Volume 122, Issue 4, Pages 1133-1584
49 total articles
Herpes simplex virus 1 (HSV-1) and HSV-2 are neurotropic viruses, which infect the host via initial infection of cells at mucocutaneous surfaces. From these sites, the viruses gain access to neurons, where latency can be established. In this issue, Reinert and colleagues have used a murine model for genital HSV-2 infection to demonstrate that defects in TLR3 or the type I IFN system render mice — like humans — susceptible to HSV infection in the CNS but not in the periphery.
Image credit: Sebastian Kaulitzki, Shutterstock.
On the occasion of the 200th anniversary of Charles Dickens’s birth, I am struck by his creative genius and by the parallel between the intellectual development of his protagonists and the evolution of peer review. Like many of his novels and serial writings, the story of the history of peer review is a bildungsroman, one that has followed a process of growing up, sought answers through a journey marked by achievement and disappointment, and ultimately matured to be accepted by a community.
Chromosomal instability (CIN) is a hallmark of human neoplasms. Despite its widespread prevalence, knowledge of the mechanisms and contributions of CIN in cancer has been elusive. It is now evident that the role of CIN in tumor initiation and growth is more complex than previously thought. Furthermore, distinguishing CIN, which consists of elevated rates of chromosome missegregation, from aneuploidy, which is a state of abnormal chromosome number, is crucial to understanding their respective contributions in cancer. Collectively, experimental evidence suggests that CIN enables tumor adaptation by allowing tumors to constantly sample the aneuploid fitness landscape. This complex relationship, together with the potential to pharmacologically influence chromosome missegregation frequencies in cancer cells, offers previously unrecognized means to limit tumor growth and its response to therapy.
Neurobiologists and immunologists study concepts often signified with identical terminology. Scientists in both fields study a structure known as the synapse, and each group analyzes a subject called memory. Is this a quirk of human language, or are there real similarities between these two physiological systems? Not only are the linguistic concepts expressed in the words “synapse” and “memory” shared between the fields, but the actual molecules of physiologic importance in one system play parallel roles in the other: complement, the major histocompatibility molecules, and even “neuro”-transmitters all have major impacts on health and on disease in both the brain and the immune system. Not only are the same molecules found in diverse roles in each system, but we have learned that there is real “hard-wired” crosstalk between nerves and lymphoid organs. This issue of the JCI highlights some of the lessons learned from experts who are working at this scintillating intersection between immunology and neuroscience.
Immunological and neural synapses share properties such as the synaptic cleft, adhesion molecules, stability, and polarity. However, the mismatch in scale has limited the utility of these comparisons. The discovery of phosphatase micro-exclusion from signaling elements in immunological synapses and innate phagocytic synapses define a common functional unit at a common sub-micron scale across synapse types. Bundling of information from multiple antigen receptor microclusters by an immunological synapse has parallels to bundling of multiple synaptic inputs into a single axonal output by neurons, allowing integration and coincidence detection. Bonafide neuroimmune synapses control the inflammatory reflex. A better understanding of the shared mechanisms between immunological and neural synapses could aid in the development of new therapeutic modalities for immunological, neurological, and neuroimmunological disorders alike.
While immune responses in neurodegeneration were regarded as little more than a curiosity a decade ago, they are now increasingly moving toward center stage. Factors driving this movement include the recognition that most of the relevant immune molecules are produced within the brain, that microglia are proficient immune cells shaping neuronal circuitry and fate, and that systemic immune responses affect brain function. We will review this complex field from the perspective of neurons, extra-neuronal brain cells, and the systemic environment and highlight the possibility that cell intrinsic innate immune molecules in neurons may function in neurodegenerative processes.
Immune responses in the CNS are common, despite its perception as a site of immune privilege. These responses can be mediated by resident microglia and astrocytes, which are innate immune cells without direct counterparts in the periphery. Furthermore, CNS immune reactions often take place in virtual isolation from the innate/adaptive immune interplay that characterizes peripheral immunity. However, microglia and astrocytes also engage in significant cross-talk with CNS-infiltrating T cells and other components of the innate immune system. Here we review the cellular and molecular basis of innate immunity in the CNS and discuss what is known about how outcomes of these interactions can lead to resolution of infection, neurodegeneration, or neural repair depending on the context.
Tissues of the CNS, such as the brain, optic nerves, and spinal cord, may be affected by a range of insults including genetic, autoimmune, infectious, or neurodegenerative diseases and cancer. The immune system is involved in the pathogenesis of many of these, either by causing tissue damage or alternatively by responding to disease and contributing to repair. It is clearly vital that cells of the immune system patrol the CNS and protect against infection. However, in contrast to other tissues, damage caused by immune pathology in the CNS can be irreparable. The nervous and immune systems have, therefore, coevolved to permit effective immune surveillance while limiting immune pathology. Here we will consider aspects of adaptive immunity in the CNS and the retina, both in the context of protection from infection as well as cancer and autoimmunity, while focusing on immune responses that compromise health and lead to significant morbidity.
Multiple sclerosis (MS) is a multifocal demyelinating disease with progressive neurodegeneration caused by an autoimmune response to self-antigens in a genetically susceptible individual. While the formation and persistence of meningeal lymphoid follicles suggest persistence of antigens to drive the continuing inflammatory and humoral response, the identity of an antigen or infectious agent leading to the oligoclonal expansion of B and T cells is unknown. In this review we examine new paradigms for understanding the immunopathology of MS, present recent data defining the common genetic variants underlying disease susceptibility, and explore how improved understanding of immune pathway disruption can inform MS prognosis and treatment decisions.
No longer do scientists look down on macrophages as “garbage men” that act “nonspecifically.” Last fall’s Nobel Prizes honored two of the few scientists who studied macrophages three decades ago. Now perhaps thousands do, and the subtypes they describe reflect ongoing discoveries of macrophages’ extraordinary plasticity.
Clinical vignette: A 59-year-old aeronautical engineer is referred to you for evaluation because of increasing difficulty with job performance over the last several years. Difficulty managing home finances, getting lost driving, and forgetting appointments have become common. Previously outgoing, he is now depressed and irritable. After appropriate neurologic assessment, including brain imaging and metabolic studies, you make the diagnosis of Alzheimer’s dementia and are asked by the patient’s family what treatment is available.
Chronic infection with the Gram-negative bacterium Helicobacter pylori is a major risk factor for the development of gastric cancer. Accumulating evidence indicates that the H. pylori virulence determinant cytotoxin-associated gene A (CagA) has a key oncogenic role in the process. Certain biological activities of CagA require its tyrosine phosphorylation by host cell kinases. In this issue of the JCI, Mueller and colleagues report their detailed kinetic and functional analysis of CagA phosphorylation, which indicates that c-Src and c-Abl kinases sequentially phosphorylate CagA. Interestingly, the two phosphorylation events need not occur on the same CagA molecule but are both required for the biological effects of CagA. The results provide a clinically relevant example of how a successful bacterial pathogen has evolved to exploit the tightly coordinated, sequential activity of host cell kinases for virulence factor activation and induction of pathology.
The hallmark of the movement disorder Parkinson’s disease (PD) is progressive degeneration of dopaminergic neurons. Mitochondrial dysfunction, impaired ubiquitin-mediated proteolysis of α-synuclein, and ER stress are each implicated in the complex and poorly understood sequence of events leading to dopaminergic neuron demise. In this issue of the JCI, Selvaraj et al. report that in a mouse neurotoxin-based model of PD, reduced Ca2+ influx through transient receptor potential C1 (TRPC1) channels in the plasma membrane of dopaminergic neurons triggers a cell death–inducing ER stress response. These new findings suggest that TRPC1 channels normally function in Ca2+-mediated signaling pathways that couple adaptive/neurotrophic responses to metabolic and oxidative stress and suggest that disruption of these pathways may contribute to PD.
Cilia are unique cellular organelles found in nearly all cell types. In recent years, the importance of these organelles has been highlighted by the discovery that mutations in genes encoding proteins related to cilia biogenesis and function cause a class of complex syndromes termed ciliopathies. Emerging evidence suggests interactions among the various ciliopathy-associated proteins, but the precise mechanisms by which these interactions generate functional networks have remained elusive. In this issue of the JCI, Rachel and colleagues have now clearly linked two ciliopathy-associated proteins (CEP290 and MKKS). Surprisingly, the effects of a hypomorphic disease-causing Cep290 allele were rescued by loss of MKKS function, suggesting that it might be possible to treat some ciliopathies by fine-tuning interactions within the expanding ciliary network.
Loss or functional impairment of p53 occurs in many human cancers, and its absence is often associated with a poor response to conventional chemotherapy. Hence, much effort is currently devoted to developing novel treatments for p53-deficient malignancies. One approach is to target pathways that are selectively required for the survival of p53-deficient cancer cells, thus exploiting a synthetic lethal interaction. Previous studies have demonstrated that inhibition of the ataxia telangiectasia and Rad3-related (ATR) and checkpoint kinase 1 (Chk1) pathway in p53-deficient cells can induce such a synthetic lethal outcome. In this issue of the JCI, Ma et al. take these findings a step closer to the clinic by demonstrating that highly specific inhibitors of Chk1 synergize with chemotherapy to stem progression of p53-deficient triple-negative breast cancers in a xenotransplant model of this disease. Together with other recent studies, this report highlights the promise of ATR and Chk1 inhibitors in targeted cancer treatment.
Blood transfusion represents the first and most prescribed cell-based therapy; however, clinical safety and efficacy trials are lacking. Clinical cohort studies have suggested that massive transfusion and/or transfusion of aged stored blood may contribute to multiorgan dysfunction in susceptible patients. In this issue of the JCI, Baek and colleagues report that aged stored blood hemolyzes after massive transfusion in a guinea pig model. Hemolysis led to vascular and kidney injury that was mediated by cell-free plasma hemoglobin and prevented by coinfusion of the specific hemoglobin scavenger protein, haptoglobin. These studies support an expanding body of research indicating that intravascular hemolysis is a pathological mechanism in several human diseases, including multiorgan dysfunction after either massive red blood cell transfusion or hemoglobin-based blood substitute therapy, the hemoglobinopathies, malaria, and other acquired and genetic hemolytic conditions.
Actin-myosin interactions provide the driving force underlying each heartbeat. The current view is that actin-bound regulatory proteins play a dominant role in the activation of calcium-dependent cardiac muscle contraction. In contrast, the relevance and nature of regulation by myosin regulatory proteins (for example, myosin light chain-2 [MLC2]) in cardiac muscle remain poorly understood. By integrating gene-targeted mouse and computational models, we have identified an indispensable role for ventricular Mlc2 (Mlc2v) phosphorylation in regulating cardiac muscle contraction. Cardiac myosin cycling kinetics, which directly control actin-myosin interactions, were directly affected, but surprisingly, Mlc2v phosphorylation also fed back to cooperatively influence calcium-dependent activation of the thin filament. Loss of these mechanisms produced early defects in the rate of cardiac muscle twitch relaxation and ventricular torsion. Strikingly, these defects preceded the left ventricular dysfunction of heart disease and failure in a mouse model with nonphosphorylatable Mlc2v. Thus, there is a direct and early role for Mlc2 phosphorylation in regulating actin-myosin interactions in striated muscle contraction, and dephosphorylation of Mlc2 or loss of these mechanisms can play a critical role in heart failure.
Early reperfusion of ischemic cardiac tissue remains the most effective intervention for improving clinical outcome following myocardial infarction. However, abnormal increases in intracellular Ca2+ during myocardial reperfusion can cause cardiomyocyte death and consequent loss of cardiac function, referred to as ischemia/reperfusion (IR) injury. Therapeutic modulation of Ca2+ handling provides some cardioprotection against the paradoxical effects of restoring blood flow to the heart, highlighting the significance of Ca2+ overload to IR injury. Cardiac IR is also accompanied by dynamic changes in the expression of microRNAs (miRNAs); for example, miR-214 is upregulated during ischemic injury and heart failure, but its potential role in these processes is unknown. Here, we show that genetic deletion of miR-214 in mice causes loss of cardiac contractility, increased apoptosis, and excessive fibrosis in response to IR injury. The cardioprotective roles of miR-214 during IR injury were attributed to repression of the mRNA encoding sodium/calcium exchanger 1 (Ncx1), a key regulator of Ca2+ influx; and to repression of several downstream effectors of Ca2+ signaling that mediate cell death. These findings reveal a pivotal role for miR-214 as a regulator of cardiomyocyte Ca2+ homeostasis and survival during cardiac injury.
Cilia are highly specialized microtubule-based organelles that have pivotal roles in numerous biological processes, including transducing sensory signals. Defects in cilia biogenesis and transport cause pleiotropic human ciliopathies. Mutations in over 30 different genes can lead to cilia defects, and complex interactions exist among ciliopathy-associated proteins. Mutations of the centrosomal protein 290 kDa (CEP290) lead to distinct clinical manifestations, including Leber congenital amaurosis (LCA), a hereditary cause of blindness due to photoreceptor degeneration. Mice homozygous for a mutant Cep290 allele (Cep290rd16 mice) exhibit LCA-like early-onset retinal degeneration that is caused by an in-frame deletion in the CEP290 protein. Here, we show that the domain deleted in the protein encoded by the Cep290rd16 allele directly interacts with another ciliopathy protein, MKKS. MKKS mutations identified in patients with the ciliopathy Bardet-Biedl syndrome disrupted this interaction. In zebrafish embryos, combined subminimal knockdown of mkks and cep290 produced sensory defects in the eye and inner ear. Intriguingly, combinations of Cep290rd16 and Mkksko alleles in mice led to improved ciliogenesis and sensory functions compared with those of either mutant alone. We propose that altered association of CEP290 and MKKS affects the integrity of multiprotein complexes at the cilia transition zone and basal body. Amelioration of the sensory phenotypes caused by specific mutations in one protein by removal of an interacting domain/protein suggests a possible novel approach for treating human ciliopathies.
Glaucoma is a common ocular disorder that is a leading cause of blindness worldwide. It is characterized by the dysfunction and loss of retinal ganglion cells (RGCs). Although many studies have implicated various molecules in glaucoma, no mechanism has been shown to be responsible for the earliest detectable damage to RGCs and their axons in the optic nerve. Here, we show that the leukocyte transendothelial migration pathway is activated in the optic nerve head at the earliest stages of disease in an inherited mouse model of glaucoma. This resulted in proinflammatory monocytes entering the optic nerve prior to detectable neuronal damage. A 1-time x-ray treatment prevented monocyte entry and subsequent glaucomatous damage. A single x-ray treatment of an individual eye in young mice provided that eye with long-term protection from glaucoma but had no effect on the contralateral eye. Localized radiation treatment prevented detectable neuronal damage and dysfunction in treated eyes, despite the continued presence of other glaucomatous stresses and signaling pathways. Injection of endothelin-2, a damaging mediator produced by the monocytes, into irradiated eyes, combined with the other glaucomatous stresses, restored neural damage with a topography characteristic of glaucoma. Together, these data support a model of glaucomatous damage involving monocyte entry into the optic nerve.
Individuals with type 2 diabetes have an increased risk of atherosclerosis. One factor underlying this is dyslipidemia, which in hyperinsulinemic subjects with early type 2 diabetes is typically characterized by increased VLDL secretion but normal LDL cholesterol levels, possibly reflecting enhanced catabolism of LDL via hepatic LDLRs. Recent studies have also suggested that hepatic insulin signaling sustains LDLR levels. We therefore sought to elucidate the mechanisms linking hepatic insulin signaling to regulation of LDLR levels. In WT mice, insulin receptor knockdown by shRNA resulted in decreased hepatic mTORC1 signaling and LDLR protein levels. It also led to increased expression of PCSK9, a known post-transcriptional regulator of LDLR expression. Administration of the mTORC1 inhibitor rapamycin caused increased expression of PCSK9, decreased levels of hepatic LDLR protein, and increased levels of VLDL/LDL cholesterol in WT but not Pcsk9–/– mice. Conversely, mice with increased hepatic mTORC1 activity exhibited decreased expression of PCSK9 and increased levels of hepatic LDLR protein levels. Pcsk9 is regulated by the transcription factor HNF1α, and our further detailed analyses suggest that increased mTORC1 activity leads to activation of PKCδ, reduced activity of HNF4α and HNF1α, decreased PCSK9 expression, and ultimately increased hepatic LDLR protein levels, which result in decreased circulating LDL levels. We therefore suggest that PCSK9 inhibition could be an effective way to reduce the adverse side effect of increased LDL levels that is observed in transplant patients taking rapamycin as immunosuppressive therapy.
The cytokine IL-12 induces IFN-γ production by T and NK cells. In preclinical models, it contributes to antitumor immunity. However, in clinical testing, it has shown limited benefit in patients with any one of a variety of malignancies. Moreover, in a clinical trial testing a combination of IL-12 and rituximab in patients with follicular B cell non-Hodgkin lymphoma (FL), those treated with IL-12 showed a lower response rate, suggesting that IL-12 actually plays a detrimental role. Here, we investigated whether the failure of IL-12 treatment for FL was due to T cell exhaustion, a condition characterized by reduced T cell differentiation, proliferation, and function, which has been observed in chronic viral infection. We found that extended exposure to IL-12 induced T cell exhaustion and contributed to the poor prognosis in FL patients. Long-term exposure of freshly isolated human CD4+ T cells to IL-12 in vitro caused T cell dysfunction and induced expression of TIM-3, a T cell immunoglobulin and mucin domain protein with a known role in T cell exhaustion, via an IFN-γ–independent mechanism. TIM-3 was required for the negative effect of IL-12 on T cell function. Importantly, TIM-3 also was highly expressed on intratumoral T cells that displayed marked functional impairment. Our findings identify IL-12– and TIM-3–mediated exhaustion of T cells as a mechanism for poor clinical outcome when IL-12 is administered to FL patients.
Malignant gliomas are the most common and the most lethal primary brain tumors in adults. Among malignant gliomas, 60%–80% show loss of P14ARF tumor suppressor activity due to somatic alterations of the INK4A/ARF genetic locus. The tumor suppressor activity of P14ARF is in part a result of its ability to prevent the degradation of P53 by binding to and sequestering HDM2. However, the subsequent finding of P14ARF loss in conjunction with TP53 gene loss in some tumors suggests the protein may have other P53-independent tumor suppressor functions. Here, we report what we believe to be a novel tumor suppressor function for P14ARF as an inhibitor of tumor-induced angiogenesis. We found that P14ARF mediates antiangiogenic effects by upregulating expression of tissue inhibitor of metalloproteinase–3 (TIMP3) in a P53-independent fashion. Mechanistically, this regulation occurred at the gene transcription level and was controlled by HDM2-SP1 interplay, where P14ARF relieved a dominant negative interaction of HDM2 with SP1. P14ARF-induced expression of TIMP3 inhibited endothelial cell migration and vessel formation in response to angiogenic stimuli produced by cancer cells. The discovery of this angiogenesis regulatory pathway may provide new insights into P53-independent P14ARF tumor-suppressive mechanisms that have implications for the development of novel therapies directed at tumors and other diseases characterized by vascular pathology.
Diagnosis of metastatic breast cancer is associated with a very poor prognosis. New therapeutic targets are urgently needed, but their development is hampered by a lack of understanding of the mechanisms leading to tumor metastasis. Exemplifying this is the fact that the approximately 30% of all breast cancers overexpressing the receptor tyrosine kinase ErbB-2 are characterized by high metastatic potential and poor prognosis, but the signaling events downstream of ErbB-2 that drive cancer cell invasion and metastasis remain incompletely understood. Here we show that overexpression of ErbB-2 in human breast cancer cell lines leads to phosphorylation and activation of the semaphorin receptor Plexin-B1. This was required for ErbB-2–dependent activation of the pro-metastatic small GTPases RhoA and RhoC and promoted invasive behavior of human breast cancer cells. In a mouse model of ErbB-2–overexpressing breast cancer, ablation of the gene encoding Plexin-B1 strongly reduced the occurrence of metastases. Moreover, in human patients with ErbB-2–overexpressing breast cancer, low levels of Plexin-B1 expression correlated with good prognosis. Our data suggest that Plexin-B1 represents a new candidate therapeutic target for treating patients with ErbB-2–positive breast cancer.
Mechanical hyperalgesia is a common and potentially disabling complication of many inflammatory and neuropathic conditions. Activation of the enzyme PKCε in primary afferent nociceptors is a major mechanism that underlies mechanical hyperalgesia, but the PKCε substrates involved downstream are not known. Here, we report that in a proteomic screen we identified the NaV1.8 sodium channel, which is selectively expressed in nociceptors, as a PKCε substrate. PKCε-mediated phosphorylation increased NaV1.8 currents, lowered the threshold voltage for activation, and produced a depolarizing shift in inactivation in wild-type — but not in PKCε-null — sensory neurons. PKCε phosphorylated NaV1.8 at S1452, and alanine substitution at this site blocked PKCε modulation of channel properties. Moreover, a specific PKCε activator peptide, ψεRACK, produced mechanical hyperalgesia in wild-type mice but not in Scn10a–/– mice, which lack NaV1.8 channels. These studies demonstrate that NaV1.8 is an important, direct substrate of PKCε that mediates PKCε-dependent mechanical hyperalgesia.
While a potential causal factor in Alzheimer’s disease (AD), brain insulin resistance has not been demonstrated directly in that disorder. We provide such a demonstration here by showing that the hippocampal formation (HF) and, to a lesser degree, the cerebellar cortex in AD cases without diabetes exhibit markedly reduced responses to insulin signaling in the IR→IRS-1→PI3K signaling pathway with greatly reduced responses to IGF-1 in the IGF-1R→IRS-2→PI3K signaling pathway. Reduced insulin responses were maximal at the level of IRS-1 and were consistently associated with basal elevations in IRS-1 phosphorylated at serine 616 (IRS-1 pS616) and IRS-1 pS636/639. In the HF, these candidate biomarkers of brain insulin resistance increased commonly and progressively from normal cases to mild cognitively impaired cases to AD cases regardless of diabetes or APOE ε4 status. Levels of IRS-1 pS616 and IRS-1 pS636/639 and their activated kinases correlated positively with those of oligomeric Aβ plaques and were negatively associated with episodic and working memory, even after adjusting for Aβ plaques, neurofibrillary tangles, and APOE ε4. Brain insulin resistance thus appears to be an early and common feature of AD, a phenomenon accompanied by IGF-1 resistance and closely associated with IRS-1 dysfunction potentially triggered by Aβ oligomers and yet promoting cognitive decline independent of classic AD pathology.
Defective brain insulin signaling has been suggested to contribute to the cognitive deficits in patients with Alzheimer’s disease (AD). Although a connection between AD and diabetes has been suggested, a major unknown is the mechanism(s) by which insulin resistance in the brain arises in individuals with AD. Here, we show that serine phosphorylation of IRS-1 (IRS-1pSer) is common to both diseases. Brain tissue from humans with AD had elevated levels of IRS-1pSer and activated JNK, analogous to what occurs in peripheral tissue in patients with diabetes. We found that amyloid-β peptide (Aβ) oligomers, synaptotoxins that accumulate in the brains of AD patients, activated the JNK/TNF-α pathway, induced IRS-1 phosphorylation at multiple serine residues, and inhibited physiological IRS-1pTyr in mature cultured hippocampal neurons. Impaired IRS-1 signaling was also present in the hippocampi of Tg mice with a brain condition that models AD. Importantly, intracerebroventricular injection of Aβ oligomers triggered hippocampal IRS-1pSer and JNK activation in cynomolgus monkeys. The oligomer-induced neuronal pathologies observed in vitro, including impaired axonal transport, were prevented by exposure to exendin-4 (exenatide), an anti-diabetes agent. In Tg mice, exendin-4 decreased levels of hippocampal IRS-1pSer and activated JNK and improved behavioral measures of cognition. By establishing molecular links between the dysregulated insulin signaling in AD and diabetes, our results open avenues for the investigation of new therapeutics in AD.
Individuals with Parkinson’s disease (PD) experience a progressive decline in motor function as a result of selective loss of dopaminergic (DA) neurons in the substantia nigra. The mechanism(s) underlying the loss of DA neurons is not known. Here, we show that a neurotoxin that causes a disease that mimics PD upon administration to mice, because it induces the selective loss of DA neurons in the substantia nigra, alters Ca2+ homeostasis and induces ER stress. In a human neuroblastoma cell line, we found that endogenous store-operated Ca2+ entry (SOCE), which is critical for maintaining ER Ca2+ levels, is dependent on transient receptor potential channel 1 (TRPC1) activity. Neurotoxin treatment decreased TRPC1 expression, TRPC1 interaction with the SOCE modulator stromal interaction molecule 1 (STIM1), and Ca2+ entry into the cells. Overexpression of functional TRPC1 protected against neurotoxin-induced loss of SOCE, the associated decrease in ER Ca2+ levels, and the resultant unfolded protein response (UPR). In contrast, silencing of TRPC1 or STIM1 increased the UPR. Furthermore, Ca2+ entry via TRPC1 activated the AKT pathway, which has a known role in neuroprotection. Consistent with these in vitro data, Trpc1–/– mice had an increased UPR and a reduced number of DA neurons. Brain lysates of patients with PD also showed an increased UPR and decreased TRPC1 levels. Importantly, overexpression of TRPC1 in mice restored AKT/mTOR signaling and increased DA neuron survival following neurotoxin administration. Overall, these results suggest that TRPC1 is involved in regulating Ca2+ homeostasis and inhibiting the UPR and thus contributes to neuronal survival.
Herpes simplex viruses (HSVs) are highly prevalent neurotropic viruses. While they can replicate lytically in cells of the epithelial lineage, causing lesions on mucocutaneous surfaces, HSVs also establish latent infections in neurons, which act as reservoirs of virus for subsequent reactivation events. Immunological control of HSV involves activation of innate immune pattern-recognition receptors such as TLR3, which detects double-stranded RNA and induces type I IFN expression. Humans with defects in the TLR3/IFN pathway have an elevated susceptibility to HSV infections of the CNS. However, it is not known what cell type mediates the role of TLR3 in the immunological control of HSV, and it is not known whether TLR3 sensing occurs prior to or after CNS entry. Here, we show that in mice TLR3 provides early control of HSV-2 infection immediately after entry into the CNS by mediating type I IFN responses in astrocytes. Tlr3–/– mice were hypersusceptible to HSV-2 infection in the CNS after vaginal inoculation. HSV-2 exhibited broader neurotropism in Tlr3–/– mice than it did in WT mice, with astrocytes being most abundantly infected. Tlr3–/– mice did not exhibit a global defect in innate immune responses to HSV, but astrocytes were defective in HSV-induced type I IFN production. Thus, TLR3 acts in astrocytes to sense HSV-2 infection immediately after entry into the CNS, possibly preventing HSV from spreading beyond the neurons mediating entry into the CNS.
In Alzheimer disease (AD), amyloid β peptide (Aβ) accumulates in plaques in the brain. Receptor for advanced glycation end products (RAGE) mediates Aβ-induced perturbations in cerebral vessels, neurons, and microglia in AD. Here, we identified a high-affinity RAGE-specific inhibitor (FPS-ZM1) that blocked Aβ binding to the V domain of RAGE and inhibited Aβ40- and Aβ42-induced cellular stress in RAGE-expressing cells in vitro and in the mouse brain in vivo. FPS-ZM1 was nontoxic to mice and readily crossed the blood-brain barrier (BBB). In aged APPsw/0 mice overexpressing human Aβ-precursor protein, a transgenic mouse model of AD with established Aβ pathology, FPS-ZM1 inhibited RAGE-mediated influx of circulating Aβ40 and Aβ42 into the brain. In brain, FPS-ZM1 bound exclusively to RAGE, which inhibited β-secretase activity and Aβ production and suppressed microglia activation and the neuroinflammatory response. Blockade of RAGE actions at the BBB and in the brain reduced Aβ40 and Aβ42 levels in brain markedly and normalized cognitive performance and cerebral blood flow responses in aged APPsw/0 mice. Our data suggest that FPS-ZM1 is a potent multimodal RAGE blocker that effectively controls progression of Aβ-mediated brain disorder and that it may have the potential to be a disease-modifying agent for AD.
The B cell–depleting IgG1 monoclonal antibody rituximab can persistently suppress disease progression in some patients with autoimmune diseases. However, the mechanism underlying these long-term beneficial effects has remained unclear. Here, we evaluated Ig gene usage in patients with anti–myelin-associated glycoprotein (anti-MAG) neuropathy, an autoimmune disease of the peripheral nervous system that is mediated by IgM autoantibodies binding to MAG antigen. Patients with anti-MAG neuropathy showed substantial clonal expansions of blood IgM memory B cells that recognized MAG antigen. The group of patients showing no clinical improvement after rituximab therapy were distinguished from clinical responders by a higher load of clonal IgM memory B cell expansions before and after therapy, by persistence of clonal expansions despite efficient peripheral B cell depletion, and by a lack of substantial changes in somatic hypermutation frequencies of IgM memory B cells. We infer from these data that the effectiveness of rituximab therapy depends on efficient depletion of noncirculating B cells and is associated with qualitative immunological changes that indicate reconfiguration of B cell memory through sustained reduction of autoreactive clonal expansions. These findings support the continued development of B cell–depleting therapies for autoimmune diseases.
The development of a broad repertoire of T cells, which is essential for effective immune function, occurs in the thymus. Although some data suggest that T cell development can occur extrathymically, many researchers remain skeptical that extrathymic T cell development has an important role in generating the T cell repertoire in healthy individuals. However, it may be important in the setting of poor thymic function or congenital deficit and in the context of autoimmunity, cancer, or regenerative medicine. Here, we report evidence that a stepwise program of T cell development occurs within the human tonsil. We identified 5 tonsillar T cell developmental intermediates: (a) CD34+CD38dimLin– cells, which resemble multipotent progenitors in the bone marrow and thymus; (b) more mature CD34+CD38brightLin– cells; (c) CD34+CD1a+CD11c– cells, which resemble committed T cell lineage precursors in the thymus; (d) CD34–CD1a+CD3–CD11c– cells, which resemble CD4+CD8+ double-positive T cells in the thymus; and (e) CD34–CD1a+CD3+CD11c– cells. The phenotype of each subset closely resembled that of its thymic counterpart. The last 4 populations expressed RAG1 and PTCRA, genes required for TCR rearrangement, and all 5 subsets were capable of ex vivo T cell differentiation. TdT+ cells found within the tonsillar fibrous scaffold expressed CD34 and/or CD1a, indicating that this distinct anatomic region contributes to pre–T cell development, as does the subcapsular region of the thymus. Thus, we provide evidence of a role for the human tonsil in a comprehensive program of extrathymic T cell development.
The bioactive lysophospholipid mediator sphingosine-1-phosphate (S1P) promotes the egress of newly formed T cells from the thymus and the release of immature B cells from the bone marrow. It has remained unclear, however, where and how S1P is released. Here, we show that in mice, the S1P transporter spinster homolog 2 (Spns2) is responsible for the egress of mature T cells and immature B cells from the thymus and bone marrow, respectively. Global Spns2-KO mice exhibited marked accumulation of mature T cells in thymi and decreased numbers of peripheral T cells in blood and secondary lymphoid organs. Mature recirculating B cells were reduced in frequency in the bone marrow as well as in blood and secondary lymphoid organs. Bone marrow reconstitution studies revealed that Spns2 was not involved in S1P release from blood cells and suggested a role for Spns2 in other cells. Consistent with these data, endothelia-specific deletion of Spns2 resulted in defects of lymphocyte egress similar to those observed in the global Spns2-KO mice. These data suggest that Spns2 functions in ECs to establish the S1P gradient required for T and B cells to egress from their respective primary lymphoid organs. Furthermore, Spns2 could be a therapeutic target for a broad array of inflammatory and autoimmune diseases.
Localized tissue hypoxia is a consequence of vascular compromise or rapid cellular proliferation and is a potent inducer of compensatory angiogenesis. The oxygen-responsive transcriptional regulator hypoxia-inducible factor 2α (HIF-2α) is highly expressed in vascular ECs and, along with HIF-1α, activates expression of target genes whose products modulate vascular functions and angiogenesis. However, the mechanisms by which HIF-2α regulates EC function and tissue perfusion under physiological and pathological conditions are poorly understood. Using mice in which Hif2a was specifically deleted in ECs, we demonstrate here that HIF-2α expression is required for angiogenic responses during hindlimb ischemia and for the growth of autochthonous skin tumors. EC-specific Hif2a deletion resulted in increased vessel formation in both models; however, these vessels failed to undergo proper arteriogenesis, resulting in poor perfusion. Analysis of cultured HIF-2α–deficient ECs revealed cell-autonomous increases in migration, invasion, and morphogenetic activity, which correlated with HIF-2α–dependent expression of specific angiogenic factors, including delta-like ligand 4 (Dll4), a Notch ligand, and angiopoietin 2. By stimulating Dll4 signaling in cultured ECs or restoring Dll4 expression in ischemic muscle tissue, we rescued most of the HIF-2α–dependent EC phenotypes in vitro and in vivo, emphasizing the critical role of Dll4/Notch signaling as a downstream target of HIF-2α in ECs. These results indicate that HIF-1α and HIF-2α fulfill complementary, but largely nonoverlapping, essential functions in pathophysiological angiogenesis.
Massive transfusion of blood can lead to clinical complications, including multiorgan dysfunction and even death. Such severe clinical outcomes have been associated with longer red blood cell (rbc) storage times. Collectively referred to as the rbc storage lesion, rbc storage results in multiple biochemical changes that impact intracellular processes as well as membrane and cytoskeletal properties, resulting in cellular injury in vitro. However, how the rbc storage lesion triggers pathophysiology in vivo remains poorly defined. In this study, we developed a guinea pig transfusion model with blood stored under standard blood banking conditions for 2 (new), 21 (intermediate), or 28 days (old blood). Transfusion with old but not new blood led to intravascular hemolysis, acute hypertension, vascular injury, and kidney dysfunction associated with pathophysiology driven by hemoglobin (Hb). These adverse effects were dramatically attenuated when the high-affinity Hb scavenger haptoglobin (Hp) was administered at the time of transfusion with old blood. Pathologies observed after transfusion with old blood, together with the favorable response to Hp supplementation, allowed us to define the in vivo consequences of the rbc storage lesion as storage-related posttransfusion hemolysis producing Hb-driven pathophysiology. Hb sequestration by Hp might therefore be a therapeutic modality for enhancing transfusion safety in severely ill or massively transfused patients.
The clinical use of niacin to treat dyslipidemic conditions is limited by noxious side effects, most commonly facial flushing. In mice, niacin-induced flushing results from COX-1–dependent formation of PGD2 and PGE2 followed by COX-2–dependent production of PGE2. Consistent with this, niacin-induced flushing in humans is attenuated when niacin is combined with an antagonist of the PGD2 receptor DP1. NSAID-mediated suppression of COX-2–derived PGI2 has negative cardiovascular consequences, yet little is known about the cardiovascular biology of PGD2. Here, we show that PGD2 biosynthesis is augmented during platelet activation in humans and, although vascular expression of DP1 is conserved between humans and mice, platelet DP1 is not present in mice. Despite this, DP1 deletion in mice augmented aneurysm formation and the hypertensive response to Ang II and accelerated atherogenesis and thrombogenesis. Furthermore, COX inhibitors in humans, as well as platelet depletion, COX-1 knockdown, and COX-2 deletion in mice, revealed that niacin evoked platelet COX-1–derived PGD2 biosynthesis. Finally, ADP-induced spreading on fibrinogen was augmented by niacin in washed human platelets, coincident with increased thromboxane (Tx) formation. However, in platelet-rich plasma, where formation of both Tx and PGD2 was increased, spreading was not as pronounced and was inhibited by DP1 activation. Thus, PGD2, like PGI2, may function as a homeostatic response to thrombogenic and hypertensive stimuli and may have particular relevance as a constraint on platelets during niacin therapy.
Breast cancers are highly heterogeneous but can be grouped into subtypes based on several criteria, including level of expression of certain markers. Claudin-low breast cancer (CLBC) is associated with early metastasis and resistance to chemotherapy, while gene profiling indicates it is characterized by the expression of markers of epithelial-mesenchymal transition (EMT) — a phenotypic conversion linked with metastasis. Although the epigenetic program controlling the phenotypic and cellular plasticity of EMT remains unclear, one contributor may be methylation of the E-cadherin promoter, resulting in decreased E-cadherin expression, a hallmark of EMT. Indeed, reduced E-cadherin often occurs in CLBC and may contribute to the early metastasis and poor patient survival associated with this disease. Here, we have determined that methylation of histone H3 on lysine 9 (H3K9me2) is critical for promoter DNA methylation of E-cadherin in three TGF-β–induced EMT model cell lines, as well as in CLBC cell lines. Further, Snail interacted with G9a, a major euchromatin methyltransferase responsible for H3K9me2, and recruited G9a and DNA methyltransferases to the E-cadherin promoter for DNA methylation. Knockdown of G9a restored E-cadherin expression by suppressing H3K9me2 and blocking DNA methylation. This resulted in inhibition of cell migration and invasion in vitro and suppression of tumor growth and lung colonization in in vivo models of CLBC metastasis. Our study not only reveals a critical mechanism underlying the epigenetic regulation of EMT but also paves a way for the development of new treatment strategies for CLBC.
Epstein-Barr virus (EBV) persistently infects more than 90% of the human population and is etiologically linked to several B cell malignancies, including Burkitt lymphoma (BL), Hodgkin lymphoma (HL), and diffuse large B cell lymphoma (DLBCL). Despite its growth transforming properties, most immune-competent individuals control EBV infection throughout their lives. EBV encodes various oncogenes, and of the 6 latency-associated EBV-encoded nuclear antigens, only EBNA3B is completely dispensable for B cell transformation in vitro. Here, we report that infection with EBV lacking EBNA3B leads to aggressive, immune-evading monomorphic DLBCL-like tumors in NOD/SCID/γc–/– mice with reconstituted human immune system components. Infection with EBNA3B-knockout EBV (EBNA3BKO) induced expansion of EBV-specific T cells that failed to infiltrate the tumors. EBNA3BKO-infected B cells expanded more rapidly and secreted less T cell–chemoattractant CXCL10, reducing T cell recruitment in vitro and T cell–mediated killing in vivo. B cell lines from 2 EBV-positive human lymphomas encoding truncated EBNA3B exhibited gene expression profiles and phenotypic characteristics similar to those of tumor-derived lines from the humanized mice, including reduced CXCL10 secretion. Screening EBV-positive DLBCL, HL, and BL human samples identified additional
Half of patients with muscle-invasive bladder cancer develop metastatic disease, and this is responsible for most of the deaths from this cancer. Low expression of RhoGTP dissociation inhibitor 2 (RhoGDI2; also known as ARHGDIB and Ly-GDI) is associated with metastatic disease in patients with muscle-invasive bladder cancer. Moreover, a reduction in metastasis is observed upon reexpression of RhoGDI2 in xenograft models of metastatic cancer. Here, we show that RhoGDI2 suppresses lung metastasis in mouse models by reducing the expression of isoforms V1 and V3 of the proteoglycan versican (VCAN; also known as chondroitin sulfate proteoglycan 2 [CSPG2]). In addition, we found that high versican levels portended poor prognosis in patients with bladder cancer. The functional importance of tumor expression of versican in promoting metastasis was established in in vitro and in vivo studies in mice that implicated a role for the chemokine CCL2 (also known as MCP1) and macrophages. Further analysis indicated that RhoGDI2 suppressed metastasis by altering inflammation in the tumor microenvironment. In summary, we demonstrate what we believe to be a new mechanism of metastasis suppression that works by reducing host responses that promote metastatic colonization of the lung. Therapeutic targeting of these interactions may provide a novel adjuvant strategy for delaying the appearance of clinical metastasis in patients.
Genetic mutations that give rise to active mutant forms of Ras are oncogenic and found in several types of tumor. However, such mutations are not clear biomarkers for disease, since they are frequently detected in healthy individuals. Instead, it has become clear that elevated levels of Ras activity are critical for Ras-induced tumorigenesis. However, the mechanisms underlying the production of pathological levels of Ras activity are unclear. Here, we show that in the presence of oncogenic Ras, inflammatory stimuli initiate a positive feedback loop involving NF-κB that further amplifies Ras activity to pathological levels. Stimulation of Ras signaling by typical inflammatory stimuli was transient and had no long-term sequelae in wild-type mice. In contrast, these stimuli generated prolonged Ras signaling and led to chronic inflammation and precancerous pancreatic lesions (PanINs) in mice expressing physiological levels of oncogenic K-Ras. These effects of inflammatory stimuli were disrupted by deletion of inhibitor of NF-κB kinase 2 (IKK2) or inhibition of Cox-2. Likewise, expression of active IKK2 or Cox-2 or treatment with LPS generated chronic inflammation and PanINs only in mice expressing oncogenic K-Ras. The data support the hypothesis that in the presence of oncogenic Ras, inflammatory stimuli trigger an NF-κB–mediated positive feedback mechanism involving Cox-2 that amplifies Ras activity to pathological levels. Because a large proportion of the adult human population possesses Ras mutations in tissues including colon, pancreas, and lung, disruption of this positive feedback loop may be an important strategy for cancer prevention.
Integrin signaling critically contributes to the progression, growth, and therapy resistance of malignant tumors. Here, we show that targeting of β1 integrins with inhibitory antibodies enhances the sensitivity to ionizing radiation and delays the growth of human head and neck squamous cell carcinoma cell lines in 3D cell culture and in xenografted mice. Mechanistically, dephosphorylation of focal adhesion kinase (FAK) upon inhibition of β1 integrin resulted in dissociation of a FAK/cortactin protein complex. This, in turn, downregulated JNK signaling and induced cell rounding, leading to radiosensitization. Thus, these findings suggest that robust and selective pharmacological targeting of β1 integrins may provide therapeutic benefit to overcome tumor cell resistance to radiotherapy.
Patients with triple-negative breast cancer (TNBC) — defined by lack of estrogen receptor and progesterone receptor expression as well as lack of human epidermal growth factor receptor 2 (HER2) amplification — have a poor prognosis. There is a need for targeted therapies to treat this condition. TNBCs frequently harbor mutations in TP53, resulting in loss of the G1 checkpoint and reliance on checkpoint kinase 1 (Chk1) to arrest cells in response to DNA damage. Previous studies have shown that inhibition of Chk1 in a p53-deficient background in response to DNA damage. We therefore tested whether inhibition of Chk1 could potentiate the cytotoxicity of the DNA damaging agent irinotecan in TNBC using xenotransplant tumor models. Tumor specimens from patients with TNBC were engrafted into humanized mammary fat pads of immunodeficient mice to create 3 independent human-in-mouse TNBC lines: 1 WT (WU-BC3) and 2 mutant for TP53 (WU-BC4 and WU-BC5). These lines were tested for their response to irinotecan and a Chk1 inhibitor (either UCN-01 or AZD7762), either as single agents or in combination. The combination therapy induced checkpoint bypass and apoptosis in WU-BC4 and WU-BC5, but not WU-BC3, tumors. Moreover, combination therapy inhibited tumor growth and prolonged survival of mice bearing the WU-BC4 line, but not the WU-BC3 line. In addition, knockdown of p53 sensitized WU-BC3 tumors to the combination therapy. These results demonstrate that p53 is a major determinant of how TNBCs respond to therapies that combine DNA damage with Chk1 inhibition.
Many bacterial pathogens inject into host cells effector proteins that are substrates for host tyrosine kinases such as Src and Abl family kinases. Phosphorylated effectors eventually subvert host cell signaling, aiding disease development. In the case of the gastric pathogen Helicobacter pylori, which is a major risk factor for the development of gastric cancer, the only known effector protein injected into host cells is the oncoprotein CagA. Here, we followed the hierarchic tyrosine phosphorylation of H. pylori CagA as a model system to study early effector phosphorylation processes. Translocated CagA is phosphorylated on Glu-Pro-Ile-Tyr-Ala (EPIYA) motifs EPIYA-A, EPIYA-B, and EPIYA-C in Western strains of H. pylori and EPIYA-A, EPIYA-B, and EPIYA-D in East Asian strains. We found that c-Src only phosphorylated EPIYA-C and EPIYA-D, whereas c-Abl phosphorylated EPIYA-A, EPIYA-B, EPIYA-C, and EPIYA-D. Further analysis revealed that CagA molecules were phosphorylated on 1 or 2 EPIYA motifs, but never simultaneously on 3 motifs. Furthermore, none of the phosphorylated EPIYA motifs alone was sufficient for inducing AGS cell scattering and elongation. The preferred combination of phosphorylated EPIYA motifs in Western strains was EPIYA-A and EPIYA-C, either across 2 CagA molecules or simultaneously on 1. Our study thus identifies a tightly regulated hierarchic phosphorylation model for CagA starting at EPIYA-C/D, followed by phosphorylation of EPIYA-A or EPIYA-B. These results provide insight for clinical H. pylori typing and clarify the role of phosphorylated bacterial effector proteins in pathogenesis.
The ability of the liver to regenerate is crucial to protect liver function after injury and during chronic disease. Increases in hepatocyte growth factor (HGF) in liver sinusoidal endothelial cells (LSECs) are thought to drive liver regeneration. However, in contrast to endothelial progenitor cells, mature LSECs express little HGF. Therefore, we sought to establish in rats whether liver injury causes BM LSEC progenitor cells to engraft in the liver and provide increased levels of HGF and to examine the relative contribution of resident and BM LSEC progenitors. LSEC label-retaining cells and progenitors were identified in liver and LSEC progenitors in BM. BM LSEC progenitors did not contribute to normal LSEC turnover in the liver. However, after partial hepatectomy, BM LSEC progenitor proliferation and mobilization to the circulation doubled. In the liver, one-quarter of the LSECs were BM derived, and BM LSEC progenitors differentiated into fenestrated LSECs. When irradiated rats underwent partial hepatectomy, liver regeneration was compromised, but infusion of LSEC progenitors rescued the defect. Further analysis revealed that BM LSEC progenitors expressed substantially more HGF and were more proliferative than resident LSEC progenitors after partial hepatectomy. Resident LSEC progenitors within their niche may play a smaller role in recovery from partial hepatectomy than BM LSEC progenitors, but, when infused after injury, these progenitors engrafted and expanded markedly over a 2-month period. In conclusion, LSEC progenitor cells are present in liver and BM, and recruitment of BM LSEC progenitors is necessary for normal liver regeneration.
Acetaminophen (APAP) overdose is the predominant cause of acute liver failure in the United States. Toxicity begins with a reactive metabolite that binds to proteins. In rodents, this leads to mitochondrial dysfunction and nuclear DNA fragmentation, resulting in necrotic cell death. While APAP metabolism is similar in humans, the later events resulting in toxicity have not been investigated in patients. In this study, levels of biomarkers of mitochondrial damage (glutamate dehydrogenase [GDH] and mitochondrial DNA [mtDNA]) and nuclear DNA fragments were measured in plasma from APAP-overdose patients. Overdose patients with no or minimal hepatic injury who had normal liver function tests (LTs) (referred to herein as the normal LT group) and healthy volunteers served as controls. Peak GDH activity and mtDNA concentration were increased in plasma from patients with abnormal LT. Peak nuclear DNA fragmentation in the abnormal LT cohort was also increased over that of controls. Parallel studies in mice revealed that these plasma biomarkers correlated well with tissue injury. Caspase-3 activity and cleaved caspase-3 were not detectable in plasma from overdose patients or mice, but were elevated after TNF-induced apoptosis, indicating that APAP overdose does not cause apoptosis. Thus, our results suggest that mitochondrial damage and nuclear DNA fragmentation are likely to be critical events in APAP hepatotoxicity in humans, resulting in necrotic cell death.
Copyright © 2014 American Society for Clinical Investigation