Research
Abstract
Immune dysregulation is characteristic of the more severe stages of SARS-CoV-2 infection. Understanding the mechanisms by which the immune system contributes to COVID-19 severity may open new avenues to treatment. Here we report that elevated interleukin-13 (IL-13) was associated with the need for mechanical ventilation in two independent patient cohorts. In addition, patients who acquired COVID-19 while prescribed Dupilumab, a mAb that blocks IL-13 and IL-4 signaling, had less severe disease. In SARS-CoV-2 infected mice, IL-13 neutralization reduced death and disease severity without affecting viral load, demonstrating an immunopathogenic role for this cytokine. Following anti-IL-13 treatment in infected mice, hyaluronan synthase 1 (Has1) was the most downregulated gene and accumulation of the hyaluronan polysaccharide was decreased in the lung. In patients with COVID-19, hyaluronan was increased in the lungs and plasma. Blockade of the hyaluronan receptor, CD44, reduced mortality in infected mice, supporting the importance of hyaluronan as a pathogenic mediator. Finally, hyaluronan was directly induced in the lungs of mice by administration of IL-13, indicating a new role for IL-13 in lung disease. Understanding the role of IL-13 and hyaluronan has important implications for therapy of COVID-19 and potentially other pulmonary diseases.
Authors
Alexandra N. Donlan, Tara E. Sutherland, Chelsea Marie, Saskia Preissner, Benjamin T. Bradley, Rebecca M. Carpenter, Jeffrey M. Sturek, Jennie Z. Ma, G. Brett Moreau, Jeffrey R. Donowitz, Gregory A. Buck, Myrna G. Serrano, Stacey L. Burgess, Mayuresh M. Abhyankar, Cameron Mura, Philip E. Bourne, Robert Preissner, Mary K. Young, Genevieve R. Lyons, Johanna J. Loomba, Sarah J. Ratcliffe, Melinda D. Poulter, Amy J. Mathers, Anthony J. Day, Barbara J Mann, Judith E. Allen, William A. Petri Jr.
Abstract
Although CD8+ T cells recognize neoantigens that arise from somatic mutations in cancer, only a small fraction of nonsynonymous mutations give rise to clinically relevant neoantigens. In this study, HLA class I ligandomes of a panel of human colorectal cancer (CRC) and matched normal tissues were analyzed using mass spectrometry-based proteogenomic analysis. Neoantigen presentation was rare; however, the analysis detected a single neoantigen in a mismatch-repair deficient (dMMR)-CRC tissue sample carrying 3,967 nonsynonymous mutations, where abundant tumor-infiltrating lymphocytes (TILs) and inflamed gene expression status were observed in the tumor microenvironment (TME). Using the HLA class I ligandome data and gene expression profiles, a set of non-mutated tumor-associated antigen (TAA) candidates was concomitantly identified. Interestingly, CD8+ TILs predominantly recognized the detected neoantigen over the array of TAA candidates. Neoantigen-reactive CD8+ TILs showed PD-1 positivity and exhibited functional and specific responses. Moreover, T cell receptor (TCR) profiling identified the sequence of the neoantigen-reactive TCR clonotype and showed its expansion in the TME. Transduction of the sequenced TCR conferred neoantigen specificity and cytotoxicity to peripheral blood lymphocytes. The proteogenomic approach revealed the antigenic and reactive T cell landscape in dMMR-CRC, demonstrating the presence of an immunogenic neoantigen and its potential therapeutic applications.
Authors
Tomomi Hirama, Serina Tokita, Munehide Nakatsugawa, Kenji Murata, Yasuhito Nannya, Kazuhiko Matsuo, Hidetoshi Inoko, Yoshihiko Hirohashi, Shinichi Hashimoto, Seishi Ogawa, Ichiro Takemasa, Noriyuki Sato, Fumitake Hata, Takayuki Kanaseki, Toshihiko Torigoe
Abstract
AAA is a disease with high morbidity and mortality, especially when ruptured. The rational of this study was to evaluate the repurposing of lenvatinib, a multi tyrosine kinase inhibitor, in limiting experimental abdominal aortic aneurysm (AAA) growth targeting vascular smooth muscle cells (VSMC) and angiogenesis. We applied systemic and local lenvatinib treatment to elastase-induced murine aortic aneurysms, and RNA profiling identified myosin heavy chain 11 (Myh11) as the most deregulated transcript. Daily oral treatment significantly reduced aneurysm formation in two independent mouse models. In addition, a large animal aneurysm model in hypercholesterolemic low-density lipoprotein receptor knockout (LDLR-/-) Yucatan minipigs was applied to endovascularly deliver Lenvatinib via drug-eluting balloons (DEB). Here, a single local endovascular delivery blocked AAA progression successfully compared to a DEB-delivered control treatment. Reduced VSMC proliferation and a restored contractile phenotype were observed in animal tissues (murine and porcine), as well as AAA patient-derived cells. Apart from increasing MYH11 levels, lenvatinib reduced downstream ERK signaling. Hence, Lenvatinib is a promising new therapy to limit aortic aneurysm expansion upon local endovascular delivery. The tyrosine kinase inhibitor was able to positively affect pathways of key relevance to human AAA disease, even in a potentially new local delivery using DEBs.
Authors
Albert Busch, Jessica Pauli, Greg Winski, Sonja Bleichert, Ekaterina Chernogubova, Susanne Metschl, Hanna Winter, Matthias Trenner, Armin Wiegering, Christoph Otto, Johannes Fischer, Judith Reiser, Julia Werner, Joy Roy, Christine Brostjan, Christoph Knappich, Hans-Henning Eckstein, Valentina Paloschi, Lars Maegdefessel
Abstract
Sepsis is a critical illness characterized by dysregulated inflammatory responses lacking counter-regulation. Specialized pro-resolving mediators are agonists for anti-inflammation and promoting resolution and are protective in preclinical sepsis models. Here, in human sepsis, we mapped resolution circuits for the specialized pro-resolving mediators resolvin D1 and resolvin D2 in peripheral blood neutrophils and monocytes, their regulation of leukocyte activation and function ex vivo, and their relationships to measures of clinical severity. Neutrophils and monocytes were isolated from healthy subjects and sepsis patients by inertial microfluidics and resolvin D1 and resolvin D2 receptor expression determined by flow cytometry. The impact of these resolvins on leukocyte activation was determined by isodielectric separation and leukocyte function by stimulated phagolysosome formation. Leukocyte pro-resolving receptor expression was significantly higher in sepsis. In nanomolar concentrations, resolvin D1 and resolvin D2 partially reversed sepsis-induced changes in leukocyte activation and function. Principal component analyses of leukocyte resolvin receptor expression and responses differentiated sepsis from health and were associated with measures of sepsis severity. These findings indicate that resolvin D1 and resolvin D2 signaling for anti-inflammation and resolution are uncoupled from leukocyte activation in early sepsis and suggest that indicators of diminished resolution signaling correlate with clinical disease severity.
Authors
Bakr Jundi, Do-Hyun Lee, Hyungkook Jeon, Melody G. Duvall, Julie Nijmeh, Raja-Elie E. Abdulnour, Mayra Pinilla-Vera, Rebecca M. Baron, Jongyoon Han, Joel Voldman, Bruce D. Levy
Abstract
The release of neutrophil extracellular traps (NETs) by hyperactive neutrophils is recognized to play an important role in the thromboinflammatory milieu inherent to severe presentations of COVID-19. At the same time, a variety of functional autoantibodies have been observed in individuals with severe COVID-19 where they likely contribute to immunopathology. Here, we aimed to determine the extent to which autoantibodies might target NETs in COVID-19 and, if detected, to elucidate their potential functions and clinical associations. We measured anti-NET antibodies in 328 individuals hospitalized with COVID-19 alongside 48 healthy controls. We found high anti-NET activity in the IgG and IgM fractions of 27% and 60% of patients, respectively. There was a strong correlation between anti-NET IgG and anti-NET IgM (r=0.4, p<0.0001). Both anti-NET IgG and IgM tracked with high levels of circulating NETs, impaired oxygenation efficiency, and high circulating D-dimer. Furthermore, patients who required mechanical ventilation had a greater burden of anti-NET antibodies than did those not requiring oxygen supplementation. Levels of anti-NET IgG (and to a lesser extent anti-NET IgM) demonstrated an inverse correlation with the efficiency of NET degradation by COVID sera. Furthermore, purified IgG from COVID sera with high levels of anti-NET antibodies impaired the ability of healthy control serum to degrade NETs. In summary, many individuals hospitalized with COVID-19 have anti-NET antibodies, which likely impair NET clearance and may potentiate SARS-CoV-2-mediated thromboinflammation.
Authors
Yu Zuo, Srilakshmi Yalavarthi, Sherwin A. Navaz, Claire K. Hoy, Alyssa Harbaugh, Kelsey Gockman, Melanie Zuo, Jacqueline A. Madison, Hui Shi, Yogendra Kanthi, Jason S. Knight
Abstract
Idiopathic nephrotic syndrome (INS) is characterized by proteinuria and renal Na retention leading to oedema. This Na retention is usually attributed to epithelial sodium channel (ENaC) activation following plasma aldosterone increase. However, most nephrotic patients show normal aldosterone levels. Using a corticosteroid-clamped rat model of INS (CC-PAN), we showed that the observed electrogenic and amiloride-sensitive Na retention could not be attributed to ENaC. We, then, identified a truncated variant of acid sensing ion channel 2b (ASIC2b) that induced sustained acid-stimulated sodium currents when co-expressed with ASIC2a. Interestingly, CC-PAN nephrotic ASIC2b-null rats did not develop sodium retention. We finally showed that expression of the truncated ASIC2b in kidney was dependent on the presence of albumin in the tubule lumen and activation of ERK in renal cells. Finally, the presence of ASIC2 mRNA was also detected in kidney biopsies from patients with INS but not in any of the patients with other renal diseases. We have, therefore, identified a novel variant of ASIC2b responsible for the renal Na retention in the pathological context of INS.
Authors
Marc Fila, Ali Sassi, Gaelle Brideau, Lydie Cheval, Luciana Morla, Pascal Houillier, Christine Walter, Michel Gennaoui, Laure Collignon, Mathilde Keck, Gabrielle Planelles, Naziha Bakouh, Michel Peuchmaur, Georges Deschenes, Ignacio Anegon, Séverine Remy, Bruno Vogt, Gilles Crambert, Alain Doucet
Abstract
Gorham-Stout disease (GSD) is a sporadically occurring lymphatic disorder. Patients with GSD develop ectopic lymphatic vessels in bone, gradually lose bone, and can have life-threatening complications such as chylothorax. The etiology of GSD is poorly understood and current treatments for this disease are inadequate for most patients. To explore the pathogenesis of GSD, we performed targeted high-throughput sequencing with samples from a GSD patient and identified an activating somatic mutation in KRAS (p.G12V). To characterize the effect of hyperactive KRAS signaling on lymphatic development, we expressed an active form of KRAS (p.G12D) in murine lymphatics (iLECKras mice). We found that iLECKras mice developed lymphatics in bone, which is a hallmark of GSD. We also found that lymphatic valve development and maintenance was altered in iLECKras mice. Because most iLECKras mice developed chylothorax and died before they had significant bone disease, we analyzed the effect of trametinib (an FDA-approved MEK1/2 inhibitor) on lymphatic valve regression in iLECKras mice. Notably, we found that trametinib suppressed this phenotype in iLECKras mice. Together, our results demonstrate that somatic activating mutations in KRAS can be associated with GSD and reveal that hyperactive KRAS signaling stimulates the formation of lymphatics in bone and impairs the development of lymphatic valves. These findings provide insight into the pathogenesis of GSD and suggest that trametinib could be an effective treatment for GSD.
Authors
Nassim Homayun Sepehr, Anna L. McCarter, Raphaël Helaers, Christine Galant, Laurence M. Boon, Pascal Brouillard, Miikka Vikkula, Michael T. Dellinger
Abstract
The majority of patients affected with lysosomal storage disorders (LSD) exhibit neurological symptoms. For mucopolysaccharidosis type IIIC (MPSIIIC), the major burdens are progressive and severe neuropsychiatric problems and dementia primarily thought to stem from neurodegeneration. Using the MPSIIIC mouse model we studied whether clinical manifestations preceding massive neurodegeneration arise from synaptic dysfunction. Reduced levels or abnormal distribution of multiple synaptic proteins were revealed in cultured hippocampal and CA1 pyramidal MPSIIIC neurons. These defects were rescued by virus-mediated gene correction. Dendritic spines were reduced in pyramidal neurons of mouse models of MPSIIIC and other (Tay-Sachs, sialidosis) LSD as early as postnatal day 10. MPSIIIC neurons also presented alterations in frequency and amplitude of miniature excitatory and inhibitory postsynaptic currents, sparse synaptic vesicles, reduced postsynaptic densities, disorganised microtubule networks and partially impaired axonal transport of synaptic proteins. Furthermore, postsynaptic densities were reduced in post-mortem cortices of human MPS patients suggesting that the pathology is a common hallmark for neurological LSD. Together, our results demonstrate that lysosomal storage defects cause early alterations in synaptic structure and abnormalities in neurotransmission originating from impaired synaptic vesicular transport, and suggest that synaptic defects could be targeted to treat behavioral and cognitive defects in neurological LSD patients.
Authors
Camila Pará, Poulomee Bose, Luigi Bruno, Erika Freemantle, Mahsa Taherzadeh, Xuefang Pan, Chanshuai Han, Peter S. McPherson, Jean-Claude Lacaille, Éric Bonneil, Pierre Thibault, Claire O'Leary, Brian Bigger, Carlos Ramon Morales, Graziella Di Cristo, Alexey V. Pshezhetsky
Abstract
SCN2A, encoding the neuronal voltage-gated Na+ channel NaV1.2, is one of the most commonly affected loci linked to autism spectrum disorders (ASDs). Most ASD-associated mutations in SCN2A are loss-of-function, but studies examining how such mutations affect neuronal function and whether Scn2a mutant mice display ASD endophenotypes have been inconsistent. We generated a protein truncation variant Scn2a mouse model (Scn2aΔ1898/+) by CRISPR that eliminates the NaV1.2 channel’s distal intracellular C-terminal domain and analyzed the molecular and cellular consequences of this variant in a heterologous expression system, in neuronal culture, in brain slices, and in vivo. We also analyzed multiple behaviors in wild type and Scn2aΔ1898/+ mice and correlated behaviors with clinical data obtained in human subjects with SCN2A variants. Expression of the NaV1.2 mutant in a heterologous expression system revealed decreased NaV1.2 channel function and cultured pyramidal neurons isolated from Scn2aΔ1898/+ forebrain showed correspondingly reduced voltage-gated Na+ channel currents without compensation from other CNS voltage-gated Na+ channels. Na+ currents in inhibitory neurons were unaffected. Consistent with loss of voltage-gated Na+ channel currents, Scn2aΔ1898/+ pyramidal neurons displayed reduced excitability in forebrain neuronal culture and reduced excitatory synaptic input onto the pyramidal neurons in brain slices. Scn2aΔ1898/+ mice displayed several behavioral abnormalities, including abnormal social interactions that reflect behavior observed in humans with ASD and with harboring loss-of-function SCN2A variants. This model and its cellular electrophysiological characterizations provide a framework for tracing how a SCN2A loss-of-function variant leads to cellular defects that result in ASD-associated behaviors.
Authors
Hong-Gang Wang, Charlotte C. Bavley, Anfei Li, Rebecca M. Jones, Jonathan E. Hackett, Yared Bayleyen, Francis S. Lee, Anjali M. Rajadhyaksha, Geoffrey S. Pitt
Abstract
Native myocardial voltage-gated sodium (NaV) channels function in macromolecular complexes comprising a pore-forming (α) subunit and multiple accessory proteins. Here, we investigated the impact of accessory NaVβ1 and NaVβ3 subunits on the functional effects of two well-known Class-Ib antiarrhythmics, lidocaine and ranolazine, on the predominant NaV channel α subunit, Nav1.5, expressed in mammalian heart. We show that both drugs stabilize the activated conformation of the voltage-sensor of in Domain-III (DIII-VSD) in NaV1.5. In the presence of NaVβ1, the effect of lidocaine on the DIII-VSD was enhanced, whereas the effect of ranolazine was abolished. Mutating the main Class-Ib drug binding site, F1760, affected but did not abolish, the modulation of drug block by Navβ1/β3. Recordings from adult mouse ventricular myocytes demonstrated that Scn1b (Navβ1) loss of differentially affected the potencies of lidocaine and ranolazine. In vivo experiments revealed distinct ECG responses to intraperitoneal injection of ranolazine or lidocaine in WT and Scn1b null animals, suggesting that NaVβ1 modulates drug responses at the whole heart level. In human heart, we found that SCN1B transcript expression is three times higher in atria than ventricles, differences that could, in combination with inherited or acquired cardiovascular disease, dramatically impact patient response to Class-Ib antiarrhythmic therapies.
Authors
Wandi Zhu, Wei Wang, Paweorn Angsutararux, Rebecca L. Mellor, Lori L. Isom, Jeanne M. Nerbonne, Jonathan R. Silva
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