Double-stranded RNA innate immune response activation from long-term adeno-associated virus vector transduction

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Abstract

Data from clinical trials for hemophilia B using adeno-associated virus (AAV) vectors have demonstrated decreased transgenic coagulation factor IX (hFIX) expression 6–10 weeks after administration of a high vector dose. While it is likely that capsid-specific cytotoxic T lymphocytes eliminate vector-transduced hepatocytes, thereby resulting in decreased hFIX, this observation is not intuitively consistent with restored hFIX levels following prednisone application. Although the innate immune response is immediately activated following AAV vector infection via TLR pathways, no studies exist regarding the role of the innate immune response at later time points after AAV vector transduction. Herein, activation of the innate immune response in cell lines, primary human hepatocytes, and hepatocytes in a human chimeric mouse model was observed at later time points following AAV vector transduction. Mechanistic analysis demonstrated that the double-stranded RNA (dsRNA) sensor MDA5 was necessary for innate immune response activation and that transient knockdown of MDA5, or MAVS, decreased IFN-β expression while increasing transgene production in AAV-transduced cells. These results both highlight the role of the dsRNA-triggered innate immune response in therapeutic transgene expression at later time points following AAV transduction and facilitate the execution of effective strategies to block the dsRNA innate immune response in future clinical trials.

Authors

Wenwei Shao, Lauriel F. Earley, Zheng Chai, Xiaojing Chen, Junjiang Sun, Ting He, Meng Deng, Matthew L. Hirsch, Jenny Ting, R. Jude Samulski, Chengwen Li

Krüppel-like factor 4 is a negative regulator of STAT3-induced glomerular epithelial cell proliferation

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Abstract

Pathologic glomerular epithelial cell (GEC) hyperplasia is characteristic of both rapidly progressive glomerulonephritis (RPGN) and subtypes of focal segmental glomerulosclerosis (FSGS). Although initial podocyte injury resulting in activation of STAT3 signals GEC proliferation in both diseases, mechanisms regulating this are unknown. Here, we show that the loss of Krüppel-like factor 4 (KLF4), a zinc-finger transcription factor, enhances GEC proliferation in both RPGN and FSGS due to dysregulated STAT3 signaling. We observed that podocyte-specific knockdown of Klf4 (C57BL/6J) increased STAT3 signaling and exacerbated crescent formation after nephrotoxic serum treatment. Interestingly, podocyte-specific knockdown of Klf4 in the FVB/N background alone was sufficient to activate STAT3 signaling, resulting in FSGS with extracapillary proliferation, as well as renal failure and reduced survival. In cultured podocytes, loss of KLF4 resulted in STAT3 activation and cell-cycle reentry, leading to mitotic catastrophe. This triggered IL-6 release into the supernatant, which activated STAT3 signaling in parietal epithelial cells. Conversely, either restoration of KLF4 expression or inhibition of STAT3 signaling improved survival in KLF4-knockdown podocytes. Finally, human kidney biopsy specimens with RPGN exhibited reduced KLF4 expression with a concomitant increase in phospho-STAT3 expression as compared with controls. Collectively, these results suggest the essential role of KLF4/STAT3 signaling in podocyte injury and its regulation of aberrant GEC proliferation.

Authors

Chelsea C. Estrada, Praharshasai Paladugu, Yiqing Guo, Jesse Pace, Monica P. Revelo, David J. Salant, Stuart J. Shankland, Vivette D. D’Agati, Anita Mehrotra, Stephanie Cardona, Agnieszka B. Bialkowska, Vincent W. Yang, John C. He, Sandeep K. Mallipattu

Murine models of Pneumocystis infection recapitulate human primary immune disorders

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Abstract

Despite the discovery of key pattern recognition receptors and CD4+ T cell subsets in laboratory mice, there is ongoing discussion of the value of murine models to reflect human disease. Pneumocystis is an AIDS-defining illness, in which risk of infection is inversely correlated with peripheral CD4+ T cell counts. Due to medical advances in the control of HIV, the current epidemiology of Pneumocystis infection is predominantly due to primary human immunodeficiencies and immunosuppressive therapies. To this end, we found that every human genetic immunodeficiency associated with Pneumocystis infection that has been tested in mice recapitulated susceptibility. For example, humans with a loss-of-function IL21R mutation are severely immunocompromised. We found that IL-21R, in addition to CD4+ T cell intrinsic STAT3 signaling, were required for generating protective antifungal class-switched antibody responses, as well as effector T cell–mediated protection. Furthermore, CD4+ T cell intrinsic IL-21R/STAT3 signaling was required for CD4+ T cell effector responses, including IL-22 production. Recombinant IL-22 administration to Il21r–/– mice induced the expression of a fungicidal peptide, cathelicidin antimicrobial peptide, which showed in vitro fungicidal activity. In conclusion, SPF laboratory mice faithfully replicate many aspects of human primary immunodeficiency and provide useful tools to understand the generation and nature of effector CD4+ T cell immunity.

Authors

Waleed Elsegeiny, Mingquan Zheng, Taylor Eddens, Richard L. Gallo, Guixiang Dai, Giraldina Trevejo-Nunez, Patricia Castillo, Kara Kracinovsky, Hillary Cleveland, William Horne, Jonathan Franks, Derek Pociask, Mark Pilarski, John F. Alcorn, Kong Chen, Jay K. Kolls

γδ T cells: an immunotherapeutic approach for HIV cure strategies

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Abstract

Current strategies aimed to cure HIV infection are based on combined efforts to reactivate the virus from latency and improve immune effector cell function to clear infected cells. These strategies are primarily focused on CD8+ T cells and approaches are challenging due to insufficient HIV antigen production from infected cells and poor HIV-specific CD8+ T cells. γδ T cells represent a unique subset of effector T cells that can traffic to tissues, and selectively target cancer or virally infected cells without requiring MHC presentation. We analyzed whether γδ T cells represent a complementary/alternative immunotherapeutic approach towards HIV cure strategies. γδ T cells from HIV-infected virologically suppressed donors were expanded with bisphosphonate pamidronate (PAM) and cells were used in autologous cellular systems ex vivo. These cells (a) are potent cytotoxic effectors able to efficiently inhibit HIV replication ex vivo, (b) degranulate in the presence of autologous infected CD4+ T cells, and (c) specifically clear latently infected cells after latency reversal with vorinostat. This is the first proof of concept to our knowledge showing that γδ T cells target and clear autologous HIV reservoirs upon latency reversal. Our results open potentially new insights into the immunotherapeutic use of γδ T cells for current interventions in HIV eradication strategies.

Authors

Carolina Garrido, Matthew L. Clohosey, Chloe P. Whitworth, Michael Hudgens, David M. Margolis, Natalia Soriano-Sarabia

Repurposing sertraline sensitizes non–small cell lung cancer cells to erlotinib by inducing autophagy

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Abstract

Lung cancer patients treated with tyrosine kinase inhibitors (TKIs) often develop resistance. More effective and safe therapeutic agents are urgently needed to overcome TKI resistance. Here, we propose a medical genetics–based approach to identify indications for over 1,000 US Food and Drug Administration–approved (FDA-approved) drugs with high accuracy. We identified a potentially novel indication for an approved antidepressant drug, sertraline, for the treatment of non–small cell lung cancer (NSCLC). We found that sertraline inhibits the viability of NSCLC cells and shows a synergy with erlotinib. Specifically, the cotreatment of sertraline and erlotinib effectively promotes autophagic flux in cells, as indicated by LC3-II accumulation and autolysosome formation. Mechanistic studies further reveal that dual treatment of sertraline and erlotinib reciprocally regulates the AMPK/mTOR pathway in NSCLC cells. The blockade of AMPK activation decreases the anticancer efficacy of either sertraline alone or the combination. Efficacy of this combination regimen is decreased by pharmacological inhibition of autophagy or genetic knockdown of ATG5 or Beclin 1. Importantly, our results suggest that sertraline and erlotinib combination suppress tumor growth and prolong mouse survival in an orthotopic NSCLC mouse model (P = 0.0005). In summary, our medical genetics–based approach facilitates discovery of new anticancer indications for FDA-approved drugs for the treatment of NSCLC.

Authors

Xingwu Jiang, Weiqiang Lu, Xiaoyang Shen, Quan Wang, Jing Lv, Mingyao Liu, Feixiong Cheng, Zhongming Zhao, Xiufeng Pang

Nasal priming by a murine coronavirus provides protective immunity against lethal heterologous virus pneumonia

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Abstract

The nasal mucosa is an important component of mucosal immunity. Immunogenic particles in inspired air are known to activate the local nasal mucosal immune system and can lead to sinonasal inflammation; however, little is known about the effect of this activation on the lung immune environment. Here, we showed that nasal inoculation of murine coronavirus (CoV) in the absence of direct lung infection primes the lung immune environment by recruiting activated monocytes (Ly6C+ inflammatory monocytes) and NK cells into the lungs. Unlike infiltration of these cells into directly infected lungs, a process that requires type I IFN signaling, nasally induced infiltration of Ly6C+ inflammatory monocytes into the lungs is IFN-I independent. These activated macrophages ingested antigen and migrated to pulmonary lymph nodes, and enhanced both innate and adaptive immunity after heterologous virus infection. Clinically, such nasal-only inoculation of MHV-1 failed to cause pneumonia but significantly reduced mortality and morbidity of lethal pneumonia caused by severe acute respiratory syndrome CoV (SARS-CoV) or influenza A virus. Together, the data indicate that the nose and upper airway remotely prime the lung immunity to protect the lungs from direct viral infections.

Authors

Xiaoyang Hua, Rahul Vijay, Rudragouda Channappanvar, Jeremiah Athmer, David K. Meyerholz, Nitin Pagedar, Stephen Tilley, Stanley Perlman

Myeloperoxidase aggravates pulmonary arterial hypertension by activation of vascular Rho-kinase

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Abstract

Pulmonary arterial hypertension (PAH) remains a disease with limited therapeutic options and dismal prognosis. Despite its etiologic heterogeneity, the underlying unifying pathophysiology is characterized by increased vascular tone and adverse remodeling of the pulmonary circulation. Myeloperoxidase (MPO), an enzyme abundantly expressed in neutrophils, has potent vasoconstrictive and profibrotic properties, thus qualifying as a potential contributor to this disease. Here, we sought to investigate whether MPO is causally linked to the pathophysiology of PAH. Investigation of 2 independent clinical cohorts revealed that MPO plasma levels were elevated in subjects with PAH and predicted adverse outcome. Experimental analyses showed that, upon hypoxia, right ventricular pressure was less increased in Mpo–/– than in WT mice. The hypoxia-induced activation of the Rho-kinase pathway, a critical subcellular signaling pathway yielding vasoconstriction and structural vascular remodeling, was blunted in Mpo–/– mice. Mice subjected to i.v. infusion of MPO revealed activation of Rho-kinase and increased right ventricular pressure, which was prevented by coinfusion of the Rho-kinase inhibitor Y-27632. In the Sugen5416/hypoxia rat model, PAH was attenuated by the MPO inhibitor AZM198. The current data demonstrate a tight mechanistic link between MPO, the activation of Rho-kinase, and adverse pulmonary vascular function, thus pointing toward a potentially novel avenue of treatment.

Authors

Anna Klinke, Eva Berghausen, Kai Friedrichs, Simon Molz, Denise Lau, Lisa Remane, Matthias Berlin, Charlotte Kaltwasser, Matti Adam, Dennis Mehrkens, Martin Mollenhauer, Kashish Manchanda, Thorben Ravekes, Gustavo A. Heresi, Metin Aytekin, Raed A. Dweik, Jan K. Hennigs, Lukas Kubala, Erik Michaëlsson, Stephan Rosenkranz, Tanja K. Rudolph, Stanley L. Hazen, Hans Klose, Ralph T. Schermuly, Volker Rudolph, Stephan Baldus

Fully automated, real-time 3D ultrasound segmentation to estimate first trimester placental volume using deep learning

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Abstract

We present a new technique to fully automate the segmentation of an organ from 3D ultrasound (3D-US) volumes, using the placenta as the target organ. Image analysis tools to estimate organ volume do exist but are too time consuming and operator dependant. Fully automating the segmentation process would potentially allow the use of placental volume to screen for increased risk of pregnancy complications. The placenta was segmented from 2,393 first trimester 3D-US volumes using a semiautomated technique. This was quality controlled by three operators to produce the “ground-truth” data set. A fully convolutional neural network (OxNNet) was trained using this ground-truth data set to automatically segment the placenta. OxNNet delivered state-of-the-art automatic segmentation. The effect of training set size on the performance of OxNNet demonstrated the need for large data sets. The clinical utility of placental volume was tested by looking at predictions of small-for-gestational-age babies at term. The receiver-operating characteristics curves demonstrated almost identical results between OxNNet and the ground-truth). Our results demonstrated good similarity to the ground-truth and almost identical clinical results for the prediction of SGA.

Authors

Pádraig Looney, Gordon N. Stevenson, Kypros H. Nicolaides, Walter Plasencia, Malid Molloholli, Stavros Natsis, Sally L. Collins

Monocyte NOTCH2 expression predicts IFN-β immunogenicity in multiple sclerosis patients

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Abstract

Multiple sclerosis (MS) is an autoimmune disease characterized by CNS inflammation leading to demyelination and axonal damage. IFN-β is an established treatment for MS; however, up to 30% of IFN-β–treated MS patients develop neutralizing antidrug antibodies (nADA), leading to reduced drug bioactivity and efficacy. Mechanisms driving antidrug immunogenicity remain uncertain, and reliable biomarkers to predict immunogenicity development are lacking. Using high-throughput flow cytometry, NOTCH2 expression on CD14+ monocytes and increased frequency of proinflammatory monocyte subsets were identified as baseline predictors of nADA development in MS patients treated with IFN-β. The association of this monocyte profile with nADA development was validated in 2 independent cross-sectional MS patient cohorts and a prospective cohort followed before and after IFN-β administration. Reduced monocyte NOTCH2 expression in nADA+ MS patients was associated with NOTCH2 activation measured by increased expression of Notch-responsive genes, polarization of monocytes toward a nonclassical phenotype, and increased proinflammatory IL-6 production. NOTCH2 activation was T cell dependent and was only triggered in the presence of serum from nADA+ patients. Thus, nADA development was driven by a proinflammatory environment that triggered activation of the NOTCH2 signaling pathway prior to first IFN-β administration.

Authors

Marsilio Adriani, Petra Nytrova, Cyprien Mbogning, Signe Hässler, Karel Medek, Poul Erik H. Jensen, Paul Creeke, Clemens Warnke, Kathleen Ingenhoven, Bernhard Hemmer, Claudia Sievers, Raija L.P. Lindberg Gasser, Nicolas Fissolo, Florian Deisenhammer, Zsolt Bocskei, Vincent Mikol, Anna Fogdell-Hahn, Eva Kubala Havrdova, Philippe Broët, Pierre Dönnes, Claudia Mauri, Elizabeth C. Jury, The ABIRISK Consortium

HSC70 is a chaperone for wild-type and mutant cardiac myosin binding protein C

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Abstract

Cardiac myosin binding protein C (MYBPC3) is the most commonly mutated gene associated with hypertrophic cardiomyopathy (HCM). Haploinsufficiency of full-length MYBPC3 and disruption of proteostasis have both been proposed as central to HCM disease pathogenesis. Discriminating the relative contributions of these 2 mechanisms requires fundamental knowledge of how turnover of WT and mutant MYBPC3 proteins is regulated. We expressed several disease-causing mutations in MYBPC3 in primary neonatal rat ventricular cardiomyocytes. In contrast to WT MYBPC3, mutant proteins showed reduced expression and failed to localize to the sarcomere. In an unbiased coimmunoprecipitation/mass spectrometry screen, we identified HSP70-family chaperones as interactors of both WT and mutant MYBPC3. Heat shock cognate 70 kDa (HSC70) was the most abundant chaperone interactor. Knockdown of HSC70 significantly slowed degradation of both WT and mutant MYBPC3, while pharmacologic activation of HSC70 and HSP70 accelerated degradation. HSC70 was expressed in discrete striations in the sarcomere. Expression of mutant MYBPC3 did not affect HSC70 localization, nor did it induce a protein folding stress response or ubiquitin proteasome dysfunction. Together these data suggest that WT and mutant MYBPC3 proteins are clients for HSC70, and that the HSC70 chaperone system plays a major role in regulating MYBPC3 protein turnover.

Authors

Amelia A. Glazier, Neha Hafeez, Dattatreya Mellacheruvu, Venkatesha Basrur, Alexey I. Nesvizhskii, Lap Man Lee, Hao Shao, Vi Tang, Jaime M. Yob, Jason E. Gestwicki, Adam S. Helms, Sharlene M. Day