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Abstract
The biology of harlequin ichthyosis (HI), a devastating skin disorder caused by loss-of-function mutations in the gene ABCA12, is poorly understood, and to date, no satisfactory treatment has been developed. We sought to investigate pathomechanisms of HI that could lead to the identification of new treatments for improving patients’ quality of life. In this study, RNA-Seq and functional assays were performed to define the effects of loss of ABCA12 using HI patient skin samples and an engineered CRISPR/Cas9 ABCA12 KO cell line. The HI living skin equivalent (3D model) recapitulated the HI skin phenotype. The cytokines IL-36α and IL-36γ were upregulated in HI skin, whereas the innate immune inhibitor IL-37 was strongly downregulated. We also identified STAT1 and its downstream target inducible nitric oxide synthase (NOS2) as being upregulated in the in vitro HI 3D model and HI patient skin samples. Inhibition of NOS2 using the inhibitor 1400W or the JAK inhibitor tofacitinib dramatically improved the in vitro HI phenotype by restoring the lipid barrier in the HI 3D model. Our study has identified dysregulated pathways in HI skin that are feasible therapeutic targets.
Authors
Florence Enjalbert, Priya Dewan, Matthew P. Caley, Eleri M. Jones, Mary A. Morse, David P. Kelsell, Anton J. Enright, Edel A. O’Toole
Abstract
The amyloid hypothesis posits that the amyloid-beta (Aβ) protein precedes and requires microtubule-associated protein tau in a sort of trigger-bullet mechanism leading to Alzheimer’s disease (AD) pathology. This sequence of events has become dogmatic in the AD field and is used to explain clinical trial failures due to a late start of the intervention when Aβ already activated tau. Here, using a multidisciplinary approach combining molecular biological, biochemical, histopathological, electrophysiological, and behavioral methods, we demonstrated that tau suppression did not protect against Aβ-induced damage of long-term synaptic plasticity and memory, or from amyloid deposition. Tau suppression could even unravel a defect in basal synaptic transmission in a mouse model of amyloid deposition. Similarly, tau suppression did not protect against exogenous oligomeric tau–induced impairment of long-term synaptic plasticity and memory. The protective effect of tau suppression was, in turn, confined to short-term plasticity and memory. Taken together, our data suggest that therapies downstream of Aβ and tau together are more suitable to combat AD than therapies against one or the other alone.
Authors
Daniela Puzzo, Elentina K. Argyrousi, Agnieszka Staniszewski, Hong Zhang, Elisa Calcagno, Elisa Zuccarello, Erica Acquarone, Mauro Fa’, Domenica D. Li Puma, Claudio Grassi, Luciano D’Adamio, Nicholas M. Kanaan, Paul E. Fraser, Ottavio Arancio
Abstract
NF-κB transcription factors, driven by the IRAK/IKK cascade, confer treatment resistance in pancreatic ductal adenocarcinoma (PDAC), a cancer characterized by near-universal KRAS mutation. Through reverse-phase protein array and RNA sequencing we discovered that IRAK4 also contributes substantially to MAPK activation in KRAS-mutant PDAC. IRAK4 ablation completely blocked RAS-induced transformation of human and murine cells. Mechanistically, expression of mutant KRAS stimulated an inflammatory, autocrine IL-1β signaling loop that activated IRAK4 and the MAPK pathway. Downstream of IRAK4, we uncovered TPL2 (also known as MAP3K8 or COT) as the essential kinase that propels both MAPK and NF-κB cascades. Inhibition of TPL2 blocked both MAPK and NF-κB signaling, and suppressed KRAS-mutant cell growth. To counter chemotherapy-induced genotoxic stress, PDAC cells upregulated TLR9, which activated prosurvival IRAK4/TPL2 signaling. Accordingly, a TPL2 inhibitor synergized with chemotherapy to curb PDAC growth in vivo. Finally, from TCGA we characterized 2 MAP3K8 point mutations that hyperactivate MAPK and NF-κB cascades by impeding TPL2 protein degradation. Cancer cell lines naturally harboring these MAP3K8 mutations are strikingly sensitive to TPL2 inhibition, underscoring the need to identify these potentially targetable mutations in patients. Overall, our study establishes TPL2 as a promising therapeutic target in RAS- and MAP3K8-mutant cancers and strongly prompts development of TPL2 inhibitors for preclinical and clinical studies.
Authors
Paarth B. Dodhiawala, Namrata Khurana, Daoxiang Zhang, Yi Cheng, Lin Li, Qing Wei, Kuljeet Seehra, Hongmei Jiang, Patrick M. Grierson, Andrea Wang-Gillam, Kian-Huat Lim
Abstract
Leber’s hereditary optic neuropathy (LHON) is a maternally inherited eye disease. X-linked nuclear modifiers were proposed to modify the phenotypic manifestation of LHON-associated mitochondrial DNA (mtDNA) mutations. By whole-exome sequencing, we identified the X-linked LHON modifier (c.157C>T, p.Arg53Trp) in PRICKLE3 encoding a mitochondrial protein linked to biogenesis of ATPase in 3 Chinese families. All affected individuals carried both ND4 11778G>A and p.Arg53Trp mutations, while subjects bearing only a single mutation exhibited normal vision. The cells carrying the p.Arg53Trp mutation exhibited defective assembly, stability, and function of ATP synthase, verified by PRICKLE3-knockdown cells. Coimmunoprecipitation indicated the direct interaction of PRICKLE3 with ATP synthase via ATP8. Strikingly, cells bearing both p.Arg53Trp and m.11778G>A mutations displayed greater mitochondrial dysfunction than those carrying only a single mutation. This finding indicated that the p.Arg53Trp mutation acted in synergy with the m.11778G>A mutation and deteriorated mitochondrial dysfunctions necessary for the expression of LHON. Furthermore, we demonstrated that Prickle3-deficient mice exhibited pronounced ATPase deficiencies. Prickle3-knockout mice recapitulated LHON phenotypes with retinal deficiencies, including degeneration of retinal ganglion cells and abnormal vasculature. Our findings provided new insights into the pathophysiology of LHON that were manifested by interaction between mtDNA mutations and X-linked nuclear modifiers.
Authors
Jialing Yu, Xiaoyang Liang, Yanchun Ji, Cheng Ai, Junxia Liu, Ling Zhu, Zhipeng Nie, Xiaofen Jin, Chenghui Wang, Juanjuan Zhang, Fuxin Zhao, Shuang Mei, Xiaoxu Zhao, Xiangtian Zhou, Minglian Zhang, Meng Wang, Taosheng Huang, Pingping Jiang, Min-Xin Guan
BET bromodomain inhibition attenuates cardiac phenotype in myocyte-specific lamin A/C–deficient mice
Abstract
Mutation in the LMNA gene, encoding lamin A/C, causes a diverse group of diseases called laminopathies. Cardiac involvement is the major cause of death and manifests as dilated cardiomyopathy, heart failure, arrhythmias, and sudden death. There is no specific therapy for LMNA-associated cardiomyopathy. We report that deletion of Lmna in cardiomyocytes in mice leads to severe cardiac dysfunction, conduction defect, ventricular arrhythmias, fibrosis, apoptosis, and premature death within 4 weeks. The phenotype is similar to LMNA-associated cardiomyopathy in humans. RNA sequencing, performed before the onset of cardiac dysfunction, led to identification of 2338 differentially expressed genes (DEGs) in Lmna-deleted cardiomyocytes. DEGs predicted activation of bromodomain-containing protein 4 (BRD4), a regulator of chromatin-associated proteins and transcription factors, which was confirmed by complementary approaches, including chromatin immunoprecipitation–sequencing. Daily injection of JQ1, a specific BET bromodomain inhibitor, partially reversed the DEGs, including those encoding secretome; improved cardiac function; abrogated cardiac arrhythmias, fibrosis, and apoptosis; and prolonged the median survival time 2-fold in the myocyte-specific Lmna-deleted mice. The findings highlight the important role of LMNA in cardiomyocytes and identify BET bromodomain inhibition as a potential therapeutic target in LMNA-associated cardiomyopathy, for which there is no specific effective therapy.
Authors
Gaelle Auguste, Leila Rouhi, Scot J. Matkovich, Cristian Coarfa, Matthew J. Robertson, Grazyna Czernuszewicz, Priyatansh Gurha, Ali J. Marian
Abstract
Cachexia, a devastating wasting syndrome characterized by severe weight loss with specific losses of muscle and adipose tissue, is driven by reduced food intake, increased energy expenditure, excess catabolism, and inflammation. Cachexia is associated with poor prognosis and high mortality and frequently occurs in patients with cancer, chronic kidney disease, infection, and many other illnesses. There is no effective treatment for this condition. Hypothalamic melanocortins have a potent and long-lasting inhibitory effect on feeding and anabolism, and pathophysiological processes increase melanocortin signaling tone, leading to anorexia, metabolic changes, and eventual cachexia. We used 3 rat models of anorexia and cachexia (LPS, methylcholanthrene sarcoma, and 5/6 subtotal nephrectomy) to evaluate efficacy of TCMCB07, a synthetic antagonist of the melanocortin-4 receptor. Our data show that peripheral treatment using TCMCB07 with intraperitoneal, subcutaneous, and oral administration increased food intake and body weight and preserved fat mass and lean mass during cachexia and LPS-induced anorexia. Furthermore, administration of TCMCB07 diminished hypothalamic inflammatory gene expression in cancer cachexia. These results suggest that peripheral TCMCB07 treatment effectively inhibits central melanocortin signaling and therefore stimulates appetite and enhances anabolism, indicating that TCMCB07 is a promising drug candidate for treating cachexia.
Authors
Xinxia Zhu, Michael F. Callahan, Kenneth A. Gruber, Marek Szumowski, Daniel L. Marks
Abstract
BACKGROUND Convalescent plasma is the only antibody-based therapy currently available for patients with coronavirus disease 2019 (COVID-19). It has robust historical precedence and sound biological plausibility. Although promising, convalescent plasma has not yet been shown to be safe as a treatment for COVID-19.METHODS Thus, we analyzed key safety metrics after transfusion of ABO-compatible human COVID-19 convalescent plasma in 5000 hospitalized adults with severe or life-threatening COVID-19, with 66% in the intensive care unit, as part of the US FDA expanded access program for COVID-19 convalescent plasma.RESULTS The incidence of all serious adverse events (SAEs), including mortality rate (0.3%), in the first 4 hours after transfusion was <1%. Of the 36 reported SAEs, there were 25 reported incidences of related SAEs, including mortality (n = 4), transfusion-associated circulatory overload (n = 7), transfusion-related acute lung injury (n = 11), and severe allergic transfusion reactions (n = 3). However, only 2 of 36 SAEs were judged as definitely related to the convalescent plasma transfusion by the treating physician. The 7-day mortality rate was 14.9%.CONCLUSION Given the deadly nature of COVID-19 and the large population of critically ill patients included in these analyses, the mortality rate does not appear excessive. These early indicators suggest that transfusion of convalescent plasma is safe in hospitalized patients with COVID-19.TRIAL REGISTRATION ClinicalTrials.gov NCT04338360.FUNDING Mayo Clinic, Biomedical Advanced Research and Development Authority (75A50120C00096), National Center for Advancing Translational Sciences (UL1TR002377), National Heart, Lung, and Blood Institute (5R35HL139854 and R01 HL059842), National Institute of Diabetes and Digestive and Kidney Diseases (5T32DK07352), Natural Sciences and Engineering Research Council of Canada (PDF-532926-2019), National Institute of Allergy and Infectious Disease (R21 AI145356, R21 AI152318, and AI152078), Schwab Charitable Fund, United Health Group, National Basketball Association, Millennium Pharmaceuticals, and Octapharma USA Inc.
Authors
Michael J. Joyner, R. Scott Wright, DeLisa Fairweather, Jonathon W. Senefeld, Katelyn A. Bruno, Stephen A. Klassen, Rickey E. Carter, Allan M. Klompas, Chad C. Wiggins, John R.A. Shepherd, Robert F. Rea, Emily R. Whelan, Andrew J. Clayburn, Matthew R. Spiegel, Patrick W. Johnson, Elizabeth R. Lesser, Sarah E. Baker, Kathryn F. Larson, Juan G. Ripoll, Kylie J. Andersen, David O. Hodge, Katie L. Kunze, Matthew R. Buras, Matthew N.P. Vogt, Vitaly Herasevich, Joshua J. Dennis, Riley J. Regimbal, Philippe R. Bauer, Janis E. Blair, Camille M. Van Buskirk, Jeffrey L. Winters, James R. Stubbs, Nigel S. Paneth, Nicole C. Verdun, Peter Marks, Arturo Casadevall
Abstract
Connexin-43 (Cx43) gap junctions provide intercellular coupling, which ensures rapid action potential propagation and synchronized heart contraction. Alterations in Cx43 localization and reductions in gap junction coupling occur in failing hearts, contributing to ventricular arrhythmias and sudden cardiac death. Recent reports have found that an internally translated Cx43 isoform, GJA1-20k, is an auxiliary subunit for the trafficking of Cx43 in heterologous expression systems. Here, we have created a mouse model by using CRISPR technology to mutate a single internal translation initiation site in Cx43 (M213L mutation), which generates full-length Cx43, but not GJA1-20k. We found that GJA1M213L/M213L mice had severely abnormal electrocardiograms despite preserved contractile function, reduced total Cx43, and reduced gap junctions, and they died suddenly at 2 to 4 weeks of age. Heterozygous GJA1M213L/WT mice survived to adulthood with increased ventricular ectopy. Biochemical experiments indicated that cytoplasmic Cx43 had a half-life that was 50% shorter than membrane-associated Cx43. Without GJA1-20k, poorly trafficked Cx43 was degraded. The data support that GJA1-20k, an endogenous entity translated independently of Cx43, is critical for Cx43 gap junction trafficking, maintenance of Cx43 protein, and normal electrical function of the mammalian heart.
Authors
Shaohua Xiao, Daisuke Shimura, Rachel Baum, Diana M. Hernandez, Sosse Agvanian, Yoshiko Nagaoka, Makoto Katsumata, Paul D. Lampe, Andre G. Kleber, TingTing Hong, Robin M. Shaw
Abstract
Globoid cell leukodystrophy (GLD; Krabbe disease) is a progressive, incurable neurodegenerative disease caused by deficient activity of the hydrolytic enzyme galactosylceramidase (GALC). The ensuing cytotoxic accumulation of psychosine results in diffuse central and peripheral nervous system (CNS, PNS) demyelination. Presymptomatic hematopoietic stem cell transplantation (HSCT) is the only treatment for infantile-onset GLD; however, clinical outcomes of HSCT recipients often remain poor, and procedure-related morbidity is high. There are no effective therapies for symptomatic patients. Herein, we demonstrate in the naturally occurring canine model of GLD that presymptomatic monotherapy with intrathecal AAV9 encoding canine GALC administered into the cisterna magna increased GALC enzyme activity, normalized psychosine concentration, improved myelination, and attenuated inflammation in both the CNS and PNS. Moreover, AAV-mediated therapy successfully prevented clinical neurological dysfunction, allowing treated dogs to live beyond 2.5 years of age, more than 7 times longer than untreated dogs. Furthermore, we found that a 5-fold lower dose resulted in an attenuated form of disease, indicating that sufficient dosing is critical. Finally, postsymptomatic therapy with high-dose AAV9 also significantly extended lifespan, signifying a treatment option for patients for whom HSCT is not applicable. If translatable to patients, these findings would improve the outcomes of patients treated either pre- or postsymptomatically.
Authors
Allison M. Bradbury, Jessica H. Bagel, Duc Nguyen, Erik A. Lykken, Jill Pesayco Salvador, Xuntian Jiang, Gary P. Swain, Charles A. Assenmacher, Ian J. Hendricks, Keiko Miyadera, Rebecka S. Hess, Arielle Ostrager, Patricia ODonnell, Mark S. Sands, Daniel S. Ory, G. Diane Shelton, Ernesto R. Bongarzone, Steven J. Gray, Charles H. Vite
Abstract
Heart failure (HF) with reduced contractile function is a common and lethal syndrome in which the heart cannot pump blood to adequately meet bodily demands, resulting in high mortality despite the current standard of care. In modern societies, the most common drivers of HF are ischemic heart disease and hypertension. However, in a substantial subset of cases, patients present with dilated and poorly contracting hearts without evidence of common inciting stressors, a syndrome called dilated cardiomyopathy (DCM). Genome sequencing has identified a host of deleterious germline variants in key cardiomyocyte genes as causes of heritable DCM, including mutations in LMNA, which encodes the nuclear lamina-associated protein lamin A/C. In this issue of the JCI, Auguste et al. generate a mouse model of DCM in which they delete Lmna in cardiomyocytes and discover that bromodomain and extraterminal (BET) protein activation is a druggable epigenetic mechanism of disease pathogenesis in this heritable HF syndrome.
Authors
Michael Alexanian, Saptarsi M. Haldar
Osteocyte necrosis triggers osteoclast-mediated bone loss through macrophage-inducible C-type lectin
Abstract
Although the control of bone-resorbing osteoclasts through osteocyte-derived RANKL is well defined, little is known about the regulation of osteoclasts by osteocyte death. Indeed, several skeletal diseases, such as bone fracture, osteonecrosis, and inflammation are characterized by excessive osteocyte death. Herein we show that osteoclasts sense damage-associated molecular patterns (DAMPs) released by necrotic osteocytes via macrophage-inducible C-type lectin (Mincle), which induced their differentiation and triggered bone loss. Osteoclasts showed robust Mincle expression upon exposure to necrotic osteocytes in vitro and in vivo. RNA sequencing and metabolic analyses demonstrated that Mincle activation triggers osteoclastogenesis via ITAM-based calcium signaling pathways, skewing osteoclast metabolism toward oxidative phosphorylation. Deletion of Mincle in vivo effectively blocked the activation of osteoclasts after induction of osteocyte death, improved fracture repair, and attenuated inflammation-mediated bone loss. Furthermore, in patients with osteonecrosis, Mincle was highly expressed at skeletal sites of osteocyte death and correlated with strong osteoclastic activity. Taken together, these data point to what we believe is a novel DAMP-mediated process that allows osteoclast activation and bone loss in the context of osteocyte death.
Authors
Darja Andreev, Mengdan Liu, Daniela Weidner, Katerina Kachler, Maria Faas, Anika Grüneboom, Ursula Schlötzer-Schrehardt, Luis E. Muñoz, Ulrike Steffen, Bettina Grötsch, Barbara Killy, Gerhard Krönke, Andreas M. Luebke, Andreas Niemeier, Falk Wehrhan, Roland Lang, Georg Schett, Aline Bozec
Abstract
No known therapies can prevent anaphylaxis. Bruton’s tyrosine kinase (BTK) is an enzyme thought to be essential for high-affinity IgE receptor (FcεRI) signaling in human cells. We tested the hypothesis that FDA-approved BTK inhibitors (BTKis) would prevent IgE-mediated responses including anaphylaxis. We showed that irreversible BTKis broadly prevented IgE-mediated degranulation and cytokine production in primary human mast cells and blocked allergen-induced contraction of isolated human bronchi. To address their efficacy in vivo, we created and used what we believe to be a novel humanized mouse model of anaphylaxis that does not require marrow ablation or human tissue implantation. After a single intravenous injection of human CD34+ cells, NSG-SGM3 mice supported the population of mature human tissue-resident mast cells and basophils. These mice showed excellent responses during passive systemic anaphylaxis using human IgE to selectively evoke human mast cell and basophil activation, and response severity was controllable by alteration of the amount of allergen used for challenge. Remarkably, pretreatment with just 2 oral doses of the BTKi acalabrutinib completely prevented moderate IgE-mediated anaphylaxis in these mice and also significantly protected against death during severe anaphylaxis. Our data suggest that BTKis may be able to prevent anaphylaxis in humans by inhibiting FcεRI-mediated signaling.
Authors
Melanie C. Dispenza, Rebecca A. Krier-Burris, Krishan D. Chhiba, Bradley J. Undem, Piper A. Robida, Bruce S. Bochner
Abstract
BACKGROUND From March 2, 2020, to April 12, 2020, New York City (NYC) experienced exponential growth of the COVID-19 pandemic due to novel coronavirus (SARS-CoV-2). Little is known regarding how physicians have been affected. We aimed to characterize the COVID-19 impact on NYC resident physicians.METHODS IRB-exempt and expedited cross-sectional analysis through survey to NYC residency program directors April 3–12, 2020, encompassing events from March 2, 2020, to April 12, 2020.RESULTS From an estimated 340 residency programs around NYC, recruitment yielded 91 responses, representing 24 specialties and 2306 residents. In 45.1% of programs, at least 1 resident with confirmed COVID-19 was reported. One hundred one resident physicians were confirmed COVID-19–positive, with an additional 163 residents presumed positive for COVID-19 based on symptoms but awaiting or unable to obtain testing. Two COVID-19–positive residents were hospitalized, with 1 in intensive care. Among specialties with more than 100 residents represented, negative binomial regression indicated that infection risk differed by specialty (P = 0.039). In 80% of programs, quarantining a resident was reported. Ninety of 91 programs reported reuse or extended mask use, and 43 programs reported that personal protective equipment (PPE) was suboptimal. Sixty-five programs (74.7%) redeployed residents elsewhere to support COVID-19 efforts.CONCLUSION Many resident physicians around NYC have been affected by COVID-19 through direct infection, quarantine, or redeployment. Lack of access to testing and concern regarding suboptimal PPE are common among residency programs. Infection risk may differ by specialty.FUNDING National Eye Institute Core Grant P30EY019007; Research to Prevent Blindness Unrestricted Grant; Parker Family Chair; University of Pennsylvania.
Authors
Mark P. Breazzano, Junchao Shen, Aliaa H. Abdelhakim, Lora R. Dagi Glass, Jason D. Horowitz, Sharon X. Xie, C. Gustavo de Moraes, Alice Chen-Plotkin, Royce W.S. Chen, on behalf of the New York City Residency Program Directors COVID-19 Research Group
Abstract
New York City has been described as the epicenter of the COVID-19 pandemic in the United States. While health care workers are notably at increased risk for COVID-19 infection, the impact on resident physicians remains unclear. In this issue of the JCI, Breazzano et al. surveyed resident physicians for their exposure to COVID-19 during the exponential phase of the COVID-19 pandemic. The researchers also assessed how personal protective equipment and COVID-19 testing protected health care workers from infection. This study highlights resident physician experiences of the first COVID-19 wave that can inform and improve preparedness for upcoming COVID-19 surges and other future epidemics.
Authors
Lauren I. Shapiro, Grace R. Kajita, Julia H. Arnsten, Yaron Tomer
Abstract
Skeletal muscle depends on the precise orchestration of contractile and metabolic gene expression programs to direct fiber-type specification and to ensure muscle performance. Exactly how such fiber type–specific patterns of gene expression are established and maintained remains unclear, however. Here, we demonstrate that histone monomethyl transferase MLL4 (KMT2D), an enhancer regulator enriched in slow myofibers, plays a critical role in controlling muscle fiber identity as well as muscle performance. Skeletal muscle–specific ablation of MLL4 in mice resulted in downregulation of the slow oxidative myofiber gene program, decreased numbers of type I myofibers, and diminished mitochondrial respiration, which caused reductions in muscle fatty acid utilization and endurance capacity during exercise. Genome-wide ChIP-Seq and mRNA-Seq analyses revealed that MLL4 directly binds to enhancers and functions as a coactivator of the myocyte enhancer factor 2 (MEF2) to activate transcription of slow oxidative myofiber genes. Importantly, we also found that the MLL4 regulatory circuit is associated with muscle fiber–type remodeling in humans. Thus, our results uncover a pivotal role for MLL4 in specifying structural and metabolic identities of myofibers that govern muscle performance. These findings provide therapeutic opportunities for enhancing muscle fitness to combat a variety of metabolic and muscular diseases.
Authors
Lin Liu, Chenyun Ding, Tingting Fu, Zhenhua Feng, Ji-Eun Lee, Liwei Xiao, Zhisheng Xu, Yujing Yin, Qiqi Guo, Zongchao Sun, Wanping Sun, Yan Mao, Likun Yang, Zheng Zhou, Danxia Zhou, Leilei Xu, Zezhang Zhu, Yong Qiu, Kai Ge, Zhenji Gan
Abstract
γ9δ2T cells play a major role in cancer immune surveillance, yet the clinical translation of their in vitro promise remains challenging. To address limitations of previous clinical attempts using expanded γ9δ2T cells, we explored the clonal diversity of γ9δ2T cell repertoires and characterized their target. We demonstrated that only a fraction of expanded γ9δ2T cells was active against cancer cells and that activity of the parental clone, or functional avidity of selected γ9δ2 T cell receptors (γ9δ2TCRs), was not associated with clonal frequency. Furthermore, we analyzed the target-receptor interface and provided a 2-receptor, 3-ligand model. We found that activation was initiated by binding of the γ9δ2TCR to BTN2A1 through the regions between CDR2 and CDR3 of the TCR γ chain and modulated by the affinity of the CDR3 region of the TCRδ chain, which was phosphoantigen independent (pAg independent) and did not depend on CD277. CD277 was secondary, serving as a mandatory coactivating ligand. We found that binding of CD277 to its putative ligand did not depend on the presence of γ9δ2TCR, did depend on usage of the intracellular CD277, created pAg-dependent proximity to BTN2A1, enhanced cell-cell conjugate formation, and stabilized the immunological synapse (IS). This process critically depended on the affinity of the γ9δ2TCR and required membrane flexibility of the γ9δ2TCR and CD277, facilitating their polarization and high-density recruitment during IS formation.
Authors
Anna Vyborova, Dennis X. Beringer, Domenico Fasci, Froso Karaiskaki, Eline van Diest, Lovro Kramer, Aram de Haas, Jasper Sanders, Anke Janssen, Trudy Straetemans, Daniel Olive, Jeanette Leusen, Lola Boutin, Steven Nedellec, Samantha L. Schwartz, Michael J. Wester, Keith A. Lidke, Emmanuel Scotet, Diane S. Lidke, Albert J.R. Heck, Zsolt Sebestyen, Jürgen Kuball
Abstract
BACKGROUND Despite an increasing appreciation of the roles that myeloid cells play in tumor progression and therapy, challenges remain in interpreting the tumor-associated myeloid response balance and its translational value. We aimed to construct a simple and reliable myeloid signature for hepatocellular carcinoma (HCC).METHODS Using in situ immunohistochemistry, we assessed the distribution of major myeloid subtypes in both peri- and intratumoral regions of HCC. A 2-feature-based, myeloid-specific prognostic signature, named the myeloid response score (MRS), was constructed using an L1-penalized Cox regression model based on data from a training subset (n = 244), a test subset (n = 244), and an independent internal (n = 341) and 2 external (n = 94; n = 254) cohorts.RESULTS The MRS and the MRS-based nomograms displayed remarkable discriminatory power, accuracy, and clinical usefulness for predicting recurrence and patient survival, superior to current staging algorithms. Moreover, an increase in MRS was associated with a shift in the myeloid response balance from antitumor to protumor activities, accompanied by enhanced CD8+ T cell exhaustion patterns. Additionally, we provide evidence that the MRS was associated with the efficacy of sorafenib treatment for recurrent HCC.CONCLUSION We identified and validated a simple myeloid signature for HCC that showed remarkable prognostic potential and may serve as a basis for the stratification of HCC immune subtypes.FUNDING This work was supported by the National Science and Technology Major Project of China, the National Natural Science Foundation of China, the Science and Information Technology of Guangzhou, the Fundamental Research Funds for the Central Universities, the Guangdong Basic and Applied Basic Research Foundation, and the China Postdoctoral Science Foundation.
Authors
Chong Wu, Jie Lin, Yulan Weng, Dan-Ni Zeng, Jing Xu, Shufeng Luo, Li Xu, Mingyu Liu, Qiaomin Hua, Chao-Qun Liu, Jin-Qing Li, Jing Liao, Cheng Sun, Jian Zhou, Min-Shan Chen, Chao Liu, Zhenhong Guo, Shi-Mei Zhuang, Jin-Hua Huang, Limin Zheng
Abstract
BACKGROUND Coronavirus disease 19 (COVID-19) is an emerging infectious disease caused by SARS-CoV-2. Antiviral immune response is crucial to achieve pathogen clearance; however, in some patients an excessive and aberrant host immune response can lead to an acute respiratory distress syndrome. The comprehension of the mechanisms that regulate pathogen elimination, immunity, and pathology is essential to better characterize disease progression and widen the spectrum of therapeutic options.METHODS We performed a flow cytometric characterization of immune cell subsets from 30 patients with COVID-19 and correlated these data with clinical outcomes.RESULTS Patients with COVID-19 showed decreased numbers of circulating T, B, and NK cells and exhibited a skewing of CD8+ T cells toward a terminally differentiated/senescent phenotype. In agreement, CD4+ T and CD8+ T, but also NK cells, displayed reduced antiviral cytokine production capability. Moreover, a reduced cytotoxic potential was identified in patients with COVID-19, particularly in those who required intensive care. The latter group of patients also showed increased serum IL-6 levels that inversely correlated to the frequency of granzyme A–expressing NK cells. Off-label treatment with tocilizumab restored the cytotoxic potential of NK cells.CONCLUSION The association between IL-6 serum levels and the impairment of cytotoxic activity suggests the possibility that targeting this cytokine may restore antiviral mechanisms.FUNDING This study was supported by funds from the Department of Experimental and Clinical Medicine of University of Florence (the ex-60% fund and the “Excellence Departments 2018–2022 Project”) derived from Ministero dell’Istruzione, dell’Università e della Ricerca (Italy).
Authors
Alessio Mazzoni, Lorenzo Salvati, Laura Maggi, Manuela Capone, Anna Vanni, Michele Spinicci, Jessica Mencarini, Roberto Caporale, Benedetta Peruzzi, Alberto Antonelli, Michele Trotta, Lorenzo Zammarchi, Luca Ciani, Leonardo Gori, Chiara Lazzeri, Andrea Matucci, Alessandra Vultaggio, Oliviero Rossi, Fabio Almerigogna, Paola Parronchi, Paolo Fontanari, Federico Lavorini, Adriano Peris, Gian Maria Rossolini, Alessandro Bartoloni, Sergio Romagnani, Francesco Liotta, Francesco Annunziato, Lorenzo Cosmi
Abstract
Oxidant stress can contribute to health and disease. Here we show that invertebrates and vertebrates share a common stereospecific redox pathway that protects against pathological responses to stress, at the cost of reduced physiological performance, by constraining Ca2+/calmodulin-dependent protein kinase II (CaMKII) activity. MICAL1, a methionine monooxygenase thought to exclusively target actin, and MSRB, a methionine reductase, control the stereospecific redox status of M308, a highly conserved residue in the calmodulin-binding (CaM-binding) domain of CaMKII. Oxidized or mutant M308 (M308V) decreased CaM binding and CaMKII activity, while absence of MICAL1 in mice caused cardiac arrhythmias and premature death due to CaMKII hyperactivation. Mimicking the effects of M308 oxidation decreased fight-or-flight responses in mice, strikingly impaired heart function in Drosophila melanogaster, and caused disease protection in human induced pluripotent stem cell–derived cardiomyocytes with catecholaminergic polymorphic ventricular tachycardia, a CaMKII-sensitive genetic arrhythmia syndrome. Our studies identify a stereospecific redox pathway that regulates cardiac physiological and pathological responses to stress across species.
Authors
Klitos Konstantinidis, Vassilios J. Bezzerides, Lo Lai, Holly M. Isbell, An-Chi Wei, Yuejin Wu, Meera C. Viswanathan, Ian D. Blum, Jonathan M. Granger, Danielle Heims-Waldron, Donghui Zhang, Elizabeth D. Luczak, Kevin R. Murphy, Fujian Lu, Daniel H. Gratz, Bruno Manta, Qiang Wang, Qinchuan Wang, Alex L. Kolodkin, Vadim N. Gladyshev, Thomas J. Hund, William T. Pu, Mark N. Wu, Anthony Cammarato, Mario A. Bianchet, Madeline A. Shea, Rodney L. Levine, Mark E. Anderson
Abstract
Desmoplasia describes the deposition of extensive extracellular matrix and defines primary pancreatic ductal adenocarcinoma (PDA). The acellular component of this stroma has been implicated in PDA pathogenesis and is being targeted therapeutically in clinical trials. By analyzing the stromal content of PDA samples from numerous annotated PDA data sets and correlating stromal content with both anatomic site and clinical outcome, we found PDA metastases in the liver, the primary cause of mortality to have less stroma, have higher tumor cellularity than primary tumors. Experimentally manipulating stromal matrix with an anti– lysyl oxidase like-2 (anti-LOXL2) antibody in syngeneic orthotopic PDA mouse models significantly decreased matrix content, led to lower tissue stiffness, lower contrast retention on computed tomography, and accelerated tumor growth, resulting in diminished overall survival. These studies suggest an important protective role of stroma in PDA and urge caution in clinically deploying stromal depletion strategies.
Authors
Honglin Jiang, Robert J. Torphy, Katja Steiger, Henry Hongo, Alexa J. Ritchie, Mark Kriegsmann, David Horst, Sarah E. Umetsu, Nancy M. Joseph, Kimberly McGregor, Michael J. Pishvaian, Edik M. Blais, Brian Lu, Mingyu Li, Michael Hollingsworth, Connor Stashko, Keith Volmar, Jen Jen Yeh, Valerie M. Weaver, Zhen J. Wang, Margaret A. Tempero, Wilko Weichert, Eric A. Collisson
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Abstract
Background:While mitochondria play an important role in innate immunity, the relationship between mitochondrial dysfunction and inflammation in heart failure (HF) is poorly understood. In this study we aimed to investigate the mechanistic link between mitochondrial dysfunction and inflammatory activation in peripheral blood mononuclear cells (PBMCs), and the potential anti-inflammatory effect of boosting NAD level.Methods:We compared the PBMC mitochondrial respiration of 19 hospitalized Stage D HF patients with 19 healthy participants. We then created an in vitro model of sterile inflammation by treating healthy PBMC with MitoDAMP (Mitochondrial Damage-Associated Molecular Patterns) isolated from human heart tissue. Lastly, we enrolled Stage D HF patients and sampled their blood before and after taking 5-9 days of oral nicotinamide riboside, an NAD precursor.Results:We demonstrated that HF is associated with both reduced respiratory capacity and elevated proinflammatory cytokine gene expressions. In our in vitro model, MitoDAMP-treated PBMCs secreted IL-6 that impaired mitochondrial respiration by reducing Complex I activity. Last, oral NR administration enhanced PBMC respiration and reduced proinflammatory cytokine gene expression in 4 HF subjects.Conclusion:These findings suggest that systemic inflammation in HF patients is causally linked to mitochondrial function of the PBMC. Increasing NAD levels may have the potential to improve mitochondrial respiration and attenuate proinflammatory activation of PBMC in HF. FundingThis study is funded by NIH R21 HL126209 (to RT and KO), NIH R01 HL144937 (to KO and RT) and University of Washington ITHS Catalyst Award (to DDW). Both BZ (18POST33990352) and DDW (18POST34030098) are funded by the AHA Postdoctoral Fellowships.
Authors
Bo Zhou, Dennis D. Wang, Yanhua Qiu, Sophia Airhart, Yaxin Liu, April Stempien-Otero, Kevin D. O’Brien, Rong Tian.
Abstract
The transcription factor, Mef2d, is important in the regulation of differentiation and adaptive responses in many cell types. Among T cells, Mef2d gains new functions in Foxp3+ T-regulatory (Treg) cells as a result of its interactions with the transcription factor, Foxp3, and its release from canonical partners, like histone/protein deacetylases. Though not necessary for the generation and maintenance of Tregs, Mef2d is required for the expression of IL-10, Ctla-4 and Icos, and for the acquisition of an effector Treg phenotype. At these loci, Mef2d acts both synergistically and additively to Foxp3, and down-stream of Blimp1. Mice with the conditional deletion in Tregs of the gene encoding Mef2d are unable to maintain long-term allograft survival despite costimulation blockade and have enhanced antitumor immunity in syngeneic models, but they display only minor evidence of autoimmunity when maintained under normal conditions. The role played by Mef2d in sustaining effector Foxp3+ Treg functions without abrogating their basal actions suggests its suitability for drug discovery efforts in cancer therapy.
Authors
Eros Di Giorgio, Liqing Wang, Yan Xiong, Tatiana Akimova, Lanette M. Christensen, Rongxiang Han, Arabinda Samanta, Matteo Trevisanut, Tricia R. Bhatti, Ulf H. Beier, Wayne W. Hancock
Abstract
No treatment for frontotemporal dementia (FTD), the second most common early-onset dementia, is available but therapeutics are being investigated to target the two main proteins associated with FTD pathological subtypes: TDP-43 (FTLD-TDP) and tau (FTLD-tau). Testing potential therapies in clinical trials is hamstrung by our inability to distinguish between patients with FTLD-TDP and FTLD-tau. Therefore, we evaluated truncated stathmin-2 (STMN2) as a proxy of TDP-43 pathology, given reports that TDP-43 dysfunction causes truncated STMN2 accumulation. Truncated STMN2 accumulated in human iPSC-derived neurons depleted of TDP-43, but not in those with pathogenic TARDBP mutations in the absence of TDP-43 aggregation or loss of nuclear protein. In RNA-seq analyses of human brain samples from the NYGC ALS cohort, truncated STMN2 RNA was confined to tissues and disease sub-types marked by TDP-43 inclusions. Lastly, we validated that truncated STMN2 RNA is elevated in the frontal cortex of a cohort of FTLD-TDP cases but not in controls or cases with progressive supranuclear palsy (PSP), a type of FTLD-tau. Further, in FTLD-TDP, we observed significant associations of truncated STMN2 RNA with phosphorylated TDP-43 levels and an earlier age of disease onset. Overall, our data uncovered truncated STMN2 as a marker for TDP-43 dysfunction in FTD.
Authors
Mercedes Prudencio, Jack Humphrey, Sarah Pickles, Anna-Leigh Brown, Sarah E. Hill, Jennifer Kachergus, Ji Shi, Michael Heckman, Matthew Spiegel, Casey Cook, Yuping Song, Mei Yue, Lillian Daughrity, Yari Carlomagno, Karen Jansen-West, Cristhoper Fernandez De Castro, Michael DeTure, Shunsuke Koga, Ying-Chih Wang, Prasanth Sivakumar, Cristian Bodo, Ana Candalija, Kevin Talbot, Bhuvaneish T. Selvaraj, Karen Burr, Siddharthan Chandran, Jia Newcombe, Tammaryn Lashley, Isabel Hubbard, Demetra Catalano, Duyang Kim, Nadia Propp, Samantha Fennessey, Delphine Fagegaltier, Hemali Phatnani, Maria Secrier, Elizabeth M.C. Fisher, Björn Oskarsson, Marka van Blitterswijk, Rosa Rademakers, Neill R. Graff-Radford, Bradley Boeve, David S. Knopman, Ronald Petersen, Keith Josephs, E. Aubrey Thompson, Towfique Raj, Michael E. Ward, Dennis Dickson, Tania F. Gendron, Pietro Fratta, Leonard Petrucelli
Abstract
Gain-of-function mutations in the WNK1 and WNK4 genes are responsible for Familial Hyperkalemic Hypertension (FHHt), a rare inherited disorder characterized by arterial hypertension and hyperkalemia with metabolic acidosis. More recently, FHHt-causing mutations in the KLHL3-CUL3 E3 ubiquitin ligase complex have shed light on the importance of WNKs cellular degradation on renal ion transport. Using full exome sequencing in a four-generation family and then targeted sequencing in other suspected cases, we have identified new missense variants at the WNK1 gene, clustering in the short conserved acidic motif known to interact with the KLHL3-CUL3 ubiquitin complex. Affected subjects had an early-onset and a marked hyperkalemic phenotype, but normal blood pressure values. Functional experiments in Xenopus laevis oocytes and HEK293T cells demonstrated that these mutations strongly decrease the ubiquitination of the kidney-specific isoform KS-WNK1 by the KLHL3-CUL3 complex, rather than the long ubiquitous catalytically active L-WNK1 isoform. A corresponding CRISPR-Cas9 engineered mouse model recapitulated both the clinical and biological phenotype. Renal investigations showed increased activation of the SPAK-NCC phosphorylation cascade, associated with impaired ROMK apical expression in the distal part of the renal tubule. Altogether, these new WNK1 genetic variants highlight the importance of the KS-WNK1 isoform abundance on potassium homeostasis.
Authors
Helene Louis-Dit-Picard, Ilektra Kouranti, Chloe Rafael, Irmine Loisel-Ferreira, Maria Chavez-Canales, Waed Abdel Khalek, Eduardo Argaiz, Stephanie Baron, Sarah Vacle, Tiffany Migeon, Richard Coleman, Marcio Do Cruzeiro, Marguerite Hureaux, Nirubiah Thurairajasingam, Stéphane Decramer, Xavier Girerd, Kevin M. O'Shaughnessy, Paolo Mulatero, Gwenaelle Roussey, Ivan Tack, Robert J. Unwin, Rosa Vargas-Poussou, Olivier Staub, P. Richard Grimm, Paul A. Welling, Gerardo Gamba, Eric Clauser, Juliette Hadchouel, Xavier Jeunemaitre
Abstract
SARS-CoV-2 is responsible for development of COVID-19 in infected individuals, who can either exhibit mild symptoms or progress towards a life-threatening acute respiratory distress syndrome (ARDS). Exacerbated inflammation and dysregulated immune responses involving T and myeloid cells occur in COVID-19 patients with severe clinical progression. However, the differential contribution of specific subsets of dendritic cells and monocytes to ARDS is still poorly understood. In addition, the role of CD8+ T cells present in the lung of COVID-19 patients and relevant for viral control has not been characterized. Here, we have studied the frequencies and activation profiles of dendritic cells and monocytes present in the blood and lung of COVID-19 patients with different clinical severity in comparison with healthy individuals. Furthermore, these subpopulations and their association with antiviral effector CD8+ T cell subsets were also characterized in lung infiltrates from critical COVID-19 patients. Our results indicate that inflammatory transitional and non-classical monocytes and CD1c+ conventional dendritic cells preferentially migrate from blood to lungs in patients with severe COVID-19. Thus, this study increases the knowledge on specific myeloid subsets involved in the pathogenesis of COVID-19 disease and could be useful for the design of therapeutic strategies to fight SARS-CoV-2 infection.
Authors
Ildefonso Sánchez-Cerrillo, Pedro Landete, Beatriz Aldave, Santiago Sánchez-Alonso, Ana Sánchez-Azofra, Ana Marcos-Jiménez, Elena Ávalos, Ana Alcaraz-Serna, Ignacio de los Santos, Tamara Mateu-Albero, Laura Esparcia, Celia López-Sanz, Pedro Martínez-Fleta, Ligia Gabrie, Luciana del Campo Guerola, Hortensia de la Fuente, María J Calzada, Isidoro González-Álvaro, Arantzazu Alfranca, Francisco Sánchez-Madrid, Cecilia Muñoz-Calleja, Joan B. Soriano, Julio Ancochea, Enrique Martín-Gayo
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August 2020
August 2020 Issue
On the cover:
“Burst of Passion II” characterizes dynamic transcriptional machinery
Prostate cancer is associated with dysregulated gene expression, but how the chromatin landscape influences RNA Polymerase II (RNA Pol II) and transcriptional output remains unclear. In this issue of the JCI, Ramanand, Chen, Yuan, and colleagues mapped the RNA Pol II/chromatin interaction of healthy or cancerous prostate cell lines. The researchers used a modified paired-end tag sequencing technique to immunoprecipitate chromatin from crosslinked cells. They revealed thousands of protein-DNA and DNA-DNA interactions. This study highlights the importance of genome structure and transcriptional dysregulation in prostate cancer. The cover image ‘Burst of Passion II’ by artist Ken Bonner exemplifies the coordinated and dynamic nature of transcriptional machinery. Image credit: Ken Bonner.
JCI This Month
August 2020 JCI This Month
JCI This Month is a digest of the research, reviews, and other features published each month.
Review Series - More
Series edited by Arturo Casadevall
Latency in Infectious Disease
Series edited by Arturo Casadevall
Latency describes the persistence of a microorganism within its host in the absence of clinical symptoms of disease. Both microorganism and host benefit from induction of latency: the microorganism establishes a stable environment that facilitates survival, and the host avoids progressive damage and disease. Latent states have been observed in bacterial, viral, fungal, and parasitic infectious diseases, though the mechanisms differ within each microorganism and host pair. In this issue, a Review Series on Latency in Infectious Disease explores the different strategies that various microorganisms use to achieve latency. Conceptualized by JCI’s Deputy Editor Arturo Casadevall, the series highlights the latency mechanisms employed by herpesviruses, HIV, Cryptococcus neoformans, and Toxoplasma gondii. In addition to describing mechanisms, the reviews outline the detrimental effects of latent disease and recent progress toward treatment and eradication.
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Copyright © 2020 American Society for Clinical Investigation
ISSN: 0021-9738 (print), 1558-8238 (online)