Issue published May 2, 2019
- Volume 4, Issue 9
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In this issue, Hammelmann et al. explore the role of cAMP in regulating hyperpolarization-activated cyclic nucleotide-gated channel type 2 (HCN2) firing patterns in the brain. They report that knockin mice with disrupted cAMP-dependent regulation of HCN2 are characterized by impaired visual learning, generalized seizures of thalamic origin, and altered sleep properties. The cover image shows merged pictures of primary hippocampal neurons transfected with HCN2, with staining of HCN2 (red), dendrites (green), and nuclei (blue).
On the cover: Abolishing cAMP sensitivity in HCN2 pacemaker channels induces generalized seizures
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Research Articles
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Abstract
The physiological process of defecation is directly controlled by colorectal motility. The transient receptor potential ankyrin 1 (TRPA1) channel is expressed in small intestine enterochromaffin cells and is involved in gastrointestinal motility via serotonin release. In the colorectum, however, enterochromaffin cell localization is largely distinct from that in the small intestine. Here, we investigated the role of lower gastrointestinal tract TRPA1 in modulating colorectal motility. We found that in colonic tissue, TRPA1 is predominantly expressed in mesenchymal cells of the lamina propria, which are clearly distinct from those in the small intestine. These cells coexpressed COX1 and microsomal prostaglandin E synthase-1. Intracolonic administration of TRPA1 agonists induced colonic contraction, which was suppressed by a prostaglandin E2 (PGE2) receptor 1 antagonist. TRPA1 activation induced calcium influx and PGE2 release from cultured human fibroblastic cells. In dextran sulfate sodium–treated animals, both TRPA1 and its endogenous agonist were dramatically increased in the colonic lamina propria, accompanied by abnormal colorectal contractions. Abnormal colorectal contractions were significantly prevented by pharmacological and genetic inhibition of TRPA1. In conclusion, in the lower gastrointestinal tract, mesenchymal TRPA1 activation results in PGE2 release and consequently promotes colorectal contraction, representing what we believe is a novel physiological and inflammatory bowel disease–associated mechanism of gastrointestinal motility.
Authors
Yanjing Yang, Shenglan Wang, Kimiko Kobayashi, Yongbiao Hao, Hirosato Kanda, Takashi Kondo, Yoko Kogure, Hiroki Yamanaka, Satoshi Yamamoto, Junxiang Li, Hiroto Miwa, Koichi Noguchi, Yi Dai
×Abstract
BACKGROUND Subgroups of patients with relapsed or refractory (R/R) chronic lymphocytic leukemia (CLL) exhibit suboptimal outcomes after standard therapies, including oral kinase inhibitors. We and others have previously reported on the safety and efficacy of autologous CD19-targeted CAR T cells for these patients. Here, we report safety and long-term follow-up of CAR T cell therapy with or without conditioning chemotherapy for patients with R/R CLL and indolent B cell non-Hodgkin lymphoma (B-NHL).METHODS We conducted a phase I clinical trial investigating CD19-targeted CAR T cells incorporating a CD28 costimulatory domain (19–28z). Seventeen of twenty patients received conditioning chemotherapy prior to CAR T cell infusion. Five patients with CLL received ibrutinib at the time of autologous T cell collection and/or CAR T cell administration.RESULTS This analysis included 16 patients with R/R CLL and 4 patients with R/R indolent B-NHL. Cytokine release syndrome (CRS) was observed in all 20 patients, but grade 3 and 4 CRS and neurological events were uncommon (10% for each). Ex vivo expansion of T cells and proportions of CAR T cells with the CD62L+CD127+ immunophenotype were significantly greater (P = 0.047; CD8 subset, P = 0.0061, CD4 subset) in patients on ibrutinib at leukapheresis. Three of twelve evaluable CLL patients receiving conditioning chemotherapy achieved complete response (CR) (2 had minimal residual disease–negative CR). All patients achieving CR remained progression free at median follow-up of 53 months.CONCLUSION Conditioning chemotherapy and 19–28z CAR T cells were acceptably tolerated across investigated dose levels in heavily pretreated patients with R/R CLL and indolent B-NHL, and a subgroup of patients achieved durable CR. Ibrutinib therapy may modulate autologous T cell phenotype.TRIAL REGISTRATION ClinicalTrials.gov NCT00466531.FUNDING Juno Therapeutics and NIH/National Cancer Institute Cancer Center Support Grant (P30-CA08748).
Authors
Mark B. Geyer, Isabelle Rivière, Brigitte Sénéchal, Xiuyan Wang, Yongzeng Wang, Terence J. Purdon, Meier Hsu, Sean M. Devlin, M. Lia Palomba, Elizabeth Halton, Yvette Bernal, Dayenne G. van Leeuwen, Michel Sadelain, Jae H. Park, Renier J. Brentjens
×Abstract
In demyelinating diseases, such as multiple sclerosis, demyelination of neuronal fibers impairs impulse conduction and causes axon degeneration. Although neuronal activity stimulates oligodendrocyte production and myelination in normal conditions, it remains unclear whether the activity of demyelinated axons restores their loss of function in a harmful environment. To investigate this question, we established a model to induce a moderate optogenetic stimulation of demyelinated axons in the corpus callosum at the level of the motor cortex in which cortical circuit activation and locomotor effects were reduced in adult freely moving mice. We demonstrate that a moderate activation of demyelinated axons enhances the differentiation of oligodendrocyte precursor cells onto mature oligodendrocytes but only under a repeated stimulation paradigm. This activity-dependent increase in the oligodendrocyte pool promotes an extensive remyelination and functional restoration of conduction, as revealed by ultrastructural analyses and compound action potential recordings. Our findings reveal the need for preserving an appropriate neuronal activity in the damaged tissue to promote oligodendrocyte differentiation and remyelination, likely by enhancing axon-oligodendroglia interactions. Our results provide new perspectives for translational research using neuromodulation in demyelinating diseases.
Authors
Fernando C. Ortiz, Chloé Habermacher, Mariana Graciarena, Pierre-Yves Houry, Akiko Nishiyama, Brahim Nait Oumesmar, María Cecilia Angulo
×Abstract
Obliterative bronchiolitis (OB) is a poorly understood airway disease characterized by the generation of fibrotic bronchiolar occlusions. In the lung transplant setting, OB is a pathological manifestation of bronchiolitis obliterans syndrome (BOS), which is a major impediment to long-term recipient survival. Club cells play a key role in bronchiolar epithelial repair, but whether they promote lung transplant tolerance through preventing OB remains unclear. We determined if OB occurs in mouse orthotopic lung transplants following conditional transgene-targeted club cell depletion. In syngeneic lung transplants club cell depletion leads to transient epithelial injury followed by rapid club cell–mediated repair. In contrast, allogeneic lung transplants develop severe OB lesions that are largely devoid of club cells despite immunosuppression treatment. Lung allograft club cell ablation also triggers the recognition of alloantigens, and pulmonary restricted self-antigens reported associated with BOS development. However, CD8+ T cell depletion restores club cell reparative responses and prevents OB. In addition, ex vivo analysis reveals a specific role for alloantigen-primed CD8+ T cells in inhibiting club cell proliferation and maintenance. Taken together, our results demonstrate a vital role for club cells in maintaining lung transplant tolerance and propose a model to identify the underlying mechanisms of OB.
Authors
Zhiyi Liu, Fuyi Liao, Davide Scozzi, Yuka Furuya, Kaitlyn N. Pugh, Ramsey Hachem, Delphine L. Chen, Marlene Cano, Jonathan M. Green, Alexander S. Krupnick, Daniel Kreisel, Anne Karina T. Perl, Howard J. Huang, Steven L. Brody, Andrew E. Gelman
×Abstract
High autophagic activity in podocytes, terminally differentiated cells that serve as main components of the kidney filtration barrier, is essential for podocyte survival under various challenges. How podocytes maintain such a high level of autophagy, however, remains unclear. Here we report that signal regulatory protein α (SIRPα) plays a key role in promoting podocyte autophagy. Unlike other glomerular cells, podocytes strongly expressed SIRPα, which was, however, downregulated in patients with focal segmental glomerulosclerosis and mice with experimental nephropathy. Podocyte SIRPα levels were inversely correlated with the severity of podocyte injury and proteinuria but positively with autophagy. Compared with WT littermates, Sirpa-deficient mice displayed greater age-related podocyte injury and proteinuria and developed more rapid and severe renal injury in various models of experimental nephropathy. Mechanistically, podocyte SIRPα strongly reduced Akt/GSK-3β/β-catenin signaling, leading to an increase in autophagic activity. Our findings thus demonstrate a critical protective role of SIRPα in podocyte survival via maintenance of autophagic activity.
Authors
Limin Li, Ying Liu, Shan Li, Rong Yang, Caihong Zeng, Weiwei Rong, Hongwei Liang, Mingchao Zhang, Xiaodong Zhu, Koby Kidder, Yuan Liu, Zhihong Liu, Ke Zen
×Abstract
The interplay among signaling events for endothelial cell (EC) senescence, apoptosis, and activation and how these pathological conditions promote atherosclerosis in the area exposed to disturbed flow (d-flow) in concert remain unclear. The aim of this study was to determine whether telomeric repeat-binding factor 2–interacting protein (TERF2IP), a member of the shelterin complex at the telomere, can regulate EC senescence, apoptosis, and activation simultaneously, and if so, by what molecular mechanisms. We found that d-flow induced p90RSK and TERF2IP interaction in a p90RSK kinase activity–dependent manner. An in vitro kinase assay revealed that p90RSK directly phosphorylated TERF2IP at the serine 205 (S205) residue, and d-flow increased TERF2IP S205 phosphorylation as well as EC senescence, apoptosis, and activation by activating p90RSK. TERF2IP phosphorylation was crucial for nuclear export of the TERF2IP-TRF2 complex, which led to EC activation by cytosolic TERF2IP-mediated NF-κB activation and also to senescence and apoptosis of ECs by depleting TRF2 from the nucleus. Lastly, using EC-specific TERF2IP-knockout (TERF2IP-KO) mice, we found that the depletion of TERF2IP inhibited d-flow–induced EC senescence, apoptosis, and activation, as well as atherosclerotic plaque formation. These findings demonstrate that TERF2IP is an important molecular switch that simultaneously accelerates EC senescence, apoptosis, and activation by S205 phosphorylation.
Authors
Sivareddy Kotla, Hang Thi Vu, Kyung Ae Ko, Yin Wang, Masaki Imanishi, Kyung-Sun Heo, Yuka Fujii, Tamlyn N. Thomas, Young Jin Gi, Hira Mazhar, Jesus Paez-Mayorga, Ji-Hyun Shin, Yunting Tao, Carolyn J. Giancursio, Jan L.M. Medina, Jack Taunton, Aldos J. Lusis, John P. Cooke, Keigi Fujiwara, Nhat-Tu Le, Jun-ichi Abe
×Abstract
We have previously reported that the carboxy-terminal proteolytic cleavage product of the COL6α3 chain that we refer to as “endotrophin” has potent effects on transformed mammary ductal epithelial cells in rodents. Endotrophin (ETP) is abundantly expressed in adipose tissue. It is a chemoattractant for macrophages, exerts effects on endothelial cells and through epithelial-mesenchymal transition (EMT) enhances progression of tumor cells. In a recombinant form, human endotrophin exerts similar effects on human macrophages and endothelial cells as its rodent counterpart. It enhances EMT in human breast cancer cells and upon overexpression in tumor cells, the cells become chemoresistant. Here, we report the identification of endotrophin from human plasma. It is circulating at higher levels in breast cancer patients. We have developed neutralizing monoclonal antibodies against human endotrophin and provide evidence for the effectiveness of these antibodies to curb tumor growth and enhance chemosensitivity in a nude mouse model carrying human tumor cell lesions. Combined, the data validate endotrophin as a viable target for anti-tumor therapy for human breast cancer and opens the possibility for further use of these new reagents for anti-fibrotic approaches in liver, kidney, bone marrow and adipose tissue.
Authors
Dawei Bu, Clair Crewe, Christine M. Kusminski, Ruth Gordillo, Alexandra L. Ghaben, Min Kim, Jiyoung Park, Hui Deng, Wei Xiong, Xiao-Zheng Liu, Per Eystein Lønning, Nils Halberg, Adan Rios, Yujun Chang, Anneliese Gonzalez, Ningyan Zhang, Zhiqiang An, Philipp E. Scherer
×Abstract
Chronic malaria is a major public health problem and significant challenge for disease eradication efforts. Despite its importance, the biological factors underpinning chronic malaria are not fully understood. Recent studies have shown that host metabolic state can influence malaria pathogenesis and transmission, but its role in chronicity is not known. Here, with the goal of identifying distinct modifications in the metabolite profiles of acute versus chronic malaria, metabolomics was performed on plasma from Plasmodium-infected humans and nonhuman primates with a range of parasitemias and clinical signs. In rhesus macaques infected with Plasmodium coatneyi, significant alterations in amines, carnitines, and lipids were detected during a high parasitemic acute phase and many of these reverted to baseline levels once a low parasitemic chronic phase was established. Plasmodium gene expression, studied in parallel in the macaques, revealed transcriptional changes in amine, fatty acid, lipid and energy metabolism genes, as well as variant antigen genes. Furthermore, a common set of amines, carnitines, and lipids distinguished acute from chronic malaria in plasma from human Plasmodium falciparum cases. In summary, distinct host-parasite metabolic environments have been uncovered that characterize acute versus chronic malaria, providing insights into the underlying host-parasite biology of malaria disease progression.
Authors
Regina Joice Cordy, Rapatbhorn Patrapuvich, Loukia N. Lili, Monica Cabrera-Mora, Jung-Ting Chien, Gregory K. Tharp, Manoj Khadka, Esmeralda V.S. Meyer, Stacey A. Lapp, Chester J. Joyner, AnaPatricia Garcia, Sophia Banton, ViLinh Tran, Viravarn Luvira, Siriwan Rungin, Teerawat Saeseu, Nattawan Rachaphaew, Suman B. Pakala, Jeremy D. DeBarry, MaHPIC Consortium, Jessica C. Kissinger, Eric A. Ortlund, Steven E. Bosinger, John W. Barnwell, Dean P. Jones, Karan Uppal, Shuzhao Li, Jetsumon Sattabongkot, Alberto Moreno, Mary R. Galinski
×Abstract
Imatinib (Gleevec) reverses type 1 diabetes (T1D) in NOD mice and is currently in clinical trials in individuals with recent-onset disease. While research has demonstrated that imatinib protects islet β cells from the harmful effects of ER stress, the role the immune system plays in its reversal of T1D has been less well understood, and specific cellular immune targets have not been identified. In this study, we demonstrate that B lymphocytes, an immune subset that normally drives diabetes pathology, are unexpectedly required for reversal of hyperglycemia in NOD mice treated with imatinib. In the presence of B lymphocytes, reversal was linked to an increase in serum insulin concentration, but not an increase in islet β cell mass or proliferation. However, improved β cell function was reflected by a partial recovery of expression of the transcription factor MafA, a sensitive marker of islet β cell stress that is important for adult β cell function. Imatinib treatment was found to increase the antioxidant capacity of B lymphocytes, improving ROS handling in NOD islets. This study reveals a mechanism through which imatinib enables B lymphocytes to orchestrate functional recovery of T1D β cells.
Authors
Christopher S. Wilson, Jason M. Spaeth, Jay Karp, Blair T. Stocks, Emilee M. Hoopes, Roland W. Stein, Daniel J. Moore
×Abstract
Breast cancer bone metastases often cause a debilitating incurable condition with osteolytic lesions, muscle weakness, and a high mortality. Current treatment comprises chemotherapy, irradiation, surgery, and antiresorptive drugs that restrict but do not revert bone destruction. In hormone receptor–negative breast cancer cell lines and human breast cancer tissue, we identified the expression of sclerostin, a soluble Wnt inhibitor that represses osteoblast differentiation and bone formation. In mice with breast cancer bone metastases, pharmacological inhibition of sclerostin using an anti-sclerostin antibody (Scl-Ab) reduced the metastatic burden. Furthermore, sclerostin inhibition prevented cancer-induced bone destruction by augmenting osteoblast-mediated bone formation and by reducing osteoclast-dependent bone resorption. During advanced disease, NF-κB and p38 signaling was increased in muscles in a TGF-β1–dependent manner, causing muscle fiber atrophy, muscle weakness, and tissue regeneration with an increase in Pax7-positive satellite cells. Scl-Ab treatment restored NF-κB and p38 signaling, the abundance of Pax7-positive cells, and muscle function. These effects improved the health and expanded the life span of cancer-bearing mice. Together, these results demonstrate that pharmacological inhibition of sclerostin reduces bone metastatic burden and muscle weakness, with a prolongation of survival time. This might provide novel options for treating musculoskeletal complications in breast cancer patients.
Authors
Eric Hesse, Saskia Schröder, Diana Brandt, Jenny Pamperin, Hiroaki Saito, Hanna Taipaleenmäki
×Abstract
Recent seminal studies have revealed that laboratory mice differ from adult humans with regard to the frequency, number, and distribution of memory T cells. Because our data show that memory T cells are more susceptible to sepsis-induced death than naive T cells, in this study we developed a model in which mice possess a memory T cell compartment more similar to that of adult humans, to better study immune responses during sepsis in the more physiologically relevant context of high frequencies of memory T cells. Using this model, we found that CD44hi memory T cells significantly upregulated the coinhibitory molecule 2B4 during sepsis, and 2B4+ memory T cells coexpressed markers of both activation and exhaustion. Genetic deficiency in 2B4 resulted in decreased mortality during sepsis. Mechanistically, this decreased mortality was associated with reduced caspase-3/7+ apoptotic T cells in 2B4–/– relative to WT, septic hosts. These results were corroborated by analysis of PBMCs isolated from human patients with sepsis, which showed increased frequencies of caspase-3/7+ apoptotic cells among 2B4+ relative to 2B4– T cells. Thus, 2B4 plays a critical role in sepsis-induced apoptosis in both murine memory T cells and those isolated from human patients with sepsis.
Authors
Jianfeng Xie, Ching-wen Chen, Yini Sun, Sonia J. Laurie, Wenxiao Zhang, Shunsuke Otani, Gregory S. Martin, Craig M. Coopersmith, Mandy L. Ford
×Abstract
Pancreatic ductal adenocarcinoma (PDA) is characterized by an activating mutation in KRAS. Direct inhibition of KRAS through pharmacological means remains a challenge; however, targeting key KRAS effectors has therapeutic potential. We investigated the contribution of TANK-binding kinase 1 (TBK1), a critical downstream effector of mutant active KRAS, to PDA progression. We report that TBK1 supports the growth and metastasis of KRAS-mutant PDA by driving an epithelial plasticity program in tumor cells that enhances invasive and metastatic capacity. Further, we identify that the receptor tyrosine kinase Axl induces TBK1 activity in a Ras-RalB–dependent manner. These findings demonstrate that TBK1 is central to an Axl-driven epithelial-mesenchymal transition in KRAS-mutant PDA and suggest that interruption of the Axl/TBK1 signaling cascade above or below KRAS has potential therapeutic efficacy in this recalcitrant disease.
Authors
Victoria H. Cruz, Emily N. Arner, Wenting Du, Alberto E. Bremauntz, Rolf A. Brekken
×Abstract
The prefrontal cortex controls food reward seeking and ingestion, playing important roles in directing attention, regulating motivation toward reward pursuit, and assigning reward salience and value. The cell types that mediate these behavioral functions, however, are not well described. We report here that optogenetic activation of vasoactive intestinal peptide–expressing (VIP-expressing) interneurons in both the infralimbic (IL) and prelimbic (PL) divisions of the medial prefrontal cortex in mice is sufficient to reduce acute, binge-like intake of high-calorie palatable food in the absence of any effect on low-calorie rodent chow intake in the sated animal. In addition, we discovered that the behavioral mechanisms associated with these changes in feeding differed between animals that underwent either IL or PL VIPergic stimulation. Although IL VIP neurons showed the ability to reduce palatable food intake, this effect was dependent upon the novelty and relative value of the food source. In addition, IL VIP neuron activation significantly reduced novel object and novel social investigative behavior. Activation of PL VIP neurons, however, produced a reduction in high-calorie palatable food intake that was independent of food novelty. Neither IL nor PL VIP excitation changed motivation to obtain food reward. Our data show how neurochemically defined populations of cortical interneurons can regulate specific aspects of food reward–driven behavior, resulting in a selective reduction in intake of highly valued food.
Authors
Brandon A. Newmyer, Ciarra M. Whindleton, Peter M. Klein, Mark P. Beenhakker, Marieke K. Jones, Michael M. Scott
×Abstract
Tregs require IL-2 signaling for signal transducer and activator of transcription 5–mediated (STAT5-mediated) induction of Foxp3. Although phosphatase 2A (PP2A) is a negative regulator of IL-2 production in effector T cells and Tregs do not produce IL-2, it is not known whether PP2A controls IL-2 signaling in Tregs. To explore the role of PP2A in IL-2 signaling in Tregs, we studied mice engineered to lack PP2A in all Foxp3-expressing cells. We report that PP2A is required to enable Foxp3 expression and to maintain sufficient numbers of Tregs in the thymus. We show for the first time to our knowledge that PP2A prevents the selective loss of surface IL-2Rβ and preserves IL-2R signaling potency in Tregs. The loss of IL-2Rβ in thymus- and spleen-derived Tregs that lack PP2A is because of increased sheddase activity. Pan-sheddase or selective ADAM10 (a disintegrin and metalloproteinase 10) inhibition, like forced expression of IL-2Rβ in PP2A-deficient Tregs, restored IL-2Rβ expression and signaling. Thus, PP2A restrains the sheddase activity of ADAM10 in Tregs to prevent the cleavage of IL-2Rβ from the cell surface to enable competent IL-2R signaling, which is essential for Tregs’ development and homeostasis.
Authors
Amir Sharabi, Hao Li, Isaac R. Kasper, Wenliang Pan, Esra Meidan, Maria G. Tsokos, Vaishali R. Moulton, George C. Tsokos
×Abstract
Skeletal muscle weakness in patients suffering from rheumatoid arthritis (RA) adds to their impaired working abilities and reduced quality of life. However, little molecular insight is available on muscle weakness associated with RA. Oxidative stress has been implicated in the disease pathogenesis of RA. Here, we show that oxidative posttranslational modifications of the contractile machinery targeted to actin result in impaired actin polymerization and reduced force production. Using mass spectrometry, we identified the actin residues targeted by oxidative 3-nitrotyrosine (3-NT) or malondialdehyde (MDA) adduct modifications in weakened skeletal muscle from mice with arthritis and patients afflicted by RA. The residues were primarily located in 3 distinct regions positioned at matching surface areas of the skeletal muscle actin molecule from arthritic mice and patients with RA. Moreover, molecular dynamics simulations revealed that these areas, here coined “hotspots,” are important for the stability of the actin molecule and its capacity to generate filaments and interact with myosin. Together, these data demonstrate how oxidative modifications on actin promote muscle weakness in RA patients and may provide novel leads for targeted therapeutic treatment to improve muscle function.
Authors
Maarten M. Steinz, Malin Persson, Bejan Aresh, Karl Olsson, Arthur J. Cheng, Emma Ahlstrand, Mats Lilja, Tommy R. Lundberg, Eric Rullman, Kristina Ängeby Möller, Katalin Sandor, Sofia Ajeganova, Takashi Yamada, Nicole Beard, Björn C.G. Karlsson, Pasi Tavi, Ellinor Kenne, Camilla I. Svensson, Dilson E. Rassier, Roger Karlsson, Ran Friedman, Thomas Gustafsson, Johanna T. Lanner
×Abstract
Hyperpolarization-activated cyclic nucleotide–gated (HCN) channels are dually gated channels that are operated by voltage and by neurotransmitters via the cAMP system. cAMP-dependent HCN regulation has been proposed to play a key role in regulating circuit behavior in the thalamus. By analyzing a knockin mouse model (HCN2EA), in which binding of cAMP to HCN2 was abolished by 2 amino acid exchanges (R591E, T592A), we found that cAMP gating of HCN2 is essential for regulating the transition between the burst and tonic modes of firing in thalamic dorsal-lateral geniculate (dLGN) and ventrobasal (VB) nuclei. HCN2EA mice display impaired visual learning, generalized seizures of thalamic origin, and altered NREM sleep properties. VB-specific deletion of HCN2, but not of HCN4, also induced these generalized seizures of the absence type, corroborating a key role of HCN2 in this particular nucleus for controlling consciousness. Together, our data define distinct pathological phenotypes resulting from the loss of cAMP-mediated gating of a neuronal HCN channel.
Authors
Verena Hammelmann, Marc Sebastian Stieglitz, Henrik Hülle, Karim Le Meur, Jennifer Kass, Manuela Brümmer, Christian Gruner, René Dominik Rötzer, Stefanie Fenske, Jana Hartmann, Benedikt Zott, Anita Lüthi, Saskia Spahn, Markus Moser, Dirk Isbrandt, Andreas Ludwig, Arthur Konnerth, Christian Wahl-Schott, Martin Biel
×Abstract
Humoral immunity is important in limiting clinical disease in malaria, yet the longitudinal B cell response to infection remains unclear. We performed a 1-year prospective study in patients treated for acute Plasmodium falciparum malaria for the first time or with previous exposure to the disease. Using an unbiased exploratory approach with mass cytometry, followed by targeted flow cytometry, we found that approximately 80% of mature B cells that proliferated in response to acute infection expressed CD11c. Only approximately 40% of CD11c+ B cells displayed an atypical B cell phenotype, with the remaining cells primarily made up of activated and resting memory B cells. The CD11c+ B cells expanded rapidly following infection, with previous exposure to malaria resulting in a significantly larger increase compared with individuals with primary infection. This was attributed to an expansion of switched CD11c+ B cells that was absent in primary infected individuals. The rate of contraction of the CD11c+ B cell compartment was independent of previous exposure to malaria and displayed a slow decay, with a half-life of approximately 300 days. Collectively, these results identify CD11c as a marker of B cells responding to malaria and further highlight differences in primary and secondary B cell responses during infection.
Authors
Christopher Sundling, Caroline Rönnberg, Victor Yman, Muhammad Asghar, Peter Jahnmatz, Tadepally Lakshmikanth, Yang Chen, Jaromir Mikes, Mattias N. Forsell, Klara Sondén, Adnane Achour, Petter Brodin, Kristina E.M. Persson, Anna Färnert
×Abstract
Dysregulation of the JAK/STAT signaling pathway is associated with multiple sclerosis (MS) and its mouse model, experimental autoimmune encephalomyelitis (EAE). Suppressors of cytokine signaling (SOCS) negatively regulate the JAK/STAT pathway. We previously reported a severe, brain-targeted, atypical form of EAE in mice lacking Socs3 in myeloid cells (Socs3ΔLysM), and that this atypical EAE is associated with cerebellar neutrophil infiltration. There is emerging evidence that neutrophils are detrimental in the pathology of MS/EAE; however, their exact function is unclear. Here we demonstrate that neutrophils from the cerebellum of Socs3ΔLysM mice show a hyperactivated phenotype with excessive production of reactive oxygen species (ROS) at the peak of EAE. Neutralization of ROS in vivo delayed the onset and reduced severity of atypical EAE. Mechanistically, Socs3-deficient neutrophils exhibited enhanced signal transducer and activator of transcription 3 (STAT3) activation, a hyperactivated phenotype in response to granulocyte colony–stimulating factor (G-CSF), and upon G-CSF priming, increased ROS production. Neutralization of G-CSF in vivo significantly reduced the incidence and severity of the atypical EAE phenotype. Overall, our work elucidates that hypersensitivity of G-CSF/STAT3 signaling in Socs3ΔLysM mice leads to atypical EAE by enhanced neutrophil activation and increased oxidative stress, which may explain the detrimental role of G-CSF in MS patients.
Authors
Zhaoqi Yan, Wei Yang, Luke Parkitny, Sara A. Gibson, Kevin S. Lee, Forrest Collins, Jessy S. Deshane, Wayne Cheng, Amy S. Weinmann, Hairong Wei, Hongwei Qin, Etty N. Benveniste
×Abstract
Human antibody-secreting cells (ASCs) triggered by immunization are globally recognized as CD19loCD38hiCD27hi. Yet, different vaccines give rise to antibody responses of different longevity, suggesting ASC populations are heterogeneous. We define circulating-ASC heterogeneity in vaccine responses using multicolor flow cytometry, morphology, VH repertoire, and RNA transcriptome analysis. We also tested differential survival using a human cell-free system that mimics the bone marrow (BM) microniche. In peripheral blood, we identified 3 CD19+ and 2 CD19– ASC subsets. All subsets contributed to the vaccine-specific responses and were characterized by in vivo proliferation and activation. The VH repertoire demonstrated strong oligoclonality with extensive interconnectivity among the 5 subsets and switched memory B cells. Transcriptome analysis showed separation of CD19+ and CD19– subsets that included pathways such as cell cycle, hypoxia, TNF-α, and unfolded protein response. They also demonstrated similar long-term in vitro survival after 48 days. In summary, vaccine-induced ASCs with different surface markers (CD19 and CD138) are derived from shared proliferative precursors yet express distinctive transcriptomes. Equal survival indicates that all ASC compartments are endowed with long-lived potential. Accordingly, in vivo survival of peripheral long-lived plasma cells may be determined in part by their homing and residence in the BM microniche.
Authors
Swetha Garimalla, Doan C. Nguyen, Jessica L. Halliley, Christopher Tipton, Alexander F. Rosenberg, Christopher F. Fucile, Celia L. Saney, Shuya Kyu, Denise Kaminski, Yu Qian, Richard H. Scheuermann, Greg Gibson, Iñaki Sanz, F. Eun-Hyung Lee
×Abstract
BACKGROUND. In sepsis, there may be dysregulation in programed cell death pathways, typified by apoptosis and necroptosis. Programmed cell death pathways may contribute to variability in the immune response. TRAIL is a potent inducer of apoptosis. Receptor-interacting serine/threonine protein kinase-3 (RIPK3) is integral to the execution of necroptosis. We explored whether plasma TRAIL levels were associated with in-hospital mortality, organ dysfunction, and septic shock. We also explored the relationship between TRAIL and RIPK3. METHODS. We performed an observational study of critically ill adults admitted to intensive care units at 3 academic medical centers across 2 continents, using 1 as derivation and the other 2 as validation cohorts. Levels of TRAIL were measured in the plasma of 570 subjects by ELISA. RESULTS. In all cohorts, lower (<28.5 pg/ml) versus higher levels of TRAIL were associated with increased organ dysfunction (P ≤ 0.002) and septic shock (P ≤ 0.004). Lower TRAIL levels were associated with in-hospital mortality in 2 of 3 cohorts (Weill Cornell-Biobank of Critical Illness, P = 0.012; Brigham and Women’s Hospital Registry of Critical Illness, P = 0.011; Asan Medical Center, P = 0.369). Lower TRAIL was also associated with increased RIPK3 (P ≤ 0.001). CONCLUSION. Lower levels of TRAIL were associated with septic shock and organ dysfunction in 3 independent ICU cohorts. TRAIL was inversely associated with RIPK3 in all cohorts. FUNDING. NIH (R01-HL055330 and KL2-TR002385).
Authors
Edward J. Schenck, Kevin C. Ma, David R. Price, Thomas Nicholson, Clara Oromendia, Eliza Rose Gentzler, Elizabeth Sanchez, Rebecca M. Baron, Laura E. Fredenburgh, Jin-Won Huh, Ilias I. Siempos, Augustine M.K. Choi
×Abstract
Extracellular mRNAs (ex-mRNAs) potentially supersede extracellular miRNAs (ex-miRNAs) and other RNA classes as biomarkers. We performed conventional small-RNA-sequencing (sRNA-seq) and sRNA-seq with T4 polynucleotide kinase (PNK) end treatment of total extracellular RNAs (exRNAs) isolated from serum and platelet-poor EDTA, acid citrate dextrose (ACD), and heparin plasma to study the effect on ex-mRNA capture. Compared with conventional sRNA-seq, PNK treatment increased the detection of informative ex-mRNAs reads up to 50-fold. The exRNA pool was dominated by RNA originating from hematopoietic cells and platelets, with additional contribution from the liver. About 60% of the 15- to 42-nt reads originated from the coding sequences, in a pattern reminiscent of ribosome profiling. Blood sample type had a considerable influence on the exRNA profile. On average approximately 350–1100 distinct ex-mRNA transcripts were detected depending on plasma type. In serum, additional transcripts from neutrophils and hematopoietic cells increased this number to near 2300. EDTA and ACD plasma showed a destabilizing effect on ex‑mRNA and noncoding RNA ribonucleoprotein complexes compared with other plasma types. In a proof-of-concept study, we investigated differences between the exRNA profiles of patients with acute coronary syndrome and healthy controls. The improved tissue resolution of ex‑mRNAs after PNK treatment enabled us to detect a neutrophil signature in ACS that escaped detection by ex‑miRNA analysis.
Authors
Kemal M. Akat, Youngmin A. Lee, Arlene Hurley, Pavel Morozov, Klaas E.A. Max, Miguel Brown, Kimberly Bogardus, Anuoluwapo Sopeyin, Kai Hildner, Thomas G. Diacovo, Markus F. Neurath, Martin Borggrefe, Thomas Tuschl
×Abstract
IgG antinuclear antibodies (ANAs) are a dominant feature of several autoimmune diseases. We previously showed that systemic lupus erythematosus (SLE) is characterized by increased ANA+ IgG plasmablasts/plasma cells (PCs) through aberrant IgG PC differentiation rather than an antigen-specific tolerance defect. Here, we aimed to understand the differentiation pathways resulting in ANA+ IgG PCs in SLE patients. We demonstrate distinct profiles of ANA+ antigen-experienced B cells in SLE patients, characterized by either a high frequency of PCs or a high frequency of IgG+ memory B cells. This classification of SLE patients was unrelated to disease activity and remained stable over time in almost all patients, suggesting minimal influence of disease activity. A similar classification applies to antigen-specific B cell subsets in mice following primary immunization with T-independent and T-dependent antigens as well as in lupus-prone mouse models (MRL/lpr and NZB/W). We further show that, in both lupus-prone mice and SLE patients, the classification correlates with the serum autoantibody profile. In this study, we identified B cell phenotypes that we propose reflect an extrafollicular pathway for PC differentiation or a germinal center pathway, respectively. The classification we propose can be used to stratify patients for longitudinal studies and clinical trials.
Authors
Jolien Suurmond, Yemil Atisha-Fregoso, Ashley N. Barlev, Silvia A. Calderon, Meggan C. Mackay, Cynthia Aranow, Betty Diamond
×Abstract
Hypercholesterolemia and hypertension are two major risk factors for coronary artery diseases, which remain the major cause of mortality in the industrialized world. Current animal models of atherosclerosis do not recapitulate coronary plaque disruption, thrombosis, and myocardial infarction occurring in humans. Recently, we demonstrated that exposure of the heart to high pressure, by transverse aortic constriction (TAC), induced coronary lesions in ApoE–/– mice on chow diet. The aim of this study was to characterize the magnitude and location of coronary lesions in ApoE–/– mice after TAC and to assess the susceptibility of coronary plaque to disruption, leading to myocardial events. Here, we describe a reliable pathological condition in mice characterized by the development of coronary lesions and its progression, leading to myocardial infarction; this model better recapitulates human disease. Following TAC surgery, about 90% of ApoE–/– mice developed coronary lesions, especially in the left anterior descending artery, with 59% of the mice manifesting a different magnitude of LAD stenosis. Myocardial events, identified in 74% of the mice, were mainly due to coronary plaque thrombosis and occlusion. That TAC-induced development and progression of coronary lesions in ApoE–/– mice, leading to myocardial events, represents a potentially novel and important tool to investigate the development of coronary lesions and its sequelae in a setting that better resemble human conditions.
Authors
Alice Marino, Yi Zhang, Luisa Rubinelli, Maria Antonietta Riemma, James E. Ip, Annarita Di Lorenzo
×Abstract
Conjugated bile acids (CBAs), such as tauroursodeoxycholic acid (TUDCA), are known to resolve the inflammatory and unfolded protein response (UPR) in inflammatory diseases, such as asthma. Whether CBAs exert their beneficial effects on allergic airway responses via 1 arm or several arms of the UPR, or alternatively through the signaling pathways for conserved bile acid receptor, remains largely unknown. We used a house dust mite–induced (HDM-induced) murine model of asthma to evaluate and compare the effects of 5 CBAs and 1 unconjugated bile acid in attenuating allergen-induced UPR and airway responses. Expression of UPR-associated transcripts was assessed in airway brushings from human patients with asthma and healthy subjects. Here we show that CBAs, such as alanyl β-muricholic acid (AβM) and TUDCA, significantly decreased inflammatory, immune, and cytokine responses; mucus metaplasia; and airway hyperresponsiveness, as compared with other CBAs in a model of allergic airway disease. CBAs predominantly bind to activating transcription factor 6α (ATF6α) compared with the other canonical transducers of the UPR, subsequently decreasing allergen-induced UPR activation and resolving allergic airway disease, without significant activation of the bile acid receptors. TUDCA and AβM also attenuated other HDM-induced ER stress markers in the lungs of allergic mice. Quantitative mRNA analysis of airway epithelial brushings from human subjects demonstrated that several ATF6α-related transcripts were significantly upregulated in patients with asthma compared with healthy subjects. Collectively, these results demonstrate that CBA-based therapy potently inhibits the allergen-induced UPR and allergic airway disease in mice via preferential binding of the canonical transducer of the UPR, ATF6α. These results potentially suggest a novel avenue to treat allergic asthma using select CBAs.
Authors
Emily M. Nakada, Nirav R. Bhakta, Bethany R. Korwin-Mihavics, Amit Kumar, Nicolas Chamberlain, Sierra R. Bruno, David G. Chapman, Sidra M. Hoffman, Nirav Daphtary, Minara Aliyeva, Charles G. Irvin, Anne E. Dixon, Prescott G. Woodruff, Shantu Amin, Matthew E. Poynter, Dhimant H. Desai, Vikas Anathy
×Abstract
Traumatic spinal cord injury (SCI) triggers an acute-phase response that leads to systemic inflammation and rapid mobilization of bone marrow (BM) neutrophils into the blood. These mobilized neutrophils then accumulate in visceral organs and the injured spinal cord where they cause inflammatory tissue damage. The receptor for complement activation product 3a, C3aR1, has been implicated in negatively regulating the BM neutrophil response to tissue injury. However, the mechanism via which C3aR1 controls BM neutrophil mobilization, and also its influence over SCI outcomes, are unknown. Here, we show that the C3a/C3aR1 axis exerts neuroprotection in SCI by acting as a physiological antagonist against neutrophil chemotactic signals. We show that C3aR1 engages phosphatase and tensin homolog (PTEN), a negative regulator of the phosphatidylinositol 3-kinase (PI3K)/AKT pathway, to restrain C-X-C chemokine receptor type 2–driven BM neutrophil mobilization following trauma. These findings are of direct clinical significance as lower circulating neutrophil numbers at presentation were identified as a marker for improved recovery in human SCI. Our work thus identifies C3aR1 and its downstream intermediary, PTEN, as therapeutic targets to broadly inhibit neutrophil mobilization/recruitment following tissue injury and reduce inflammatory pathology.
Authors
Faith H. Brennan, Trisha Jogia, Ellen R. Gillespie, Linda V. Blomster, Xaria X. Li, Bianca Nowlan, Gail M. Williams, Esther Jacobson, Geoff W. Osborne, Frederic A. Meunier, Stephen M. Taylor, Kate E. Campbell, Kelli P.A. MacDonald, Jean-Pierre Levesque, Trent M. Woodruff, Marc J. Ruitenberg
×Abstract
Following injury, leukocytes are released from hematopoietic organs and migrate to the site of damage to regulate tissue inflammation and repair; however, leukocytes lacking β2-adrenergic receptor (β2-AR) expression have marked impairments in these processes. Beta blockade is a common strategy for the treatment of many cardiovascular etiologies; therefore, the objective of our study was to assess the impact of prior beta blocker treatment on baseline leukocyte parameters and their responsiveness to acute injury. In a temporal and β-adrenergic receptor isoform–dependent manner, chronic beta blocker infusion increased splenic vascular cell adhesion molecule 1 expression and leukocyte accumulation (monocytes/macrophages, mast cells, and neutrophils) and decreased chemokine receptor 2 (CCR2) expression and migration of bone marrow and peripheral blood leukocytes (PBLs) to, as well as infiltration into, the heart following acute cardiac injury. Further, CCR2 expression and migratory responsiveness were significantly reduced in the PBLs of patients receiving beta blocker therapy compared with beta blocker–naive patients. These results highlight the ability of chronic beta blocker treatment to alter baseline leukocyte characteristics that decrease leukocytes’ responsiveness to acute injury and suggest that prior beta blockade may act to reduce the severity of innate immune responses.
Authors
Laurel A. Grisanti, Claudio de Lucia, Toby P. Thomas, Aron Stark, John T. Strony, Valerie D. Myers, Remus Beretta, Daohai Yu, Celestino Sardu, Raffaele Marfella, Erhe Gao, Steven R. Houser, Walter J. Koch, Eman A. Hamad, Douglas G. Tilley
