependent intestinal tumorigenesis in mice.

Chronic inflammation is an important cancer risk factor but the molecular pathways linking inflammation and cancer are incompletely understood. The transcription factor c-Jun/AP-1 (activator protein 1) is involved in inflammatory responses and tumorigenesis and has been proposed as an essential mediator of oncogenic β-catenin signaling in the intestine. Here, we examined the functions of c-Jun in two distinct mouse models of conditional and intestine-specific activation of β-catenin. c-Jun is strongly expressed in the small intestine of mutant mice. However, β-catenin-dependent cell proliferation is surprisingly not affected in mice lacking c-jun in intestinal epithelium, suggesting that c-Jun is not an essential immediate target of β-catenin signaling in the small intestine. To examine the functions of Jun and Fos proteins during inflammation and cancer in the colon, colitis-associated tumors were induced chemically in the respective knockout mice. Tumors were characterized by activated β-catenin and strongly expressed c-Jun and JunB. However, tumorigenesis was not affected by inactivation of c-Jun in either intestinal epithelium or myeloid cells. Moreover, tumorigenesis was not altered in mice lacking junB, junD, c-fos, fra-1 or fra-2, suggesting that inhibition of c-Jun or other single AP-1 proteins is not a determining factor in colitis-associated cancer in mice.

The transcription factor c-JUN/AP-1 promotes HBV-related liver tumorigenesis in mice

Hepatocellular carcinoma (HCC) develops as a consequence of chronic inflammatory liver diseases such as chronic hepatitis B virus (HBV) infection. The transcription factor c-Jun/activator protein 1 (AP-1) is strongly expressed in response to inflammatory stimuli, promotes hepatocyte survival during acute hepatitis and acts as an oncogene during chemically induced liver carcinogenesis in mice. Here, we therefore aimed to characterize the functions of c-Jun during HBV-related liver tumorigenesis. To this end, transgenic mice expressing all HBV envelope proteins (HBV+), an established model of HBV-related HCC, were crossed with knockout mice lacking c-Jun specifically in hepatocytes and tumorigenesis was analyzed. Hepatic expression of c-Jun was strongly induced at several time points during tumorigenesis in HBV+ mice, whereas expression of other AP-1 components remained unchanged. Importantly, formation of premalignant foci and tumors was strongly reduced in HBV+ mice lacking c-Jun. This phenotype correlated with impaired hepatocyte proliferation and increased expression of the cell cycle inhibitor p21, whereas hepatocyte survival was not affected. Progression and prognosis of HBV-related HCC correlates with the expression of the cytokine osteopontin (Opn), an established AP-1 target gene. Opn expression was strongly reduced in HBV+ livers and primary mouse hepatocytes lacking c-Jun, demonstrating that c-Jun regulates hepatic Opn expression in a cell-autonomous manner. These findings indicate that c-Jun has important functions during HBV-associated tumorigenesis by promoting hepatocyte proliferation as well as progression of dysplasia. Therefore, targeting c-Jun may be a useful strategy to prevent hepatitis-associated tumorigenesis.