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Early B cell changes predict autoimmunity following combination immune checkpoint blockade
Combination checkpoint blockade (CCB) targeting inhibitory CTLA4 and PD1 receptors holds promise for cancer therapy. Immune-related adverse events (IRAEs) remain a major obstacle for the optimal application of CCB in cancer. Here, we analyzed B cell changes in patients with melanoma following treatment with either anti-CTLA4 or anti-PD1, or in combination. CCB therapy led to changes in circulating B cells that were detectable after the first cycle of therapy and characterized by a decline in circulating B cells and an increase in CD21lo B cells and plasmablasts. PD1 expression was higher in the CD21lo B cells, and B cell receptor sequencing of these cells demonstrated greater clonality and a higher frequency of clones compared with CD21hi cells. CCB induced proliferation in the CD21lo compartment, and single-cell RNA sequencing identified B cell activation in cells with genomic profiles of CD21lo B cells in vivo. Increased clonality of circulating B cells following CCB occurred in some patients. Treatment-induced changes in B cells preceded and correlated with both the frequency and timing of IRAEs. Patients with early B cell changes experienced higher rates of grade 3 or higher IRAEs 6 months after CCB. Thus, early changes in B cells following CCB may identify patients who are at increased risk of IRAEs, and preemptive strategies targeting B cells may reduce toxicities in these patients.



B cells as biomarkers: predicting immune checkpoint therapy adverse events
Immune checkpoint inhibitors are becoming a cornerstone of cancer immunotherapy as a result of their clinical success in relieving immune suppression and driving durable antitumor T cell responses in certain subsets of patients. Unfortunately, checkpoint inhibition is also associated with treatment-related toxicities that result in a myriad of side effects, ranging from mild and manageable to severe and debilitating. In this issue of the JCI, Das and colleagues report an association between early therapy-induced changes in circulating B cells and an increased risk of high-grade immune-related adverse events (IRAEs) in patients treated with checkpoint inhibitors that target cytotoxic T lymphocyte–associated antigen-4 (CTLA4) and programmed cell death protein 1 (PD1). These findings identify potential predictive biomarkers for high-grade IRAEs that may be leveraged to improve patient monitoring and may prompt new treatment strategies to prevent IRAEs.



Hepatitis B virus–specific T cells associate with viral control upon nucleos(t)ide-analogue therapy discontinuation
BACKGROUND. The clinical management of chronic hepatitis B virus (HBV) patients is based exclusively on virological parameters that cannot independently determine in which patients nucleos(t)ide-analogue (NUC) therapy can be safely discontinued. NUCs efficiently suppress viral replication, but do not eliminate HBV. Thus, therapy discontinuation can be associated with virological and biochemical relapse and, consequently, therapy in the majority is life-long. METHODS. Since antiviral immunity is pivotal for HBV control, we investigated potential biomarkers for the safe discontinuation of NUCs within immune profiles of chronic HBV patients by utilizing traditional immunological assays (ELISPOT, flow cytometry) in conjunction with analyses of global non–antigen-specific immune populations (NanoString and CyTOF). Two distinct cohorts of 19 and 27 chronic HBV patients, respectively, were analyzed longitudinally prior to and after discontinuation of 2 different NUC therapy strategies. RESULTS. Absence of hepatic flares following discontinuation of NUC treatment correlated with the presence, during NUC viral suppression, of HBV core and polymerase-specific T cells that were contained within the ex vivo PD-1+ population. CONCLUSIONS. This study identifies the presence of functional HBV-specific T cells as a candidate immunological biomarker for safe therapy discontinuation in chronic HBV patients. Furthermore, the persistent and functional antiviral activity of PD-1+ HBV–specific T cells highlights the potential beneficial role of the expression of T cell exhaustion markers during human chronic viral infection. FUNDING. This work was funded by a Singapore Translational Research Investigator Award (NMRC/STaR/013/2012), the Eradication of HBV TCR Program (NMRC/TCR/014-NUHS/2015), the Singapore Immunology Network, the Wellcome Trust (107389/Z/15/Z), and a Barts and The London Charity (723/1795) grant.



Insulin exits skeletal muscle capillaries by fluid-phase transport
Before insulin can stimulate myocytes to take up glucose, it must first move from the circulation to the interstitial space. The continuous endothelium of skeletal muscle (SkM) capillaries restricts insulin’s access to myocytes. The mechanism by which insulin crosses this continuous endothelium is critical to understand insulin action and insulin resistance; however, methodological obstacles have limited understanding of endothelial insulin transport in vivo. Here, we present an intravital microscopy technique to measure the rate of insulin efflux across the endothelium of SkM capillaries. This method involves development of a fully bioactive, fluorescent insulin probe, a gastrocnemius preparation for intravital microscopy, an automated vascular segmentation algorithm, and the use of mathematical models to estimate endothelial transport parameters. We combined direct visualization of insulin efflux from SkM capillaries with modeling of insulin efflux kinetics to identify fluid-phase transport as the major mode of transendothelial insulin efflux in mice. Model-independent experiments demonstrating that insulin movement is neither saturable nor affected by insulin receptor antagonism supported this result. Our finding that insulin enters the SkM interstitium by fluid-phase transport may have implications in the pathophysiology of SkM insulin resistance as well as in the treatment of diabetes with various insulin analogs.



Unravelling the fate of functional PD1+ T cells in chronic viral hepatitis
Hepatitis B virus (HBV) infection can be managed clinically with nucleos(t)ide therapy, which suppresses viral replication; however, these drugs must often be used long term, as they are unable to fully eliminate the virus. For many patients, discontinuation of treatment results in viral resurgence and hepatic flare, and there is not a reliable way to identify those individuals that can be successfully taken off nucleos(t)ide therapy. In this issue of the JCI, Rivino and colleagues report on their use of a multipronged approach to investigate potential biomarkers indicative of HBV-infected patients who can safely stop nucleos(t)ide therapy. The authors identified a population of HBV-specific, PD1-positive T cells that was present in HBV-infected patients who successfully discontinued treatment without hepatic flare, but not in those that developed flare upon treatment cessation. Together, these results support the concept that PD1+ cells may play an important role in viral control, the further evaluation of this T cell subset in preventing hepatic flare, and the development of assays to better detect this PD1+ T cell population in HBV-infected patients on nucleos(t)ide therapy.



Critical roles of αII spectrin in brain development and epileptic encephalopathy
The nonerythrocytic α-spectrin-1 (SPTAN1) gene encodes the cytoskeletal protein αII spectrin. Mutations in SPTAN1 cause early infantile epileptic encephalopathy type 5 (EIEE5); however, the role of αII spectrin in neurodevelopment and EIEE5 pathogenesis is unknown. Prior work suggests that αII spectrin is absent in the axon initial segment (AIS) and contributes to a diffusion barrier in the distal axon. Here, we have shown that αII spectrin is expressed ubiquitously in rodent and human somatodendritic and axonal domains. CRISPR-mediated deletion of Sptan1 in embryonic rat forebrain by in utero electroporation caused altered dendritic and axonal development, loss of the AIS, and decreased inhibitory innervation. Overexpression of human EIEE5 mutant SPTAN1 in embryonic rat forebrain and mouse hippocampal neurons led to similar developmental defects that were also observed in EIEE5 patient-derived neurons. Additionally, patient-derived neurons displayed aggregation of spectrin complexes. Taken together, these findings implicate αII spectrin in critical aspects of dendritic and axonal development and synaptogenesis, and support a dominant-negative mechanism of SPTAN1 mutations in EIEE5.



PD-L1 on host cells is essential for PD-L1 blockade–mediated tumor regression
Programmed death–ligand 1 (PD-L1) expression on tumor cells is essential for T cell impairment, and PD-L1 blockade therapy has shown unprecedented durable responses in several clinical studies. Although higher expression of PD-L1 on tumor cells is associated with a better immune response after Ab blockade, some PD-L1–negative patients also respond to this therapy. In the current study, we explored whether PD-L1 on tumor or host cells was essential for anti–PD-L1–mediated therapy in 2 different murine tumor models. Using real-time imaging in whole tumor tissues, we found that anti–PD-L1 Ab accumulates in tumor tissues regardless of the status of PD-L1 expression on tumor cells. We further observed that, while PD-L1 on tumor cells was largely dispensable for the response to checkpoint blockade, PD-L1 in host myeloid cells was essential for this response. Additionally, PD-L1 signaling in defined antigen presenting cells (APCs) negatively regulated and inhibited T cell activation. PD-L1 blockade inside tumors was not sufficient to mediate regression, as limiting T cell trafficking reduced the efficacy of the blockade. Together, these findings demonstrate that PD-L1 expressed in APCs, rather than on tumor cells, plays an essential role in checkpoint blockade therapy, providing an insight into the mechanisms of this therapy.



The host protecting the tumor from the host — targeting PD‑L1 expressed by host cells
Tumors frequently escape from immune surveillance by hijacking the natural control mechanisms that regulate normal immune responses. The programmed death-1 receptor (PD‑1) on T cells normally helps limit excessive immune activation, but it can also suppress beneficial antitumor immunity. In the clinic, blocking either PD‑1 or one of its principal counterligands, programmed death–ligand 1 (PD‑L1), can lead to dramatic responses in certain patients. Because PD‑L1 can be expressed by both the tumor cells themselves and also the host cells, including host immune cells, the actual mechanistic target of therapy has remained unclear. In the current issue of the JCI, two papers, one by Tang and colleagues and the other by Lin and colleagues, used a variety of mouse tumor models to demonstrate that the relevant target for therapy in each case was the PD‑L1 molecules expressed by host cells and not by tumor cells. If this finding is generalized to humans, then it would suggest that the tumor persuades the host to actively suppress its own attempted immune response against the tumor cells.



Host expression of PD-L1 determines efficacy of PD-L1 pathway blockade–mediated tumor regression
Programmed death-1 receptor (PD-L1, B7-H1) and programmed cell death protein 1 (PD-1) pathway blockade is a promising therapy for treating cancer. However, the mechanistic contribution of host and tumor PD-L1 and PD-1 signaling to the therapeutic efficacy of PD-L1 and PD-1 blockade remains elusive. Here, we evaluated 3 tumor-bearing mouse models that differ in their sensitivity to PD-L1 blockade and demonstrated a loss of therapeutic efficacy of PD-L1 blockade in immunodeficient mice and in PD-L1– and PD-1–deficient mice. In contrast, neither knockout nor overexpression of PD-L1 in tumor cells had an effect on PD-L1 blockade efficacy. Human and murine studies showed high levels of functional PD-L1 expression in dendritic cells and macrophages in the tumor microenvironments and draining lymph nodes. Additionally, expression of PD-L1 on dendritic cells and macrophages in ovarian cancer and melanoma patients correlated with the efficacy of treatment with either anti–PD-1 alone or in combination with anti–CTLA-4. Thus, PD-L1–expressing dendritic cells and macrophages may mechanistically shape and therapeutically predict clinical efficacy of PD-L1/PD-1 blockade.



Zinc transporter Slc39a8 is essential for cardiac ventricular compaction
Isolated left ventricular noncompaction (LVNC) results from excessive trabeculation and impaired myocardial compaction during heart development. The extracellular matrix (ECM) that separates endocardium from myocardium plays a critical but poorly understood role in ventricular trabeculation and compaction. In an attempt to characterize solute carrier family 39 member 8–null (Slc39a8-null) mice, we discovered that homozygous null embryos do not survive embryogenesis, and exhibit a cardiac phenotype similar to human LVNC. Slc39a8 encodes a divalent metal cation importer that has been implicated in ECM degradation through the zinc/metal regulatory transcription factor 1 (Zn/MTF1) axis, which promotes the expression of ECM-degrading enzymes, including Adamts metalloproteinases. Here, we have shown that Slc39a8 is expressed by endothelial cells in the developing mouse heart, where it serves to maintain cellular Zn levels. Furthermore, Slc39a8-null hearts exhibited marked ECM accumulation and reduction of several Adamts metalloproteinases. Consistent with the in vivo observations, knockdown of SLC39A8 in HUVECs decreased ADAMTS1 transcription by decreasing cellular Zn uptake, and as a result, MTF1 transcriptional activity. Our study thus identifies a gene underlying ventricular trabeculation and compaction development, and a pathway regulating ECM during myocardial morphogenesis.



A double negative: inhibition of hepatic Gi signaling improves glucose homeostasis
Hepatic glucose production (HGP) is a key determinant of glucose homeostasis. Glucagon binding to its cognate seven-transmembrane Gs-coupled receptor in hepatocytes stimulates cAMP production, resulting in increased HGP. In this issue of the JCI, Rossi and colleagues tested the hypothesis that activation of hepatic Gi–coupled receptors, which should inhibit cAMP production, would oppose the cAMP-inducing action of glucagon and thereby decrease HGP. Surprisingly, however, the opposite occurred: activation of Gi signaling increased HGP via a novel mechanism, while inhibition of Gi signaling reduced HGP. These results define a new physiologic role for hepatic Gi signaling and identify a potential therapeutic target for HGP regulation.



RAGE binds preamyloid IAPP intermediates and mediates pancreatic β cell proteotoxicity
Islet amyloidosis is characterized by the aberrant accumulation of islet amyloid polypeptide (IAPP) in pancreatic islets, resulting in β cell toxicity, which exacerbates type 2 diabetes and islet transplant failure. It is not fully clear how IAPP induces cellular stress or how IAPP-induced toxicity can be prevented or treated. We recently defined the properties of toxic IAPP species. Here, we have identified a receptor-mediated mechanism of islet amyloidosis–induced proteotoxicity. In human diabetic pancreas and in cellular and mouse models of islet amyloidosis, increased expression of the receptor for advanced glycation endproducts (RAGE) correlated with human IAPP–induced (h-IAPP–induced) β cell and islet inflammation, toxicity, and apoptosis. RAGE selectively bound toxic intermediates, but not nontoxic forms of h-IAPP, including amyloid fibrils. The isolated extracellular ligand–binding domains of soluble RAGE (sRAGE) blocked both h-IAPP toxicity and amyloid formation. Inhibition of the interaction between h-IAPP and RAGE by sRAGE, RAGE-blocking antibodies, or genetic RAGE deletion protected pancreatic islets, β cells, and smooth muscle cells from h-IAPP–induced inflammation and metabolic dysfunction. sRAGE-treated h-IAPP Tg mice were protected from amyloid deposition, loss of β cell area, β cell inflammation, stress, apoptosis, and glucose intolerance. These findings establish RAGE as a mediator of IAPP-induced toxicity and suggest that targeting the IAPP/RAGE axis is a potential strategy to mitigate this source of β cell dysfunction in metabolic disease.



Role for VGLUT2 in selective vulnerability of midbrain dopamine neurons
Parkinson’s disease is characterized by the loss of dopamine (DA) neurons in the substantia nigra pars compacta (SNc). DA neurons in the ventral tegmental area are more resistant to this degeneration than those in the SNc, though the mechanisms for selective resistance or vulnerability remain poorly understood. A key to elucidating these processes may lie within the subset of DA neurons that corelease glutamate and express the vesicular glutamate transporter VGLUT2. Here, we addressed the potential relationship between VGLUT expression and DA neuronal vulnerability by overexpressing VGLUT in DA neurons of flies and mice. In Drosophila, VGLUT overexpression led to loss of select DA neuron populations. Similarly, expression of VGLUT2 specifically in murine SNc DA neurons led to neuronal loss and Parkinsonian behaviors. Other neuronal cell types showed no such sensitivity, suggesting that DA neurons are distinctively vulnerable to VGLUT2 expression. Additionally, most DA neurons expressed VGLUT2 during development, and coexpression of VGLUT2 with DA markers increased following injury in the adult. Finally, conditional deletion of VGLUT2 made DA neurons more susceptible to Parkinsonian neurotoxins. These data suggest that the balance of VGLUT2 expression is a crucial determinant of DA neuron survival. Ultimately, manipulation of this VGLUT2-dependent process may represent an avenue for therapeutic development.



OATP1B2 deficiency protects against paclitaxel-induced neurotoxicity
Paclitaxel is among the most widely used anticancer drugs and is known to cause a dose-limiting peripheral neurotoxicity, the initiating mechanisms of which remain unknown. Here, we identified the murine solute carrier organic anion–transporting polypeptide B2 (OATP1B2) as a mediator of paclitaxel-induced neurotoxicity. Additionally, using established tests to assess acute and chronic paclitaxel-induced neurotoxicity, we found that genetic or pharmacologic knockout of OATP1B2 protected mice from mechanically induced allodynia, thermal hyperalgesia, and changes in digital maximal action potential amplitudes. The function of this transport system was inhibited by the tyrosine kinase inhibitor nilotinib through a noncompetitive mechanism, without compromising the anticancer properties of paclitaxel. Collectively, our findings reveal a pathway that explains the fundamental basis of paclitaxel-induced neurotoxicity, with potential implications for its therapeutic management.



Dinaciclib induces immunogenic cell death and enhances anti–PD-1–mediated tumor suppression
Blockade of the checkpoint inhibitor programmed death 1 (PD1) has demonstrated remarkable success in the clinic for the treatment of cancer; however, a majority of tumors are resistant to anti-PD1 monotherapy. Numerous ongoing clinical combination therapy studies will likely reveal additional therapeutics that complement anti-PD1 blockade. Recent studies found that immunogenic cell death (ICD) improves T cell responses against different tumors, thus indicating that ICD may further augment antitumor immunity elicited by anti-PD1. Here, we observed antitumor activity following combinatorial therapy with anti-PD1 Ab and the cyclin-dependent kinase inhibitor dinaciclib in immunocompetent mouse tumor models. Dinaciclib induced a type I IFN gene signature within the tumor, leading us to hypothesize that dinaciclib potentiates the effects of anti-PD1 by eliciting ICD. Indeed, tumor cells treated with dinaciclib showed the hallmarks of ICD including surface calreticulin expression and release of high mobility group box 1 (HMGB1) and ATP. Mice treated with both anti-PD1 and dinaciclib showed increased T cell infiltration and DC activation within the tumor, indicating that this combination improves the overall quality of the immune response generated. These findings identify a potential mechanism for the observed benefit of combining dinaciclib and anti-PD1, in which dinaciclib induces ICD, thereby converting the tumor cell into an endogenous vaccine and boosting the effects of anti-PD1.



Long noncoding RNA BLACAT2 promotes bladder cancer–associated lymphangiogenesis and lymphatic metastasis
The prognosis for bladder cancer patients with lymph node (LN) metastasis is dismal and only minimally improved by current treatment modalities. Elucidation of the molecular mechanisms that underlie LN metastasis may provide clinical therapeutic strategies for LN-metastatic bladder cancer. Here, we report that a long noncoding RNA LINC00958, which we have termed bladder cancer–associated transcript 2 (BLACAT2), was markedly upregulated in LN-metastatic bladder cancer and correlated with LN metastasis. Overexpression of BLACAT2 promoted bladder cancer–associated lymphangiogenesis and lymphatic metastasis in both cultured bladder cancer cell lines and mouse models. Furthermore, we demonstrate that BLACAT2 epigenetically upregulated VEGF-C expression by directly associating with WDR5, a core subunit of human H3K4 methyltransferase complexes. Importantly, administration of an anti–VEGF-C antibody inhibited LN metastasis in BLACAT2-overexpressing bladder cancer. Taken together, these findings uncover a molecular mechanism in the lymphatic metastasis of bladder cancer and indicate that BLACAT2 may represent a target for clinical intervention in LN-metastatic bladder cancer.



JAK2/IDH-mutant–driven myeloproliferative neoplasm is sensitive to combined targeted inhibition
Patients with myeloproliferative neoplasms (MPNs) frequently progress to bone marrow failure or acute myeloid leukemia (AML), and mutations in epigenetic regulators such as the metabolic enzyme isocitrate dehydrogenase (IDH) are associated with poor outcomes. Here, we showed that combined expression of Jak2V617F and mutant IDH1R132H or Idh2R140Q induces MPN progression, alters stem/progenitor cell function, and impairs differentiation in mice. Jak2V617F Idh2R140Q–mutant MPNs were sensitive to small-molecule inhibition of IDH. Combined inhibition of JAK2 and IDH2 normalized the stem and progenitor cell compartments in the murine model and reduced disease burden to a greater extent than was seen with JAK inhibition alone. In addition, combined JAK2 and IDH2 inhibitor treatment also reversed aberrant gene expression in MPN stem cells and reversed the metabolite perturbations induced by concurrent JAK2 and IDH2 mutations. Combined JAK2 and IDH2 inhibitor therapy also showed cooperative efficacy in cells from MPN patients with both JAK2mut and IDH2mut mutations. Taken together, these data suggest that combined JAK and IDH inhibition may offer a therapeutic advantage in this high-risk MPN subtype.



Aberrant TGF-β activation in bone tendon insertion induces enthesopathy-like disease
Enthesopathy is a disorder of bone, tendon, or ligament insertion. It represents one-fourth of all tendon-ligament diseases and is one of the most difficult tendon-ligament disorders to treat. Despite its high prevalence, the exact pathogenesis of this condition remains unknown. Here, we show that TGF-β was activated in both a semi-Achilles tendon transection (SMTS) mouse model and in a dorsiflexion immobilization (DI) mouse model of enthesopathy. High concentrations of active TGF-β recruited mesenchymal stromal stem cells (MSCs) and led to excessive vessel formation, bone deterioration, and fibrocartilage calcification. Transgenic expression of active TGF-β1 in bone also induced enthesopathy with a phenotype similar to that observed in SMTS and DI mice. Systemic inhibition of TGF-β activity by injection of 1D11, a TGF-β–neutralizing antibody, but not a vehicle antibody, attenuated the excessive vessel formation and restored uncoupled bone remodeling in SMTS mice. 1D11-treated SMTS fibrocartilage had increased proteoglycan and decreased collagen X and matrix metalloproteinase 13 expression relative to control antibody treatment. Notably, inducible knockout of the TGF-β type II receptor in mouse MSCs preserved the bone microarchitecture and fibrocartilage composition after SMTS relative to the WT littermate controls. Thus, elevated levels of active TGF-β in the enthesis bone marrow induce the initial pathological changes of enthesopathy, indicating that TGF-β inhibition could be a potential therapeutic strategy.



Latent HIV reservoirs exhibit inherent resistance to elimination by CD8+ T cells
The presence of persistent, latent HIV reservoirs in CD4+ T cells obstructs current efforts to cure infection. The so-called kick-and-kill paradigm proposes to purge these reservoirs by combining latency-reversing agents with immune effectors such as cytotoxic T lymphocytes. Support for this approach is largely based on success in latency models, which do not fully reflect the makeup of latent reservoirs in individuals on long-term antiretroviral therapy (ART). Recent studies have shown that CD8+ T cells have the potential to recognize defective proviruses, which comprise the vast majority of all infected cells, and that the proviral landscape can be shaped over time due to in vivo clonal expansion of infected CD4+ T cells. Here, we have shown that treating CD4+ T cells from ART-treated individuals with combinations of potent latency-reversing agents and autologous CD8+ T cells consistently reduced cell-associated HIV DNA, but failed to deplete replication-competent virus. These CD8+ T cells recognized and potently eliminated CD4+ T cells that were newly infected with autologous reservoir virus, ruling out a role for both immune escape and CD8+ T cell dysfunction. Thus, our results suggest that cells harboring replication-competent HIV possess an inherent resistance to CD8+ T cells that may need to be addressed to cure infection.



Endothelial Tie1–mediated angiogenesis and vascular abnormalization promote tumor progression and metastasis
The endothelial tyrosine kinase receptor Tie1 remains poorly characterized, largely owing to its orphan receptor status. Global Tie1 inactivation causes late embryonic lethality, thereby reflecting its importance during development. Tie1 also plays pivotal roles during pathologies such as atherosclerosis and tumorigenesis. In order to study the contribution of Tie1 to tumor progression and metastasis, we conditionally deleted Tie1 in endothelial cells at different stages of tumor growth and metastatic dissemination. Tie1 deletion during primary tumor growth in mice led to a decrease in microvessel density and an increase in mural cell coverage with improved vessel perfusion. Reduced angiogenesis and enhanced vascular normalization resulted in a progressive increase of intratumoral necrosis that caused a growth delay only at later stages of tumor progression. Concomitantly, surgical removal of the primary tumor decreased the number of circulating tumor cells, reduced metastasis, and prolonged overall survival. Additionally, Tie1 deletion in experimental murine metastasis models prevented extravasation of tumor cells into the lungs and reduced metastatic foci. Taken together, the data support Tie1 as a therapeutic target by defining its regulatory functions during angiogenesis and vascular abnormalization and identifying its role during metastasis.



Erythrocyte efferocytosis modulates macrophages towards recovery after intracerebral hemorrhage
Macrophages are a source of both proinflammatory and restorative functions in damaged tissue through complex dynamic phenotypic changes. Here, we sought to determine whether monocyte-derived macrophages (MDMs) contribute to recovery after acute sterile brain injury. By profiling the transcriptional dynamics of MDMs in the murine brain after experimental intracerebral hemorrhage (ICH), we found robust phenotypic changes in the infiltrating MDMs over time and demonstrated that MDMs are essential for optimal hematoma clearance and neurological recovery. Next, we identified the mechanism by which the engulfment of erythrocytes with exposed phosphatidylserine directly modulated the phenotype of both murine and human MDMs. In mice, loss of receptor tyrosine kinases AXL and MERTK reduced efferocytosis of eryptotic erythrocytes and hematoma clearance, worsened neurological recovery, exacerbated iron deposition, and decreased alternative activation of macrophages after ICH. Patients with higher circulating soluble AXL had poor 1-year outcomes after ICH onset, suggesting that therapeutically augmenting efferocytosis may improve functional outcomes by both reducing tissue injury and promoting the development of reparative macrophage responses. Thus, our results identify the efferocytosis of eryptotic erythrocytes through AXL/MERTK as a critical mechanism modulating macrophage phenotype and contributing to recovery from ICH.



HIF-1α promotes autophagic proteolysis of Dicer and enhances tumor metastasis
HIF-1α, one of the most extensively studied oncogenes, is activated by a variety of microenvironmental factors. The resulting biological effects are thought to depend on its transcriptional activity. The RNAse enzyme Dicer is frequently downregulated in human cancers, which has been functionally linked to enhanced metastatic properties; however, current knowledge of the upstream mechanisms regulating Dicer is limited. In the present study, we identified Dicer as a HIF-1α–interacting protein in multiple types of cancer cell lines and different human tumors. HIF-1α downregulated Dicer expression by facilitating its ubiquitination by the E3 ligase Parkin, thereby enhancing autophagy-mediated degradation of Dicer, which further suppressed the maturation of known tumor suppressors, such as the microRNA let-7 and microRNA-200b. Consequently, expression of HIF-1α facilitated epithelial-mesenchymal transition (EMT) and metastasis in tumor-bearing mice. Thus, this study uncovered a connection between oncogenic HIF-1α and the tumor-suppressive Dicer. This function of HIF-1α is transcription independent and occurs through previously unrecognized protein interaction–mediated ubiquitination and autophagic proteolysis.



Cancer-associated fibroblasts regulate endothelial adhesion protein LPP to promote ovarian cancer chemoresistance
The molecular mechanism by which cancer-associated fibroblasts (CAFs) confer chemoresistance in ovarian cancer is poorly understood. The purpose of the present study was to evaluate the roles of CAFs in modulating tumor vasculature, chemoresistance, and disease progression. Here, we found that CAFs upregulated the lipoma-preferred partner (LPP) gene in microvascular endothelial cells (MECs) and that LPP expression levels in intratumoral MECs correlated with survival and chemoresistance in patients with ovarian cancer. Mechanistically, LPP increased focal adhesion and stress fiber formation to promote endothelial cell motility and permeability. siRNA-mediated LPP silencing in ovarian tumor–bearing mice improved paclitaxel delivery to cancer cells by decreasing intratumoral microvessel leakiness. Further studies showed that CAFs regulate endothelial LPP via a calcium-dependent signaling pathway involving microfibrillar-associated protein 5 (MFAP5), focal adhesion kinase (FAK), ERK, and LPP. Thus, our findings suggest that targeting endothelial LPP enhances the efficacy of chemotherapy in ovarian cancer. Our data highlight the importance of CAF–endothelial cell crosstalk signaling in cancer chemoresistance and demonstrate the improved efficacy of using LPP-targeting siRNA in combination with cytotoxic drugs.



PBX transcription factors drive pulmonary vascular adaptation to birth
A critical event in the adaptation to extrauterine life is relaxation of the pulmonary vasculature at birth, allowing for a rapid increase in pulmonary blood flow that is essential for efficient gas exchange. Failure of this transition leads to pulmonary hypertension (PH), a major cause of newborn mortality associated with preterm birth, infection, hypoxia, and malformations including congenital diaphragmatic hernia (CDH). While individual vasoconstrictor and dilator genes have been identified, the coordination of their expression is not well understood. Here, we found that lung mesenchyme–specific deletion of CDH-implicated genes encoding pre–B cell leukemia transcription factors (Pbx) led to lethal PH in mice shortly after birth. Loss of Pbx genes resulted in the misexpression of both vasoconstrictors and vasodilators in multiple pathways that converge to increase phosphorylation of myosin in vascular smooth muscle (VSM) cells, causing persistent constriction. While targeting endothelin and angiotensin, which are upstream regulators that promote VSM contraction, was not effective, treatment with the Rho-kinase inhibitor Y-27632 reduced vessel constriction and PH in Pbx-mutant mice. These results demonstrate a lung-intrinsic, herniation-independent cause of PH in CDH. More broadly, our findings indicate that neonatal PH can result from perturbation of multiple pathways and suggest that targeting the downstream common effectors may be a more effective treatment for neonatal PH.



Microglia ablation alleviates myelin-associated catatonic signs in mice
The underlying cellular mechanisms of catatonia, an executive “psychomotor” syndrome that is observed across neuropsychiatric diseases, have remained obscure. In humans and mice, reduced expression of the structural myelin protein CNP is associated with catatonic signs in an age-dependent manner, pointing to the involvement of myelin-producing oligodendrocytes. Here, we showed that the underlying cause of catatonic signs is the low-grade inflammation of white matter tracts, which marks a final common pathway in Cnp-deficient and other mutant mice with minor myelin abnormalities. The inhibitor of CSF1 receptor kinase signaling, PLX5622, depleted microglia and alleviated the catatonic symptoms of Cnp mutants. Thus, microglia and low-grade inflammation of myelinated tracts emerged as the trigger of a previously unexplained mental condition. We observed a very high (25%) prevalence of individuals with catatonic signs in a deeply phenotyped schizophrenia sample (n = 1095). Additionally, we found the loss-of-function allele of a myelin-specific gene (CNP rs2070106-AA) associated with catatonia in 2 independent schizophrenia cohorts and also associated with white matter hyperintensities in a general population sample. Since the catatonic syndrome is likely a surrogate marker for other executive function defects, we suggest that microglia-directed therapies may be considered in psychiatric disorders associated with myelin abnormalities.



Micro(glial)-managing executive function: white matter inflammation drives catatonia
White matter abnormalities are prevalent in neuropsychiatric disorders such as schizophrenia, but it is unclear whether these abnormalities represent a cause or consequence of these disorders. Reduced levels of the myelin protein 2′-3′-cyclic nucleotide 3′-phosphodiesterase (CNP) are associated with the schizophrenic symptom catatonia in both humans and mouse models. In this issue of the JCI, Janova et al. show that reduced CNP levels correlate with catatonia and white matter inflammation in human subjects. Furthermore, they demonstrate that microglial ablation prevents and alleviates catatonic signs in Cnp–/– mice, indicating that microglial-mediated inflammation causes catatonia. Together, this study identifies a cellular mechanism by which subtle myelin abnormalities cause low-grade neuroinflammation and catatonic behavior.



Influenza-specific lung-resident memory T cells are proliferative and polyfunctional and maintain diverse TCR profiles
The human lung harbors a large population of resident memory T cells (Trm cells). These cells are perfectly positioned to mediate rapid protection against respiratory pathogens such as influenza virus, a highly contagious respiratory pathogen that continues to be a major public health burden. Animal models show that influenza-specific lung CD8+ Trm cells are indispensable for crossprotection against pulmonary infection with different influenza virus strains. However, it is not known whether influenza-specific CD8+ Trm cells present within the human lung have the same critical role in modulating the course of the disease. Here, we showed that human lung contains a population of CD8+ Trm cells that are highly proliferative and have polyfunctional progeny. We observed that different influenza virus–specific CD8+ T cell specificities differentiated into Trm cells with varying efficiencies and that the size of the influenza-specific CD8+ T cell population persisting in the lung directly correlated with the efficiency of differentiation into Trm cells. To our knowledge, we provide the first ex vivo dissection of paired T cell receptor (TCR) repertoires of human influenza–specific CD8+ Trm cells. Our data reveal diverse TCR profiles within the human lung Trm cells and a high degree of clonal sharing with other CD8+ T cell populations, a feature important for effective T cell function and protection against the generation of viral-escape mutants.



Hepatic Gi signaling regulates whole-body glucose homeostasis
An increase in hepatic glucose production (HGP) is a key feature of type 2 diabetes. Excessive signaling through hepatic Gs–linked glucagon receptors critically contributes to pathologically elevated HGP. Here, we tested the hypothesis that this metabolic impairment can be counteracted by enhancing hepatic Gi signaling. Specifically, we used a chemogenetic approach to selectively activate Gi-type G proteins in mouse hepatocytes in vivo. Unexpectedly, activation of hepatic Gi signaling triggered a pronounced increase in HGP and severely impaired glucose homeostasis. Moreover, increased Gi signaling stimulated glucose release in human hepatocytes. A lack of functional Gi-type G proteins in hepatocytes reduced blood glucose levels and protected mice against the metabolic deficits caused by the consumption of a high-fat diet. Additionally, we delineated a signaling cascade that links hepatic Gi signaling to ROS production, JNK activation, and a subsequent increase in HGP. Taken together, our data support the concept that drugs able to block hepatic Gi–coupled GPCRs may prove beneficial as antidiabetic drugs.



Fructose metabolism and metabolic disease
Increased sugar consumption is increasingly considered to be a contributor to the worldwide epidemics of obesity and diabetes and their associated cardiometabolic risks. As a result of its unique metabolic properties, the fructose component of sugar may be particularly harmful. Diets high in fructose can rapidly produce all of the key features of the metabolic syndrome. Here we review the biology of fructose metabolism as well as potential mechanisms by which excessive fructose consumption may contribute to cardiometabolic disease.



Blood will out: vascular contributions to Alzheimer’s disease
The fundamental pathology in Alzheimer’s disease (AD) is neuronal dysfunction leading to cognitive impairment. The amyloid-β peptide (Aβ), derived from amyloid precursor protein, is one driver of AD, but how it leads to neuronal dysfunction is not established. In this Review, I discuss the complexity of AD and possible cause-and-effect relationships between Aβ and the vascular and hemostatic systems. AD can be considered a multifactorial syndrome with various contributing pathological mechanisms. Therefore, as is routinely done with cancer, it will be important to classify patients with respect to their disease signature so that specific pathologies, including vascular pathways, can be therapeutically targeted.



Micro(glial)-managing executive function: white matter inflammation drives catatonia
White matter abnormalities are prevalent in neuropsychiatric disorders such as schizophrenia, but it is unclear whether these abnormalities represent a cause or consequence of these disorders. Reduced levels of the myelin protein 2′-3′-cyclic nucleotide 3′-phosphodiesterase (CNP) are associated with the schizophrenic symptom catatonia in both humans and mouse models. In this issue of the JCI, Janova et al. show that reduced CNP levels correlate with catatonia and white matter inflammation in human subjects. Furthermore, they demonstrate that microglial ablation prevents and alleviates catatonic signs in Cnp–/– mice, indicating that microglial-mediated inflammation causes catatonia. Together, this study identifies a cellular mechanism by which subtle myelin abnormalities cause low-grade neuroinflammation and catatonic behavior.



A double negative: inhibition of hepatic Gi signaling improves glucose homeostasis
Hepatic glucose production (HGP) is a key determinant of glucose homeostasis. Glucagon binding to its cognate seven-transmembrane Gs-coupled receptor in hepatocytes stimulates cAMP production, resulting in increased HGP. In this issue of the JCI, Rossi and colleagues tested the hypothesis that activation of hepatic Gi–coupled receptors, which should inhibit cAMP production, would oppose the cAMP-inducing action of glucagon and thereby decrease HGP. Surprisingly, however, the opposite occurred: activation of Gi signaling increased HGP via a novel mechanism, while inhibition of Gi signaling reduced HGP. These results define a new physiologic role for hepatic Gi signaling and identify a potential therapeutic target for HGP regulation.



The host protecting the tumor from the host — targeting PD‑L1 expressed by host cells
Tumors frequently escape from immune surveillance by hijacking the natural control mechanisms that regulate normal immune responses. The programmed death-1 receptor (PD‑1) on T cells normally helps limit excessive immune activation, but it can also suppress beneficial antitumor immunity. In the clinic, blocking either PD‑1 or one of its principal counterligands, programmed death–ligand 1 (PD‑L1), can lead to dramatic responses in certain patients. Because PD‑L1 can be expressed by both the tumor cells themselves and also the host cells, including host immune cells, the actual mechanistic target of therapy has remained unclear. In the current issue of the JCI, two papers, one by Tang and colleagues and the other by Lin and colleagues, used a variety of mouse tumor models to demonstrate that the relevant target for therapy in each case was the PD‑L1 molecules expressed by host cells and not by tumor cells. If this finding is generalized to humans, then it would suggest that the tumor persuades the host to actively suppress its own attempted immune response against the tumor cells.



Unravelling the fate of functional PD1+ T cells in chronic viral hepatitis
Hepatitis B virus (HBV) infection can be managed clinically with nucleos(t)ide therapy, which suppresses viral replication; however, these drugs must often be used long term, as they are unable to fully eliminate the virus. For many patients, discontinuation of treatment results in viral resurgence and hepatic flare, and there is not a reliable way to identify those individuals that can be successfully taken off nucleos(t)ide therapy. In this issue of the JCI, Rivino and colleagues report on their use of a multipronged approach to investigate potential biomarkers indicative of HBV-infected patients who can safely stop nucleos(t)ide therapy. The authors identified a population of HBV-specific, PD1-positive T cells that was present in HBV-infected patients who successfully discontinued treatment without hepatic flare, but not in those that developed flare upon treatment cessation. Together, these results support the concept that PD1+ cells may play an important role in viral control, the further evaluation of this T cell subset in preventing hepatic flare, and the development of assays to better detect this PD1+ T cell population in HBV-infected patients on nucleos(t)ide therapy.



B cells as biomarkers: predicting immune checkpoint therapy adverse events
Immune checkpoint inhibitors are becoming a cornerstone of cancer immunotherapy as a result of their clinical success in relieving immune suppression and driving durable antitumor T cell responses in certain subsets of patients. Unfortunately, checkpoint inhibition is also associated with treatment-related toxicities that result in a myriad of side effects, ranging from mild and manageable to severe and debilitating. In this issue of the JCI, Das and colleagues report an association between early therapy-induced changes in circulating B cells and an increased risk of high-grade immune-related adverse events (IRAEs) in patients treated with checkpoint inhibitors that target cytotoxic T lymphocyte–associated antigen-4 (CTLA4) and programmed cell death protein 1 (PD1). These findings identify potential predictive biomarkers for high-grade IRAEs that may be leveraged to improve patient monitoring and may prompt new treatment strategies to prevent IRAEs.



PD-L1 on host cells is essential for PD-L1 blockade–mediated tumor regression
Programmed death–ligand 1 (PD-L1) expression on tumor cells is essential for T cell impairment, and PD-L1 blockade therapy has shown unprecedented durable responses in several clinical studies. Although higher expression of PD-L1 on tumor cells is associated with a better immune response after Ab blockade, some PD-L1–negative patients also respond to this therapy. In the current study, we explored whether PD-L1 on tumor or host cells was essential for anti–PD-L1–mediated therapy in 2 different murine tumor models. Using real-time imaging in whole tumor tissues, we found that anti–PD-L1 Ab accumulates in tumor tissues, regardless of the status of PD-L1 expression on tumor cells. We further observed that, while PD-L1 on tumor cells was largely dispensable for the response to checkpoint blockade, PD-L1 in host myeloid cells was essential for this response. Additionally, PD-L1 signaling in defined antigen-presenting cells (APCs) negatively regulated and inhibited T cell activation. PD-L1 blockade inside tumors was not sufficient to mediate regression, as limiting T cell trafficking reduced the efficacy of the blockade. Together, these findings demonstrate that PD-L1 expressed in APCs, rather than on tumor cells, plays an essential role in checkpoint blockade therapy, providing an insight into the mechanisms of this therapy.



Cancer-associated fibroblasts regulate endothelial adhesion protein LPP to promote ovarian cancer chemoresistance
The molecular mechanism by which cancer-associated fibroblasts (CAFs) confer chemoresistance in ovarian cancer is poorly understood. The purpose of the present study was to evaluate the roles of CAFs in modulating tumor vasculature, chemoresistance, and disease progression. Here, we found that CAFs upregulated the lipoma-preferred partner (LPP) gene in microvascular endothelial cells (MECs) and that LPP expression levels in intratumoral MECs correlated with survival and chemoresistance in patients with ovarian cancer. Mechanistically, LPP increased focal adhesion and stress fiber formation to promote endothelial cell motility and permeability. siRNA-mediated LPP silencing in ovarian tumor–bearing mice improved paclitaxel delivery to cancer cells by decreasing intratumoral microvessel leakiness. Further studies showed that CAFs regulate endothelial LPP via a calcium-dependent signaling pathway involving microfibrillar-associated protein 5 (MFAP5), focal adhesion kinase (FAK), ERK, and LPP. Thus, our findings suggest that targeting endothelial LPP enhances the efficacy of chemotherapy in ovarian cancer. Our data highlight the importance of CAF–endothelial cell crosstalk signaling in cancer chemoresistance and demonstrate the improved efficacy of using LPP-targeting siRNA in combination with cytotoxic drugs.



Erythrocyte efferocytosis modulates macrophages towards recovery after intracerebral hemorrhage
Macrophages are a source of both proinflammatory and restorative functions in damaged tissue through complex dynamic phenotypic changes. Here, we sought to determine whether monocyte-derived macrophages (MDMs) contribute to recovery after acute sterile brain injury. By profiling the transcriptional dynamics of MDMs in the murine brain after experimental intracerebral hemorrhage (ICH), we found robust phenotypic changes in the infiltrating MDMs over time and demonstrated that MDMs are essential for optimal hematoma clearance and neurological recovery. Next, we identified the mechanism by which the engulfment of erythrocytes with exposed phosphatidylserine directly modulated the phenotype of both murine and human MDMs. In mice, loss of receptor tyrosine kinases AXL and MERTK reduced efferocytosis of eryptotic erythrocytes and hematoma clearance, worsened neurological recovery, exacerbated iron deposition, and decreased alternative activation of macrophages after ICH. Patients with higher circulating soluble AXL had poor 1-year outcomes after ICH onset, suggesting that therapeutically augmenting efferocytosis may improve functional outcomes by both reducing tissue injury and promoting the development of reparative macrophage responses. Thus, our results identify the efferocytosis of eryptotic erythrocytes through AXL/MERTK as a critical mechanism modulating macrophage phenotype and contributing to recovery from ICH.



HIF-1α promotes autophagic proteolysis of Dicer and enhances tumor metastasis
HIF-1α, one of the most extensively studied oncogenes, is activated by a variety of microenvironmental factors. The resulting biological effects are thought to depend on its transcriptional activity. The RNAse enzyme Dicer is frequently downregulated in human cancers, which has been functionally linked to enhanced metastatic properties; however, current knowledge of the upstream mechanisms regulating Dicer is limited. In the present study, we identified Dicer as a HIF-1α–interacting protein in multiple types of cancer cell lines and different human tumors. HIF-1α downregulated Dicer expression by facilitating its ubiquitination by the E3 ligase Parkin, thereby enhancing autophagy-mediated degradation of Dicer, which further suppressed the maturation of known tumor suppressors, such as the microRNA let-7 and microRNA-200b. Consequently, expression of HIF-1α facilitated epithelial-mesenchymal transition (EMT) and metastasis in tumor-bearing mice. Thus, this study uncovered a connection between oncogenic HIF-1α and the tumor-suppressive Dicer. This function of HIF-1α is transcription independent and occurs through previously unrecognized protein interaction–mediated ubiquitination and autophagic proteolysis.



Dinaciclib induces immunogenic cell death and enhances anti-PD1–mediated tumor suppression
Blockade of the checkpoint inhibitor programmed death 1 (PD1) has demonstrated remarkable success in the clinic for the treatment of cancer; however, a majority of tumors are resistant to anti-PD1 monotherapy. Numerous ongoing clinical combination therapy studies will likely reveal additional therapeutics that complement anti-PD1 blockade. Recent studies found that immunogenic cell death (ICD) improves T cell responses against different tumors, thus indicating that ICD may further augment antitumor immunity elicited by anti-PD1. Here, we observed antitumor activity following combinatorial therapy with anti-PD1 Ab and the cyclin-dependent kinase inhibitor dinaciclib in immunocompetent mouse tumor models. Dinaciclib induced a type I IFN gene signature within the tumor, leading us to hypothesize that dinaciclib potentiates the effects of anti-PD1 by eliciting ICD. Indeed, tumor cells treated with dinaciclib showed the hallmarks of ICD including surface calreticulin expression and release of high mobility group box 1 (HMGB1) and ATP. Mice treated with both anti-PD1 and dinaciclib showed increased T cell infiltration and DC activation within the tumor, indicating that this combination improves the overall quality of the immune response generated. These findings identify a potential mechanism for the observed benefit of combining dinaciclib and anti-PD1, in which dinaciclib induces ICD, thereby converting the tumor cell into an endogenous vaccine and boosting the effects of anti-PD1.



PBX transcription factors drive pulmonary vascular adaptation to birth
A critical event in the adaptation to extrauterine life is relaxation of the pulmonary vasculature at birth, allowing for a rapid increase in pulmonary blood flow that is essential for efficient gas exchange. Failure of this transition leads to pulmonary hypertension (PH), a major cause of newborn mortality associated with preterm birth, infection, hypoxia, and malformations including congenital diaphragmatic hernia (CDH). While individual vasoconstrictor and dilator genes have been identified, the coordination of their expression is not well understood. Here, we found that lung mesenchyme–specific deletion of CDH-implicated genes encoding pre–B cell leukemia transcription factors (Pbx) led to lethal PH in mice shortly after birth. Loss of Pbx genes resulted in the misexpression of both vasoconstrictors and vasodilators in multiple pathways that converge to increase phosphorylation of myosin in vascular smooth muscle (VSM) cells, causing persistent constriction. While targeting endothelin and angiotensin, which are upstream regulators that promote VSM contraction, was not effective, treatment with the Rho-kinase inhibitor Y-27632 reduced vessel constriction and PH in Pbx-mutant mice. These results demonstrate a lung-intrinsic, herniation-independent cause of PH in CDH. More broadly, our findings indicate that neonatal PH can result from perturbation of multiple pathways and suggest that targeting the downstream common effectors may be a more effective treatment for neonatal PH.



Hepatitis B virus–specific T cells associate with viral control upon nucleos(t)ide-analogue therapy discontinuation
BACKGROUND. The clinical management of chronic hepatitis B virus (HBV) patients is based exclusively on virological parameters that cannot independently determine in which patients nucleos(t)ide-analogue (NUC) therapy can be safely discontinued. NUCs efficiently suppress viral replication, but do not eliminate HBV. Thus, therapy discontinuation can be associated with virological and biochemical relapse and, consequently, therapy in the majority is life-long. METHODS. Since antiviral immunity is pivotal for HBV control, we investigated potential biomarkers for the safe discontinuation of NUCs within immune profiles of chronic HBV patients by utilizing traditional immunological assays (ELISPOT, flow cytometry) in conjunction with analyses of global non–antigen-specific immune populations (NanoString and CyTOF). Two distinct cohorts of 19 and 27 chronic HBV patients, respectively, were analyzed longitudinally prior to and after discontinuation of 2 different NUC therapy strategies. RESULTS. Absence of hepatic flares following discontinuation of NUC treatment correlated with the presence, during NUC viral suppression, of HBV core and polymerase-specific T cells that were contained within the ex vivo PD-1+ population. CONCLUSIONS. This study identifies the presence of functional HBV-specific T cells as a candidate immunological biomarker for safe therapy discontinuation in chronic HBV patients. Furthermore, the persistent and functional antiviral activity of PD-1+ HBV–specific T cells highlights the potential beneficial role of the expression of T cell exhaustion markers during human chronic viral infection. FUNDING. This work was funded by a Singapore Translational Research Investigator Award (NMRC/STaR/013/2012), the Eradication of HBV TCR Program (NMRC/TCR/014-NUHS/2015), the Singapore Immunology Network, the Wellcome Trust (107389/Z/15/Z), and a Barts and The London Charity (723/1795) grant.



RAGE binds preamyloid IAPP intermediates and mediates pancreatic β cell proteotoxicity
Islet amyloidosis is characterized by the aberrant accumulation of islet amyloid polypeptide (IAPP) in pancreatic islets, resulting in β cell toxicity, which exacerbates type 2 diabetes and islet transplant failure. It is not fully clear how IAPP induces cellular stress or how IAPP-induced toxicity can be prevented or treated. We recently defined the properties of toxic IAPP species. Here, we have identified a receptor-mediated mechanism of islet amyloidosis–induced proteotoxicity. In human diabetic pancreas and in cellular and mouse models of islet amyloidosis, increased expression of the receptor for advanced glycation endproducts (RAGE) correlated with human IAPP–induced (h-IAPP–induced) β cell and islet inflammation, toxicity, and apoptosis. RAGE selectively bound toxic intermediates, but not nontoxic forms of h-IAPP, including amyloid fibrils. The isolated extracellular ligand–binding domains of soluble RAGE (sRAGE) blocked both h-IAPP toxicity and amyloid formation. Inhibition of the interaction between h-IAPP and RAGE by sRAGE, RAGE-blocking antibodies, or genetic RAGE deletion protected pancreatic islets, β cells, and smooth muscle cells from h-IAPP–induced inflammation and metabolic dysfunction. sRAGE-treated h-IAPP Tg mice were protected from amyloid deposition, loss of β cell area, β cell inflammation, stress, apoptosis, and glucose intolerance. These findings establish RAGE as a mediator of IAPP-induced toxicity and suggest that targeting the IAPP/RAGE axis is a potential strategy to mitigate this source of β cell dysfunction in metabolic disease.



Insulin exits skeletal muscle capillaries by fluid-phase transport
Before insulin can stimulate myocytes to take up glucose, it must first move from the circulation to the interstitial space. The continuous endothelium of skeletal muscle (SkM) capillaries restricts insulin’s access to myocytes. The mechanism by which insulin crosses this continuous endothelium is critical to understand insulin action and insulin resistance; however, methodological obstacles have limited understanding of endothelial insulin transport in vivo. Here, we present an intravital microscopy technique to measure the rate of insulin efflux across the endothelium of SkM capillaries. This method involves development of a fully bioactive, fluorescent insulin probe, a gastrocnemius preparation for intravital microscopy, an automated vascular segmentation algorithm, and the use of mathematical models to estimate endothelial transport parameters. We combined direct visualization of insulin efflux from SkM capillaries with modeling of insulin efflux kinetics to identify fluid-phase transport as the major mode of transendothelial insulin efflux in mice. Model-independent experiments demonstrating that insulin movement is neither saturable nor affected by insulin receptor antagonism supported this result. Our finding that insulin enters the SkM interstitium by fluid-phase transport may have implications in the pathophysiology of SkM insulin resistance as well as in the treatment of diabetes with various insulin analogs.



Early B cell changes predict autoimmunity following combination immune checkpoint blockade
Combination checkpoint blockade (CCB) targeting inhibitory CTLA4 and PD1 receptors holds promise for cancer therapy. Immune-related adverse events (IRAEs) remain a major obstacle for the optimal application of CCB in cancer. Here, we analyzed B cell changes in patients with melanoma following treatment with either anti-CTLA4 or anti-PD1, or in combination. CCB therapy led to changes in circulating B cells that were detectable after the first cycle of therapy and characterized by a decline in circulating B cells and an increase in CD21lo B cells and plasmablasts. PD1 expression was higher in the CD21lo B cells, and B cell receptor sequencing of these cells demonstrated greater clonality and a higher frequency of clones compared with CD21hi cells. CCB induced proliferation in the CD21lo compartment, and single-cell RNA sequencing identified B cell activation in cells with genomic profiles of CD21lo B cells in vivo. Increased clonality of circulating B cells following CCB occurred in some patients. Treatment-induced changes in B cells preceded and correlated with both the frequency and timing of IRAEs. Patients with early B cell changes experienced higher rates of grade 3 or higher IRAEs 6 months after CCB. Thus, early changes in B cells following CCB may identify patients who are at increased risk of IRAEs, and preemptive strategies targeting B cells may reduce toxicities in these patients.



Influenza-specific lung-resident memory T cells are proliferative and polyfunctional and maintain diverse TCR profiles
The human lung harbors a large population of resident memory T cells (Trm cells). These cells are perfectly positioned to mediate rapid protection against respiratory pathogens such as influenza virus, a highly contagious respiratory pathogen that continues to be a major public health burden. Animal models show that influenza-specific lung CD8+ Trm cells are indispensable for crossprotection against pulmonary infection with different influenza virus strains. However, it is not known whether influenza-specific CD8+ Trm cells present within the human lung have the same critical role in modulating the course of the disease. Here, we showed that human lung contains a population of CD8+ Trm cells that are highly proliferative and have polyfunctional progeny. We observed that different influenza virus–specific CD8+ T cell specificities differentiated into Trm cells with varying efficiencies and that the size of the influenza-specific CD8+ T cell population persisting in the lung directly correlated with the efficiency of differentiation into Trm cells. To our knowledge, we provide the first ex vivo dissection of paired T cell receptor (TCR) repertoires of human influenza–specific CD8+ Trm cells. Our data reveal diverse TCR profiles within the human lung Trm cells and a high degree of clonal sharing with other CD8+ T cell populations, a feature important for effective T cell function and protection against the generation of viral-escape mutants.



Microglia ablation alleviates myelin-associated catatonic signs in mice
The underlying cellular mechanisms of catatonia, an executive “psychomotor” syndrome that is observed across neuropsychiatric diseases, have remained obscure. In humans and mice, reduced expression of the structural myelin protein CNP is associated with catatonic signs in an age-dependent manner, pointing to the involvement of myelin-producing oligodendrocytes. Here, we showed that the underlying cause of catatonic signs is the low-grade inflammation of white matter tracts, which marks a final common pathway in Cnp-deficient and other mutant mice with minor myelin abnormalities. The inhibitor of CSF1 receptor kinase signaling PLX5622 depleted microglia and alleviated the catatonic symptoms of Cnp mutants. Thus, microglia and low-grade inflammation of myelinated tracts emerged as the trigger of a previously unexplained mental condition. We observed a very high (25%) prevalence of individuals with catatonic signs in a deeply phenotyped schizophrenia sample (n = 1095). Additionally, we found the loss-of-function allele of a myelin-specific gene (CNP rs2070106-AA) associated with catatonia in 2 independent schizophrenia cohorts and also associated with white matter hyperintensities in a general population sample. Since the catatonic syndrome is likely a surrogate marker for other executive function defects, we suggest that microglia-directed therapies may be considered in psychiatric disorders associated with myelin abnormalities.



Hepatic Gi signaling regulates whole-body glucose homeostasis
An increase in hepatic glucose production (HGP) is a key feature of type 2 diabetes. Excessive signaling through hepatic Gs–linked glucagon receptors critically contributes to pathologically elevated HGP. Here, we tested the hypothesis that this metabolic impairment can be counteracted by enhancing hepatic Gi signaling. Specifically, we used a chemogenetic approach to selectively activate Gi-type G proteins in mouse hepatocytes in vivo. Unexpectedly, activation of hepatic Gi signaling triggered a pronounced increase in HGP and severely impaired glucose homeostasis. Moreover, increased Gi signaling stimulated glucose release in human hepatocytes. A lack of functional Gi-type G proteins in hepatocytes reduced blood glucose levels and protected mice against the metabolic deficits caused by the consumption of a high-fat diet. Additionally, we delineated a signaling cascade that links hepatic Gi signaling to ROS production, JNK activation, and a subsequent increase in HGP. Taken together, our data support the concept that drugs able to block hepatic Gi–coupled GPCRs may prove beneficial as antidiabetic drugs.



Critical roles of αII spectrin in brain development and epileptic encephalopathy
The nonerythrocytic α-spectrin-1 (SPTAN1) gene encodes the cytoskeletal protein αII spectrin. Mutations in SPTAN1 cause early infantile epileptic encephalopathy type 5 (EIEE5); however, the role of αII spectrin in neurodevelopment and EIEE5 pathogenesis is unknown. Prior work suggests that αII spectrin is absent in the axon initial segment (AIS) and contributes to a diffusion barrier in the distal axon. Here, we have shown that αII spectrin is expressed ubiquitously in rodent and human somatodendritic and axonal domains. CRISPR-mediated deletion of Sptan1 in embryonic rat forebrain by in utero electroporation caused altered dendritic and axonal development, loss of the AIS, and decreased inhibitory innervation. Overexpression of human EIEE5 mutant SPTAN1 in embryonic rat forebrain and mouse hippocampal neurons led to similar developmental defects that were also observed in EIEE5 patient-derived neurons. Additionally, patient-derived neurons displayed aggregation of spectrin complexes. Taken together, these findings implicate αII spectrin in critical aspects of dendritic and axonal development and synaptogenesis, and support a dominant-negative mechanism of SPTAN1 mutations in EIEE5.



Role for VGLUT2 in selective vulnerability of midbrain dopamine neurons
Parkinson’s disease is characterized by the loss of dopamine (DA) neurons in the substantia nigra pars compacta (SNc). DA neurons in the ventral tegmental area are more resistant to this degeneration than those in the SNc, though the mechanisms for selective resistance or vulnerability remain poorly understood. A key to elucidating these processes may lie within the subset of DA neurons that corelease glutamate and express the vesicular glutamate transporter VGLUT2. Here, we addressed the potential relationship between VGLUT expression and DA neuronal vulnerability by overexpressing VGLUT in DA neurons of flies and mice. In Drosophila, VGLUT overexpression led to loss of select DA neuron populations. Similarly, expression of VGLUT2 specifically in murine SNc DA neurons led to neuronal loss and Parkinsonian behaviors. Other neuronal cell types showed no such sensitivity, suggesting that DA neurons are distinctively vulnerable to VGLUT2 expression. Additionally, most DA neurons expressed VGLUT2 during development, and coexpression of VGLUT2 with DA markers increased following injury in the adult. Finally, conditional deletion of VGLUT2 made DA neurons more susceptible to Parkinsonian neurotoxins. These data suggest that the balance of VGLUT2 expression is a crucial determinant of DA neuron survival. Ultimately, manipulation of this VGLUT2-dependent process may represent an avenue for therapeutic development.



JAK2/IDH-mutant–driven myeloproliferative neoplasm is sensitive to combined targeted inhibition
Patients with myeloproliferative neoplasms (MPNs) frequently progress to bone marrow failure or acute myeloid leukemia (AML), and mutations in epigenetic regulators such as the metabolic enzyme isocitrate dehydrogenase (IDH) are associated with poor outcomes. Here, we showed that combined expression of Jak2V617F and mutant IDH1R132H or Idh2R140Q induces MPN progression, alters stem/progenitor cell function, and impairs differentiation in mice. Jak2V617F Idh2R140Q–mutant MPNs were sensitive to small-molecule inhibition of IDH. Combined inhibition of JAK2 and IDH2 normalized the stem and progenitor cell compartments in the murine model and reduced disease burden to a greater extent than was seen with JAK inhibition alone. In addition, combined JAK2 and IDH2 inhibitor treatment also reversed aberrant gene expression in MPN stem cells and reversed the metabolite perturbations induced by concurrent JAK2 and IDH2 mutations. Combined JAK2 and IDH2 inhibitor therapy also showed cooperative efficacy in cells from MPN patients with both JAK2mut and IDH2mut mutations. Taken together, these data suggest that combined JAK and IDH inhibition may offer a therapeutic advantage in this high-risk MPN subtype.



Host expression of PD-L1 determines efficacy of PD-L1 pathway blockade–mediated tumor regression
Programmed death-1 receptor (PD-L1, B7-H1) and programmed cell death protein 1 (PD-1) pathway blockade is a promising therapy for treating cancer. However, the mechanistic contribution of host and tumor PD-L1 and PD-1 signaling to the therapeutic efficacy of PD-L1 and PD-1 blockade remains elusive. Here, we evaluated 3 tumor-bearing mouse models that differ in their sensitivity to PD-L1 blockade and demonstrated a loss of therapeutic efficacy of PD-L1 blockade in immunodeficient mice and in PD-L1– and PD-1–deficient mice. In contrast, neither knockout nor overexpression of PD-L1 in tumor cells had an effect on PD-L1 blockade efficacy. Human and murine studies showed high levels of functional PD-L1 expression in dendritic cells and macrophages in the tumor microenvironments and draining lymph nodes. Additionally, expression of PD-L1 on dendritic cells and macrophages in ovarian cancer and melanoma patients correlated with the efficacy of treatment with either anti–PD-1 alone or in combination with anti–CTLA-4. Thus, PD-L1–expressing dendritic cells and macrophages may mechanistically shape and therapeutically predict clinical efficacy of PD-L1/PD-1 blockade.



OATP1B2 deficiency protects against paclitaxel-induced neurotoxicity
Paclitaxel is among the most widely used anticancer drugs and is known to cause a dose-limiting peripheral neurotoxicity, the initiating mechanisms of which remain unknown. Here, we identified the murine solute carrier organic anion–transporting polypeptide B2 (OATP1B2) as a mediator of paclitaxel-induced neurotoxicity. Additionally, using established tests to assess acute and chronic paclitaxel-induced neurotoxicity, we found that genetic or pharmacologic knockout of OATP1B2 protected mice from mechanically induced allodynia, thermal hyperalgesia, and changes in digital maximal action potential amplitudes. The function of this transport system was inhibited by the tyrosine kinase inhibitor nilotinib through a noncompetitive mechanism, without compromising the anticancer properties of paclitaxel. Collectively, our findings reveal a pathway that explains the fundamental basis of paclitaxel-induced neurotoxicity, with potential implications for its therapeutic management.



Zinc transporter Slc39a8 is essential for cardiac ventricular compaction
Isolated left ventricular noncompaction (LVNC) results from excessive trabeculation and impaired myocardial compaction during heart development. The extracellular matrix (ECM) that separates endocardium from myocardium plays a critical but poorly understood role in ventricular trabeculation and compaction. In an attempt to characterize solute carrier family 39 member 8–null (Slc39a8-null) mice, we discovered that homozygous null embryos do not survive embryogenesis and exhibit a cardiac phenotype similar to human LVNC. Slc39a8 encodes a divalent metal cation importer that has been implicated in ECM degradation through the zinc/metal regulatory transcription factor 1 (Zn/MTF1) axis, which promotes the expression of ECM-degrading enzymes, including Adamts metalloproteinases. Here, we have shown that Slc39a8 is expressed by endothelial cells in the developing mouse heart, where it serves to maintain cellular Zn levels. Furthermore, Slc39a8-null hearts exhibited marked ECM accumulation and reduction of several Adamts metalloproteinases. Consistent with the in vivo observations, knockdown of SLC39A8 in HUVECs decreased ADAMTS1 transcription by decreasing cellular Zn uptake and, as a result, MTF1 transcriptional activity. Our study thus identifies a gene underlying ventricular trabeculation and compaction development, and a pathway regulating ECM during myocardial morphogenesis.



Endothelial Tie1–mediated angiogenesis and vascular abnormalization promote tumor progression and metastasis
The endothelial tyrosine kinase receptor Tie1 remains poorly characterized, largely owing to its orphan receptor status. Global Tie1 inactivation causes late embryonic lethality, thereby reflecting its importance during development. Tie1 also plays pivotal roles during pathologies such as atherosclerosis and tumorigenesis. In order to study the contribution of Tie1 to tumor progression and metastasis, we conditionally deleted Tie1 in endothelial cells at different stages of tumor growth and metastatic dissemination. Tie1 deletion during primary tumor growth in mice led to a decrease in microvessel density and an increase in mural cell coverage with improved vessel perfusion. Reduced angiogenesis and enhanced vascular normalization resulted in a progressive increase of intratumoral necrosis that caused a growth delay only at later stages of tumor progression. Concomitantly, surgical removal of the primary tumor decreased the number of circulating tumor cells, reduced metastasis, and prolonged overall survival. Additionally, Tie1 deletion in experimental murine metastasis models prevented extravasation of tumor cells into the lungs and reduced metastatic foci. Taken together, the data support Tie1 as a therapeutic target by defining its regulatory functions during angiogenesis and vascular abnormalization and identifying its role during metastasis.



Aberrant TGF-β activation in bone tendon insertion induces enthesopathy-like disease
Enthesopathy is a disorder of bone, tendon, or ligament insertion. It represents one-fourth of all tendon-ligament diseases and is one of the most difficult tendon-ligament disorders to treat. Despite its high prevalence, the exact pathogenesis of this condition remains unknown. Here, we show that TGF-β was activated in both a semi-Achilles tendon transection (SMTS) mouse model and in a dorsiflexion immobilization (DI) mouse model of enthesopathy. High concentrations of active TGF-β recruited mesenchymal stromal stem cells (MSCs) and led to excessive vessel formation, bone deterioration, and fibrocartilage calcification. Transgenic expression of active TGF-β1 in bone also induced enthesopathy with a phenotype similar to that observed in SMTS and DI mice. Systemic inhibition of TGF-β activity by injection of 1D11, a TGF-β–neutralizing antibody, but not a vehicle antibody, attenuated the excessive vessel formation and restored uncoupled bone remodeling in SMTS mice. 1D11-treated SMTS fibrocartilage had increased proteoglycan and decreased collagen X and matrix metalloproteinase 13 expression relative to control antibody treatment. Notably, inducible knockout of the TGF-β type II receptor in mouse MSCs preserved the bone microarchitecture and fibrocartilage composition after SMTS relative to the WT littermate controls. Thus, elevated levels of active TGF-β in the enthesis bone marrow induce the initial pathological changes of enthesopathy, indicating that TGF-β inhibition could be a potential therapeutic strategy.



Long noncoding RNA BLACAT2 promotes bladder cancer–associated lymphangiogenesis and lymphatic metastasis
The prognosis for bladder cancer patients with lymph node (LN) metastasis is dismal and only minimally improved by current treatment modalities. Elucidation of the molecular mechanisms that underlie LN metastasis may provide clinical therapeutic strategies for LN-metastatic bladder cancer. Here, we report that a long noncoding RNA LINC00958, which we have termed bladder cancer–associated transcript 2 (BLACAT2), was markedly upregulated in LN-metastatic bladder cancer and correlated with LN metastasis. Overexpression of BLACAT2 promoted bladder cancer–associated lymphangiogenesis and lymphatic metastasis in both cultured bladder cancer cell lines and mouse models. Furthermore, we demonstrate that BLACAT2 epigenetically upregulated VEGF-C expression by directly associating with WDR5, a core subunit of human H3K4 methyltransferase complexes. Importantly, administration of an anti–VEGF-C antibody inhibited LN metastasis in BLACAT2-overexpressing bladder cancer. Taken together, these findings uncover a molecular mechanism in the lymphatic metastasis of bladder cancer and indicate that BLACAT2 may represent a target for clinical intervention in LN-metastatic bladder cancer.



Latent HIV reservoirs exhibit inherent resistance to elimination by CD8+ T cells
The presence of persistent, latent HIV reservoirs in CD4+ T cells obstructs current efforts to cure infection. The so-called kick-and-kill paradigm proposes to purge these reservoirs by combining latency-reversing agents with immune effectors such as cytotoxic T lymphocytes. Support for this approach is largely based on success in latency models, which do not fully reflect the makeup of latent reservoirs in individuals on long-term antiretroviral therapy (ART). Recent studies have shown that CD8+ T cells have the potential to recognize defective proviruses, which comprise the vast majority of all infected cells, and that the proviral landscape can be shaped over time due to in vivo clonal expansion of infected CD4+ T cells. Here, we have shown that treating CD4+ T cells from ART-treated individuals with combinations of potent latency-reversing agents and autologous CD8+ T cells consistently reduced cell-associated HIV DNA, but failed to deplete replication-competent virus. These CD8+ T cells recognized and potently eliminated CD4+ T cells that were newly infected with autologous reservoir virus, ruling out a role for both immune escape and CD8+ T cell dysfunction. Thus, our results suggest that cells harboring replication-competent HIV possess an inherent resistance to CD8+ T cells that may need to be addressed to cure infection.