Neurons in the ventral premammillary nucleus (PMV) that control the onset of puberty through leptin signaling. A series of studies have shown that Kiss1 neurons mediate leptin’s effect on pubertal development, but Donato and colleagues (Page 355) use multiple genetic mouse models to find that leptin signaling in Kiss1 neurons is not required for the onset of puberty. Rather, the PMV, a previously unrecognized hypothalamic nucleus, serves as a key link between leptin and pubertal development.
Influenza A viruses cause recurrent, seasonal epidemics and occasional global pandemics with devastating levels of morbidity and mortality. The ability of influenza A viruses to adapt to various hosts and undergo reassortment events ensures constant generation of new strains with unpredictable degrees of pathogenicity, transmissibility, and pandemic potential. Currently, the combination of factors that drives the emergence of pandemic influenza is unclear, making it impossible to foresee the details of a future outbreak. Identification and characterization of influenza A virus virulence determinants may provide insight into genotypic signatures of pathogenicity as well as a more thorough understanding of the factors that give rise to pandemics.
Dysregulated Hedgehog (Hh) signaling has been implicated in a growing number of human cancers. To date, most antagonists of this signaling pathway that have been developed target the Hh receptor Smoothened. However, these are predicted to have minimal effect when the pathway is activated as a result of dysregulation downstream of this receptor. In this issue of the JCI, Beauchamp and colleagues provide preclinical evidence that arsenic trioxide, a drug FDA approved for the treatment of acute promyelocytic leukemia, inhibits the growth of Ewing sarcoma and medulloblastoma cells by targeting GLI family zinc finger (GLI) proteins, which are Hh signaling pathway components downstream of Smoothened.
Advances in DNA sequencing technologies have increased attention on genetic variation in somatic tissues. Although long known to cause neoplastic diseases, somatic variation is now being investigated as a pathogenetic mechanism for other diseases. Somatic changes are genomic DNA variations that were not inherited but arise in tissues throughout life. In this issue of the JCI, Magerus-Chatinet et al. explore somatic changes in patients with autoimmune lymphoproliferative syndrome (ALPS), a congenital disease of defective apoptosis and autoimmunity that is usually associated with germline heterozygous mutations in the gene encoding the Fas death receptor. They explain why certain individuals have severe disease manifestations by documenting somatic alterations in the germline normal FAS allele in an unusual population of “double-negative” T cells found in ALPS. Thus, the oncological concept of somatic loss of heterozygosity leading to selected cell expansion also applies to autoimmune diseases.
Vaccines remain one of the most cost-effective public health measures. Despite ongoing efforts, protective vaccines against cancer and many infectious diseases, including malaria, tuberculosis, and HIV/AIDS, are still not in hand. Most investigators believe that to succeed against these difficult targets, vaccines that generate potent T cell responses are needed. In this issue of the JCI, Salek-Ardakani et al. show how the relative virulence of a virus/vaccine vector affects the memory CD8+ T cells generated and how the response may be enhanced. The work has important implications for the development of future vaccines that aim to trigger CD8+ T cell responses.
Chronic myeloid leukemia (CML) is a hematopoietic disease characterized by expansion of myeloid blood cells. It is caused by the t(9;22) chromosomal translocation that results in the expression of the fusion tyrosine kinase BCR-ABL. Tyrosine kinase inhibitor (TKI) therapy has led to long-term remissions, but patients remain BCR-ABL+. There is agreement that TKIs do not kill CML stem cells; however, it is controversial whether this is because of a lack of BCR-ABL kinase inhibition in CML stem cells or because CML stem cells do not require BCR-ABL for survival. In this issue of the JCI, Corbin and colleagues provide definitive evidence that BCR-ABL is kinase active in CML stem cells and that TKIs inhibit this kinase activity without affecting CML stem cell survival. Rather, CML stem cells revert to a normal dependence on cytokines for survival and proliferation. These results demonstrate that the CML stem cell is not BCR-ABL addicted and have important implications for developing curative therapeutic approaches to CML.
Immune responses to hepatitis C virus (HCV) fail to clear the virus in most individuals. Why patients who are less likely to clear HCV infection have high plasma levels of CXCL10 (also known as IP-10), a chemokine that directs T cells to sites of infection, has long been unclear. In this issue of the JCI, Casrouge and colleagues shed light on this paradox by showing that CXCL10 in the plasma of many HCV patients is enzymatically processed to produce a CXCL10 receptor antagonist. These findings introduce a role for chemokine antagonism during HCV infection and unveil new avenues for improved HCV diagnosis and therapy.
The hallmarks of the rare inherited disorder Fanconi anemia (FA) are progressive bone marrow failure and susceptibility to cancer. The former is the major cause of death for patients with FA, as it usually occurs earlier in life than cancer development. Despite spectacular advances in unraveling the molecular details of FA, the origin of the bone marrow failure that is central to this condition for most patients has long been puzzling and controversial. Two studies recently published in the JCI, including one in this issue, will add to the debate. They also highlight the fact that studying rare disorders can elucidate important new clinical and biological principles.
The blood-brain barrier (BBB) presents a significant obstacle to delivery of targeted therapies to brain tumors. In this issue of the JCI, Staquicini and colleagues apply an in vivo phage-displayed library of random peptides to identify differentially expressed peptides that can be used to transport targeted agents across the intact BBB. The authors uncover a non-canonical, peptide-mediated iron-mimicry mechanism to induce transport of the transferrin/transferrin receptor complex across the BBB. They then demonstrate the ability of phage-targeting approaches to deliver therapeutic cargo and molecular imaging reporters across the BBB in an intracranial glioblastoma mouse model.
Leptin exerts a permissive action on puberty by stimulating release of gonadotropin-releasing hormone (GnRH) in the hypothalamus. However, GnRH neurons lack leptin receptor (LepR), indicating that leptin must indirectly regulate these neurons. The Kiss1 gene produces kisspeptins that stimulate GnRH secretion. Because Kiss1 neurons express LepR and inactivation of Kiss1 causes hypogonadotropic hypogonadism, Donato et al., in this issue of the JCI, assessed whether deletion of LepR from Kiss1 neurons would prevent sexual maturation. Unexpectedly, mice lacking LepR in Kiss1 neurons had normal pubertal development and fertility. In contrast, deletion of LepR from the ventral premammillary nucleus, a region of the brain involved in sexual behavior, prevented puberty and fertility. These findings highlight the complex biology of leptin in reproduction.
Autophagy, a process for catabolizing cytoplasmic components, has been implicated in the modulation of interactions between RNA viruses and their host. However, the mechanism underlying the functional role of autophagy in the viral life cycle still remains unclear. Hepatitis C virus (HCV) is a single-stranded, positive-sense, membrane-enveloped RNA virus that can cause chronic liver disease. Here we report that HCV induces the unfolded protein response (UPR), which in turn activates the autophagic pathway to promote HCV RNA replication in human hepatoma cells. Further analysis revealed that the entire autophagic process through to complete autolysosome maturation was required to promote HCV RNA replication and that it did so by suppressing innate antiviral immunity. Gene silencing or activation of the UPR-autophagy pathway activated or repressed, respectively, IFN-β activation mediated by an HCV-derived pathogen-associated molecular pattern (PAMP). Similar results were achieved with a PAMP derived from Dengue virus (DEV), indicating that HCV and DEV may both exploit the UPR-autophagy pathway to escape the innate immune response. Taken together, these results not only define the physiological significance of HCV-induced autophagy, but also shed light on the knowledge of host cellular responses upon HCV infection as well as on exploration of therapeutic targets for controlling HCV infection.
Infection with influenza A virus represents a major public health threat worldwide, particularly in patients with asthma. However, immunity induced by influenza A virus may have beneficial effects, particularly in young children, that might protect against the later development of asthma, as suggested by the hygiene hypothesis. Herein, we show that infection of suckling mice with influenza A virus protected the mice as adults against allergen-induced airway hyperreactivity (AHR), a cardinal feature of asthma. The protective effect was associated with the preferential expansion of CD4–CD8–, but not CD4+, NKT cells and required T-bet and TLR7. Adoptive transfer of this cell population into allergen-sensitized adult mice suppressed the development of allergen-induced AHR, an effect associated with expansion of the allergen-specific forkhead box p3+ (Foxp3+) Treg cell population. Influenza-induced protection was mimicked by treating suckling mice with a glycolipid derived from Helicobacter pylori (a bacterium associated with protection against asthma) that activated NKT cells in a CD1d-restricted fashion. These findings suggest what we believe to be a novel pathway that can regulate AHR, and a new therapeutic strategy (treatment with glycolipid activators of this NKT cell population) for asthma.
Muscle contraction relies on a highly organized intracellular network of membrane organelles and cytoskeleton proteins. Among the latter are the intermediate filaments (IFs), a large family of proteins mutated in more than 30 human diseases. For example, mutations in the DES gene, which encodes the IF desmin, lead to desmin-related myopathy and cardiomyopathy. Here, we demonstrate that myotubularin (MTM1), which is mutated in individuals with X-linked centronuclear myopathy (XLCNM; also known as myotubular myopathy), is a desmin-binding protein and provide evidence for direct regulation of desmin by MTM1 in vitro and in vivo. XLCNM-causing mutations in MTM1 disrupted the MTM1-desmin complex, resulting in abnormal IF assembly and architecture in muscle cells and both mouse and human skeletal muscles. Adeno-associated virus–mediated ectopic expression of WT MTM1 in Mtm1-KO muscle reestablished normal desmin expression and localization. In addition, decreased MTM1 expression and XLCNM-causing mutations induced abnormal mitochondrial positioning, shape, dynamics, and function. We therefore conclude that MTM1 is a major regulator of both the desmin cytoskeleton and mitochondria homeostasis, specifically in skeletal muscle. Defects in IF stabilization and mitochondrial dynamics appear as common physiopathological features of centronuclear myopathies and desmin-related myopathies.
Bile acid homeostasis is tightly regulated via a feedback loop operated by the nuclear receptors farnesoid X receptor (FXR) and small heterodimer partner (SHP). Contrary to current models, which place FXR upstream of SHP in a linear regulatory pathway, here we show that the phenotypic consequences in mice of the combined loss of both receptors are much more severe than the relatively modest impact of the loss of either Fxr or Shp alone. Fxr–/–Shp–/– mice exhibited cholestasis and liver injury as early as 3 weeks of age, and this was linked to the dysregulation of bile acid homeostatic genes, particularly cytochrome P450, family 7, subfamily a, polypeptide 1 (Cyp7a1). In addition, double-knockout mice showed misregulation of genes in the C21 steroid biosynthesis pathway, with strong induction of cytochrome P450, family 17, subfamily a, polypeptide 1 (Cyp17a1), resulting in elevated serum levels of its enzymatic product 17-hydroxyprogesterone (17-OHP). Treatment of WT mice with 17-OHP was sufficient to induce liver injury that reproduced many of the histopathological features observed in the double-knockout mice. Therefore, our data indicate a pathologic role for increased production of 17-hydroxy steroid metabolites in liver injury and suggest that Fxr–/–Shp–/– mice could provide a model for juvenile onset cholestasis.
The white adipose organ is composed of both subcutaneous and several intra-abdominal depots. Excess abdominal adiposity is a major risk factor for metabolic disease in rodents and humans, while expansion of subcutaneous fat does not carry the same risks. Brown adipose produces heat as a defense against hypothermia and obesity, and the appearance of brown-like adipocytes within white adipose tissue depots is associated with improved metabolic phenotypes. Thus, understanding the differences in cell biology and function of these different adipose cell types and depots may be critical to the development of new therapies for metabolic disease. Here, we found that Prdm16, a brown adipose determination factor, is selectively expressed in subcutaneous white adipocytes relative to other white fat depots in mice. Transgenic expression of Prdm16 in fat tissue robustly induced the development of brown-like adipocytes in subcutaneous, but not epididymal, adipose depots. Prdm16 transgenic mice displayed increased energy expenditure, limited weight gain, and improved glucose tolerance in response to a high-fat diet. shRNA-mediated depletion of Prdm16 in isolated subcutaneous adipocytes caused a sharp decrease in the expression of thermogenic genes and a reduction in uncoupled cellular respiration. Finally, Prdm16 haploinsufficiency reduced the brown fat phenotype in white adipose tissue stimulated by β-adrenergic agonists. These results demonstrate that Prdm16 is a cell-autonomous determinant of a brown fat–like gene program and thermogenesis in subcutaneous adipose tissues.
Autoimmune diseases develop in approximately 5% of humans. They can arise when self-tolerance checkpoints of the immune system are bypassed as a consequence of inherited mutations of key genes involved in lymphocyte activation, survival, or death. For example, autoimmune lymphoproliferative syndrome (ALPS) results from defects in self-tolerance checkpoints as a consequence of mutations in the death receptor–encoding gene TNF receptor superfamily, member 6 (TNFRSF6; also known as FAS). However, some mutation carriers remain asymptomatic throughout life. We have now demonstrated in 7 ALPS patients that the disease develops as a consequence of an inherited TNFRSF6 heterozygous mutation combined with a somatic genetic event in the second TNFRSF6 allele. Analysis of the patients’ CD4–CD8– (double negative) T cells — accumulation of which is a hallmark of ALPS — revealed that in these cells, 3 patients had somatic mutations in their second TNFRSF6 allele, while 4 patients had loss of heterozygosity by telomeric uniparental disomy of chromosome 10. This observation provides the molecular bases of a nonmalignant autoimmune disease development in humans and may shed light on the mechanism underlying the occurrence of other autoimmune diseases.
The pituitary-specific transcriptional factor-1 (PIT-1, also known as POU1F1), is an essential factor for multiple hormone-secreting cell types. A genetic defect in the PIT-1 gene results in congenital growth hormone (GH), prolactin (PRL), and thyroid-stimulating hormone (TSH) deficiency. Here, we investigated 3 cases of adult-onset combined GH, PRL, and TSH deficiencies and found that the endocrinological phenotype in each was linked to autoimmunity directed against the PIT-1 protein. We detected anti–PIT-1 antibody along with various autoantibodies in the patients’ sera. An ELISA-based screening revealed that this antibody was highly specific to the disease and absent in control subjects. Immunohistochemical analysis revealed that PIT-1–, GH-, PRL-, and TSH-positive cells were absent in the pituitary of patient 2, who also had a range of autoimmune endocrinopathies. These clinical manifestations were compatible with the definition of autoimmune polyendocrine syndrome (APS). However, the main manifestations of APS-I — hypoparathyroidism and Candida infection — were not observed and the pituitary abnormalities were obviously different from the hypophysitis associated with APS. These data suggest that these patients define a unique “anti–PIT-1 antibody syndrome,” related to APS.
In antiphospholipid syndrome (APS), antiphospholipid antibodies (aPL) binding to β2 glycoprotein I (β2GPI) induce endothelial cell–leukocyte adhesion and thrombus formation via unknown mechanisms. Here we show that in mice both of these processes are caused by the inhibition of eNOS. In studies of cultured human, bovine, and mouse endothelial cells, the promotion of monocyte adhesion by aPL entailed decreased bioavailable NO, and aPL fully antagonized eNOS activation by diverse agonists. Similarly, NO-dependent, acetylcholine-induced increases in carotid vascular conductance were impaired in aPL-treated mice. The inhibition of eNOS was caused by antibody recognition of domain I of β2GPI and β2GPI dimerization, and it was due to attenuated eNOS S1179 phosphorylation mediated by protein phosphatase 2A (PP2A). Furthermore, LDL receptor family member antagonism with receptor-associated protein (RAP) prevented aPL inhibition of eNOS in cell culture, and ApoER2–/– mice were protected from aPL inhibition of eNOS in vivo. Moreover, both aPL-induced increases in leukocyte–endothelial cell adhesion and thrombus formation were absent in eNOS–/– and in ApoER2–/– mice. Thus, aPL-induced leukocyte–endothelial cell adhesion and thrombosis are caused by eNOS antagonism, which is due to impaired S1179 phosphorylation mediated by β2GPI, apoER2, and PP2A. Our results suggest that novel therapies for APS can now be developed targeting these mechanisms.
Many patients with advanced bladder cancer develop lethal metastases to the lung. The vasoconstricting protein endothelin-1 (ET-1) has been implicated in this process, although the mechanism(s) by which it promotes metastasis remains unclear. Here, we have evaluated whether tumor ET-1 expression can serve as a biomarker for lung metastasis and whether it is required for metastatic disease. Evaluation of ET-1 mRNA and protein expression in four patient cohorts revealed that levels of ET-1 are higher in patients with muscle-invasive bladder cancers, which are associated with higher incidence of metastasis, and that high ET-1 levels are associated with decreased disease-specific survival. Consistent with its proinflammatory activity, we found that tumor-derived ET-1 acts through endothelin-1 receptor A (ETAR) to enhance migration and invasion of both tumor cells and macrophages and induces expression of inflammatory cytokines and proteases. Using human and mouse cancer cells depleted of ET-1 and pharmacologic blockade of ET receptors in lung metastasis models, we found that tumor ET-1 expression and ETAR activity are necessary for metastatic lung colonization and that this process is preceded by and dependent on macrophage infiltration of the lung. In contrast, tumor ET-1 expression and ETAR activity appeared less important in established primary or metastatic tumor growth. These findings strongly suggest that ETAR inhibitors might be more effective as adjuvant therapeutic agents than as initial treatment for advanced primary or metastatic disease.
The Hedgehog (Hh) pathway is activated in some human cancers, including medulloblastoma. The glioma-associated oncogene homolog (GLI) transcription factors are critical mediators of the activated Hh pathway, and their expression may be elevated in some tumors independent of upstream Hh signaling. Thus, therapies targeting GLI transcription factors may benefit a wide spectrum of patients with mutations at different nodal points of the Hh pathway. In this study, we present evidence that arsenic trioxide (ATO) suppresses human cancer cell growth and tumor development in mice by inhibiting GLI1. Mechanistically, ATO directly bound to GLI1 protein, inhibited its transcriptional activity, and decreased expression of endogenous GLI target genes. Consistent with this, ATO inhibited the growth of human cancer cell lines that depended on upregulated GLI expression in vitro and in vivo in a xenograft model of Ewing sarcoma. Furthermore, ATO improved survival of a clinically relevant spontaneous mouse model of medulloblastoma with activated Hh pathway signaling. Our results establish ATO as a Hh pathway inhibitor acting at the level of GLI1 both in vitro and in vivo. These results warrant the clinical investigation of ATO for tumors with activated Hh/GLI signaling, in particular patients who develop resistance to current therapies targeting the Hh pathway upstream of GLI.
The management of CNS tumors is limited by the blood-brain barrier (BBB), a vascular interface that restricts the passage of most molecules from the blood into the brain. Here we show that phage particles targeted with certain ligand motifs selected in vivo from a combinatorial peptide library can cross the BBB under normal and pathological conditions. Specifically, we demonstrated that phage clones displaying an iron-mimic peptide were able to target a protein complex of transferrin and transferrin receptor (TfR) through a non-canonical allosteric binding mechanism and that this functional protein complex mediated transport of the corresponding viral particles into the normal mouse brain. We also showed that, in an orthotopic mouse model of human glioblastoma, a combination of TfR overexpression plus extended vascular permeability and ligand retention resulted in remarkable brain tumor targeting of chimeric adeno-associated virus/phage particles displaying the iron-mimic peptide and carrying a gene of interest. As a proof of concept, we delivered the HSV thymidine kinase gene for molecular-genetic imaging and targeted therapy of intracranial xenografted tumors. Finally, we established that these experimental findings might be clinically relevant by determining through human tissue microarrays that many primary astrocytic tumors strongly express TfR. Together, our combinatorial selection system and results may provide a translational avenue for the targeted detection and treatment of brain tumors.
Wilms tumor (WT) is a genetically heterogeneous childhood kidney tumor. Several genetic alterations have been identified in WT patients, including inactivating mutations in WT1 and loss of heterozygosity or loss of imprinting at 11p15, which results in biallelic expression of IGF2. However, the mechanisms by which one or a combination of genetic alterations results in tumorigenesis has remained challenging to determine, given the lack of a mouse model of WT. Here, we engineered mice to sustain mosaic, somatic ablation of Wt1 and constitutional Igf2 upregulation, mimicking a subset of human tumors. Mice with this combination of genetic alterations developed tumors at an early age. Mechanistically, Wt1 ablation blocked mesenchyme differentiation, and increased Igf2 expression upregulated ERK1/2 phosphorylation. Importantly, a subset of human tumors similarly displayed upregulation of ERK1/2 phosphorylation, which suggests ERK signaling might contribute to WT development. Thus, we have generated a biologically relevant mouse model of WT and defined one combination of driver alterations for WT. This mouse model will provide a powerful tool to study the biology of WT initiation and progression and to investigate therapeutic strategies for cancers with IGF pathway dysregulation.
DNA damage checkpoints in the cell cycle may be important barriers against cancer progression in human cells. Fanconi anemia (FA) is an inherited DNA instability disorder that is associated with bone marrow failure and a strong predisposition to cancer. Although FA cells experience constitutive chromosomal breaks, cell cycle arrest at the G2 DNA damage checkpoint, and an excess of cell death, some patients do become clinically stable, and the mechanisms underlying this, other than spontaneous reversion of the disease-causing mutation, are not well understood. Here we have defined a clonal phenotype, termed attenuation, in which FA patients acquire an abrogation of the G2 checkpoint arrest. Attenuated cells expressed lower levels of CHK1 (also known as CHEK1) and p53. The attenuation could be recapitulated by modulating the ATR/CHK1 pathway, and CHK1 inhibition protected FA cells from cell death. FA patients who expressed the attenuated phenotype had mild bone marrow deficiency and reached adulthood, but several of them eventually developed myelodysplasia or leukemia. Better understanding of attenuation might help predict a patient’s clinical course and guide choice of treatment. Our results also highlight the importance of evaluating the cellular DNA damage checkpoint and repair pathways in cancer therapies in general.
DNA damage is a well-known initiator of tumorigenesis. Studies have shown that most cancer cells rely on aerobic glycolysis for their bioenergetics. We sought to identify a molecular link between genomic mutations and metabolic alterations in neoplastic transformation. We took advantage of the intrinsic genomic instability arising in xeroderma pigmentosum C (XPC). The XPC protein plays a key role in recognizing DNA damage in nucleotide excision repair, and patients with XPC deficiency have increased incidence of skin cancer and other malignancies. In cultured human keratinocytes, we showed that lentivirus-mediated knockdown of XPC reduced mitochondrial oxidative phosphorylation and increased glycolysis, recapitulating cancer cell metabolism. Accumulation of unrepaired DNA following XPC silencing increased DNA-dependent protein kinase activity, which subsequently activated AKT1 and NADPH oxidase-1 (NOX1), resulting in ROS production and accumulation of specific deletions in mitochondrial DNA (mtDNA) over time. Subcutaneous injection of XPC-deficient keratinocytes into immunodeficient mice led to squamous cell carcinoma formation, demonstrating the tumorigenic potential of transduced cells. Conversely, simultaneous knockdown of either NOX1 or AKT1 blocked the neoplastic transformation induced by XPC silencing. Our results demonstrate that genomic instability resulting from XPC silencing results in activation of AKT1 and subsequently NOX1 to induce ROS generation, mtDNA deletions, and neoplastic transformation in human keratinocytes.
Cancer cells have an efficient antioxidant system to counteract their increased generation of ROS. However, whether this ability to survive high levels of ROS has an important role in the growth and metastasis of tumors is not well understood. Here, we demonstrate that the redox protein thioredoxin-like 2 (TXNL2) regulates the growth and metastasis of human breast cancer cells through a redox signaling mechanism. TXNL2 was found to be overexpressed in human cancers, including breast cancers. Knockdown of TXNL2 in human breast cancer cell lines increased ROS levels and reduced NF-κB activity, resulting in inhibition of in vitro proliferation, survival, and invasion. In addition, TXNL2 knockdown inhibited tumorigenesis and metastasis of these cells upon transplantation into immunodeficient mice. Furthermore, analysis of primary breast cancer samples demonstrated that enhanced TXNL2 expression correlated with metastasis to the lung and brain and with decreased overall patient survival. Our studies provided insight into redox-based mechanisms underlying tumor growth and metastasis and suggest that TXNL2 could be a target for treatment of breast cancer.
Integrins regulate cell-cell and cell-matrix adhesion and thereby play critical roles in tumor progression and metastasis. Although work in preclinical models suggests that β1 integrins may stimulate metastasis of a number of cancers, expression of the β1 subunit alone has not been shown to be a useful prognostic indicator in human cancer patients. Here we have demonstrated that the α2β1 integrin suppresses metastasis in a clinically relevant spontaneous mouse model of breast cancer. These data are consistent with previous studies indicating high expression of α2β1 integrin in normal breast epithelium and loss of α2β1 in poorly differentiated breast cancer. They are also consistent with our systematic analysis of microarray databases of human breast and prostate cancer, which revealed that decreased expression of the gene encoding α2 integrin, but not genes encoding α1, α3, or β1 integrin, was predictive of metastatic dissemination and decreased survival. The predictive value of α2 expression persisted within both good-risk and poor-risk cohorts defined by estrogen receptor and lymph node status. Thus, the α2β1 integrin functionally inhibits breast tumor metastasis, and α2 expression may serve as an important biomarker of metastatic potential and patient survival.
Biofilms are surface-attached agglomerations of microorganisms embedded in an extracellular matrix. Biofilm-associated infections are difficult to eradicate and represent a significant reservoir for disseminating and recurring serious infections. Infections involving biofilms frequently develop on indwelling medical devices in hospitalized patients, and Staphylococcus epidermidis is the leading cause of infection in this setting. However, the molecular determinants of biofilm dissemination are unknown. Here we have demonstrated that specific secreted, surfactant-like S. epidermidis peptides — the β subclass of phenol-soluble modulins (PSMs) — promote S. epidermidis biofilm structuring and detachment in vitro and dissemination from colonized catheters in a mouse model of device-related infection. Our study establishes in vivo significance of biofilm detachment mechanisms for the systemic spread of biofilm-associated infection and identifies the effectors of biofilm maturation and detachment in a premier biofilm-forming pathogen. Furthermore, by demonstrating that antibodies against PSMβ peptides inhibited bacterial spread from indwelling medical devices, we have provided proof of principle that interfering with biofilm detachment mechanisms may prevent dissemination of biofilm-associated infection.
NKT cells in the mouse recognize antigen in the context of the MHC class I–like molecule CD1d and play an important role in peripheral tolerance and protection against autoimmune and other diseases. NKT cells are usually activated by CD1d-presented lipid antigens. However, peptide recognition in the context of CD1 has also been documented, although no self-peptide ligands have been reported to date. Here, we have identified an endogenous peptide that is presented by CD1d to activate mouse NKT cells. This peptide, the immunodominant epitope from mouse collagen type II (mCII707–721), was not associated with either MHC class I or II. Activation of CD1d-restricted mCII707–721–specific NKT cells was induced via TCR signaling and classical costimulation. In addition, mCII707–721–specific NKT cells induced T cell death through Fas/FasL, in an IL-17A–independent fashion. Moreover, mCII707–721–specific NKT cells suppressed a range of in vivo inflammatory conditions, including delayed-type hypersensitivity, antigen-induced airway inflammation, collagen-induced arthritis, and EAE, which were all ameliorated by mCII707-721 vaccination. The findings presented here offer new insight into the intrinsic roles of NKT cells in health and disease. Given the results, endogenous collagen peptide activators of NKT cells may offer promise as novel therapeutics in tissue-specific autoimmune and inflammatory diseases.
Granulocytes are pivotal regulators of tissue injury. However, the transcriptional mechanisms that regulate granulopoiesis under inflammatory conditions are poorly understood. Here we show that the transcriptional coregulator B cell leukemia/lymphoma 3 (Bcl3) limits granulopoiesis under emergency (i.e., inflammatory) conditions, but not homeostatic conditions. Treatment of mouse myeloid progenitors with G-CSF — serum concentrations of which rise under inflammatory conditions — rapidly increased Bcl3 transcript accumulation in a STAT3-dependent manner. Bcl3-deficient myeloid progenitors demonstrated an enhanced capacity to proliferate and differentiate into granulocytes following G-CSF stimulation, whereas the accumulation of Bcl3 protein attenuated granulopoiesis in an NF-κB p50–dependent manner. In a clinically relevant model of transplant-mediated lung ischemia reperfusion injury, expression of Bcl3 in recipients inhibited emergency granulopoiesis and limited acute graft damage. These data demonstrate a critical role for Bcl3 in regulating emergency granulopoiesis and suggest that targeting the differentiation of myeloid progenitors may be a therapeutic strategy for preventing inflammatory lung injury.
Idiopathic pulmonary fibrosis (IPF) is a chronic fibroproliferative pulmonary disorder for which there are currently no treatments. Although the etiology of IPF is unknown, dysregulated TGF-β signaling has been implicated in its pathogenesis. Recent studies also suggest a central role for abnormal epithelial repair. In this study, we sought to elucidate the function of epithelial TGF-β signaling via TGF-β receptor II (TβRII) and its contribution to fibrosis by generating mice in which TβRII was specifically inactivated in mouse lung epithelium. These mice, which are referred to herein as TβRIINkx2.1-cre mice, were used to determine the impact of TβRII inactivation on (a) embryonic lung morphogenesis in vivo; and (b) the epithelial cell response to TGF-β signaling in vitro and in a bleomycin-induced, TGF-β–mediated mouse model of pulmonary fibrosis. Although postnatally viable with no discernible abnormalities in lung morphogenesis and epithelial cell differentiation, TβRIINkx2.1-cre mice developed emphysema, suggesting a requirement for epithelial TβRII in alveolar homeostasis. Absence of TβRII increased phosphorylation of Smad2 and decreased, but did not entirely block, phosphorylation of Smad3 in response to endogenous/physiologic TGF-β. However, TβRIINkx2.1-cre mice exhibited increased survival and resistance to bleomycin-induced pulmonary fibrosis. To our knowledge, these findings are the first to demonstrate a specific role for TGF-β signaling in the lung epithelium in the pathogenesis of pulmonary fibrosis.
Characterizing the TCRα and TCRβ chains expressed by T cells responding to a given pathogen or underlying autoimmunity helps in the development of vaccines and immunotherapies, respectively. However, our understanding of complementary TCRα and TCRβ chain utilization is very limited for pathogen- and autoantigen-induced immunity. To address this problem, we have developed a multiplex nested RT-PCR method for the simultaneous amplification of transcripts encoding the TCRα and TCRβ chains from single cells. This multiplex method circumvented the lack of antibodies specific for variable regions of mouse TCRα chains and the need for prior knowledge of variable region usage in the TCRβ chain, resulting in a comprehensive, unbiased TCR repertoire analysis with paired coexpression of TCRα and TCRβ chains with single-cell resolution. Using CD8+ CTLs specific for an influenza epitope recovered directly from the pneumonic lungs of mice, this technique determined that 25% of such effectors expressed a dominant, nonproductively rearranged Tcra transcript. T cells with these out-of-frame Tcra mRNAs also expressed an alternate, in-frame Tcra, whereas approximately 10% of T cells had 2 productive Tcra transcripts. The proportion of cells with biallelic transcription increased over the course of a response, a finding that has implications for immune memory and autoimmunity. This technique may have broad applications in mouse models of human disease.
Induction of CD8+ T cell immunity is a key characteristic of an effective vaccine. For safety reasons, human vaccination strategies largely use attenuated nonreplicating or weakly replicating poxvirus-based vectors, but these often elicit poor CD8+ T cell immunity and might not result in optimal protection. Recent studies have suggested that virulence is directly linked to immunogenicity, but the molecular mechanisms underlying optimal CD8+ T cell responses remain to be defined. Here, using natural and recombinant vaccinia virus (VACV) strains, we have shown in mice that VACV strains of differing virulence induce distinct levels of T cell memory because of the differential use of TNF receptor (TNFR) family costimulatory receptors. With strongly replicating (i.e., virulent) VACV, the TNFR family costimulatory receptors OX40 (also known as CD134) and CD27 were engaged and promoted the generation of high numbers of memory CD8+ T cells, which protected against a lethal virus challenge in the absence of other mechanisms, including antibody and help from CD4+ T cells. In contrast, weakly replicating (i.e., low-virulence) VACV strains were poor at eliciting protective CD8+ T cell memory, as only the Ig family costimulatory receptor CD28 was engaged, and not OX40 or CD27. Our results suggest that the virulence of a virus dictates costimulatory receptor usage to determine the level of protective CD8+ T cell immunity.
Chronic infection with hepatitis C virus (HCV) is a major public health problem, with nearly 170 million infected individuals worldwide. Current treatment for chronic infection is a combination of pegylated IFN-α2 and ribavirin (RBV); however, this treatment is effective in fewer than 50% of patients infected with HCV genotype 1 or 4. Recent studies identified the chemokine CXCL10 (also known as IP-10) as an important negative prognostic biomarker. Given that CXCL10 mediates chemoattraction of activated lymphocytes, it is counterintuitive that this chemokine correlates with therapeutic nonresponsiveness. Herein, we offer new insight into this paradox and provide evidence that CXCL10 in the plasma of patients chronically infected with HCV exists in an antagonist form, due to in situ amino-terminal truncation of the protein. We further demonstrated that dipeptidyl peptidase IV (DPP4; also known as CD26), possibly in combination with other proteases, mediates the generation of the antagonist form(s) of CXCL10. These data offer what we believe to be the first evidence for CXCL10 antagonism in human disease and identify a possible factor contributing to the inability of patients to clear HCV.
During intrauterine life, the mammalian embryo survives via its physical connection to the mother. The uterine decidua, which differentiates from stromal cells after implantation in a process known as decidualization, plays essential roles in supporting embryonic growth before establishment of the placenta. Here we show that female mice lacking death effector domain–containing protein (DEDD) are infertile owing to unsuccessful decidualization. In uteri of Dedd–/– mice, development of the decidual zone and the surrounding edema after embryonic implantation was defective. This was subsequently accompanied by disintegration of implantation site structure, leading to embryonic death before placentation. Polyploidization, a hallmark of mature decidual cells, was attenuated in DEDD-deficient cells during decidualization. Such inefficient decidualization appeared to be caused by decreased Akt levels, since polyploidization was restored in DEDD-deficient decidual cells by overexpression of Akt. In addition, we showed that DEDD associates with and stabilizes cyclin D3, an important element in polyploidization, and that overexpression of cyclin D3 in DEDD-deficient cells improved polyploidization. These results indicate that DEDD is indispensable for the establishment of an adequate uterine environment to support early pregnancy in mice.
Sex in mammals is genetically determined and is defined at the cellular level by sex chromosome complement (XY males and XX females). The Y chromosome–linked gene sex-determining region Y (SRY) is believed to be the master initiator of male sex determination in almost all eutherian and metatherian mammals, functioning to upregulate expression of its direct target gene Sry-related HMG box–containing gene 9 (SOX9). Data suggest that SRY evolved from SOX3, although there is no direct functional evidence to support this hypothesis. Indeed, loss-of-function mutations in SOX3 do not affect sex determination in mice or humans. To further investigate Sox3 function in vivo, we generated transgenic mice overexpressing Sox3. Here, we report that in one of these transgenic lines, Sox3 was ectopically expressed in the bipotential gonad and that this led to frequent complete XX male sex reversal. Further analysis indicated that Sox3 induced testis differentiation in this particular line of mice by upregulating expression of Sox9 via a similar mechanism to Sry. Importantly, we also identified genomic rearrangements within the SOX3 regulatory region in three patients with XX male sex reversal. Together, these data suggest that SOX3 and SRY are functionally interchangeable in sex determination and support the notion that SRY evolved from SOX3 via a regulatory mutation that led to its de novo expression in the early gonad.
E3 ubiquitin ligase complexes of the SCF type consist of ring-box 1 (Rbx1), cullin 1 (Cul1), S-phase kinase-associated protein 1 (Skp1), and a member of the F-box family of proteins. The identity of the F-box protein determines the substrate specificity of the complex. The F-box family member F-box– and WD repeat domain–containing 7 (Fbxw7; also known as Fbw7, SEL-10, hCdc4, and hAgo) targets for degradation proteins with wide-ranging functions, and uncovering its in vivo role has been difficult, because Fbxw7–/– embryos die in utero. Using two different Cre-loxP systems (Mx1-Cre and Alb-Cre), we generated mice with liver-specific null mutations of Fbxw7. Hepatic ablation of Fbxw7 resulted in hepatomegaly and steatohepatitis, with massive deposition of triglyceride, a phenotype similar to that observed in humans with nonalcoholic steatohepatitis. Both cell proliferation and the abundance of Fbxw7 substrates were increased in the Fbxw7-deficient liver. Long-term Fbxw7 deficiency resulted in marked proliferation of the biliary system and the development of hamartomas. Fbxw7 deficiency also skewed the differentiation of liver stem cells toward the cholangiocyte lineage rather than the hepatocyte lineage in vitro. This bias was corrected by additional loss of the Notch cofactor RBP-J, suggesting that Notch accumulation triggered the abnormal proliferation of the biliary system. Together, our results suggest that Fbxw7 plays key roles, regulating lipogenesis and cell proliferation and differentiation in the liver.
Studies in humans and rodents indicate that a minimum amount of stored energy is required for normal pubertal development. The adipocyte-derived hormone leptin is a key metabolic signal to the neuroendocrine reproductive axis. Humans and mice lacking leptin or the leptin receptor (LepR) (ob/ob and db/db mice, respectively) are infertile and fail to enter puberty. Leptin administration to leptin-deficient subjects and ob/ob mice induces puberty and restores fertility, but the exact site or sites of leptin action are unclear. Here, we found that genetic deletion of LepR selectively from hypothalamic Kiss1 neurons in mice had no effect on puberty or fertility, indicating that direct leptin signaling in Kiss1 neurons is not required for these processes. However, bilateral lesions of the ventral premammillary nucleus (PMV) of ob/ob mice blunted the ability of exogenous leptin to induce sexual maturation. Moreover, unilateral reexpression of endogenous LepR in PMV neurons was sufficient to induce puberty and improve fertility in female LepR-null mice. This LepR reexpression also normalized the increased hypothalamic GnRH content characteristic of leptin-signaling deficiency. These data suggest that the PMV is a key site for leptin’s permissive action at the onset of puberty and support the hypothesis that the multiple actions of leptin to control metabolism and reproduction are anatomically dissociated.
Retinal pigment epithelial (RPE) cell dysfunction plays a central role in various retinal degenerative diseases, but knowledge is limited regarding the pathways responsible for adult RPE stress responses in vivo. RPE mitochondrial dysfunction has been implicated in the pathogenesis of several forms of retinal degeneration. Here we have shown that postnatal ablation of RPE mitochondrial oxidative phosphorylation in mice triggers gradual epithelium dedifferentiation, typified by reduction of RPE-characteristic proteins and cellular hypertrophy. The electrical response of the retina to light decreased and photoreceptors eventually degenerated. Abnormal RPE cell behavior was associated with increased glycolysis and activation of, and dependence upon, the hepatocyte growth factor/met proto-oncogene pathway. RPE dedifferentiation and hypertrophy arose through stimulation of the AKT/mammalian target of rapamycin (AKT/mTOR) pathway. Administration of an oxidant to wild-type mice also caused RPE dedifferentiation and mTOR activation. Importantly, treatment with the mTOR inhibitor rapamycin blunted key aspects of dedifferentiation and preserved photoreceptor function for both insults. These results reveal an in vivo response of the mature RPE to diverse stressors that prolongs RPE cell survival at the expense of epithelial attributes and photoreceptor function. Our findings provide a rationale for mTOR pathway inhibition as a therapeutic strategy for retinal degenerative diseases involving RPE stress.
Human leukemic stem cells, like other cancer stem cells, are hypothesized to be rare, capable of incomplete differentiation, and restricted to a phenotype associated with early hematopoietic progenitors or stem cells. However, recent work in other types of tumors has challenged the cancer stem cell model. Using a robust model of xenotransplantation based on NOD/SCID/IL2Rγc-deficient mice, we confirmed that human leukemic stem cells, functionally defined by us as SCID leukemia-initiating cells (SL-ICs), are rare in acute myelogenous leukemia (AML). In contrast to previous results, SL-ICs were found among cells expressing lineage markers (i.e., among Lin+ cells), CD38, or CD45RA, all markers associated with normal committed progenitors. Remarkably, each engrafting fraction consistently recapitulated the original phenotypic diversity of the primary AML specimen and contained self-renewing leukemic stem cells, as demonstrated by secondary transplants. While SL-ICs were enriched in the Lin–CD38– fraction compared with the other fractions analyzed, SL-ICs in this fraction represented only one-third of all SL-ICs present in the unfractionated specimen. These results indicate that human AML stem cells are rare and enriched but not restricted to the phenotype associated with normal primitive hematopoietic cells. These results suggest a plasticity of the cancer stem cell phenotype that we believe has not been previously described.
Imatinib therapy, which targets the oncogene product BCR-ABL, has transformed chronic myeloid leukemia (CML) from a life-threatening disease into a chronic condition. Most patients, however, harbor residual leukemia cells, and disease recurrence usually occurs when imatinib is discontinued. Although various mechanisms to explain leukemia cell persistence have been proposed, the critical question from a therapeutic standpoint — whether disease persistence is BCR-ABL dependent or independent — has not been answered. Here, we report that human CML stem cells do not depend on BCR-ABL activity for survival and are thus not eliminated by imatinib therapy. Imatinib inhibited BCR-ABL activity to the same degree in all stem (CD34+CD38–, CD133+) and progenitor (CD34+CD38+) cells and in quiescent and cycling progenitors from newly diagnosed CML patients. Although short-term in vitro imatinib treatment reduced the expansion of CML stem/progenitors, cytokine support permitted growth and survival in the absence of BCR-ABL activity that was comparable to that of normal stem/progenitor counterparts. Our findings suggest that primitive CML cells are not oncogene addicted and that therapies that biochemically target BCR-ABL will not eliminate CML stem cells.
Dysregulated angiogenesis is a hallmark of chronic inflammatory diseases, including psoriasis, a common skin disorder that affects approximately 2% of the population. Studying both human psoriasis in 2 complementary xenotransplantation models and psoriasis-like skin lesions in transgenic mice with epidermal expression of human TGF-β1, we have demonstrated that antiangiogenic non-viral somatic gene therapy reduces the cutaneous microvasculature and alleviates chronic inflammatory skin disorders. Transient muscular expression of the recombinant disintegrin domain (RDD) of metargidin (also known as ADAM-15) by in vivo electroporation reduced cutaneous angiogenesis and vascularization in all 3 models. As demonstrated using red fluorescent protein–coupled RDD, the treatment resulted in muscular expression of the gene product and its deposition within the cutaneous hyperangiogenic connective tissue. High-resolution ultrasound revealed reduced cutaneous blood flow in vivo after electroporation with RDD but not with control plasmids. In addition, angiogenesis- and inflammation-related molecular markers, keratinocyte proliferation, epidermal thickness, and clinical disease scores were downregulated in all models. Thus, non-viral antiangiogenic gene therapy can alleviate psoriasis and may do so in other angiogenesis-related inflammatory skin disorders.
Congenital anomalies of the aortic valve are common and are associated with progressive valvular insufficiency and/or stenosis. In addition, aneurysm, coarctation, and dissection of the ascending aorta and aortic arch are often associated conditions that complicate patient management and increase morbidity and mortality. These associated aortopathies are commonly attributed to turbulent hemodynamic flow through the malformed valve leading to focal defects in the vessel wall. However, numerous surgical and pathological studies have identified widespread cystic medial necrosis and smooth muscle apoptosis throughout the aortic arch in affected patients. Here, we provide experimental evidence for an alternative model to explain the association of aortic vessel and valvular disease. Using mice with primary and secondary cardiac neural crest deficiencies, we have shown that neural crest contribution to the outflow endocardial cushions (the precursors of the semilunar valves) is required for late gestation valvular remodeling, mesenchymal apoptosis, and proper valve architecture. Neural crest was also shown to contribute to the smooth muscle layer of the wall of the ascending aorta and aortic arch. Hence, defects of cardiac neural crest can result in functionally abnormal semilunar valves and concomitant aortic arch artery abnormalities.
Although widely prescribed for their potent antiinflammatory actions, glucocorticoid drugs (e.g., dexamethasone) cause undesirable side effects that are features of the metabolic syndrome, including hyperglycemia, fatty liver, insulin resistance, and type II diabetes. Liver x receptors (LXRs) are nuclear receptors that respond to cholesterol metabolites and regulate the expression of a subset of glucocorticoid target genes. Here, we show LXRβ is required to mediate many of the negative side effects of glucocorticoids. Mice lacking LXRβ (but not LXRα) were resistant to dexamethasone-induced hyperglycemia, hyperinsulinemia, and hepatic steatosis, but remained sensitive to dexamethasone-dependent repression of the immune system. In vivo, LXRα/β knockout mice demonstrated reduced dexamethasone-induced expression of the key hepatic gluconeogenic gene, phosphoenolpyruvate carboxykinase (PEPCK). In perfused liver and primary mouse hepatocytes, LXRβ was required for glucocorticoid-induced recruitment of the glucocorticoid receptor to the PEPCK promoter. These findings suggest a new avenue for the design of safer glucocorticoid drugs through a mechanism of selective glucocorticoid receptor transactivation.
Type 1A diabetes (T1D) is an autoimmune disease characterized by leukocyte infiltration of the pancreatic islets of Langerhans. A major impediment to advances in understanding, preventing, and curing T1D has been the inability to “see” the disease initiate, progress, or regress, especially during the occult phase. Here, we report the development of a noninvasive method to visualize T1D at the target organ level in patients with active insulitis. Specifically, we visualized islet inflammation, manifest by microvascular changes and monocyte/macrophage recruitment and activation, using magnetic resonance imaging of magnetic nanoparticles (MNPs). As a proof of principle for this approach, imaging of infused ferumoxtran-10 nanoparticles permitted effective visualization of the pancreas and distinction of recent-onset diabetes patients from nondiabetic controls. The observation that MNPs accumulate in the pancreas of T1D patients opens the door to exploiting this noninvasive imaging method to follow T1D progression and monitoring the ability of immunomodulatory agents to clear insulitis.
Solute carrier family 1, member 1 (SLC1A1; also known as EAAT3 and EAAC1) is the major epithelial transporter of glutamate and aspartate in the kidneys and intestines of rodents. Within the brain, SLC1A1 serves as the predominant neuronal glutamate transporter and buffers the synaptic release of the excitatory neurotransmitter glutamate within the interneuronal synaptic cleft. Recent studies have also revealed that polymorphisms in SLC1A1 are associated with obsessive-compulsive disorder (OCD) in early-onset patient cohorts. Here we report that SLC1A1 mutations leading to substitution of arginine to tryptophan at position 445 (R445W) and deletion of isoleucine at position 395 (I395del) cause human dicarboxylic aminoaciduria, an autosomal recessive disorder of urinary glutamate and aspartate transport that can be associated with mental retardation. These mutations of conserved residues impeded or abrogated glutamate and cysteine transport by SLC1A1 and led to near-absent surface expression in a canine kidney cell line. These findings provide evidence that SLC1A1 is the major renal transporter of glutamate and aspartate in humans and implicate SLC1A1 in the pathogenesis of some neurological disorders.
Copyright © 2014 American Society for Clinical Investigation