Deep venous thrombi (DVTs) result from dysregulated coagulation that may be exacerbated by trauma, infection, or other inflammatory conditions. In this issue of the JCI, Yadav, Chi, et al. identify a role for the ATP-hydrolyzing enzyme CD39 (also known as ENTPD-1) in venous thrombogenesis. In a model of flow-restricted venous thrombosis, they show that CD39 deficiency enhances inflammasome activation and elevates circulating IL-1β, mediating increases in clot incidence, size, and fibrin content. These insights into pivotal roles of CD39 and the inflammasome in regulating vascular thrombosis and inflammation point to potential interventions for DVT risk.
Immune cell populations determine the balance between ongoing damage and repair following tissue injury. Cells responding to a tissue-damaged environment have significant bioenergetic and biosynthetic needs. In addition to supporting these needs, metabolic pathways govern the function of pro-repair immune cells, including regulatory T cells and tissue macrophages. In this Review, we explore how specific features of the tissue-damaged environment such as hypoxia, oxidative stress, and nutrient depletion serve as metabolic cues to promote or impair the reparative functions of immune cell populations. Hypoxia, mitochondrial DNA stress, and altered redox balance each contribute to mechanisms regulating the response to tissue damage. For example, hypoxia induces changes in regulatory T cell and macrophage metabolic profiles, including generation of 2-hydroxyglutarate, which inhibits demethylase reactions to modulate cell fate and function. Reactive oxygen species abundant in oxidative environments cause damage to mitochondrial DNA, initiating signaling pathways that likewise control pro-repair cell function. Nutrient depletion following tissue damage also affects pro-repair cell function through metabolic signaling pathways, specifically those sensitive to the redox state of the cell. The study of immunometabolism as an immediate sensor and regulator of the tissue-damaged environment provides opportunities to consider mechanisms that facilitate healthy repair of tissue injury.
Benjamin D. Singer, Navdeep S. Chandel
Acute organ injuries such as acute cerebrovascular accidents, myocardial infarction, acute kidney injury, acute lung injury, and others are among the leading causes of death worldwide. Dysregulated or insufficient organ repair mechanisms limit restoration of homeostasis and contribute to chronic organ failure. Studies reveal that both humans and mice harness potent non-stem cells that are capable of directly or indirectly promoting tissue repair. Specific populations of T lymphocytes have emerged as important reparative cells with context-specific actions. These T cells can resolve inflammation and secrete reparative cytokines and growth factors as well as interact with other immune and stromal cells to promote the complex and active process of tissue repair. This Review focuses on the major populations of T lymphocytes known to mediate tissue repair, their reparative mechanisms, and the diseases in which they have been implicated. Elucidating and harnessing the mechanisms that promote the reparative functions of these T cells could greatly improve organ dysfunction after acute injury.
Franco R. D’Alessio, Johanna T. Kurzhagen, Hamid Rabb
Macrophages are tissue-resident or infiltrated immune cells critical for innate immunity, normal tissue development, homeostasis, and repair of damaged tissue. Macrophage function is a sum of their ontogeny, the local environment in which they reside, and the type of injuries or pathogen to which they are exposed. In this Review, we discuss the role of macrophages in the restoration of tissue function after injury, highlighting important questions about how they respond to and modify the local microenvironment to restore homeostasis.
Satoshi Watanabe, Michael Alexander, Alexander V. Misharin, G.R. Scott Budinger
Neutrophils are the most abundant immune cells in humans and serve as first responders to a myriad of host perturbations. Equipped with a plethora of antimicrobial molecules, neutrophils invade sites of inflammation to eradicate pathogens and clear debris. Traditionally, neutrophils were thought to cause collateral tissue damage before dying at the site. However, the presence of neutrophil infiltration into sterile injuries (in the absence of infections) suggests additional roles for these cells. Now, the view of neutrophils as indiscriminate killers seems to be changing as evolving evidence suggests that neutrophils actively orchestrate resolution of inflammation and contribute to tissue repair. Novel concepts include the idea that neutrophils are key to revascularization and subsequently reverse-transmigrate back to the vasculature, actively leaving sites of tissue damage to re-home to functional niches in the lung and bone marrow. This Review scrutinizes the role of neutrophils in tissue damage and repair, discussing recent findings and raising unresolved questions around this intriguing immune cell.
Moritz Peiseler, Paul Kubes
Over the last ten years, immunologists have recognized the central importance of an emerging group of innate lymphoid cells (ILCs) in health and disease. Characterization of these cells has provided a molecular definition of ILCs and their tissue-specific functions. Although the lineage-defining transcription factors, cytokine production, and nomenclature parallel those of T helper cells, ILCs do not require adaptive immune programming. Both environmental and host-derived signals shape the function of these evolutionarily ancient cells, which provide pathogen protection and promote tissue restoration. As such, ILCs function as a double-edged sword, balancing the inflammatory and reparative responses that arise during injury and disease. This Review highlights our recent understanding of tissue-resident ILCs and the signals that regulate their contribution to inflammation and tissue repair in health and disease.
Jim G. Castellanos, Randy S. Longman
As the opioid addiction crisis reaches epidemic levels, the identification of opioid analgesics that lack abuse potential may provide a path to safer treatment of chronic pain. Preclinical studies have demonstrated that galanin affects physical dependence and rewarding actions associated with morphine. In the brain and periphery, galanin and opioids signal through their respective GPCRs, GalR1–3 and the μ-opioid receptor (MOR). In this issue of the JCI, Cai and collaborators reveal that heteromers between GalR1 and MOR in the rat ventral tegmental area attenuate the potency of methadone, but not other opioids, in stimulating the dopamine release that produces euphoria. These studies help us understand why some synthetic opioids, such as methadone, do not trigger the release of dopamine in the mesolimbic system but still possess strong analgesic properties.
Randal A. Serafini, Venetia Zachariou
Anemia is defined by low levels of circulating hemoglobin, resulting in insufficient tissue oxygenation. This condition results from both genetic and nutritional factors and affects more than a billion people worldwide. For the inherited anemias, progress made over the last 40 years has increased our understanding of the structural basis for normal red cell membrane function and allowed definition of the genetic and pathophysiological bases of many human RBC membrane disorders. Despite these advances, there are continued uncertainties in the genotype-phenotype relationship in cases of severe, membrane-linked anemia. In this issue of the JCI, Gallagher and colleagues have identified a severe form of inherited anemia that results from aberrant splicing of α-spectrin, which in turn leads to abnormal erythrocyte membrane structure and function. The identification and characterization of this splicing-associated genetic disease will facilitate diagnosis and treatment of severe anemia in affected patients. These findings not only improve understanding of red cell disorders, they are likely to impact many disciplines, as the disease-associated alternate branch point utilization defined in the report may be the underlying etiology for many other inherited or acquired disorders.
Autophagy is a Greek-derived concept that means “self-eating” and is increasingly recognized as an important regulator of homeostasis and disease. In this issue of the JCI, Yeganeh et al. report the important finding that intrinsic autophagy is required for normal progression of lung development. Conditional deletion of the beclin 1–encoding gene (Becn1) specifically within lung epithelial cells of embryonic mice resulted in neonatal lethal respiratory distress that was associated with negative impacts on airway branching and differentiation of airway epithelial cell lineages. The authors draw speculative parallels with the alveolar simplification phenotype of bronchopulmonary dysplasia in premature human infants and suggest that stimulation of autophagy by AMP-dependent kinase activation might conceivably rescue these phenotypes.
David Warburton, Saverio Bellusci
Multiple myeloma (MM) is a relatively common hematologic malignancy, and up to half of patients with MM present with renal dysfunction at the time of diagnosis. MM-associated renal injury has been linked to an excess level of monoclonal immunoglobulin free light chains (FLCs) in the circulation; however, it is not clear how these FLCs drive renal pathology. In this issue of the JCI, Ying et al. unravel a novel mechanism by which FLCs mediate renal injury in MM by inducing fibrotic and inflammatory pathways in the kidney. Specifically, FLC-mediated production of H2O2 was shown to activate JAK2/STAT1 signaling, increase production of IL-1β via induction of capsase-1, and promote activation of TGF-β via αvβ6 integrin. Moreover, the authors identified a tryptophan residue within a specific monoclonal FLC that was required for optimal H2O2 production and downstream signaling. A better understanding of the drivers of MM-associated renal injury has potential for the identification of promising therapeutic targets.
Erin B. Taylor, Michael J. Ryan
In this issue of the JCI, Panigrahy et al. demonstrate that preoperative administration of the antiinflammatory drug ketorolac or specialized proresolving mediators (SPM) called resolvins increases disease-free survival rates and prevents metastasis after surgery and chemotherapy in mouse models of cancer. The antitumor response was partially mediated by tumor-specific T cell immunity and immunological memory.
Esra Güç, Jeffrey W. Pollard
Motile cilia provide propulsion, and immotile ones are enriched with receptors. Both are required to establish left-right identity in the developing embryo and are also implicated in a wide range of human diseases. Abnormalities in cilial function underlie heterotaxy congenital heart disease (CHD) occurring in individuals with laterality disturbance. Mitochondrial function and cellular energetics, through mTOR and autophagy, are now linked with cilial function, revealing new mechanisms and candidate genes for syndromic human disease. In the current issue of the JCI, Burkhalter et al. ask the question: Can mitochondrial disturbances produce ciliopathy and does this explain some cases of heterotaxy?
Bill Chaudhry, Deborah J. Henderson
The polarization of macrophages is regulated by transcription factors, such as NF-κB and activator protein 1 (AP-1). In this manuscript, we delineated the role of the transcription factor Fos-related antigen 1 (Fra-1) during macrophage activation and development of arthritis. Network level interaction analysis of microarray data derived from Fra-1– or Fra-2–deficient macrophages revealed a central role of Fra-1, but not of Fra-2, in orchestrating the expression of genes related to wound response, Toll-like receptor activation, and interleukin signaling. ChIP sequencing and standard ChIP analyses of macrophages identified arginase 1 (Arg1) as a target of Fra-1. Luciferase reporter assays revealed that Fra-1 downregulated Arg1 expression by direct binding to the promoter region. Using macrophage-specific Fra-1– or Fra-2–deficient mice, we observed enhanced expression and activity of Arg1 and a reduction of arthritis in the absence of Fra-1, but not of Fra-2. This phenotype was reversed by treatment with the arginase inhibitor Nω-hydroxy-nor-ʟ-arginine, while ʟ-arginine supplementation increased arginase activity and alleviated arthritis, supporting the notion that reduced arthritis in macrophage-specific Fra-1–deficient mice resulted from enhanced Arg1 expression and activity. Moreover, patients with active rheumatoid arthritis (RA) showed increased Fra-1 expression in the peripheral blood and elevated Fra-1 protein in synovial macrophages compared with RA patients in remission. In addition, the Fra-1/ARG1 ratio in synovial macrophages was related to RA disease activity. In conclusion, these data suggest that Fra-1 orchestrates the inflammatory state of macrophages by inhibition of Arg1 expression and thereby impedes the resolution of inflammation.
Nicole Hannemann, Shan Cao, Daniel Eriksson, Anne Schnelzer, Jutta Jordan, Martin Eberhardt, Ulrike Schleicher, Jürgen Rech, Andreas Ramming, Steffen Uebe, Arif Ekici, Juan D. Cañete, Xiaoxiang Chen, Tobias Bäuerle, Julio Vera, Christian Bogdan, Georg Schett, Aline Bozec
Chronic myeloid leukemia (CML) results from hematopoietic stem cell transformation by the BCR-ABL kinase. Despite the success of BCR-ABL tyrosine kinase inhibitors (TKIs) in treating CML patients, leukemia stem cells (LSCs) resist elimination and persist as a major barrier to cure. Previous studies suggest that overexpression of the sirtuin 1 (SIRT1) deacetylase may contribute to LSC maintenance in CML. Here, by genetically deleting SIRT1 in transgenic CML mice, we definitively demonstrated an important role for SIRT1 in leukemia development. We identified a previously unrecognized role for SIRT1 in mediating increased mitochondrial oxidative phosphorylation in CML LSCs. We showed that mitochondrial alterations were kinase independent and that TKI treatment enhanced inhibition of CML hematopoiesis in SIRT1-deleted mice. We further showed that the SIRT1 substrate PGC-1α contributed to increased oxidative phosphorylation and TKI resistance in CML LSCs. These results reveal an important role for SIRT1 and downstream signaling mechanisms in altered mitochondrial respiration in CML LSCs.
Ajay Abraham, Shaowei Qiu, Balu K. Chacko, Hui Li, Andrew Paterson, Jianbo He, Puneet Agarwal, Mansi Shah, Robert Welner, Victor M. Darley-Usmar, Ravi Bhatia
The development of metastatic melanoma is thought to require the dynamic shifting of neoplastic cells between proliferative and invasive phenotypes. Contrary to this conventional “phenotype switching” model, we now show that disease progression can involve malignant melanoma cells simultaneously displaying proliferative and invasive properties. Using a genetic mouse model of melanoma in combination with in vitro analyses of melanoma cell lines, we found that conditional deletion of the downstream signaling molecule Smad4, which abrogates all canonical TGF-β signaling, indeed inhibited both tumor growth and metastasis. Conditional deletion of the inhibitory signaling factor Smad7, however, generated cells that are both highly invasive and proliferative, indicating that invasiveness is compatible with a high proliferation rate. In fact, conditional Smad7 deletion led to sustained melanoma growth and at the same time promoted massive metastasis formation, a result consistent with data indicating that low SMAD7 levels in patient tumors are associated with a poor survival. Our findings reveal that modulation of SMAD7 levels can overcome the need for phenotype switching during tumor progression and may thus represent a therapeutic target in metastatic disease.
Eylul Tuncer, Raquel R. Calçada, Daniel Zingg, Sandra Varum, Phil Cheng, Sandra N. Freiberger, Chu-Xia Deng, Ingo Kleiter, Mitchell P. Levesque, Reinhard Dummer, Lukas Sommer
Invasive fungal infection is a serious health threat with high morbidity and mortality. Current antifungal drugs only demonstrate partial success in improving prognosis. Furthermore, mechanisms regulating host defense against fungal pathogens remain elusive. Here, we report that the downstream of kinase 3 (Dok3) adaptor negatively regulates antifungal immunity in neutrophils. Our data revealed that Dok3 deficiency increased phagocytosis, proinflammatory cytokine production, and netosis in neutrophils, thereby enhancing mutant mouse survival against systemic infection with a lethal dose of the pathogenic fungus Candida albicans. Biochemically, Dok3 recruited protein phosphatase 1 (PP1) to dephosphorylate Card9, an essential player in innate antifungal defense, to dampen downstream NF-κB and JNK activation and immune responses. Thus, Dok3 suppresses Card9 signaling, and disrupting Dok3-Card9 interaction or inhibiting PP1 activity represents therapeutic opportunities to develop drugs to combat candidaemia.
Jia Tong Loh, Shengli Xu, Jian Xin Huo, Susana Soo-Yeon Kim, Yue Wang, Kong-Peng Lam
Identifying nonaddictive opioid medications is a high priority in medical science, but μ-opioid receptors (MORs) mediate both the analgesic and addictive effects of opioids. We found a significant pharmacodynamic difference between morphine and methadone that is determined entirely by heteromerization of MORs with galanin Gal1 receptors (Gal1Rs), rendering a profound decrease in the potency of methadone. This finding was explained by the weaker proficiency of methadone in activating the dopaminergic system as compared with morphine and predicted a dissociation of the therapeutic and euphoric effects of methadone, which was corroborated by a significantly lower incidence of self-reports of feeling “high” in methadone-medicated patients. These results suggest that μ-opioid–Gal1R heteromers mediate the dopaminergic effects of opioids. The results further suggest a lower addictive liability of some opioids, such as methadone, due to their selective low potency for the μ-opioid–Gal1R heteromer.
Ning-Sheng Cai, César Quiroz, Jordi Bonaventura, Alessandro Bonifazi, Thomas O. Cole, Julia Purks, Amy S. Billing, Ebonie Massey, Michael Wagner, Eric D. Wish, Xavier Guitart, William Rea, Sherry Lam, Estefanía Moreno, Verònica Casadó-Anguera, Aaron D. Greenblatt, Arthur E. Jacobson, Kenner C. Rice, Vicent Casadó, Amy H. Newman, John W. Winkelman, Michael Michaelides, Eric Weintraub, Nora D. Volkow, Annabelle M. Belcher, Sergi Ferré
Mobilized peripheral blood has become the primary source of hematopoietic stem and progenitor cells (HSPCs) for stem cell transplantation, with a 5-day course of granulocyte colony-stimulating factor (G-CSF) as the most common regimen used for HSPC mobilization. The CXCR4 inhibitor plerixafor is a more rapid mobilizer, yet not potent enough when used as a single agent, thus emphasizing the need for faster acting agents with more predictable mobilization responses and fewer side effects. We sought to improve hematopoietic stem cell transplantation by developing a new mobilization strategy in mice through combined targeting of the chemokine receptor CXCR2 and the very late antigen 4 (VLA4) integrin. Rapid and synergistic mobilization of HSPCs along with an enhanced recruitment of true HSCs was achieved when a CXCR2 agonist was coadministered in conjunction with a VLA4 inhibitor. Mechanistic studies revealed involvement of CXCR2 expressed on BM stroma in addition to stimulation of the receptor on granulocytes in the regulation of HSPC localization and egress. Given the rapid kinetics and potency of HSPC mobilization achieved by the VLA4 inhibitor and CXCR2 agonist combination in mice compared with currently approved HSPC mobilization methods, the combination represents an exciting potential strategy for clinical development in the future.
Darja Karpova, Michael P. Rettig, Julie Ritchey, Daniel Cancilla, Stephanie Christ, Leah Gehrs, Ezhilarasi Chendamarai, Moses O. Evbuomwan, Matthew Holt, Jingzhu Zhang, Grazia Abou-Ezzi, Hamza Celik, Eliza Wiercinska, Wei Yang, Feng Gao, Linda G. Eissenberg, Richard F. Heier, Stacy D. Arnett, Marvin J. Meyers, Michael J. Prinsen, David W. Griggs, Andreas Trumpp, Peter G. Ruminski, Dwight M. Morrow, Halvard B. Bonig, Daniel C. Link, John F. DiPersio
Oxidative stress is elevated in the recipients of allogeneic hematopoietic cell transplantation (allo-HCT) and likely contributes to the development of graft-versus-host disease (GVHD). GVHD is characterized by activation, expansion, cytokine production, and migration of alloreactive donor T cells, and remains a major cause of morbidity and mortality after allo-HCT. Hence, strategies to limit oxidative stress in GVHD are highly desirable. Thioredoxin-1 (Trx1) counteracts oxidative stress by scavenging ROS and regulating other enzymes that metabolize H2O2. The present study sought to elucidate the role of Trx1 in the pathophysiology of GVHD. Using murine and xenograft models of allogeneic bone marrow transplantation (allo-BMT) and genetic (human Trx1-Tg) as well as pharmacological (human recombinant Trx1 [RTrx1]) strategies, we found that Trx1-Tg donor T cells or administration of RTrx1 to the recipients significantly reduced GVHD severity. Mechanistically, we observed that RTrx1 reduced ROS accumulation and cytokine production of mouse and human T cells in response to alloantigen stimulation in vitro. In allo-BMT settings, we found that Trx1-Tg or RTrx1 decreased downstream signaling molecules, including NF-κB activation and T-bet expression, and reduced proliferation, IFN-γ production, and ROS accumulation in donor T cells within GVHD target organs. More importantly, administration of RTrx1 did not impair the graft-versus-leukemia effect. Taken together, the current work provides a strong rationale for, and demonstrates the feasibility of, targeting the ROS pathway, which can be readily translated to the clinic.
M. Hanief Sofi, Yongxia Wu, Steven D. Schutt, Min Dai, Anusara Daenthanasanmak, Jessica Heinrichs Voss, Hung Nguyen, David Bastian, Supinya Iamsawat, Shanmugam Panneer Selvam, Chen Liu, Nilanjana Maulik, Besim Ogretmen, Junfei Jin, Shikhar Mehrotra, Xue-Zhong Yu
Hypertension is a primary risk factor for cardiovascular diseases including myocardial infarction and stroke. Major determinants of blood pressure are vasodilatory factors such as nitric oxide (NO) released from the endothelium under the influence of fluid shear stress exerted by the flowing blood. Several endothelial signaling processes mediating fluid shear stress–induced formation and release of vasodilatory factors have been described. It is, however, still poorly understood how fluid shear stress induces these endothelial responses. Here we show that the endothelial mechanosensitive cation channel PIEZO1 mediated fluid shear stress–induced release of adrenomedullin, which in turn activated its Gs-coupled receptor. The subsequent increase in cAMP levels promoted the phosphorylation of endothelial NO synthase (eNOS) at serine 633 through protein kinase A (PKA), leading to the activation of the enzyme. This Gs/PKA-mediated pathway synergized with the AKT-mediated pathways leading to eNOS phosphorylation at serine 1177. Mice with endothelium-specific deficiency of adrenomedullin, the adrenomedullin receptor, or Gαs showed reduced flow-induced eNOS activation and vasodilation and developed hypertension. Our data identify fluid shear stress–induced PIEZO1 activation as a central regulator of endothelial adrenomedullin release and establish the adrenomedullin receptor and subsequent Gs-mediated formation of cAMP as a critical endothelial mechanosignaling pathway regulating basal endothelial NO formation, vascular tone, and blood pressure.
Andras Iring, Young-June Jin, Julián Albarrán-Juárez, Mauro Siragusa, ShengPeng Wang, Péter T. Dancs, Akiko Nakayama, Sarah Tonack, Min Chen, Carsten Künne, Anna M. Sokol, Stefan Günther, Alfredo Martínez, Ingrid Fleming, Nina Wettschureck, Johannes Graumann, Lee S. Weinstein, Stefan Offermanns
Because of the less-than-robust response to therapy and impact on choice of optimal chemotherapy and prognosis, chronic kidney disease has drawn attention in the treatment of multiple myeloma, a malignant hematologic disorder that can produce significant amounts of monoclonal immunoglobulin free light chains (FLCs). These low-molecular-weight proteins are relatively freely filtered through the glomerulus and are reabsorbed by the proximal tubule. The present study demonstrated that during the process of metabolism of immunoglobulin FLCs, ROS activated the STAT1 pathway in proximal tubule epithelium. STAT1 activation served as the seminal signaling molecule that produced the proinflammatory molecule IL-1β, as well as the profibrotic agent TGF-β by this portion of the nephron. These effects occurred in vivo and were produced specifically by the generation of hydrogen peroxide by the VL domain of the light chain. To the extent that the experiments reflect the human condition, these studies offer insights into the pathogenesis of progressive kidney failure in the setting of lymphoproliferative disorders, such as multiple myeloma, that feature increased circulating levels of monoclonal immunoglobulin fragments that require metabolism by the kidney.
Wei-Zhong Ying, Xingsheng Li, Sunil Rangarajan, Wenguang Feng, Lisa M. Curtis, Paul W. Sanders
Phosphorylation of dynamin-related protein 1 (Drp1) represents an important regulatory mechanism for mitochondrial fission. Here, we established the role of Drp1 serine 600 (Drp1S600) phosphorylation in mitochondrial fission in vivo and assessed the functional consequences of targeted elimination of the Drp1S600 phosphorylation site in the progression of diabetic nephropathy (DN). We generated a knockin mouse in which S600 was mutated to alanine (Drp1S600A). We found that diabetic Drp1S600A mice exhibited improved biochemical and histological features of DN along with reduced mitochondrial fission and diminished mitochondrial ROS in vivo. Importantly, we observed that the effect of Drp1S600 phosphorylation on mitochondrial fission in the diabetic milieu was stimulus dependent but not cell type dependent. Mechanistically, we show that mitochondrial fission in high-glucose conditions occurs through concomitant binding of phosphorylated Drp1S600 with mitochondrial fission factor (MFF) and actin-related protein 3 (Arp3), ultimately leading to accumulation of F-actin and Drp1 on the mitochondria. Taken together, these findings establish the idea that a single phosphorylation site in Drp1 can regulate mitochondrial fission and progression of DN in vivo and highlight the stimulus-specific consequences of Drp1S600 phosphorylation in mitochondrial dynamics.
Daniel L. Galvan, Jianyin Long, Nathanael Green, Benny H. Chang, Jamie S. Lin, Paul Schumacker, Luan D. Truong, Paul Overbeek, Farhad R. Danesh
Impaired intestinal epithelial barrier (IEB) function with loss of desmosomal junctional protein desmoglein 2 (DSG2) is a hallmark in the pathogenesis of inflammatory bowel disease (IBD). While previous studies have reported that glial cell line–derived neurotrophic factor (GDNF) promotes IEB function, the mechanisms are poorly understood. We hypothesized that GDNF is involved in the loss of DSG2, resulting in impaired IEB function as seen in IBD. In the inflamed intestine of patients with IBD, there was a decrease in GDNF concentrations accompanied by a loss of DSG2, changes of the intermediate filament system, and increased phosphorylation of p38 MAPK and cytokeratins. DSG2-deficient and RET-deficient Caco2 cells revealed that GDNF specifically recruits DSG2 to the cell borders, resulting in increased DSG2-mediated intercellular adhesion via the RET receptor. Challenge of Caco2 cells and enteroids with proinflammatory cytokines as well as dextran sulfate sodium–induced (DSS-induced) colitis in C57Bl/6 mice led to impaired IEB function with reduced DSG2 mediated by p38 MAPK–dependent phosphorylation of cytokeratins. GDNF blocked all inflammation-induced changes in the IEB. GDNF attenuates inflammation-induced impairment of IEB function caused by the loss of DSG2 through p38 MAPK–dependent phosphorylation of cytokeratin. The reduced GDNF in patients with IBD indicates a disease-relevant contribution to the development of IEB dysfunction.
Michael Meir, Natalie Burkard, Hanna Ungewiß, Markus Diefenbacher, Sven Flemming, Felix Kannapin, Christoph-Thomas Germer, Matthias Schweinlin, Marco Metzger, Jens Waschke, Nicolas Schlegel
About 1% of all newborns are affected by congenital heart disease (CHD). Recent findings identify aberrantly functioning cilia as a possible source for CHD. Faulty cilia also prevent the development of proper left-right asymmetry and cause heterotaxy, the incorrect placement of visceral organs. Intriguingly, signaling cascades such as mTOR that influence mitochondrial biogenesis also affect ciliogenesis, and can cause heterotaxy-like phenotypes in zebrafish. Here, we identify levels of mitochondrial function as a determinant for ciliogenesis and a cause for heterotaxy. We detected reduced mitochondrial DNA content in biopsies of heterotaxy patients. Manipulation of mitochondrial function revealed a reciprocal influence on ciliogenesis and affected cilia-dependent processes in zebrafish, human fibroblasts and Tetrahymena thermophila. Exome analysis of heterotaxy patients revealed an increased burden of rare damaging variants in mitochondria-associated genes as compared with 1000 Genome controls. Knock down of such candidate genes caused cilia elongation and ciliopathy-like phenotypes in zebrafish, which could not be rescued by RNA encoding damaging rare variants identified in heterotaxy patients. Our findings suggest that ciliogenesis is coupled to the abundance and function of mitochondria. Our data further reveal disturbed mitochondrial function as an underlying cause for heterotaxy-linked CHD and provide a mechanism for unexplained phenotypes of mitochondrial disease.
Martin D. Burkhalter, Arthi Sridhar, Pedro Sampaio, Raquel Jacinto, Martina S. Burczyk, Cornelia Donow, Max Angenendt, Competence Network for Congenital Heart Defects Investigators, Maja Hempel, Paul Walther, Petra Pennekamp, Heymut Omran, Susana S. Lopes, Stephanie M. Ware, Melanie Philipp
Calcineurin acts as a calcium-activated phosphatase that dephosphorylates various substrates, including members of the nuclear factor of activated T cells (NFAT) family, to trigger their nuclear translocation and transcriptional activity. However, the detailed mechanism regulating the recruitment of NFATs to calcineurin remains poorly understood. Here, we report that calcineurin A (CNA), encoded by PPP3CB or PPP3CC, is constitutively ubiquitinated on lysine 327, and this polyubiquitin chain is rapidly removed by ubiquitin carboxyl-terminal hydrolase 16 (USP16) in response to intracellular calcium stimulation. The K29-linked ubiquitination of CNA impairs NFAT recruitment and transcription of NFAT-targeted genes. USP16 deficiency prevents calcium-triggered deubiquitination of CNA in a manner consistent with defective maintenance and proliferation of peripheral T cells. T cell–specific USP16 knockout mice exhibit reduced severity of experimental autoimmune encephalitis and inflammatory bowel disease. Our data reveal the physiological function of CNA ubiquitination and its deubiquitinase USP16 in peripheral T cells. Notably, our results highlight a critical mechanism for the regulation of calcineurin activity and a novel immunosuppressive drug target for the treatment of autoimmune diseases.
Yu Zhang, Rong-bei Liu, Qian Cao, Ke-qi Fan, Ling-jie Huang, Jian-shuai Yu, Zheng-jun Gao, Tao Huang, Jiang-yan Zhong, Xin-tao Mao, Fei Wang, Peng Xiao, Yuan Zhao, Xin-hua Feng, Yi-yuan Li, Jin Jin
Deep vein thrombosis (DVT), caused by alterations in venous homeostasis, is the third most common cause of cardiovascular mortality, however, key molecular determinants in venous thrombosis have not been fully elucidated. Several lines of evidence indicate that DVT occurs at the intersection of dysregulated inflammation and coagulation. The enzyme ectonucleoside tri(di)phosphohydrolase (ENTPD1, also known as CD39) is a vascular ecto-apyrase on the surface of leukocytes and the endothelium that inhibits intravascular inflammation and thrombosis by hydrolysis of phosphodiester bonds from nucleotides released by activated cells. Here, we evaluated the contribution of CD39 to venous thrombosis in a restricted-flow model of murine inferior vena cava stenosis. CD39 deficiency conferred a greater than 2-fold increase in venous thrombogenesis, characterized by increased leukocyte engagement, neutrophil extracellular trap formation, fibrin, and local activation of tissue factor in the thrombotic milieu. This venous thrombogenesis was orchestrated by increased phosphorylation of the p65 subunit of NF-κB, activation of the NLR family pyrin domain–containing 3 (NLRP3) inflammasome, and IL-1β release in CD39-deficient mice. Substantiating these findings, an IL-1β–neutralizing antibody or the IL-1 receptor inhibitor anakinra attenuated the thrombosis risk in CD39-deficient mice. These data demonstrate that IL-1β is a key accelerant of venous thrombo-inflammation, which can be suppressed by CD39. CD39 inhibits in vivo crosstalk between inflammation and coagulation pathways and is a critical vascular checkpoint in venous thrombosis.
Vinita Yadav, Liguo Chi, Raymond Zhao, Benjamin E. Tourdot, Srilakshmi Yalavarthi, Benjamin N. Jacobs, Alison Banka, Hui Liao, Sharon Koonse, Anuli C. Anyanwu, Scott H. Visovatti, Michael A. Holinstat, J. Michelle Kahlenberg, Jason S. Knight, David J. Pinsky, Yogendra Kanthi
The etiology of severe hemolytic anemia in most patients with recessive hereditary spherocytosis (rHS) and the related disorder hereditary pyropoikilocytosis (HPP) is unknown. Whole-exome sequencing of DNA from probands of 24 rHS or HPP kindreds identified numerous mutations in erythrocyte membrane α-spectrin (SPTA1). Twenty-eight mutations were novel, with null alleles frequently found in trans to missense mutations. No mutations were identified in a third of SPTA1 alleles (17/48). WGS revealed linkage disequilibrium between the common rHS-linked αBH polymorphism and a rare intron 30 variant in all 17 mutation-negative alleles. In vitro minigene studies and in vivo splicing analyses revealed the intron 30 variant changes a weak alternate branch point (BP) to a strong BP. This change leads to increased utilization of an alternate 3′ splice acceptor site, perturbing normal α-spectrin mRNA splicing and creating an elongated mRNA transcript. In vivo mRNA stability studies revealed the newly created termination codon in the elongated transcript activates nonsense-mediated decay leading to spectrin deficiency. These results demonstrate that a unique mechanism of human genetic disease contributes to the etiology of a third of rHS cases, facilitating diagnosis and treatment of severe anemia and identifying a new target for therapeutic manipulation.
Patrick G. Gallagher, Yelena Maksimova, Kimberly Lezon-Geyda, Peter E. Newburger, Desiree Medeiros, Robin D. Hanson, Jennifer Rothman, Sara Israels, Donna A. Wall, Robert F. Sidonio Jr., Colin Sieff, L. Kate Gowans, Nupur Mittal, Roland Rivera-Santiago, David W. Speicher, Susan J. Baserga, Vincent P. Schulz
Influenza A virus–specific (IAV-specific) T cell responses are important correlates of protection during primary and subsequent infections. The generation and maintenance of robust IAV-specific T cell responses relies on T cell interactions with dendritic cells (DCs). In this study, we explore the role of the nucleotide-binding domain leucine-rich repeat–containing receptor family member NLRC4 in modulating the DC phenotype during IAV infection. Nlrc4–/– mice had worsened survival and increased viral titers during infection, normal innate immune cell recruitment, and IAV-specific CD8+ T cell responses, but severely blunted IAV-specific CD4+ T cell responses compared with WT mice. The defect in the pulmonary IAV–specific CD4+ T cell response was not a result of defective priming or migration of these cells in Nlrc4–/– mice but was instead due to an increase in FasL+ DCs, resulting in IAV-specific CD4+ T cell death. Together, our data support a role for NLRC4 in regulating the phenotype of lung DCs during a respiratory viral infection and thereby influencing the magnitude of protective T cell responses.
Emma E. Hornick, Jargalsaikhan Dagvadorj, Zeb R. Zacharias, Ann M. Miller, Ryan A. Langlois, Peter Chen, Kevin L. Legge, Gail A. Bishop, Fayyaz S. Sutterwala, Suzanne L. Cassel
Ritonavir (RTV) is on the World Health Organization’s list of essential medicines for antiretroviral therapy, but can cause hepatotoxicity by unknown mechanisms. Multiple clinical studies found that hepatotoxicity occurred in 100% of participants who were pretreated with rifampicin or efavirenz followed by RTV-containing regimens. Both rifampicin and efavirenz are activators of the pregnane X receptor (PXR), a transcription factor with marked interspecies differences in ligand-dependent activation. Using PXR-humanized mouse models, we recapitulated the RTV hepatotoxicity observed in the clinic. PXR was found to modulate RTV hepatotoxicity through CYP3A4-dependent pathways involved in RTV bioactivation, oxidative stress, and endoplasmic reticulum stress. In summary, the current work demonstrated the essential roles of human PXR and CYP3A4 in RTV hepatotoxicity, which can be applied to guide the safe use of RTV-containing regimens in the clinic.
Amina I. Shehu, Jie Lu, Pengcheng Wang, Junjie Zhu, Yue Wang, Da Yang, Deborah McMahon, Wen Xie, Frank J. Gonzalez, Xiaochao Ma
Bronchopulmonary dysplasia (BPD) remains a major respiratory illness in extremely premature infants. The biological mechanisms leading to BPD are not fully understood, although an arrest in lung development has been implicated. The current study aimed to investigate the occurrence of autophagy in the developing mouse lung and its regulatory role in airway branching and terminal sacculi formation. We found 2 windows of epithelial autophagy activation in the developing mouse lung, both resulting from AMPK activation. Inhibition of AMPK-mediated autophagy led to reduced lung branching in vitro. Conditional deletion of beclin 1 (Becn1) in mouse lung epithelial cells (Becn1Epi-KO), either at early (E10.5) or late (E16.5) gestation, resulted in lethal respiratory distress at birth or shortly after. E10.5 Becn1Epi-KO lungs displayed reduced airway branching and sacculi formation accompanied by impaired vascularization, excessive epithelial cell death, reduced mesenchymal thinning of the interstitial walls, and delayed epithelial maturation. E16.5 Becn1Epi-KO lungs had reduced terminal air sac formation and vascularization and delayed distal epithelial differentiation, a pathology similar to that seen in infants with BPD. Taken together, our findings demonstrate that intrinsic autophagy is an important regulator of lung development and morphogenesis and may contribute to the BPD phenotype when impaired.
Behzad Yeganeh, Joyce Lee, Leonardo Ermini, Irene Lok, Cameron Ackerley, Martin Post
A resident population of dendritic cells (DCs) has been identified in murine bone marrow, but its contribution to the regulation of hematopoiesis and establishment of the stem cell niche is largely unknown. Here, we show that murine bone marrow DCs are perivascular and have a type 2 conventional DC (cDC2) immunophenotype. RNA expression analysis of sorted bone marrow DCs showed that expression of many chemokines and chemokine receptors is distinct from that observed in splenic cDC2s, suggesting that bone marrow DCs might represent a unique DC population. A similar population of DCs was present in human bone marrow. Ablation of conventional DCs (cDCs) results in hematopoietic stem/progenitor cell (HSPC) mobilization that was greater than that seen with ablation of bone marrow macrophages, and cDC ablation also synergizes with granulocyte–colony stimulating factor to mobilize HSPCs. Ablation of cDCs was associated with an expansion of bone marrow endothelial cells and increased vascular permeability. CXCR2 expression in sinusoidal endothelial cells and the expression of 2 CXCR2 ligands, CXCL1 and CXCL2, in the bone marrow were markedly increased following cDC ablation. Treatment of endothelial cells in vitro with CXCL1 induced increased vascular permeability and HSPC transmigration. Finally, we showed that HSPC mobilization after cDC ablation is attenuated in mice lacking CXCR2 expression. Collectively, these data suggest that bone marrow DCs play an important role in regulating HSPC trafficking, in part, through regulation of sinusoidal CXCR2 signaling and vascular permeability.
Jingzhu Zhang, Teerawit Supakorndej, Joseph R. Krambs, Mahil Rao, Grazia Abou-Ezzi, Rachel Y. Ye, Sidan Li, Kathryn Trinkaus, Daniel C. Link
T cell therapy is a promising means to treat chronic hepatitis B virus (HBV) infection and HBV-associated hepatocellular carcinoma. T cells engineered to express an HBV-specific T cell receptor (TCR) may cure an HBV infection upon adoptive transfer. We investigated the therapeutic potential and safety of T cells stably expressing high-affinity HBV envelope– or core–specific TCRs recognizing European and Asian HLA-A2 subtypes. Both CD8+ and CD4+ T cells from healthy donors and patients with chronic hepatitis B became polyfunctional effector cells when grafted with HBV-specific TCRs and eliminated HBV from infected HepG2-NTCP cell cultures. A single transfer of TCR-grafted T cells into HBV-infected, humanized mice controlled HBV infection, and virological markers declined by 4 to 5 log or below the detection limit. Engineered T cells specifically cleared infected hepatocytes without damaging noninfected cells when, as in a typical clinical setting, only a minority of hepatocytes were infected. Cell death was compensated by hepatocyte proliferation, and alanine amino transferase levels peaking between days 5 and 7 normalized again thereafter. Cotreatment with the entry inhibitor myrcludex B ensured long-term control of HBV infection. Thus, T cells stably transduced with highly functional TCRs have the potential to mediate clearance of HBV-infected cells, causing limited liver injury.
Karin Wisskirchen, Janine Kah, Antje Malo, Theresa Asen, Tassilo Volz, Lena Allweiss, Jochen M. Wettengel, Marc Lütgehetmann, Stephan Urban, Tanja Bauer, Maura Dandri, Ulrike Protzer
Transfer RNAs (tRNAs) are a major class of noncoding RNA. Stress-induced cleavage of tRNA is highly conserved and results in tRNA fragments. Here, we found that specific tRNA fragments in plasma are associated with epilepsy. Small RNA-Seq of plasma samples collected during video EEG monitoring of patients with focal epilepsy identified significant differences in 3 tRNA fragments (5′GlyGCC, 5′AlaTGC, and 5′GluCTC) compared with samples from healthy controls. The levels of these tRNA fragments were higher in pre-seizure than in post-seizure samples, suggesting that they may serve as biomarkers of seizure risk in patients with epilepsy. In vitro studies confirmed that production and extracellular release of tRNA fragments were lower after epileptiform-like activity in hippocampal neurons. We designed PCR-based assays to quantify tRNA fragments in a cohort of pre- and post-seizure plasma samples from patients with focal epilepsy and from healthy controls. Receiver operating characteristic analysis indicated that tRNA fragments potently distinguished pre- from post-seizure patients. Elevated levels of tRNA fragments were not detected in patients with psychogenic nonepileptic seizures and did not result from medication tapering. This study potentially identifies a new class of epilepsy biomarker and reveals the possible existence of prodromal molecular patterns in blood that could be used to predict seizure risk.
Marion C. Hogg, Rana Raoof, Hany El Naggar, Naser Monsefi, Norman Delanty, Donncha F. O’Brien, Sebastian Bauer, Felix Rosenow, David C. Henshall, Jochen H.M. Prehn
Recent studies have demonstrated that CD4+ T cells can efficiently reject MHC-II–negative tumors. This requires indirect presentation of tumor-associated antigens on surrounding antigen-presenting cells. We hypothesized that intercellular transfer of proteins is not the sole consequence of cell death–mediated protein release, but depends on heat-shock cognate protein 70 (HSC70) and its KFERQ-like binding motif on substrate proteins. Using human Y chromosome antigen DBY, we showed that mutation of one of its 2 putative binding motifs markedly diminished T cell activation after indirect presentation and reduced protein-protein interaction with HSC70. Intercellular antigen transfer was shown to be independent of cell-cell contact, but relied on engulfment within secreted microvesicles. In vivo, alterations of the homologous KFERQ-like motif in murine DBY hampered tumor rejection, T cell activation, and migration into the tumor and substantially impaired survival. Collectively, we show that intercellular antigen transfer of DBY is tightly regulated via binding to HSC70 and that this mechanism influences recognition and rejection of MHC-II–negative tumors in vivo.
Sascha Kretschmann, Stefanie Herda, Heiko Bruns, Josefine Russ, Edith D. van der Meijden, Ursula Schlötzer-Schrehardt, Marieke Griffioen, Il-Kang Na, Andreas Mackensen, Anita N. Kremer
Cancer therapy is a double-edged sword, as surgery and chemotherapy can induce an inflammatory/immunosuppressive injury response that promotes dormancy escape and tumor recurrence. We hypothesized that these events could be altered by early blockade of the inflammatory cascade and/or by accelerating the resolution of inflammation. Preoperative, but not postoperative, administration of the nonsteroidal antiinflammatory drug ketorolac and/or resolvins, a family of specialized proresolving autacoid mediators, eliminated micrometastases in multiple tumor-resection models, resulting in long-term survival. Ketorolac unleashed anticancer T cell immunity that was augmented by immune checkpoint blockade, negated by adjuvant chemotherapy, and dependent on inhibition of the COX-1/thromboxane A2 (TXA2) pathway. Preoperative stimulation of inflammation resolution via resolvins (RvD2, RvD3, and RvD4) inhibited metastases and induced T cell responses. Ketorolac and resolvins exhibited synergistic antitumor activity and prevented surgery- or chemotherapy-induced dormancy escape. Thus, simultaneously blocking the ensuing proinflammatory response and activating endogenous resolution programs before surgery may eliminate micrometastases and reduce tumor recurrence.
Dipak Panigrahy, Allison Gartung, Jun Yang, Haixia Yang, Molly M. Gilligan, Megan L. Sulciner, Swati S. Bhasin, Diane R. Bielenberg, Jaimie Chang, Birgitta A. Schmidt, Julia Piwowarski, Anna Fishbein, Dulce Soler-Ferran, Matthew A. Sparks, Steven J. Staffa, Vidula Sukhatme, Bruce D. Hammock, Mark W. Kieran, Sui Huang, Manoj Bhasin, Charles N. Serhan, Vikas P. Sukhatme
Pardeep Kumar, F. Matthew Kuhlmann, Subhra Chakraborty, A. Louis Bourgeois, Jennifer Foulke-Abel, Brunda Tumala, Tim J. Vickers, David A. Sack, Barbara DeNearing, Clayton D. Harro, W. Shea Wright, Jeffrey C. Gildersleeve, Matthew A. Ciorba, Srikanth Santhanam, Chad K. Porter, Ramiro L. Gutierrez, Michael G. Prouty, Mark S. Riddle, Alexander Polino, Alaullah Sheikh, Mark Donowitz, James M. Fleckenstein