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Citation Information: J Clin Invest. 2023. https://doi.org/10.1172/JCI162870.
Abstract
SIPRα on macrophages binds with CD47 to resist pro-engulfment signals, but how the downstream signal of SIPRα controls tumor-infiltrating macrophages (TIMs) is still poorly clarified. Here we reported that the CD47/SIRPα axis requires the deneddylation of tyrosine phosphatase SHP2. Mechanistically, SHP2 is constitutively neddylated on K358 and K364 sites, thus its auto-inhibited conformation is maintained. In response to CD47-liganded SIRPα, SHP2 is deneddylated by SENP8, which leads to the dephosphorylation of relevant substrates at the phagocytic cup and subsequent inhibition of macrophage phagocytosis. Furthermore, neddylation inactivated myeloid-SHP2 and greatly boosted the efficacy of colorectal cancer (CRC) immunotherapy. Importantly, we observed that the supplementation with SHP2 allosteric inhibitors sensitized the immune treatment-resistant CRC to immunotherapy. Our results emphasized that the CRC subtype which is unresponsive to immunotherapy relies on SIRPαhiSHP2hiNEDD8lo TIMs, and highlighted the need to further combine the strategy of SHP2 targeting in colorectal cancer therapy.
Authors
Yiqing Li, Hui Zhou, Pan Liu, Dandan Lv, Yichun Shi, Bufu Tang, Jiaqi Xu, Tingting Zhong, Wangting Xu, Jie Zhang, Jianying Zhou, Kejing Ying, Yongchao Zhao, YI Sun, Zhinong Jiang, Hongqiang Cheng, Xue Zhang, Yue-Hai Ke
Citation Information: J Clin Invest. 2023. https://doi.org/10.1172/JCI158352.
Abstract
The role of tumor-associated macrophages (TAMs) along with the regulatory mechanisms underlying distinct macrophage activation states remain poorly understood in prostate cancer (PCa). Herein, we reported that PCa growth in macrophage-specific Ubc9 deficient mice is substantially suppressed compared to their wild-type littermates, an effect partially ascribed to the augmented CD8+ T cell response. Biochemical and molecular analyses revealed that the signal transducer and activator of transcription 4 (STAT4) is a crucial UBC9-mediated SUMOylation target, with lysine residue 350 (K350) as the major modification site. Site-directed mutation of STAT4 (K350R) enhanced its nuclear translocation and stability, thereby facilitating the proinflammatory activation of macrophages. Importantly, administration of UBC9 inhibitor, 2-D08, promoted the antitumor effect of TAMs and increased the expression of PD-1 on CD8+ T cells, supporting a synergistic antitumor efficacy once it combined with the immune checkpoint blockade (ICB) therapy. Together, our results demonstrated that ablation of UBC9 could reverse the immunosuppressive phenotype of TAMs via promoting STAT4 mediated macrophage activation and macrophage-CD8+ T cell crosstalk, which provides valuable insights to halt the pathogenic process of tumorigenesis.
Authors
Jun Xiao, Fei Sun, Ya-Nan Wang, Bo Liu, Peng Zhou, Fa-Xi Wang, Hai-Feng Zhou, Yue Ge, Tian-Tian Yue, Jia-Hui Luo, Chun-Liang Yang, Shan-Jie Rong, Ze-Zhong Xiong, Sheng Ma, Qi Zhang, Yang Xun, Chun-Guang Yang, Yang Luan, Shao-Gang Wang, Cong-Yi Wang, Zhi-Hua Wang
Citation Information: J Clin Invest. 2023. https://doi.org/10.1172/JCI162190.
Abstract
Aberrant immune responses to resident microbes promote inflammatory bowel disease and other chronic inflammatory conditions. However, how microbiota-specific immunity is controlled in mucosal tissues remains poorly understood. Here, we find that mice lacking epithelial expression of microbiota-sensitive histone deacetylase 3 (HDAC3) exhibit increased accumulation of commensal-specific CD4+ T cells in the intestine, provoking the hypothesis that epithelial HDAC3 may instruct local microbiota-specific immunity. Consistent with this, microbiota-specific CD4+ T cells and epithelial HDAC3 expression were concurrently induced following early-life microbiota colonization. Further, epithelial-intrinsic ablation of HDAC3 decreased commensal-specific Tregs, increased commensal-specific Th17 cells, and promoted T cell-driven colitis. Mechanistically, HDAC3 was essential for NFκB-dependent regulation of epithelial MHC class II (MHCII). Epithelial-intrinsic MHCII dampened local accumulation of commensal-specific Th17 cells in adult mice, and protected against microbiota-triggered inflammation. Remarkably, HDAC3 enabled the microbiota to induce MHCII on epithelial cells and limit the number of commensal-specific T cells in the intestine. Collectively, these data reveal a central role for an epithelial histone deacetylase in directing the dynamic balance of tissue-intrinsic CD4+ T cell subsets that recognize commensal microbes and control inflammation.
Authors
Emily M. Eshleman, Tzu-Yu Shao, Vivienne Woo, Taylor Rice, Laura Engleman, Bailey J. Didriksen, Jordan Whitt, David B. Haslam, Sing Sing Way, Theresa Alenghat
Citation Information: J Clin Invest. 2023. https://doi.org/10.1172/JCI160036.
Abstract
Genetic variants in the third intron of the PRDM6 gene have been associated with blood pressure traits in multiple genome-wide association studies (GWAS). By combining fine mapping, massive-ly parallel reporter assays, and gene editing we identified the causal variants for hypertension as super-enhancers that drive the expression of PRDM6 and are partly regulated by STAT1. The het-erozygous SMC-specific Prdm6 knockout mice (Prdm6fl/+ Sm22Cre) exhibited a markedly high-er number of renin-producing cells in the kidneys at embryonic day 18.5 compared to wild-type littermates and developed salt-induced systemic hypertension that was completely responsive to the renin inhibitor aliskiren. Strikingly, RNA-seq analysis of the mice aorta identified a network of PRDM6-regulated genes that are located in GWAS-associated loci for blood pressure, most nota-bly Sox6, which modulates renin-expression in the kidney. Accordingly, the smooth muscle cell-specific disruption of Sox6 in Prdm6fl/+ Sm22Cre mice resulted in a dramatic reduction of renin. Fate mapping and histological studies also showed increased numbers of neural crest-derived cells accompanied by increased collagen deposition in the kidneys of Prdm6fl/+ Wnt1Cre-ZsGreen1Cre compared to wild-type mice. These findings establish the role of PRDM6 as a regulator of renin-producing cells and an attractive target for the development of antihypertensive drugs.
Authors
Kushan L. Gunawardhana, Lingjuan Hong, Trojan Rugira, Severin Uebbing, Joanna Kucharczak, Sameet Mehta, Dineth R. Karunamuni, Brenda Cabrera-Mendoza, Renato Polimanti, James P. Noonan, Arya Mani
Citation Information: J Clin Invest. 2023. https://doi.org/10.1172/JCI164919.
Abstract
Mutations in the human Progranulin (GRN) gene are a leading cause of frontotemporal lobar degeneration (FTLD). While previous studies implicate aberrant microglial activation as a disease-driving factor in neurodegeneration in the thalamocortical circuit in Grn-/- mice, the exact mechanism for neurodegeneration in FTLD-GRN remains unclear. By performing comparative single-cell transcriptomics in the thalamus and frontal cortex of Grn-/- mice and patients with FTLD-GRN, we have uncovered a highly conserved astroglial pathology characterized by upregulation of gap junction protein GJA1, water channel AQP4, and lipid-binding protein APOE, and downregulation of glutamate transporter SLC1A2 that promoted profound synaptic degeneration across the two species. This astroglial toxicity could be recapitulated in mouse astrocyte-neuron cocultures and by transplanting induced pluripotent stem cell-derived astrocytes to cortical organoids, where Progranulin-deficient astrocytes promoted synaptic degeneration, neuronal stress, and TDP-43 proteinopathy. Together, these results reveal previously unappreciated astroglial pathology as a key mechanism in neurodegeneration in FTLD-GRN.
Authors
Elise Marsan, Dmitry Velmeshev, Arren Ramsey, Ravi K. Patel, Jiasheng Zhang, Mark Koontz, Madeline G. Andrews, Martina de Majo, Cristina Mora, Jessica Blumenfeld, Alissa N. Li, Salvatore Spina, Lea T. Grinberg, William Seeley, Bruce L. Miller, Erik M. Ullian, Matthew F. Krummel, Arnold Kriegstein, Eric J. Huang
Citation Information: J Clin Invest. 2023. https://doi.org/10.1172/JCI166408.
Abstract
Cortical neural dynamics mediate information processing for the cerebral cortex, implicated in fundamental biological processes, such as vision and olfaction, in addition to neurological and psychiatric diseases. Spontaneous pain is a key feature of human neuropathic pain. Whether spontaneous pain pushes cortical network into an aberrant state, and if so, whether it can be brought back to a ‘normal’ operating range to ameliorate pain are unknown. Using a clinically relevant mouse model of neuropathic pain with spontaneous pain-like behavior, we report that orofacial spontaneous pain activated a specific area within the primary somatosensory cortex (S1), displaying synchronized neural dynamics revealed by intravital two-photon calcium imaging. This synchronization was underpinned by local GABAergic interneuron hypoactivity. Pain-induced cortical synchronization could be attenuated by manipulating local S1 networks or clinically effective pain therapies. Specifically, both chemogenetic inhibition of pain-related c-Fos-expressing neurons, and selective activation of GABAergic interneurons, significantly attenuated S1 synchronization. Clinically effective pain therapies including carbamazepine and nerve root decompression could also dampen S1 synchronization. More importantly, restoring a ‘normal’ range of neural dynamics, through attenuating pain-induced S1 synchronization, alleviated pain-like behavior. These results suggest spontaneous pain pushes S1 regional network into a synchronized state, whereas reversal of this synchronization alleviates pain.
Authors
Weihua Ding, Lukas Fischer, Qian Chen, Ziyi Li, Liuyue Yang, Zerong You, Kun Hu, Xinbo Wu, Xue Zhou, Wei Chao, Peter Hu, Tewodros Mulugeta Dagnew, Daniel M. DuBreuil, Shiyu Wang, Suyun Xia, Caroline Bao, Shengmei Zhu, Lucy Chen, Changning Wang, Brian Wainger, Peng Jin, Jianren Mao, Guoping Feng, Mark T. Harnett, Shiqian Shen
Citation Information: J Clin Invest. 2023. https://doi.org/10.1172/JCI165245.
Abstract
BACKGROUND. Antiretroviral therapy (ART) halts HIV-1 replication, decreasing viremia to below the detection limit of clinical assays. However, some individuals experience persistent nonsuppressible viremia (NSV) originating from CD4+ T cell clones carrying infectious proviruses. Defective proviruses represent over 90% of all proviruses persisting during ART and can express viral genes, but whether they can cause NSV and complicate ART management is unknown. METHODS. We carried an in-depth characterization of proviruses causing NSV in 4 study participants with optimal adherence and no drug resistance. We investigated the impact of the observed defects on 5’-Leader RNA properties, virus infectivity, and gene expression. Integration-site specific assays were used to track these proviruses over time and among cell subsets. RESULTS. Clones carrying proviruses with 5’-Leader defects can cause persistent NSV up to ~103 copies/mL. These proviruses had small, often identical deletions or point mutations involving the major splicing donor site (MSD) and showed partially reduced RNA dimerization and nucleocapsid binding. Nevertheless, they were inducible and produced non-infectious virions containing viral RNA but lacking Envelope. CONCLUSION. These findings show that proviruses with 5’-Leader defects in CD4+ T cell clones can give rise to NSV, affecting clinical care. Sequencing of the 5’-Leader can help understanding failure to completely suppress viremia. FUNDING. Office of the NIH Director and National Institute of Dental & Craniofacial Research, NIH; Howard Hughes Medical Institute; Johns Hopkins University Center for AIDS Research; National Institute for Allergy and Infectious Diseases, NIH, to the PAVE, BEAT-HIV and DARE Martin Delaney collaboratories.
Authors
Jennifer A. White, Fengting Wu, Saif Yasin, Milica Moskovljevic, Joseph Varriale, Filippo Dragoni, Angelica Camilo Contreras, Jiayi Duan, Mei Y. Zheng, Ndeh F. Tadzong, Heer B. Patel, Jeanelle Mae C. Quiambao, Kyle Rhodehouse, Hao Zhang, Jun Lai, Subul A. Beg, Michael Delannoy, Christin Kilcrease, Christopher J. Hoffmann, Sébastien Poulin, Frédéric Chano, Cecile Tremblay, Jerald Cherian, Patricia Barditch-Crovo, Natasha Chida, Richard D. Moore, Michael F. Summers, Robert F. Siliciano, Janet D. Siliciano, Francesco R. Simonetti
Citation Information: J Clin Invest. 2022. https://doi.org/10.1172/JCI165578.
Abstract
Visceral pain (VP) is a global problem with complex etiologies and limited therapeutic options. Guanylyl cyclase C (GUCY2C), an intestinal receptor producing cyclic GMP which regulates luminal fluid secretion, has emerged as a therapeutic target for VP. Indeed, FDA-approved GUCY2C agonists ameliorate VP in patients with chronic constipation syndromes, although analgesic mechanisms remain obscure. Here, we reveal that intestinal GUCY2C is selectively enriched in neuropod cells, a type of enteroendocrine cell that synapses with submucosal neurons in mice and humans. GUCY2CHigh neuropod cells associate with co-cultured dorsal root ganglia neurons and induce hyperexcitability, reducing the rheobase and increasing the resulting number of evoked action potentials. Conversely, the GUCY2C agonist linaclotide eliminated neuronal hyperexcitability produced by GUCY2C-sufficient, but not GUCY2C-deficient, neuropod cells, an effect independent of bulk epithelial cells or extracellular cGMP. Genetic elimination of intestinal GUCY2C amplified nociceptive signaling and VP that was comparable to chemically-induced VP but refractory to linaclotide. Importantly, eliminating GUCY2C selectively in neuropod cells also increased nociceptive signaling and VP that was refractory to linaclotide. In the context of loss of GUCY2C hormones in patients with VP, these observations suggest a specific role for neuropod GUCY2C signaling in the pathophysiology and treatment of these pain syndromes.
Authors
Joshua R. Barton, Annie K. Londregan, Tyler D. Alexander, Ariana A. Entezari, Shely Bar-Ad, Lan Cheng, Angelo C. Lepore, Adam E. Snook, Manuel Covarrubias, Scott A. Waldman
Citation Information: J Clin Invest. 2022. https://doi.org/10.1172/JCI161472.
Abstract
Insulin and IGF-1 receptors (IR/IGF1R) are highly homologous and share similar signaling systems, but each has a unique physiological role, with IR primarily regulating metabolic homeostasis and IGF1R regulating mitogenic control and growth. Here, we showed that replacement of a single amino acid at position 973, just distal to the NPEY motif in the intracellular juxtamembrane region, from leucine, which is highly-conserved in IRs, to phenylalanine, the highly-conserved homologous residue in IGF1Rs, resulted in decreased IRS-1-PI3K-Akt-mTORC1 signaling and increased of Shc-Gab1-MAPK-cell cycle signaling. As a result, cells expressing L973F-IR exhibited decreased insulin-induced glucose uptake, increased cell growth and impaired receptor internalization. Mice with knockin of the L973F-IR showed similar alterations in signaling in vivo, and this leaded to decreased insulin sensitivity, a modest increase in growth and decreased weight gain when challenged with high-fat diet. Thus, leucine973 in the juxtamembrane region of the IR acts as a crucial residue differentiating IR signaling from IGF1R signaling.
Authors
Hirofumi Nagao, Weikang Cai, Bruna Brasil Brandão, Nicolai J. Wewer Albrechtsen, Martin Steger, Arijeet K. Gattu, Hui Pan, Jonathan M. Dreyfuss, F. Thomas Wunderlich, Matthias Mann, C. Ronald Kahn
Citation Information: J Clin Invest. 2022. https://doi.org/10.1172/JCI156436.
Abstract
Despite major advances in acute interventions of myocardial infarction (MI), adverse cardiac remodeling and excess fibrosis post MI causing ischemic heart failure (IHF) remains a leading cause of death worldwide. Here we identify a pro-fibrotic coagulation signaling pathway that can be targeted for improved cardiac function following MI with persistent ischemia. Quantitative phospho-proteomics of cardiac tissue revealed an up-regulated mitogen activated protein kinase (MAPK) pathway in human IHF. Intervention in this pathway with trametinib improves myocardial function and prevents fibrotic remodeling in a murine model of non-reperfused MI. MAPK activation in MI requires myeloid cell signaling of protease activated receptor 2 linked to the cytoplasmic domain of the coagulation initiator tissue factor (TF). They act upstream of pro-oxidant NOX2 NADPH oxidase, ERK1/2 phosphorylation, and activation of pro-fibrotic transforming growth factor β1 (TGF-β1). Specific targeting with the TF inhibitor nematode anticoagulant protein c2 (NAPc2) starting one day after established experimental MI averts IHF. Increased TF cytoplasmic domain phosphorylation in circulating monocytes from patients with sub-acute MI identifies a potential thrombo-inflammatory biomarker reflective of increased risk for IHF and suitable for patient selection to receive targeted TF inhibition therapy.
Authors
Venkata Garlapati, Michael Molitor, Thomas Michna, Gregory S. Harms, Stefanie Finger, Rebecca Jung, Jeremy Lagrange, Panagiotis Efentakis, Johannes Wild, Maike Knorr, Susanne Karbach, Sabine Wild, Ksenija Vujacic-Mirski, Thomas Münzel, Andreas Daiber, Moritz Brandt, Tommaso Gori, Hendrik Milting, Stefan Tenzer, Wolfram Ruf, Philip Wenzel
Citation Information: J Clin Invest. 2022. https://doi.org/10.1172/JCI162434.
Abstract
Ubiquitin-conjugating enzyme E2C (UBE2C) mediates the ubiquitylation chain formation via the K11 linkage. While previous in vitro studies showed that UBE2C plays a growth-promoting role in cancer cell lines, the underlying mechanism remains elusive. Still unknown is whether and how UBE2C plays a promoting role in vivo. Here we reported that UBE2C is indeed essential for growth and survival of lung cancer cells harboring Kras mutations, and UBE2C is required for KrasG12D-induced lung tumorigenesis, since Ube2c deletion significantly inhibits tumor formation and extends the life-span of mice. Mechanistically, KrasG12D induces expression of UBE2C, which couples with APC/CCDH1 E3 ligase to promote ubiquitylation and degradation of DEPTOR, leading to activation of the mTORC signals. Importantly, DEPTOR levels are fluctuated during cell cycle progression in a manner dependent of UBE2C and CDH1, indicating their physiological connection. Finally, Deptor deletion fully rescues the tumor inhibitory effect of Ube2c deletion in the KrasG12D lung tumor model, indicating a causal role of Deptor. Taken together, our study shows that the UBE2C/CDH1/DEPTOR axis forms an oncogene-tumor suppressor cascade that regulates cell cycle progression and autophagy and validates that UBE2C is an attractive target for lung cancer associated with Kras mutations.
Authors
Shizhen Zhang, Xiahong You, Yawen Zheng, Yanwen Shen, Xiufang Xiong, Yi Sun
Citation Information: J Clin Invest. 2022. https://doi.org/10.1172/JCI164915.
Abstract
Disorders of isolated mineralocorticoid deficiency causing potentially life-threatening salt-wasting crisis early in life have been associated with gene variants of aldosterone biosynthesis or resistance, but in some patients no such variants are found. WNT/β-catenin signaling is crucial for differentiation and maintenance of the aldosterone producing adrenal zona glomerulosa (zG). We describe a highly consanguineous family with multiple perinatal deaths or infants presenting at birth with failure to thrive, severe salt-wasting crises associated with isolated hypoaldosteronism, nail anomalies, short stature, and deafness. Whole exome sequencing revealed a homozygous splice variant in the R-SPONDIN receptor LGR4 gene (c.618-1G>C) regulating WNT signaling. The resulting transcripts affected protein function and stability, and resulted in loss of Wnt/β-catenin signaling in vitro. The impact of LGR4 inactivation was analyzed by adrenal cortex specific ablation of Lgr4, using Lgr4Flox/Flox mated with Sf1:Cre mice. Inactivation of Lgr4 within the adrenal cortex in the mouse model caused decreased WNT signaling, aberrant zonation with deficient zG and reduced aldosterone production. Thus, human LGR4 mutations establish a direct link between LGR4 inactivation and decreased canonical WNT signaling with abnormal zG differentiation and endocrine function. Therefore, variants in WNT signaling and its regulators should systematically be considered in familial hyperreninemic hypoaldosteronism.
Authors
Cécily Lucas, Kay-Sara Sauter, Michael Steigert, Delphine Mallet, James Wilmouth Jr., Julie Olabe, Ingrid Plotton, Yves Morel, Daniel Aeberli, Franca Wagner, Hans Clevers, Amit V. Pandey, Pierre Val, Florence Roucher-Boulez, Christa E. Fluck
Citation Information: J Clin Invest. 2022. https://doi.org/10.1172/JCI162775.
Abstract
BACKGROUND. Assessing circadian rhythmicity from infrequently sampled data is challenging, however this type of data is often encountered when measuring circadian transcripts in hospitalised patients. METHODS. We present ClinCirc. This method combines two existing mathematical methods (Lomb-Scargle periodogram and cosinor) sequentially, and is designed to measure circadian oscillations from infrequently sampled clinical data. The accuracy of this method was compared against 9 other methods using simulated and frequently sampled biological data. ClinCirc was then evaluated in 13 ICU patients as well as in a separate cohort of 29 kidney transplant recipients. Finally, the consequences of circadian alterations were investigated in a retrospective cohort of 726 kidney transplant recipients. RESULTS. ClinCirc had comparable performance to existing methods for analysing simulated data or clock transcript expression of healthy volunteers. It had improved accuracy compared to the cosinor method in evaluating circadian parameters in PER2::luc cell lines. In ICU patients, it was the only method investigated to suggest that loss of circadian oscillations in the peripheral oscillator was associated with inflammation, a feature widely reported in animal models. Additionally, ClinCirc was able to detect other circadian alterations, including a phase shift following kidney transplantation that was associated with the administration of glucocorticoids. This phase shift could explain why a significant complication of kidney transplantation (delayed graft dysfunction) oscillates according to the time-of-day kidney transplantation is performed. CONCLUSION. ClinCirc analysis of the peripheral oscillator reveals important clinical associations in hospitalised patients. FUNDING. UKRI, NIHR, EPSRC, NIAA, Asthma+Lung UK, Kidneys for Life.
Authors
Peter S. Cunningham, Gareth B. Kitchen, Callum Jackson, Stavros Papachristos, Thomas Springthorpe, David van Dellen, Julie E. Gibbs, Timothy W. Felton, Anthony J. Wilson, Jonathan Bannard-Smith, Martin K. Rutter, Thomas House, Paul Dark, Titus Augustine, Ozgur E. Akman, Andrew L. Hazel, John F. Blaikley
Citation Information: J Clin Invest. 2022. https://doi.org/10.1172/JCI160807.
Abstract
Our understanding of neuropathic itch is limited, due to the lack of relevant animal models. Patients with cutaneous T-cell lymphoma (CTCL) suffer from severe itching. Here we characterize a mouse model of chronic itch with remarkable lymphoma growth, immune cell accumulation, and persistent pruritus. Intradermal CTCL inoculation produces time-dependent changes in nerve innervations in lymphoma-bearing skin. In the early-phase (20 days), CTCL causes hyper-innervations in the epidermis. However, chronic itch is associated with loss of epidermal nerve fibers in the late-phases (40 and 60 days). CTCL is also characterized by marked nerve innervations in mouse lymphoma. Blockade of C-fibers reduced pruritus at early- and late-phases, whereas blockade of A-fibers only suppressed late-phase itch. Intrathecal gabapentin injection reduced late-phase but not early-phase pruritus. IL-31 is upregulated in mouse lymphoma, while its receptor Il31ra was persistently upregulated in Trpv1-expressing sensory neurons in CTCL mice. Intratumoral anti-IL-31 treatment effectively suppressed CTCL-induced scratching and alloknesis (mechanical itch). Finally, intrathecal administration of TLR4 antagonist attenuated pruritus in early and late phases and in both sexes. Collectively, we have established a mouse model of neuropathic and cancer itch with relevance to human disease. Our findings also suggest distinct mechanisms underlying acute, chronic, and neuropathic itch.
Authors
Ouyang Chen, Qianru He, Qingjian Han, Kenta Furutani, Yun Gu, Madelynne Olexa, Ru-Rong Ji
Citation Information: J Clin Invest. 2022. https://doi.org/10.1172/JCI162809.
Abstract
Approximately 10% of monogenic diseases are caused by nonsense point mutations that generate premature termination codons (PTCs), resulting in a truncated protein and nonsense-mediated decay of the mutant mRNAs. Here, we demonstrate a mini-dCas13X-mediated RNA adenine base editing (mxABE) strategy to treat nonsense mutation-related monogenic diseases via A-to-G editing in a genetically humanized mouse model of Duchenne muscular dystrophy (DMD). Initially, we identified a nonsense point mutation (c.4174C>T, p.Gln1392*) in the DMD gene of a patient and validated its pathogenicity in humanized mice. In this model, single adeno-associated virus (AAV)-packaged mxABE reached A-to-G editing rates up to 84% in vivo, which is at least 20-fold greater compared to rates reported in previous studies using other RNA-editing modalities. Furthermore, mxABE restored robust expression of dystrophin protein to over 50% of wild-type (WT) levels by enabling PTC read-through in multiple muscle tissues. Importantly, systemic delivery of mxABE by AAV also rescued dystrophin expression to averages of 37%, 6%, and 54% of WT levels in the diaphragm, tibialis anterior, and heart muscle, respectively, as well as rescued muscle function. Our data strongly suggest that mxABE-based strategies may be a viable new treatment modality for DMD and other monogenic diseases.
Authors
Guoling Li, Ming Jin, Zhifang Li, Qingquan Xiao, Jiajia Lin, Dong Yang, Yuanhua Liu, Xing Wang, Long Xie, Wenqin Ying, Haoqiang Wang, Erwei Zuo, Linyu Shi, Ning Wang, Wanjin Chen, Chunlong Xu, Hui Yang
Citation Information: J Clin Invest. 2022. https://doi.org/10.1172/JCI164508.
Abstract
Disuse osteoporosis is a metabolic bone disease resulted from skeletal unloading (e.g., during extended bed rest, limb immobilization, and spaceflight), and the slow and insufficient bone recovery during re-ambulation remains an unresolved medical challenge. Here, we demonstrated that loading-induced increase in bone architecture/strength was suppressed in skeletons previously exposed to unloading. This reduction in bone mechanosensitivity was directly associated with attenuated osteocytic Ca2+ oscillatory dynamics. The unloading-induced compromised osteocytic Ca2+ response to reloading resulted from the HIF-1α/PDK1 axis-mediated increase in glycolysis, and a subsequent reduction in ATP synthesis. HIF-1α also transcriptionally induced substantial glutaminase 2 expression and thereby glutamine addiction in osteocytes. Inhibition of glycolysis by blocking PDK1 or glutamine supplementation restored the mechanosensitivity in those skeletons with previous unloading by fueling the tricarboxylic acid cycle and rescuing subsequent Ca2+ oscillations in osteocytes. Thus, we provide a mechanistic insight into disuse-induced deterioration of bone mechanosensitivity and a promising therapeutic approach to accelerate bone recovery after long-duration disuse.
Authors
Xiyu Liu, Zedong Yan, Jing Cai, Dan Wang, Yongqing Yang, Yuanjun Ding, Xi Shao, Xiaoxia Hao, Erping Luo, X. Edward Guo, Peng Luo, Liangliang Shen, Da Jing
Citation Information: J Clin Invest. 2022. https://doi.org/10.1172/JCI156722.
Abstract
Leptin exerts its biological actions by activating LepRb. LepRb signaling impairment and leptin resistance are believed to cause obesity. Transcription factor Slug (also known as Snai2) recruits epigenetic modifiers and regulates gene expression by an epigenetic mechanism; however, its epigenetic action has not been explored in leptin resistance. Here, we uncover a pro-obesity function of neuronal Slug. Hypothalamic Slug was upregulated in obese mice. LepRb cell-specific Slug knockout (SlugΔLepRb) mice were resistant to diet-induced obesity, type 2 diabetes, and liver steatosis, accompanied by decreased food intake and increased fat thermogenesis. Leptin stimulated hypothalamic Stat3 phosphorylation and weight loss to a significantly higher level in SlugΔLepRb than in Slugf/f mice even before their body weight divergence. Conversely, hypothalamic LepRb neuron-specific overexpression of Slug, mediated by AAV-DIO-Slug transduction, induced leptin resistance, obesity, and metabolic disorders in mice on a chow diet. At the genomic level, Slug bound to and repressed the LepRb promoter, thereby inhibiting LepRb transcription. Consistently, Slug deficiency decreased LepRb promoter histone 3 lysine-27 methylations, repressive epigenetic marks, and increased LepRb mRNA levels in the hypothalamus. Collectively, these results unravel a previously-unrecognized hypothalamic neuronal Slug/epigenetic reprogramming/leptin resistance axis that promotes energy imbalance, obesity, and metabolic disease.
Authors
Min-Hyun Kim, Yuan Li, Qiantao Zheng, Lin Jiang, Martin G. Myers, Wen-Shu Wu, Liangyou Rui
Citation Information: J Clin Invest. 2022. https://doi.org/10.1172/JCI161759.
Abstract
Impaired angiogenesis in diabetes is a key process contributing to ischemic diseases such as peripheral arterial disease. Epigenetic mechanisms, including those mediated by long non-coding RNAs are crucial links connecting diabetes and the related chronic tissue ischemia. Here we identify the LncRNA that Enhances Endothelial Nitric oxide synthase Expression (LEENE) as a regulator of angiogenesis and ischemic response. LEENE expression is decreased in diabetic conditions in cultured endothelial cells (EC), mouse hindlimb muscles, and human arteries. Inhibition of LEENE in human microvascular ECs reduces their angiogenic capacity with a dysregulated angiogenic gene program. Diabetic mice deficient in leene demonstrate impaired angiogenesis and perfusion following hindlimb ischemia. Importantly, overexpression of human LEENE rescues the impaired ischemic response in leene knockout mice at tissue functional and single-cell transcriptomic levels. Mechanistically, LEENE RNA promotes transcription of pro-angiogenic genes in ECs, such as KDR and eNOS, potentially by interacting with LEO1, a key component of RNA Polymerase II-associated factor complex and MYC, a crucial transcription factor for angiogenesis. Taken together, our findings demonstrate an essential role for LEENE in the regulation of angiogenesis and tissue perfusion. Functional enhancement of LEENE to restore angiogenesis for tissue repair and regeneration may represent a potential strategy to tackle ischemic vascular diseases.
Authors
Xiaofang Tang, Yingjun Luo, Dongqiang Yuan, Riccardo Calandrelli, Naseeb Kaur Malhi, Kiran Sriram, Yifei Miao, Chih Hong Lou, Walter Tsark, Alonso Tapia, Aleysha T. Chen, Guangyu Zhang, Daniel Roeth, Markus Kalkum, Zhao V. Wang, Shu Chien, Rama Natarajan, John P. Cooke, Sheng Zhong, Zhen Bouman Chen
Citation Information: J Clin Invest. 2022. https://doi.org/10.1172/JCI157907.
Abstract
Autologous stem cell transplantation (ASCT), with subsequent lenalidomide maintenance is standard consolidation therapy for multiple myeloma and a subset of patients achieve durable progression-free survival that is suggestive of long-term immune control. Nonetheless, most patients ultimately relapse, suggesting immune escape. TIGIT appears a potent inhibitor of myeloma-specific immunity and represents a promising checkpoint target. Here we demonstrate high expression of TIGIT on activated CD8 T cells in mobilized peripheral blood stem cell grafts from patients with myeloma. To guide clinical application of TIGIT inhibition, we evaluated identical TIGIT Abs that do or do not engage FcγR and demonstrated that anti-TIGIT activity is dependent on FcγR binding. We subsequently used CRBN mice to investigate the efficacy of anti-TIGIT in combination with lenalidomide maintenance after transplantation. Notably, the combination of anti-TIGIT with lenalidomide provided synergistic, CD8 T cell-dependent, anti-myeloma efficacy. Analysis of bone marrow (BM) CD8 T cells demonstrated that combination therapy suppressed T cell exhaustion, enhanced effector function, and expanded central memory subsets. Importantly, these immune phenotypes were specific to the BM tumor microenvironment. Collectively, these data provide a logical rationale for combining TIGIT inhibition with immunomodulatory drugs to prevent myeloma progression after stem cell transplantation.
Authors
Simone A. Minnie, Olivia G. Waltner, Kathleen S. Ensbey, Stuart D. Olver, Alika D. Collinge, David P. Sester, Christine R. Schmidt, Samuel R.W. Legg, Shuichiro Takahashi, Nicole S. Nemychenkov, Tomoko Sekiguchi, Gregory Driessens, Ping Zhang, Motoko Koyama, Andrew Spencer, Leona A. Holmberg, Scott N. Furlan, Antiopi Varelias, Geoffrey R. Hill
Citation Information: J Clin Invest. 2022. https://doi.org/10.1172/JCI159498.
Abstract
Innate immune cells play important roles in tissue injury and repair following acute myocardial infarction (MI). Although reprogramming of macrophage metabolism has been observed during inflammation and resolution phases, the mechanistic link to macrophage phenotype is not fully understood. In this study, we found myeloid specific deletion of mitochondrial Complex I protein Ndufs4 (mKO) reproduced the proinflammatory metabolic profile in macrophages and exaggerated the response to lipopolysacharride. Moreover, mKO mice showed increased mortality, poor scar formation and worsened cardiac function 30 days post-MI. We observed a greater inflammatory response in mKO on day 1 followed by increased cell death of infiltrating macrophages and blunted transition to reparative phase during day 3-7 post-MI. Efferocytosis is markedly impaired in mKO macrophages leading to lower expression of anti-inflammatory cytokine and tissue repair factors, which suppressed the proliferation/activation of myofibroblasts in the infarct area. Mitochondria-targeted ROS scavenging rescued these impairments and improved myofibroblast function in vivo and reduced post-MI mortality in mKO mice. Together these results reveal a novel role of mitochondria in inflammation resolution and tissue repair via modulating efferocytosis and crosstalk with fibroblasts. The findings are significant for post-MI recovery as well as for other inflammatory conditions.
Authors
Shanshan Cai, Mingyue Zhao, Bo Zhou, Akira Yoshii, Darrian Bugg, Outi Villet, Anita Sahu, Gregory S. Olson, Jennifer Davis, Rong Tian
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ISSN: 0021-9738 (print), 1558-8238 (online)