51 total articles
Since it seems many of the articles we’re accepting continue to have image rearrangements and changes in them that are not consistent with JCI policy, we thought it might be a good idea to spell out exactly what is, and more importantly what isn’t, allowed.
Ushma Neill, Laurence A. Turka
Lung cancer is the leading cause of cancer death worldwide. The disease is particularly difficult to detect, and patients often present at an advanced stage. Current treatments have limited effectiveness, and unfortunately, the prognosis remains poor. Recent insights into the molecular pathogenesis and biologic behavior of lung cancer have led to the development of rationally designed methods of early detection, prevention, and treatment of this disease. This article will review the important clinical implications of these advances, with a focus on new molecularly targeted therapies currently in development.
Sophie Sun, Joan H. Schiller, Monica Spinola, John D. Minna
Much of the mortality following myocardial infarction results from remodeling of the heart after the acute ischemic event. Cardiomyocyte apoptosis has been thought to play a key role in this remodeling process. In this issue of the JCI, Diwan and colleagues present evidence that Bnip3, a proapoptotic Bcl2 family protein, mediates cardiac enlargement, reshaping, and dysfunction in mice without influencing infarct size (see the related article beginning on page 2825).
Russell S. Whelan, Kartik Mani, Richard N. Kitsis
In this issue of the JCI, Morioka et al. report on mice with a whole-pancreas knockout of the leptin receptor that exhibit improved glucose tolerance due to enhanced insulin secretion (see the related article beginning on page 2860). At first glance, their findings are very different from those reported in another recent study in which β cell–specific and hypothalamic knockout of the same gene caused obesity and impaired β cell function. The differences, which are understandable when one considers the body weights of the animals studied, provide new insight into the links among insulin, leptin action, and β cell function.
Kevin D. Niswender, Mark A. Magnuson
Factors involved in determining whether infectious mononucleosis occurs after primary EBV infection may include age, dose of virus received, and various genetic markers. A study by McAulay and colleagues reported in this issue of the JCI shows that the presence of certain HLA class I alleles correlates with the incidence and severity of infectious mononucleosis (see the related article beginning on page 3042). These same HLA alleles are also risk factors for EBV-associated Hodgkin lymphoma (HL), supporting recent epidemiology that indicates that a history of infectious mononucleosis predisposes to HL. Recent studies suggest that an EBV vaccine might help to prevent infectious mononucleosis, and further development of this should now be considered.
Paul J. Farrell
Age-related macular degeneration (AMD), the most common cause of blindness in the elderly, is characterized by degeneration of the macula and can lead to loss of fine color vision. Alterations in inflammatory and immune system pathways, which arise from genetic differences, predispose individuals to AMD. Yet the mechanism of disease progression with respect to inflammation is not fully understood. In this issue of the JCI, the study by Combadière and colleagues shows that CX3C chemokine receptor 1–deficient (CX3CR1-deficient) mice have abnormal microglia that accumulate beneath the retina and contribute to the progression of AMD (see the related article beginning on page 2920).
Jing Chen, Kip M. Connor, Lois E.H. Smith
In this issue of the JCI, Nissen et al. report that a reciprocal interaction exists between the growth factors FGF2 and PDGF-BB, causing tumors to exhibit increased angiogenesis and metastatic potential (see the related article beginning on page 2766). Both FGF2 and PDGF-BB signal through tyrosine kinase receptors, which have been the target of tyrosine kinase inhibitors for cancer therapies. These inhibitors are usually small molecules that inhibit the kinase activity of a receptor or nonreceptor tyrosine kinase, preventing downstream signaling. The results of this study shed light on why tyrosine kinase inhibitors have been useful for the treatment of only a small number of advanced cancers. Currently, a major focus of pharmaceutical companies is to develop ever more potent and specific tyrosine kinases. The results presented here suggest that this approach may not be successful.
Jack L. Arbiser
Tumors produce multiple growth factors, but little is known about the interplay between various angiogenic factors in promoting tumor angiogenesis, growth, and metastasis. Here we show that 2 angiogenic factors frequently upregulated in tumors, PDGF-BB and FGF2, synergistically promote tumor angiogenesis and pulmonary metastasis. Simultaneous overexpression of PDGF-BB and FGF2 in murine fibrosarcomas led to the formation of high-density primitive vascular plexuses, which were poorly coated with pericytes and VSMCs. Surprisingly, overexpression of PDGF-BB alone in tumor cells resulted in dissociation of VSMCs from tumor vessels and decreased recruitment of pericytes. In the absence of FGF2, capillary ECs lacked response to PDGF-BB. However, FGF2 triggers PDGFR-α and -β expression at the transcriptional level in ECs, which acquire hyperresponsiveness to PDGF-BB. Similarly, PDGF-BB–treated VSMCs become responsive to FGF2 stimulation via upregulation of FGF receptor 1 (FGFR1) promoter activity. These findings demonstrate that PDGF-BB and FGF2 reciprocally increase their EC and mural cell responses, leading to disorganized neovascularization and metastasis. Our data suggest that intervention of this non-VEGF reciprocal interaction loop for the tumor vasculature could be an important therapeutic target for the treatment of cancer and metastasis.
Lars Johan Nissen ... Ebba Bråkenhielm, Yihai Cao
The decline in adaptive immunity, T lymphocyte output, and the contraction of the TCR repertoire with age is largely attributable to thymic involution. The loss of thymic function with age may be due to diminished numbers of progenitors and the loss of critical cytokines and hormones from the thymic microenvironment. We have previously demonstrated that the orexigenic hormone ghrelin is expressed by immune cells and regulates T cell activation and inflammation. Here we report that ghrelin and ghrelin receptor expression within the thymus diminished with progressive aging. Infusion of ghrelin into 14-month-old mice significantly improved the age-associated changes in thymic architecture and thymocyte numbers, increasing recent thymic emigrants and improving TCR diversity of peripheral T cell subsets. Ghrelin-induced thymopoiesis during aging was associated with enhanced early thymocyte progenitors and bone marrow–derived Lin–Sca1+cKit+ cells, while ghrelin- and growth hormone secretagogue receptor–deficient (GHS-R–deficient) mice displayed enhanced age-associated thymic involution. Leptin also enhanced thymopoiesis in aged but not young mice. Our findings demonstrate what we believe to be a novel role for ghrelin and its receptor in thymic biology and suggest a possible therapeutic benefit of harnessing this pathway in the reconstitution of thymic function in immunocompromised subjects.
Vishwa Deep Dixit ... Roy G. Smith, Dennis D. Taub
Three forms of PPARs are expressed in the heart. In animal models, PPARγ agonist treatment improves lipotoxic cardiomyopathy; however, PPARγ agonist treatment of humans is associated with peripheral edema and increased heart failure. To directly assess effects of increased PPARγ on heart function, we created transgenic mice expressing PPARγ1 in the heart via the cardiac α–myosin heavy chain (α-MHC) promoter. PPARγ1-transgenic mice had increased cardiac expression of fatty acid oxidation genes and increased lipoprotein triglyceride (TG) uptake. Unlike in cardiac PPARα-transgenic mice, heart glucose transporter 4 (GLUT4) mRNA expression and glucose uptake were not decreased. PPARγ1-transgenic mice developed a dilated cardiomyopathy associated with increased lipid and glycogen stores, distorted architecture of the mitochondrial inner matrix, and disrupted cristae. Thus, while PPARγ agonists appear to have multiple beneficial effects, their direct actions on the myocardium have the potential to lead to deterioration in heart function.
Ni-Huiping Son ... Li-Shin Huang, Ira J. Goldberg
Myotonic dystrophy type 1 (DM1) is caused by a CTG trinucleotide expansion in the 3′ untranslated region (3′ UTR) of DM protein kinase (DMPK). The key feature of DM1 pathogenesis is nuclear accumulation of RNA, which causes aberrant alternative splicing of specific pre-mRNAs by altering the functions of CUG-binding proteins (CUGBPs). Cardiac involvement occurs in more than 80% of individuals with DM1 and is responsible for up to 30% of disease-related deaths. We have generated an inducible and heart-specific DM1 mouse model expressing expanded CUG RNA in the context of DMPK 3′ UTR that recapitulated pathological and molecular features of DM1 including dilated cardiomyopathy, arrhythmias, systolic and diastolic dysfunction, and misregulated alternative splicing. Combined in situ hybridization and immunofluorescent staining for CUGBP1 and CUGBP2, the 2 CUGBP1 and ETR-3 like factor (CELF) proteins expressed in heart, demonstrated elevated protein levels specifically in nuclei containing foci of CUG repeat RNA. A time-course study demonstrated that colocalization of MBNL1 with RNA foci and increased CUGBP1 occurred within hours of induced expression of CUG repeat RNA and coincided with reversion to embryonic splicing patterns. These results indicate that CUGBP1 upregulation is an early and primary response to expression of CUG repeat RNA.
Guey-Shin Wang, Debra L. Kearney, Mariella De Biasi, George Taffet, Thomas A. Cooper
Marked sarcomere disorganization is a well-documented characteristic of cardiomyocytes in the failing human myocardium. Myosin regulatory light chain 2, ventricular/cardiac muscle isoform (MLC2v), which is involved in the development of human cardiomyopathy, is an important structural protein that affects physiologic cardiac sarcomere formation and heart development. Integrated cDNA expression analysis of failing human myocardia uncovered a novel protein kinase, cardiac-specific myosin light chain kinase (cardiac-MLCK), which acts on MLC2v. Expression levels of cardiac-MLCK were well correlated with the pulmonary arterial pressure of patients with heart failure. In cultured cardiomyocytes, knockdown of cardiac-MLCK by specific siRNAs decreased MLC2v phosphorylation and impaired epinephrine-induced activation of sarcomere reassembly. To further clarify the physiologic roles of cardiac-MLCK in vivo, we cloned the zebrafish ortholog z–cardiac-MLCK. Knockdown of z–cardiac-MLCK expression using morpholino antisense oligonucleotides resulted in dilated cardiac ventricles and immature sarcomere structures. These results suggest a significant role for cardiac-MLCK in cardiogenesis.
Osamu Seguchi ... Naoki Mochizuki, Masafumi Kitakaze
Following myocardial infarction, nonischemic myocyte death results in infarct expansion, myocardial loss, and ventricular dysfunction. Here, we demonstrate that a specific proapoptotic gene, Bnip3, minimizes ventricular remodeling in the mouse, despite having no effect on early or late infarct size. We evaluated the effects of ablating Bnip3 on cardiomyocyte death, infarct size, and ventricular remodeling after surgical ischemia/reperfusion (IR) injury in mice. Immediately following IR, no significant differences were observed between Bnip3–/– and WT mice. However, at 2 days after IR, apoptosis was diminished in Bnip3–/– periinfarct and remote myocardium, and at 3 weeks after IR, Bnip3–/– mice exhibited preserved LV systolic performance, diminished LV dilation, and decreased ventricular sphericalization. These results suggest myocardial salvage by inhibition of apoptosis. Forced cardiac expression of Bnip3 increased cardiomyocyte apoptosis in unstressed mice, causing progressive LV dilation and diminished systolic function. Conditional Bnip3 overexpression prior to coronary ligation increased apoptosis and infarct size. These studies identify postischemic apoptosis by myocardial Bnip3 as a major determinant of ventricular remodeling in the infarcted heart, suggesting that Bnip3 may be an attractive therapeutic target.
Abhinav Diwan ... W. Keith Jones, Gerald W. Dorn II
The fact that adenoviral vectors activate innate immunity and induce type I IFNs has not been fully appreciated in the context of cancer gene therapy. Type I IFNs influence different aspects of human immune response and are believed to be crucial for efficient tumor rejection. We performed transcriptional profiling to characterize the response of cutaneous lymphomas to intralesional adenovirus-mediated IFN-γ (Ad-IFN-γ) gene transfer. Gene expression profiles of skin lesions obtained from 19 cutaneous lymphoma patients before and after treatment with Ad-IFN-γ revealed a distinct gene signature consisting of IFN-γ– and numerous IFN-α–inducible genes (type II– and type I–inducible genes, respectively). The type I IFN response appears to have been induced by the vector itself, and its complexity, in terms of immune activation, was potentiated by the IFN-γ gene insert. Intralesional IFN-γ expression together with the induction of a combined type I/II IFN response to Ad-IFN-γ gene transfer seem to underlie the objective (measurable) clinical response of the treated lesions. Biological effects of type I IFNs seem to enhance those set in motion by the transgene, in our case IFN-γ. This combination may prove to be of therapeutic importance in cytokine gene transfer using Ads.
Mirjana Urosevic ... Bruce Acres, Reinhard Dummer
Ischemia/reperfusion injury (IRI) may activate innate immunity through the engagement of TLRs by endogenous ligands. TLR4 expressed within the kidney is a potential mediator of innate activation and inflammation. Using a mouse model of kidney IRI, we demonstrated a significant increase in TLR4 expression by tubular epithelial cells (TECs) and infiltrating leukocytes within the kidney following ischemia. TLR4 signaling through the MyD88-dependent pathway was required for the full development of kidney IRI, as both TLR4–/– and MyD88–/– mice were protected against kidney dysfunction, tubular damage, neutrophil and macrophage accumulation, and expression of proinflammatory cytokines and chemokines. In vitro, WT kidney TECs produced proinflammatory cytokines and chemokines and underwent apoptosis after ischemia. These effects were attenuated in TLR4–/– and MyD88–/– TECs. In addition, we demonstrated upregulation of the endogenous ligands high-mobility group box 1 (HMGB1), hyaluronan, and biglycan, providing circumstantial evidence that one or more of these ligands may be the source of TLR4 activation. To determine the relative contribution of TLR4 expression by parenchymal cells or leukocytes to kidney damage during IRI, we generated chimeric mice. TLR4–/– mice engrafted with WT hematopoietic cells had significantly lower serum creatinine and less tubular damage than WT mice reconstituted with TLR4–/– BM, suggesting that TLR4 signaling in intrinsic kidney cells plays the dominant role in mediating kidney damage.
Huiling Wu ... Alexandra F. Sharland, Steven J. Chadban
Obesity is characterized by hyperinsulinemia, hyperleptinemia, and an increase in islet volume. While the mechanisms that hasten the onset of diabetes in obese individuals are not known, it is possible that the adipose-derived hormone leptin plays a role. In addition to its central actions, leptin exerts biological effects by acting in peripheral tissues including the endocrine pancreas. To explore the impact of disrupting leptin signaling in the pancreas on β cell growth and/or function, we created pancreas-specific leptin receptor (ObR) KOs using mice expressing Cre recombinase under the control of the pancreatic and duodenal homeobox 1 (Pdx1) promoter. The KOs exhibited improved glucose tolerance due to enhanced early-phase insulin secretion, and a greater β cell mass secondary to increased β cell size and enhanced expression and phosphorylation of p70S6K. Similar effects on p70S6K were observed in MIN6 β cells with knockdown of the ObR gene, suggesting crosstalk between leptin and insulin signaling pathways. Surprisingly, challenging the KOs with a high-fat diet led to attenuated acute insulin secretory response to glucose, poor compensatory islet growth, and glucose intolerance. Together, these data provide direct genetic evidence, from a unique mouse model lacking ObRs only in the pancreas, for a critical role for leptin signaling in islet biology and suggest that altered leptin action in islets is one factor that contributes to obesity-associated diabetes.
Tomoaki Morioka ... Robert T. Kennedy, Rohit N. Kulkarni
Diabetes results from an inadequate mass of functional β cells, due to either β cell loss caused by immune assault or the lack of compensation to overcome insulin resistance. Elucidating the mechanisms that regulate β cell mass has important ramifications for fostering β cell regeneration and the treatment of diabetes. We report here that Skp2, a substrate recognition component of Skp1–Cul1–F-box (SCF) ubiquitin ligase, played an essential and specific role in regulating the cellular abundance of p27 and was a critical determinant of β cell proliferation. In Skp2–/– mice, accumulation of p27 resulted in enlarged polyploid β cells as a result of endoreduplication replacing proliferation. Despite β cell hypertrophy, Skp2–/– mice exhibited diminished β cell mass, hypoinsulinemia, and glucose intolerance. Increased insulin resistance resulting from diet-induced obesity caused Skp2–/– mice to become overtly diabetic, because β cell growth in the absence of cell division was insufficient to compensate for increased metabolic demand. These results indicate that the Skp2-mediated degradation pathway regulating the cellular degradation of p27 is essential for establishing β cell mass and to respond to increased metabolic demand associated with insulin resistance.
Lingwen Zhong, Senta Georgia, Shuen-ing Tschen, Keiko Nakayama, Keiichi Nakayama, Anil Bhushan
Obesity is associated with a state of chronic, low-grade inflammation characterized by abnormal cytokine production and macrophage infiltration into adipose tissue, which may contribute to the development of insulin resistance. During immune responses, tissue infiltration by macrophages is dependent on the expression of osteopontin, an extracellular matrix protein and proinflammatory cytokine that promotes monocyte chemotaxis and cell motility. In the present study, we used a murine model of diet-induced obesity to examine the role of osteopontin in the accumulation of adipose tissue macrophages and the development of insulin resistance during obesity. Mice exposed to a high-fat diet exhibited increased plasma osteopontin levels, with elevated expression in macrophages recruited into adipose tissue. Obese mice lacking osteopontin displayed improved insulin sensitivity in the absence of an effect on diet-induced obesity, body composition, or energy expenditure. These mice further demonstrated decreased macrophage infiltration into adipose tissue, which may reflect both impaired macrophage motility and attenuated monocyte recruitment by stromal vascular cells. Finally, obese osteopontin-deficient mice exhibited decreased markers of inflammation, both in adipose tissue and systemically. Taken together, these results suggest that osteopontin may play a key role in linking obesity to the development of insulin resistance by promoting inflammation and the accumulation of macrophages in adipose tissue.
Takashi Nomiyama ... Matthias H. Tschöp, Dennis Bruemmer
Ependymal overexpression of brain-derived neurotrophic factor (BDNF) stimulates neuronal addition to the adult striatum, from subependymal progenitor cells. Noggin, by suppressing subependymal gliogenesis and increasing progenitor availability, potentiates this process. We asked whether BDNF/Noggin overexpression might be used to recruit new striatal neurons in R6/2 huntingtin transgenic mice. R6/2 mice injected with adenoviral BDNF and adenoviral Noggin (AdBDNF/AdNoggin) recruited BrdU+βIII-tubulin+ neurons, which developed as DARPP-32+ and GABAergic medium spiny neurons that expressed either enkephalin or substance P and extended fibers to the globus pallidus. Only AdBDNF/AdNoggin-treated R6/2 mice harbored migrating doublecortin-defined neuroblasts in their striata, and the new neurons expressed p27 as a marker of mitotic quiescence after parenchymal integration. AdBDNF/AdNoggin-treated R6/2 mice sustained their rotarod performance and open-field activity and survived longer than did AdNull-treated and untreated controls. Neither motor performance nor survival improved in R6/2 mice treated only with AdBDNF, and intraventricular infusion of the mitotic inhibitor Ara-C completely blocked the performance and survival effects of AdBDNF/AdNoggin, suggesting that the benefits of AdBDNF/AdNoggin derived from neuronal addition. Thus, BDNF and Noggin induced striatal neuronal regeneration, delayed motor impairment, and extended survival in R6/2 mice, suggesting a new therapeutic strategy in Huntington disease.
Sung-Rae Cho ... Aris Economides, Steven A. Goldman
The slow-channel myasthenic syndrome (SCS) is a hereditary disorder of the acetylcholine receptor (AChR) of the neuromuscular junction (NMJ) that leads to prolonged AChR channel opening, Ca2+ overload, and degeneration of the NMJ. We used an SCS transgenic mouse model to investigate the role of the calcium-activated protease calpain in the pathogenesis of synaptic dysfunction in SCS. Cleavage of a fluorogenic calpain substrate was increased at the NMJ of dissociated muscle fibers. Inhibition of calpain using a calpastatin (CS) transgene improved strength and neuromuscular transmission. CS caused a 2-fold increase in the frequency of miniature endplate currents (MEPCs) and an increase in NMJ size, but MEPC amplitudes remained reduced. Persistent degeneration of the NMJ was associated with localized activation of the non-calpain protease caspase-3. This study suggests that calpain may act presynaptically to impair NMJ function in SCS but further reveals a role for other cysteine proteases whose inhibition may be of additional therapeutic benefit in SCS and other excitotoxic disorders.
Jason S. Groshong ... Richard J. Miller, Christopher M. Gomez
Amyotrophic lateral sclerosis (ALS), one of the most common adult-onset neurodegenerative diseases, has no known cure. Enhanced redox stress and inflammation have been associated with the pathoprogression of ALS through a poorly defined mechanism. Here we determined that dysregulated redox stress in ALS mice caused by NADPH oxidases Nox1 and Nox2 significantly influenced the progression of motor neuron disease caused by mutant SOD1G93A expression. Deletion of either Nox gene significantly slowed disease progression and improved survival. However, 50% survival rates were enhanced significantly more by Nox2 deletion than by Nox1 deletion. Interestingly, female ALS mice containing only 1 active X-linked Nox1 or Nox2 gene also had significantly delayed disease onset, but showed normal disease progression rates. Nox activity in spinal cords from Nox2 heterozygous female ALS mice was approximately 50% that of WT female ALS mice, suggesting that random X-inactivation was not influenced by Nox2 gene deletion. Hence, chimerism with respect to Nox-expressing cells in the spinal cord significantly delayed onset of motor neuron disease in ALS. These studies define what we believe to be new modifier gene targets for treatment of ALS.
Jennifer J. Marden ... Henry Paulson, John F. Engelhardt
The role of retinal microglial cells (MCs) in age-related macular degeneration (AMD) is unclear. Here we demonstrated that all retinal MCs express CX3C chemokine receptor 1 (CX3CR1) and that homozygosity for the CX3CR1 M280 allele, which is associated with impaired cell migration, increases the risk of AMD. In humans with AMD, MCs accumulated in the subretinal space at sites of retinal degeneration and choroidal neovascularization (CNV). In CX3CR1-deficient mice, MCs accumulated subretinally with age and albino background and after laser impact preceding retinal degeneration. Raising the albino mice in the dark prevented both events. The appearance of lipid-bloated subretinal MCs was drusen-like on funduscopy of senescent mice, and CX3CR1-dependent MC accumulation was associated with an exacerbation of experimental CNV. These results show that CX3CR1-dependent accumulation of subretinal MCs evokes cardinal features of AMD. These findings reveal what we believe to be a novel pathogenic process with important implications for the development of new therapies for AMD.
Christophe Combadière ... Francine Behar-Cohen, Florian Sennlaub
PTEN is a tumor suppressor gene mutated in many human cancers. We generated a bronchioalveolar epithelium–specific null mutation of Pten in mice [SP-C-rtTA/(tetO)7-Cre/Ptenflox/flox (SOPtenflox/flox) mice] that was under the control of doxycycline. Ninety percent of SOPtenflox/flox mice that received doxycycline in utero [SOPtenflox/flox(E10–16) mice] died of hypoxia soon after birth. Surviving SOPtenflox/flox(E10–16) mice and mice that received doxycycline postnatally [SOPtenflox/flox(P21–27) mice] developed spontaneous lung adenocarcinomas. Urethane treatment accelerated number and size of lung tumors developing in SOPtenflox/flox mice of both ages. Histological and biochemical examinations of the lungs of SOPtenflox/flox(E10–16) mice revealed hyperplasia of bronchioalveolar epithelial cells and myofibroblast precursors, enlarged alveolar epithelial cells, and impaired production of surfactant proteins. Numbers of bronchioalveolar stem cells (BASCs), putative initiators of lung adenocarcinomas, were increased. Lungs of SOPtenflox/flox(E10–16) mice showed increased expression of Spry2, which inhibits the maturation of alveolar epithelial cells. Levels of Akt, c-Myc, Bcl-2, and Shh were also elevated in SOPtenflox/flox(E10–16) and SOPtenflox/flox(P21–27) lungs. Furthermore, K-ras was frequently mutated in adenocarcinomas observed in SOPtenflox/flox(P21–27) lungs. These results indicate that Pten is essential for both normal lung morphogenesis and the prevention of lung carcinogenesis, possibly because this tumor suppressor is required for BASC homeostasis.
Shigehisa Yanagi ... Masamitsu Nakazato, Akira Suzuki
Development of persistent Th2 responses in asthma and chronic helminth infections are a major health concern. IL-10 has been identified as a critical regulator of Th2 immunity, but mechanisms for controlling Th2 effector function remain unclear. IL-10 also has paradoxical effects on Th2-associated pathology, with IL-10 deficiency resulting in increased Th2-driven inflammation but also reduced airway hyperreactivity (AHR), mucus hypersecretion, and fibrosis. We demonstrate that increased IL-13 receptor α 2 (IL-13Rα2) expression is responsible for the reduced AHR, mucus production, and fibrosis in BALB/c IL-10–/– mice. Using models of allergic asthma and chronic helminth infection, we demonstrate that IL-10 and IL-13Rα2 coordinately suppress Th2-mediated inflammation and pathology, respectively. Although IL-10 was identified as the dominant antiinflammatory mediator, studies with double IL-10/IL-13Rα2–deficient mice illustrate an indispensable role for IL-13Rα2 in the suppression of AHR, mucus production, and fibrosis. Thus, IL-10 and IL-13Rα2 are both required to control chronic Th2-driven pathological responses.
Mark S. Wilson ... Allen W. Cheever, Thomas A. Wynn
The mechanisms by which exposure to particulate matter increases the risk of cardiovascular events are not known. Recent human and animal data suggest that particulate matter may induce alterations in hemostatic factors. In this study we determined the mechanisms by which particulate matter might accelerate thrombosis. We found that mice treated with a dose of well characterized particulate matter of less than 10 μM in diameter exhibited a shortened bleeding time, decreased prothrombin and partial thromboplastin times (decreased plasma clotting times), increased levels of fibrinogen, and increased activity of factor II, VIII, and X. This prothrombotic tendency was associated with increased generation of intravascular thrombin, an acceleration of arterial thrombosis, and an increase in bronchoalveolar fluid concentration of the prothrombotic cytokine IL-6. Knockout mice lacking IL-6 were protected against particulate matter–induced intravascular thrombin formation and the acceleration of arterial thrombosis. Depletion of macrophages by the intratracheal administration of liposomal clodronate attenuated particulate matter–induced IL-6 production and the resultant prothrombotic tendency. Our findings suggest that exposure to particulate matter triggers IL-6 production by alveolar macrophages, resulting in reduced clotting times, intravascular thrombin formation, and accelerated arterial thrombosis. These results provide a potential mechanism linking ambient particulate matter exposure and thrombotic events.
Gökhan M. Mutlu ... Jacob I. Sznajder, G.R. Scott Budinger
The actin filament–associated protein AFAP-110 is an actin cross-linking protein first identified as a substrate of the viral oncogene v-Src. AFAP-110 regulates actin cytoskeleton integrity but also functions as an adaptor protein that affects crosstalk between Src and PKC. Here we investigated the roles of AFAP-110 in the tumorigenic process of prostate carcinoma. Using immunohistochemistry of human tissue arrays, we found that AFAP-110 was absent or expressed at very low levels in normal prostatic epithelium and benign prostatic hyperplasia but significantly increased in prostate carcinomas. The level of AFAP-110 in carcinomas correlated with the Gleason scores. Downregulation of AFAP-110 in PC3 prostate cancer cells inhibited cell proliferation in vitro and tumorigenicity and growth in orthotopic nude mouse models. Furthermore, downmodulation of AFAP-110 resulted in decreased cell-matrix adhesion and cell migration, defective focal adhesions, and reduced integrin β1 expression. Reintroduction of avian AFAP-110 or a mutant disabling its interaction with Src restored these properties. However, expression of an AFAP-110 lacking the PKC-interacting domain failed to restore properties of parental cells. Thus, increased expression of AFAP-110 is associated with progressive stages of prostate cancer and is critical for tumorigenic growth, in part by regulating focal contacts in a PKC-dependent mechanism.
Jing Zhang ... Daniel C. Flynn, Gary E. Gallick
T lymphocyte responses promote proatherogenic inflammatory events, which are influenced by costimulatory molecules of the B7 family. Effects of negative regulatory members of the B7 family on atherosclerosis have not been described. Programmed death–ligand 1 (PD-L1) and PD-L2 are B7 family members expressed on several cell types, which inhibit T cell activation via binding to programmed death–1 (PD-1) on T cells. In order to test whether the PD-1/PD-L pathway regulates proatherogenic T cell responses, we compared atherosclerotic lesion burden and phenotype in hypercholesterolemic PD-L1/2–/–LDLR–/– mice and LDLR–/– controls. PD-L1/2 deficiency led to significantly increased atherosclerotic burden throughout the aorta and increased numbers of lesional CD4+ and CD8+ T cells. Compared with controls, PD-L1/2–/–LDLR–/– mice had iliac lymphadenopathy and increased numbers of activated CD4+ T cells. Serum levels of TNF-α were higher in PD-L1/2–/–LDLR–/– mice than in controls. PD-L1/2–deficient APCs were more effective than control APCs in activating CD4+ T cells in vitro, with or without cholesterol loading. Freshly isolated APCs from hypercholesterolemic PD-L1/2–/–LDLR–/– mice stimulated greater T cell responses than did APCs from hypercholesterolemic controls. Our findings indicate that the PD-1/PD-L pathway has an important role in downregulating proatherogenic T cell response and atherosclerosis by limiting APC-dependent T cell activation.
Israel Gotsman, Nir Grabie, Rosa Dacosta, Galina Sukhova, Arlene Sharpe, Andrew H. Lichtman
Accumulation of cholesteryl esters (CEs) in macrophage foam cells, central to atherosclerotic plaque formation, occurs as a result of imbalance between the cholesterol influx and efflux pathways. While the uptake, or influx, of modified lipoproteins is largely unregulated, extracellular acceptor-mediated free cholesterol (FC) efflux is rate limited by the intracellular hydrolysis of CE. We previously identified and cloned a neutral CE hydrolase (CEH) from human macrophages and demonstrated its role in cellular CE mobilization. In the present study, we examined the hypothesis that macrophage-specific overexpression of CEH in atherosclerosis-susceptible Ldlr–/– mice will result in reduction of diet-induced atherosclerosis. Transgenic mice overexpressing this CEH specifically in the macrophages (driven by scavenger receptor promoter/enhancer) were developed and crossed into the Ldlr–/– background (Ldlr–/–CEHTg mice). Macrophage-specific overexpression of CEH led to a significant reduction in the lesion area and cholesterol content of high-fat, high-cholesterol diet–induced atherosclerotic lesions. The lesions from Ldlr–/–CEHTg mice did not have increased FC, were less necrotic, and contained significantly higher numbers of viable macrophage foam cells. Higher CEH-mediated FC efflux resulted in enhanced flux of FC from macrophages to gall bladder bile and feces in vivo. These studies demonstrate that by enhancing cholesterol efflux and reverse cholesterol transport, macrophage-specific overexpression of CEH is antiatherogenic.
Bin Zhao, Jingmei Song, Woon N. Chow, Richard W. St. Clair, Lawrence L. Rudel, Shobha Ghosh
Uterine decidualization, a process that occurs in response to embryo implantation, is critical for embryonic survival and thus is a key event for successful pregnancy. Here we show that the sphingolipid metabolic pathway is highly activated in the deciduum during pregnancy and disturbance of the pathway by disruption of sphingosine kinase (Sphk) genes causes defective decidualization with severely compromised uterine blood vessels, leading to early pregnancy loss. Sphk-deficient female mice (Sphk1–/–Sphk2+/–) exhibited both an enormous accumulation of dihydrosphingosine and sphingosine and a reduction in phosphatidylethanolamine levels in pregnant uteri. These mice also revealed increased cell death in decidual cells, decreased cell proliferation in undifferentiated stromal cells, and massive breakage of decidual blood vessels, leading to uterine hemorrhage and early embryonic lethality. Thus, sphingolipid metabolism regulates proper uterine decidualization and blood vessel stability. Our findings also suggest that disturbance in sphingolipid metabolism may be considered as a cause of pregnancy loss in humans.
Kiyomi Mizugishi ... Chu-Xia Deng, Richard L. Proia
The discovery of fetal mRNA transcripts in the maternal circulation holds great promise for noninvasive prenatal diagnosis. To identify potential fetal biomarkers, we studied whole blood and plasma gene transcripts that were common to 9 term pregnant women and their newborns but absent or reduced in the mothers postpartum. RNA was isolated from peripheral or umbilical blood and hybridized to gene expression arrays. Gene expression, paired Student’s t test, and pathway analyses were performed. In whole blood, 157 gene transcripts met statistical significance. These fetal biomarkers included 27 developmental genes, 5 sensory perception genes, and 22 genes involved in neonatal physiology. Transcripts were predominantly expressed or restricted to the fetus, the embryo, or the neonate. Real-time RT-PCR amplification confirmed the presence of specific gene transcripts; SNP analysis demonstrated the presence of 3 fetal transcripts in maternal antepartum blood. Comparison of whole blood and plasma samples from the same pregnant woman suggested that placental genes are more easily detected in plasma. We conclude that fetal and placental mRNA circulates in the blood of pregnant women. Transcriptional analysis of maternal whole blood identifies a unique set of biologically diverse fetal genes and has a multitude of clinical applications.
Jill L. Maron ... Zinger Yang, Diana W. Bianchi
Reduced capacity to produce ROS increases the severity of T cell–dependent arthritis in both mice and rats with polymorphisms in neutrophil cytosolic factor 1 (Ncf1) (p47phox). Since T cells cannot exert oxidative burst, we hypothesized that T cell responsiveness is downregulated by ROS produced by APCs. Macrophages have the highest burst capacity among APCs, so to study the effect of macrophage ROS on T cell activation, we developed transgenic mice expressing functional Ncf1 restricted to macrophages. Macrophage-restricted expression of functional Ncf1 restored arthritis resistance to the level of that of wild-type mice in a collagen-induced arthritis model but not in a T cell–independent anti-collagen antibody–induced arthritis model. T cell activation was downregulated and skewed toward Th2 in transgenic mice. In vitro, IL-2 production and T cell proliferation were suppressed by macrophage ROS, irrespective of T cell origin. IFN-γ production, however, was independent of macrophage ROS but dependent on T cell origin. These effects were antigen dependent but not restricted to collagen type II. In conclusion, macrophage-derived ROS play a role in T cell selection, maturation, and differentiation, and also a suppressive role in T cell activation, and thereby mediate protection against autoimmune diseases like arthritis.
Kyra A. Gelderman ... Ragnar Mattsson, Rikard Holmdahl
CD137 is expressed on activated T cells and ligands to this costimulatory molecule have clinical potential for amplifying CD8 T cell immunity to tumors and viruses, while suppressing CD4 autoimmune T cell responses. To understand the basis for this dichotomy in T cell function, CD4 and CD8 antiviral immunity was measured in lymphocytic choriomeningitis virus (LCMV) Armstrong– or A/PR8/34 influenza–infected mice injected with anti-CD137 mAbs. We found that the timing of administration of anti-CD137 mAbs profoundly altered the nature of the antiviral immune response during acute infection. Antiviral immunity progressed normally for the first 72 hours when the mAb was administered early in infection before undergoing complete collapse by day 8 postinfection. Anti-CD137–injected LCMV-infected mice became tolerant to, and persistently infected with, LCMV Armstrong. Elevated levels of IL-10 early in the response was key to the loss of CD4+ T cells, whereas CD8+ T cell deletion was dependent on a prolonged TNF-α response, IL-10, and upregulation of Fas. Blocking IL-10 function rescued CD4 antiviral immunity but not CD8+ T cell deletion. Anti-CD137 treatment given beyond 72 hours after infection significantly enhanced antiviral immunity. Mice treated with anti-CD137 mAb 1 day before infection with A/PR8/34 virus experienced 80% mortality compared with 40% mortality of controls. When treatment was delayed until day 1 postinfection, 100% of the infected mice survived. These data show that anti-CD137 mAbs can induce T cell activation–induced cell death or enhance antiviral immunity depending on the timing of treatment, which may be important for vaccine development.
Benyue Zhang ... Joshy Jacob, Robert S. Mittler
Infectious mononucleosis (IM) is an immunopathological disease caused by EBV that occurs in young adults and is a risk factor for Hodgkin lymphoma (HL). An association between EBV-positive HL and genetic markers in the HLA class I locus has been identified, indicating that genetic differences in the HLA class I locus may alter disease phenotypes associated with EBV infection. To further determine whether HLA class I alleles may affect development of EBV-associated diseases, we analyzed 2 microsatellite markers and 2 SNPs located near the HLA class I locus in patients with acute IM and in asymptomatic EBV-seropositive and -seronegative individuals. Alleles of both microsatellite markers were significantly associated with development of IM. Specific alleles of the 2 SNPs were also significantly more frequent in patients with IM than in EBV-seronegative individuals. IM patients possessing the associated microsatellite allele had fewer lymphocytes and increased neutrophils relative to IM patients lacking the allele. These patients also displayed higher EBV titers and milder IM symptoms. The results of this study indicate that HLA class I polymorphisms may predispose patients to development of IM upon primary EBV infection, suggesting that genetic variation in T cell responses can influence the nature of primary EBV infection and the level of viral persistence.
Karen A. McAulay ... Hilary Williams, Dorothy H. Crawford
Candida parapsilosis is a major cause of human disease, yet little is known about the pathogen’s virulence. We have developed an efficient gene deletion system for C. parapsilosis based on the repeated use of the dominant nourseothricin resistance marker (caSAT1) and its subsequent deletion by FLP-mediated, site-specific recombination. Using this technique, we deleted the lipase locus in the C. parapsilosis genome consisting of adjacent genes CpLIP1 and CpLIP2. Additionally we reconstructed the CpLIP2 gene, which restored lipase activity. Lipolytic activity was absent in the null mutants, whereas the WT, heterozygous, and reconstructed mutants showed similar lipase production. Biofilm formation was inhibited with lipase-negative mutants and their growth was significantly reduced in lipid-rich media. The knockout mutants were more efficiently ingested and killed by J774.16 and RAW 264.7 macrophage-like cells. Additionally, the lipase-negative mutants were significantly less virulent in infection models that involve inoculation of reconstituted human oral epithelium or murine intraperitoneal challenge. These studies represent what we believe to be the first targeted disruption of a gene in C. parapsilosis and show that C. parapsilosis–secreted lipase is involved in disease pathogenesis. This efficient system for targeted gene deletion holds great promise for rapidly enhancing our knowledge of the biology and virulence of this increasingly common invasive fungal pathogen.
Attila Gácser, David Trofa, Wilhelm Schäfer, Joshua D. Nosanchuk
The flavivirus West Nile virus (WNV) is an emerging pathogen that causes life-threatening encephalitis in susceptible individuals. We investigated the role of the proinflammatory cytokine macrophage migration inhibitory factor (MIF), which is an upstream mediator of innate immunity, in WNV immunopathogenesis. We found that patients suffering from acute WNV infection presented with increased MIF levels in plasma and in cerebrospinal fluid. MIF expression also was induced in WNV-infected mice. Remarkably, abrogation of MIF action by 3 distinct approaches (antibody blockade, small molecule pharmacologic inhibition, and genetic deletion) rendered mice more resistant to WNV lethality. Mif–/– mice showed a reduced viral load and inflammatory response in the brain when compared with wild-type mice. Our results also indicate that MIF favors viral neuroinvasion by compromising the integrity of the blood-brain barrier. In conclusion, the data obtained from this study provide direct evidence for the involvement of MIF in viral pathogenesis and suggest that pharmacotherapeutic approaches targeting MIF may hold promise for the treatment of WNV encephalitis.
Alvaro Arjona ... Erol Fikrig, Richard Bucala
HIV-2 infection in the majority of infected subjects follows an attenuated disease course that distinguishes it from infection with HIV-1. Antigen-specific T cells are pivotal in the management of chronic viral infections but are not sufficient to control viral replication in HIV-1–positive subjects, and their function in HIV-2 infection is not fully established. In a community-based cohort of HIV-2 long-term nonprogressors in rural Guinea-Bissau, we performed what we believe is the first comprehensive analysis of HIV-2–specific immune responses. We demonstrate that Gag is the most immunogenic protein. The magnitude of the IFN-γ immune response to the HIV-2 proteome was inversely correlated with HIV-2 viremia, and this relationship was specifically due to the targeting of Gag. Furthermore, patients with undetectable viremia had greater Gag-specific responses compared with patients with high viral replication. The most frequently recognized peptides clustered within a defined region of Gag, and responses to a single peptide in this region were associated with low viral burden. The consistent relationship between Gag-specific immune responses and viremia control suggests that T cell responses are vital in determining the superior outcome of HIV-2 infection. A better understanding of how HIV-2 infection is controlled may identify correlates of effective protective immunity essential for the design of HIV vaccines.
Aleksandra Leligdowicz ... Hilton Whittle, Sarah Rowland-Jones
In humans, loss-of-function mutations in the gene encoding Wnt1 inducible signaling pathway protein 3 (WISP3) cause the autosomal-recessive skeletal disorder progressive pseudorheumatoid dysplasia (PPD). However, in mice there is no apparent phenotype caused by Wisp3 deficiency or overexpression. Consequently, the in vivo activities of Wisp3 have remained elusive. We cloned the zebrafish ortholog of Wisp3 and investigated its biologic activity in vivo using gain-of-function and loss-of-function approaches. Overexpression of zebrafish Wisp3 protein inhibited bone morphogenetic protein (BMP) and Wnt signaling in developing zebrafish. Conditioned medium–containing zebrafish and human Wisp3 also inhibited BMP and Wnt signaling in mammalian cells by binding to BMP ligand and to the Wnt coreceptors low-density lipoprotein receptor–related protein 6 (LRP6) and Frizzled, respectively. Wisp3 proteins containing disease-causing amino acid substitutions found in patients with PPD had reduced activity in these assays. Morpholino-mediated inhibition of zebrafish Wisp3 protein expression in developing zebrafish affected pharyngeal cartilage size and shape. These data provide a biologic assay for Wisp3, reveal a role for Wisp3 during zebrafish cartilage development, and suggest that dysregulation of BMP and/or Wnt signaling contributes to cartilage failure in humans with PPD.
Yukio Nakamura ... Randall T. Moon, Matthew L. Warman
The exploitation of the physiologic processing and presenting machinery of DCs by in vivo loading of tumor-associated antigens may improve the immunogenic potential and clinical efficacy of DC-based cancer vaccines. Here we show that lymphocytes genetically modified to express self/tumor antigens, acting as antigen carriers, efficiently target DCs in vivo in tumor-bearing mice. The infusion of tyrosinase-related protein 2–transduced (TRP-2–transduced) lymphocytes induced the establishment of protective immunity and long-term memory in tumor-bearing mice. Analysis of the mechanism responsible for the induction of such an immune response allowed us to demonstrate that cross-presentation of the antigen mediated by the CD11c+CD8α+ DC subset had occurred. Furthermore, we demonstrated in vivo and in vitro that DCs had undergone activation upon phagocytosis of genetically modified lymphocytes, a process mediated by a cell-to-cell contact mechanism independent of CD40 triggering. Targeting and activation of secondary lymphoid organ–resident DCs endowed antigen-specific T cells with full effector functions, which ultimately increased tumor growth control and animal survival in a therapeutic tumor setting. We conclude that the use of transduced lymphocytes represents an efficient method for the in vivo loading of tumor-associated antigens on DCs.
Vincenzo Russo ... Claudio Bordignon, Catia Traversari
Triggering receptor expressed on myeloid cells–1 (TREM-1) potently amplifies acute inflammatory responses by enhancing degranulation and secretion of proinflammatory mediators. Here we demonstrate that TREM-1 is also crucially involved in chronic inflammatory bowel diseases (IBD). Myeloid cells of the normal intestine generally lack TREM-1 expression. In experimental mouse models of colitis and in patients with IBD, however, TREM-1 expression in the intestine was upregulated and correlated with disease activity. TREM-1 significantly enhanced the secretion of relevant proinflammatory mediators in intestinal macrophages from IBD patients. Blocking TREM-1 by the administration of an antagonistic peptide substantially attenuated clinical course and histopathological alterations in experimental mouse models of colitis. This effect was also seen when the antagonistic peptide was administered only after the first appearance of clinical signs of colitis. Hence, TREM-1–mediated amplification of inflammation contributes not only to the exacerbation of acute inflammatory disorders but also to the perpetuation of chronic inflammatory disorders. Furthermore, interfering with TREM-1 engagement leads to the simultaneous reduction of production and secretion of a variety of pro-inflammatory mediators such as TNF, IL-6, IL-8 (CXCL8), MCP-1 (CCL2), and IL-1β. Therefore, TREM-1 may also represent an attractive target for the treatment of chronic inflammatory disorders.
Mirjam Schenk, Axel Bouchon, Frank Seibold, Christoph Mueller
The effectiveness of IFN-α2b for human multiple myeloma has been variable. TRAIL has been proposed to mediate IFN-α2b apoptosis in myeloma. In this study we assessed the effects of IFN-α2b signaling on the apoptotic activity of TRAIL and human myeloma cell survival. While TRAIL was one of the most potently induced proapoptotic genes in myeloma cells following IFN-α2b treatment, less than 20% of myeloma cells underwent apoptosis. Thus, we hypothesized that an IFN-stimulated gene (ISG) with prosurvival activity might suppress TRAIL-mediated apoptosis. Consistent with this, IFN-α2b stabilized mitochondria and inhibited caspase-3 activation, which antagonized TRAIL-mediated apoptosis and cytotoxicity after 24 hours of cotreatment in cell lines and in fresh myeloma cells, an effect not evident after 72 hours. Induced expression of G1P3, an ISG with largely unknown function, was correlated with the antiapoptotic activity of IFN-α2b. Ectopically expressed G1P3 localized to mitochondria and antagonized TRAIL-mediated mitochondrial potential loss, cytochrome c release, and apoptosis, suggesting specificity of G1P3 for the intrinsic apoptosis pathway. Furthermore, RNAi-mediated downregulation of G1P3 restored IFN-α2b–induced apoptosis. Our data identify the direct role of a mitochondria-localized prosurvival ISG in antagonizing the effect of TRAIL. Curtailing G1P3-mediated antiapoptotic signals could improve therapies for myeloma or other malignancies.
Venugopalan Cheriyath ... Mohamad A. Hussein, Ernest C. Borden
Cystic fibrosis (CF) is caused by dysfunction of the CF transmembrane conductance regulator (CFTR), an anion channel whose dysfunction leads to chronic bacterial and fungal airway infections via a pathophysiological cascade that is incompletely understood. Airway glands, which produce most airway mucus, do so in response to both acetylcholine (ACh) and vasoactive intestinal peptide (VIP). CF glands fail to secrete mucus in response to VIP, but do so in response to ACh. Because vagal cholinergic pathways still elicit strong gland mucus secretion in CF subjects, it is unclear whether VIP-stimulated, CFTR-dependent gland secretion participates in innate defense. It was recently hypothesized that airway intrinsic neurons, which express abundant VIP and ACh, are normally active and stimulate low-level gland mucus secretion that is a component of innate mucosal defenses. Here we show that low levels of VIP and ACh produced significant mucus secretion in human glands via strong synergistic interactions; synergy was lost in glands of CF patients. VIP/ACh synergy also existed in pig glands, where it was CFTR dependent, mediated by both Cl– and HCO3–, and clotrimazole sensitive. Loss of “housekeeping” gland mucus secretion in CF, in combination with demonstrated defects in surface epithelia, may play a role in the vulnerability of CF airways to bacterial infections.
Jae Young Choi ... John W. Hanrahan, Jeffrey J. Wine
John I. Gallin
Anthony S. Fauci
Chrystal M. Paulos ... Steven A. Rosenberg, Nicholas P. Restifo
Yi Dai ... Hiroki Yamanaka, Koichi Noguchi
Robert V. Farese ... Ursula Braun, Michael Leitges
Copyright © 2014 American Society for Clinical Investigation