Treatment with anti–PD-1 and anti-PD-L1 therapies has shown durable clinical benefit in non–small cell lung cancer (NSCLC). However, patients with NSCLC with epidermal growth factor receptor (EGFR) mutations do not respond as well to treatment as patients without an EGFR mutation. We show that EGFR-mutated NSCLC expressed higher levels of CD73 compared with EGFR WT tumors and that CD73 expression was regulated by EGFR signaling. EGFR-mutated cell lines were significantly more resistant to T cell killing compared with WT cell lines through suppression of T cell proliferation and function. In a xenograft mouse model of EGFR-mutated NSCLC, neither anti–PD-L1 nor anti-CD73 antibody alone inhibited tumor growth compared with the isotype control. In contrast, the combination of both antibodies significantly inhibited tumor growth, increased the number of tumor-infiltrating CD8+ T cells, and enhanced IFN-γ and TNF-α production of these T cells. Consistently, there were increases in gene expression that corresponded to inflammation and T cell function in tumors treated with the combination of anti–PD-L1 and anti-CD73. Together, these results further support the combination of anti-CD73 and anti–PD-L1 therapies in treating EGFR-mutated NSCLC, while suggesting that increased T cell activity may play a role in response to therapy.
Eric Tu, Kelly McGlinchey, Jixin Wang, Philip Martin, Steven L.K. Ching, Nicolas Floc’h, James Kurasawa, Jacqueline H. Starrett, Yelena Lazdun, Leslie Wetzel, Barrett Nuttall, Felicia S.L. Ng, Karen T. Coffman, Paul D. Smith, Katerina Politi, Zachary A. Cooper, Katie Streicher
There is a high prevalence of ventricular arrhythmias related to sudden cardiac death in patients with chronic kidney disease (CKD). To explored the possible mechanism of CKD-related ventricular arrhythmias, a CKD rat model was created, and indoxyl sulfate (IS) was further used in vivo and in vitro. This project used the following methods: patch clamp, electrocardiogram, and some molecular biology experimental techniques. IS was found to be significantly elevated in the serum of CKD rats. Interestingly, the expression levels of the fast transient outward potassium current–related (Ito,f-related) proteins (Kv4.2, Kv4.3, and KChIP2) in the heart of CKD rats and rats treated with IS decreased. IS dose-dependently reduced Ito,f density, accompanied by the decreases in Kv4.2, Kv4.3, and KChIP2 proteins in vitro. IS also prolonged the action potential duration and QT interval, and paroxysmal ventricular tachycardia could be induced by IS. In-depth studies have shown that ROS/p38MAPK, ROS–p44/42 MAPK, and NF-κB signaling pathways play key roles in the reduction of Ito,f density and Ito,f-related proteins caused by IS. These data suggest that IS reduces Ito,f-related proteins and Ito,f density by activating ROS/MAPK and NF-κB signaling pathways, and the action potential duration and QT interval are subsequently prolonged, which contributes to increasing the susceptibility to arrhythmia in CKD.
Jing Yang, Hongxia Li, Chi Zhang, Yafeng Zhou
Exchange proteins directly activated by cAMP (Epacs) are abundantly expressed in the renal tubules. We used genetic and pharmacological tools in combination with balance, electrophysiological, and biochemical approaches to examine the role of Epac1 and Epac2 in renal sodium handling. We demonstrate that Epac1–/– and Epac2–/– mice exhibit a delayed anti-natriuresis to dietary sodium restriction despite augmented aldosterone levels. This was associated with a significantly lower response to the epithelial Na+ channel (ENaC) blocker amiloride, reduced ENaC activity in split-opened collecting ducts, and defective posttranslational processing of α and γENaC subunits in the KO mice fed with a Na+-deficient diet. Concomitant deletion of both isoforms led to a marginally greater natriuresis but further increased aldosterone levels. Epac2 blocker ESI-05 and Epac1&2 blocker ESI-09 decreased ENaC activity in Epac WT mice kept on the Na+-deficient diet but not on the regular diet. ESI-09 injections led to natriuresis in Epac WT mice on the Na+-deficient diet, which was caused by ENaC inhibition. In summary, our results demonstrate similar but nonredundant actions of Epac1 and Epac2 in stimulation of ENaC activity during variations in dietary salt intake. We speculate that inhibition of Epac signaling could be instrumental in treatment of hypertensive states associated with ENaC overactivation.
Viktor N. Tomilin, Kyrylo Pyrshev, Anna Stavniichuk, Naghmeh Hassanzadeh Khayyat, Guohui Ren, Oleg Zaika, Sherif Khedr, Alexander Staruschenko, Fang C. Mei, Xiaodong Cheng, Oleh Pochynyuk
Increased adipose tissue macrophages (ATMs) correlate with metabolic dysfunction in humans and are causal in development of insulin resistance in mice. Recent bulk and single-cell transcriptomics studies reveal a wide spectrum of gene expression signatures possible for macrophages that depends on context, but the signatures of human ATM subtypes are not well defined in obesity and diabetes. We profiled 3 prominent ATM subtypes from human adipose tissue in obesity and determined their relationship to type 2 diabetes. Visceral adipose tissue (VAT) and s.c. adipose tissue (SAT) samples were collected from diabetic and nondiabetic obese participants to evaluate cellular content and gene expression. VAT CD206+CD11c− ATMs were increased in diabetic participants, were scavenger receptor–rich with low intracellular lipids, secreted proinflammatory cytokines, and diverged significantly from 2 CD11c+ ATM subtypes, which were lipid-laden, were lipid antigen presenting, and overlapped with monocyte signatures. Furthermore, diabetic VAT was enriched for CD206+CD11c− ATM and inflammatory signatures, scavenger receptors, and MHC II antigen presentation genes. VAT immunostaining found CD206+CD11c– ATMs concentrated in vascularized lymphoid clusters adjacent to CD206–CD11c+ ATMs, while CD206+CD11c+ were distributed between adipocytes. Our results show ATM subtype–specific profiles that uniquely contribute to the phenotypic variation in obesity.
Lindsey A. Muir, Kae Won Cho, Lynn M. Geletka, Nicki A. Baker, Carmen G. Flesher, Anne P. Ehlers, Niko Kaciroti, Stephen Lindsly, Scott Ronquist, Indika Rajapakse, Robert W. O’Rourke, Carey N. Lumeng
The fibrous annulus of the mitral valve plays an important role in valvular function and cardiac physiology, while normal variation in the size of cardiovascular anatomy may share a genetic link with common and rare disease. We derived automated estimates of mitral valve annular diameter in the 4-chamber view from 32,220 MRI images from the UK Biobank at ventricular systole and diastole as the basis for GWAS. Mitral annular dimensions corresponded to previously described anatomical norms, and GWAS inclusive of 4 population strata identified 10 loci, including possibly novel loci (GOSR2, ERBB4, MCTP2, MCPH1) and genes related to cardiac contractility (BAG3, TTN, RBFOX1). ATAC-Seq of primary mitral valve tissue localized multiple variants to regions of open chromatin in biologically relevant cell types and rs17608766 to an algorithmically predicted enhancer element in GOSR2. We observed strong genetic correlation with measures of contractility and mitral valve disease and clinical correlations with heart failure, cerebrovascular disease, and ventricular arrhythmias. Polygenic scoring of mitral valve annular diameter in systole was predictive of risk mitral valve prolapse across 4 cohorts. In summary, genetic and clinical studies of mitral valve annular diameter revealed genetic determinants of mitral valve biology, while highlighting clinical associations. Polygenic determinants of mitral valve annular diameter may represent an independent risk factor for mitral prolapse. Overall, computationally estimated phenotypes derived at scale from medical imaging represent an important substrate for genetic discovery and clinical risk prediction.
Mengyao Yu, Catherine Tcheandjieu, Adrien Georges, Ke Xiao, Helio Tejeda, Christian Dina, Thierry Le Tourneau, Madalina Fiterau, Renae Judy, Noah L. Tsao, Dulguun Amgalan, Chad J. Munger, Jesse M. Engreitz, Scott M. Damrauer, Nabila Bouatia-Naji, James R. Priest
Norrie disease is caused by mutation of the NDP gene, presenting as congenital blindness followed by later onset of hearing loss. Protecting patients from hearing loss is critical for maintaining their quality of life. This study aimed to understand the onset of pathology in cochlear structure and function. By investigating patients and juvenile Ndp-mutant mice, we elucidated the sequence of onset of physiological changes (in auditory brainstem responses, distortion product otoacoustic emissions, endocochlear potential, blood-labyrinth barrier integrity) and determined the cellular, histological, and ultrastructural events leading to hearing loss. We found that cochlear vascular pathology occurs earlier than previously reported and precedes sensorineural hearing loss. The work defines a disease mechanism whereby early malformation of the cochlear microvasculature precedes loss of vessel integrity and decline of endocochlear potential, leading to hearing loss and hair cell death while sparing spiral ganglion cells. This provides essential information on events defining the optimal therapeutic window and indicates that early intervention is needed. In an era of advancing gene therapy and small-molecule technologies, this study establishes Ndp-mutant mice as a platform to test such interventions and has important implications for understanding the progression of hearing loss in Norrie disease.
Dale Bryant, Valda Pauzuolyte, Neil J. Ingham, Aara Patel, Waheeda Pagarkar, Lucy A. Anderson, Katie E. Smith, Dale A. Moulding, Yeh C. Leong, Daniyal J. Jafree, David A. Long, Amina Al-Yassin, Karen P. Steel, Daniel J. Jagger, Andrew Forge, Wolfgang Berger, Jane C. Sowden, Maria Bitner-Glindzicz
CMV infection remains an important cause of morbidity and mortality after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Several investigators have reported that adaptive NKG2C+ NK cells persistently expand during CMV reactivation. In our study, 2 cohorts were enrolled to explore the relationships among the NKG2C genotype, NKG2C+ NK cell reconstitution, and CMV infection. Multivariate analysis showed that donor NKG2C gene deletion was an independent prognostic factor for CMV reactivation and refractory CMV reactivation. Furthermore, adaptive NKG2C+ NK cells’ quantitative and qualitative reconstitution, along with their anti-CMV function after transplantation, was significantly lower in patients grafted with NKG2Cwt/del donor cells than in those grafted with NKG2Cwt/wt donor cells. At day 30 after transplantation, quantitative reconstitution of NKG2C+ NK cells was significantly lower in patients with treatment-refractory CMV reactivation than in patients without CMV reactivation and those with nonrefractory CMV reactivation. In humanized CMV-infected mice, we found that, compared with those from NKG2Cwt/del donors, adaptive NKG2C+ NK cells from NKG2Cwt/wt donors induced earlier and stronger expansion of NKG2C+ NK cells as well as earlier and stronger CMV clearance in vivo. In conclusion, donor NKG2C homozygosity contributes to CMV clearance by promoting the quantitative and qualitative reconstruction of adaptive NKG2C+ NK cells after haploidentical allo-HSCT.
Xing-Xing Yu, Qian-Nan Shang, Xue-Fei Liu, Mei He, Xu-Ying Pei, Xiao-Dong Mo, Meng Lv, Ting-Ting Han, Ming-Rui Huo, Xiao-Su Zhao, Ying-Jun Chang, Yu Wang, Xiao-Hui Zhang, Lan-Ping Xu, Kai-Yan Liu, Xiang-Yu Zhao, Xiao-Jun Huang
Short stature is a major skeletal phenotype in osteogenesis imperfecta (OI), a genetic disorder mainly caused by mutations in genes encoding type I collagen. However, the underlying mechanism is poorly understood, and no effective treatment is available. In OI mice that carry a G610C mutation in COL1A2, we previously found that mature hypertrophic chondrocytes (HCs) are exposed to cell stress due to accumulation of misfolded mutant type I procollagen in the endoplasmic reticulum (ER). By fate mapping analysis of HCs in G610C OI mice, we found that HCs stagnate in the growth plate, inhibiting translocation of HC descendants to the trabecular area and their differentiation to osteoblasts. Treatment with 4-phenylbutyric acid (4PBA), a chemical chaperone, restored HC ER structure and rescued this inhibition, resulting in enhanced longitudinal bone growth in G610C OI mice. Interestingly, the effects of 4PBA on ER dilation were limited in osteoblasts, and the bone fragility was not ameliorated. These results highlight the importance of targeting HCs to treat growth deficiency in OI. Our findings demonstrate that HC dysfunction induced by ER disruption plays a critical role in the pathogenesis of OI growth deficiency, which lays the foundation for developing new therapies for OI.
Amanda L. Scheiber, Kevin J. Wilkinson, Akiko Suzuki, Motomi Enomoto-Iwamoto, Takashi Kaito, Kathryn S.E. Cheah, Masahiro Iwamoto, Sergey Leikin, Satoru Otsuru
Acute respiratory distress syndrome (ARDS) results in catastrophic lung failure and has an urgent, unmet need for improved early recognition and therapeutic development. Neutrophil influx is a hallmark of ARDS and is associated with the release of tissue-destructive immune effectors, such as matrix metalloproteinases (MMPs) and membrane-anchored metalloproteinase disintegrins (ADAMs). Here, we observed using intravital microscopy that Adam8–/– mice had impaired neutrophil transmigration. In mouse pneumonia models, both genetic deletion and pharmacologic inhibition of ADAM8 attenuated neutrophil infiltration and lung injury while improving bacterial containment. Unexpectedly, the alterations of neutrophil function were not attributable to impaired proteolysis but resulted from reduced intracellular interactions of ADAM8 with the actin-based motor molecule Myosin1f that suppressed neutrophil motility. In 2 ARDS cohorts, we analyzed lung fluid proteolytic signatures and identified that ADAM8 activity was positively correlated with disease severity. We propose that in acute inflammatory lung diseases such as pneumonia and ARDS, ADAM8 inhibition might allow fine-tuning of neutrophil responses for therapeutic gain.
Catharina Conrad, Daniela Yildiz, Simon J. Cleary, Andreas Margraf, Lena Cook, Uwe Schlomann, Barry Panaretou, Jessica L. Bowser, Harry Karmouty-Quintana, Jiwen Li, Nathaniel K. Berg, Samuel C. Martin, Ahmad Aljohmani, S. Farshid Moussavi-Harami, Kristin M. Wang, Jennifer J. Tian, Mélia Magnen, Colin Valet, Longhui Qiu, Jonathan P. Singer, Holger K. Eltzschig, CAPSys Study Group, Wilhelm Bertrams, Susanne Herold, Norbert Suttorp, Bernd Schmeck, Zachary T. Ball, Alexander Zarbock, Mark R. Looney, Jörg W. Bartsch
Pancreatic ductal adenocarcinoma (PDA) is an extremely metastatic and lethal disease. Here, in both murine and human PDA, we demonstrate that extracellular matrix architecture regulates cell extrusion and subsequent invasion from intact ductal structures through tumor-associated collagen signatures (TACS). This results in early dissemination from histologically premalignant lesions and continual invasion from well-differentiated disease, and it suggests TACS as a biomarker to aid in the pathologic assessment of early disease. Furthermore, we show that pancreatitis results in invasion-conducive architectures, thus priming the stroma prior to malignant disease. Analysis in potentially novel microfluidic-derived microtissues and in vivo demonstrates decreased extrusion and invasion following focal adhesion kinase (FAK) inhibition, consistent with decreased metastasis. Thus, data suggest that targeting FAK or strategies to reengineer and normalize tumor microenvironments may have roles not only in very early disease, but also for limiting continued dissemination from unresectable disease. Likewise, it may be beneficial to employ stroma-targeting strategies to resolve precursor diseases such as pancreatitis in order to remove stromal architectures that increase risk for early dissemination.
Arja Ray, Mackenzie K. Callaway, Nelson J. Rodríguez-Merced, Alexandra L. Crampton, Marjorie Carlson, Kenneth B. Emme, Ethan A. Ensminger, Alexander A. Kinne, Jonathan H. Schrope, Haley R. Rasmussen, Hong Jiang, David G. DeNardo, David K. Wood, Paolo P. Provenzano
Colorectal cancer (CRC) severely threatens human health and life span. An effective therapeutic strategy has not been established because we do not clearly know its pathogenesis. Here, we report that ceramide and sterol O-acyltransferase 1 (SOAT1) have roles in both spontaneous and chemical-induced intestinal cancers. We first found that miRNA-148a deficiency dramatically increased mouse gut dysbiosis through upregulating ceramide synthase 5 (Cers5) expression, which promoted ceramide synthesis afterward. The newly generated ceramide further promoted both azoxymethane/dextran sodium sulfate–induced (AOM/DSS-induced) and ApcMin/+ spontaneous intestinal tumorigenesis via increasing mouse gut dysbiosis. Meanwhile, increased level of ceramide correlated with the significant enhancements of both β-catenin activity and colorectal tumorigenesis in a TLR4-dependent fashion. Next, we found a direct binding of β-catenin to SOAT1 promoter to activate transcriptional expression of SOAT1, which further induced cholesterol esterification and colorectal tumorigenesis. In human patients with CRC, the same CERS5/TLR4/β-catenin/SOAT1 axis was also found to be dysregulated. Finally, the SOAT1 inhibitor (avasimibe) showed significant levels of therapeutic effects on both AOM/DSS-induced and ApcMin/+ spontaneous intestinal cancer. Our study clarified that ceramide promoted CRC development through increasing gut dysbiosis, further resulting in the increase of cholesterol esterification in a SOAT1-dependent way. Treatment with avasimibe to specifically decrease cholesterol esterification could be considered as a clinical strategy for effective CRC therapy in a future study.
Yahui Zhu, Li Gu, Xi Lin, Jinmiao Zhang, Yi Tang, Xinyi Zhou, Bingjun Lu, Xingrong Lin, Cheng Liu, Edward V. Prochownik, Youjun Li
The anatomical routes for the clearance of cerebrospinal fluid (CSF) remain incompletely understood. However, recent evidence has given strong support for routes leading to lymphatic vessels. A current debate centers upon the routes through which CSF can access lymphatics, with evidence emerging for either direct routes to meningeal lymphatics or along cranial nerves to reach lymphatics outside the skull. Here, a method was established to infuse contrast agent into the ventricles using indwelling cannulae during imaging of mice at 2 and 12 months of age by magnetic resonance imaging. As expected, a substantial decline in overall CSF turnover was found with aging. Quantifications demonstrated that the bulk of the contrast agent flowed from the ventricles to the subarachnoid space in the basal cisterns. Comparatively little contrast agent signal was found at the dorsal aspect of the skull. The imaging dynamics from the 2 cohorts revealed that the contrast agent was cleared from the cranium through the cribriform plate to the nasopharyngeal lymphatics. On decalcified sections, we confirmed that fluorescently labeled ovalbumin drained through the cribriform plate and could be found within lymphatics surrounding the nasopharynx. In conclusion, routes leading to nasopharyngeal lymphatics appear to be a major efflux pathway for cranial CSF.
Yann Decker, Jonas Krämer, Li Xin, Andreas Müller, Anja Scheller, Klaus Fassbender, Steven T. Proulx
Discoidin domain receptor 1 (DDR1), a receptor tyrosine kinase activated by collagen, contributes to chronic kidney disease. However, its role in acute kidney injury and subsequent development of kidney fibrosis is not clear. Thus, we performed a model of severe ischemia/reperfusion-induced acute kidney injury that progressed to kidney fibrosis in WT and Ddr1-null mice. We showed that Ddr1-null mice had reduced acute tubular injury, inflammation, and tubulointerstitial fibrosis with overall decreased renal monocyte chemoattractant protein (MCP-1) levels and STAT3 activation. We identified breakpoint cluster region (BCR) protein as a phosphorylated target of DDR1 that controls MCP-1 production in renal proximal tubule epithelial cells. DDR1-induced BCR phosphorylation or BCR downregulation increased MCP-1 secretion, suggesting that BCR negatively regulates the levels of MCP-1. Mechanistically, phosphorylation or downregulation of BCR increased β-catenin activity and in turn MCP-1 production. Finally, we showed that DDR1-mediated STAT3 activation was required to stimulate the secretion of TGF-β. Thus, DDR1 contributes to acute and chronic kidney injury by regulating BCR and STAT3 phosphorylation and in turn the production of MCP-1 and TGF-β. These findings identify DDR1 an attractive therapeutic target for ameliorating both proinflammatory and profibrotic signaling in kidney disease.
Corina M. Borza, Gema Bolas, Fabian Bock, Xiuqi Zhang, Favour C. Akabogu, Ming-Zhi Zhang, Mark de Caestecker, Min Yang, Haichun Yang, Ethan Lee, Leslie Gewin, Agnes B. Fogo, W. Hayes McDonald, Roy Zent, Ambra Pozzi
Immune cells express an array of inhibitory checkpoint receptors that are upregulated upon activation and limit tissue damage associated with excessive response to pathogens or allergens. Mouse leukocyte immunoglobulin like receptor B4 (LILRB4), also known as glycoprotein 49B (gp49B), is an inhibitory checkpoint receptor constitutively expressed in myeloid cells and upregulated in B cells, T cells, and NK cells upon activation. Here, we report that expression of LILRB4, which binds Zika virus (ZIKV), was increased in microglia and myeloid cells infiltrating the brains of neonatal mice with ZIKV-associated meningoencephalitis. Importantly, while C57BL/6 mice developed transient neurological symptoms but survived infection, mice lacking LILRB4/gp49B (LILRB4 KO) exhibited more severe signs of neurological disease and succumbed to disease. Their brains showed increased cellular infiltration but reduced control of viral burden. The reduced viral clearance was associated with altered NK cell function in the absence of LILRB4/gp49B. In naive animals, this manifested as reduced granzyme B responses to stimulation, but in ZIKV-infected animals, NK cells showed phenotypic changes that suggested altered maturation, diminished glucose consumption, reduced IFN-γ and granzyme B production, and impaired cytotoxicity. Together, our data reveal LILRB4/gp49B as an important regulator of NK cell function during viral infections.
Ha-Na Lee, Mohanraj Manangeeswaran, Aaron P. Lewkowicz, Kaliroi Engel, Monica Chowdhury, Mamatha Garige, Michael A. Eckhaus, Carole Sourbier, Derek D.C. Ireland, Daniela Verthelyi
Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an arrhythmia syndrome caused by gene mutations that render RYR2 Ca release channels hyperactive, provoking spontaneous Ca release and delayed afterdepolarizations (DADs). What remains unknown is the cellular source of ventricular arrhythmia triggered by DADs: Purkinje cells in the conduction system or ventricular cardiomyocytes in the working myocardium. To answer this question, we used a genetic approach in mice to knock out cardiac calsequestrin either in Purkinje cells or in ventricular cardiomyocytes. Total loss of calsequestrin in the heart causes a severe CPVT phenotype in mice and humans. We found that loss of calsequestrin only in ventricular myocytes produced a full-blown CPVT phenotype, whereas mice with loss of calsequestrin only in Purkinje cells were comparable to WT mice. Subendocardial chemical ablation or restoration of calsequestrin expression in subendocardial cardiomyocytes neighboring Purkinje cells was sufficient to protect against catecholamine-induced arrhythmias. In silico modeling demonstrated that DADs in ventricular myocardium can trigger full action potentials in the Purkinje fiber, but not vice versa. Hence, ectopic beats in CPVT are likely generated at the Purkinje–myocardial junction via a heretofore unrecognized tissue mechanism, whereby DADs in the ventricular myocardium trigger full action potentials in adjacent Purkinje cells.
Daniel J. Blackwell, Michela Faggioni, Matthew J. Wleklinski, Nieves Gomez-Hurtado, Raghav Venkataraman, Chelsea E. Gibbs, Franz J. Baudenbacher, Shiaoching Gong, Glenn I. Fishman, Patrick M. Boyle, Karl Pfeifer, Bjorn C. Knollmann
Stromal cells are emerging as key drivers of autoimmunity, partially because they produce inflammatory chemokines that orchestrate inflammation. Chemokine expression is regulated transcriptionally but also through posttranscriptional mechanisms, the specific drivers of which are still incompletely defined. CCL2 (MCP1) is a multifunctional chemokine that drives myeloid cell recruitment. During experimental autoimmune encephalomyelitis (EAE), an IL-17–driven model of multiple sclerosis, CCL2 produced by lymph node (LN) stromal cells was essential for immunopathology. Here, we showed that Ccl2 mRNA upregulation in human stromal fibroblasts in response to IL-17 required the RNA-binding protein IGF-2 mRNA-binding protein 2 (IGF2BP2, IMP2), which is expressed almost exclusively in nonhematopoietic cells. IMP2 binds directly to CCL2 mRNA, markedly extending its transcript half-life, and is thus required for efficient CCL2 secretion. Consistent with this, Imp2−/− mice showed reduced CCL2 production in LNs during EAE, causing impairments in monocyte recruitment and Th17 cell polarization. Imp2–/– mice were fully protected from CNS inflammation. Moreover, deletion of IMP2 after EAE onset was sufficient to mitigate disease severity. These data showed that posttranscriptional control of Ccl2 in stromal cells by IMP2 was required to permit IL-17–driven progression of EAE pathogenesis.
Rami Bechara, Nilesh Amatya, Saikat Majumder, Chunsheng Zhou, Yang Li, Qixing Liu, Mandy J. McGeachy, Sarah L. Gaffen
Ca2+/calmodulin-stimulated group I adenylyl cyclase (AC) isoforms AC1 and AC8 have been involved in nociceptive processing and morphine responses. However, whether AC3, another member of group I ACs, is involved in nociceptive transmission and regulates opioid receptor signaling remains elusive. Here, we report that conditional KO of AC3 (AC3 CKO) in L3 and L4 DRGs robustly facilitated the mouse nociceptive responses, decreased voltage-gated potassium (Kv) channel currents, and increased neuronal excitability. Furthermore, we report AC3 CKO eliminated the analgesic effect of κ-opioid receptor (KOR) agonist and its inhibition on Kv channel by classical Gαi/o signaling or nonclassical direct interaction of KOR and AC3 proteins. Interestingly, significantly upregulated AC1 level and cAMP concentration were detected in AC3-deficient DRGs. Inhibition of AC1 completely reversed cAMP upregulation, neuronal excitability enhancement, and nociceptive behavioral hypersensitivity in AC3-CKO mice. Our findings suggest a crucial role of peripheral AC3 in nociceptive modulation and KOR opioid analgesia.
Wen-Wen Zhang, Hong Cao, Yang Li, Xian-Jun Fu, Yu-Qiu Zhang
Cells recognize both foreign and host-derived double-stranded RNA (dsRNA) via a signaling pathway that is usually studied in the context of viral infection. It has become increasingly clear that the sensing and handling of endogenous dsRNA is also critical for cellular differentiation and development. The adenosine RNA deaminase, ADAR1, has been implicated as a central regulator of the dsRNA response, but how regulation of the dsRNA response might mediate cell fate during injury and whether such signaling is cell intrinsic remain unclear. Here, we show that the ADAR1-mediated response to dsRNA was dramatically induced in 2 distinct injury models of gastric metaplasia. Mouse organoid and in vivo genetic models showed that ADAR1 coordinated a cell-intrinsic, epithelium-autonomous, and interferon signaling–independent dsRNA response. In addition, dsRNA accumulated within a differentiated epithelial population (chief cells) in mouse and human stomachs as these cells reprogrammed to a proliferative, reparative (metaplastic) state. Finally, chief cells required ADAR1 to reenter the cell cycle during metaplasia. Thus, cell-intrinsic ADAR1 signaling is critical for the induction of metaplasia. Because metaplasia increases cancer risk, these findings support roles for ADAR1 and the response to dsRNA in oncogenesis.
José B. Sáenz, Nancy Vargas, Charles J. Cho, Jason C. Mills
The discovery of the oncometabolite 2-hydroxyglutarate in isocitrate dehydrogenase 1–mutated (IDH1-mutated) tumor entities affirmed the role of metabolism in cancer. However, large databases with tissue metabolites that are modulated by IDH1 mutation remain an area of development. Here, we present an unprecedented and valuable resource for tissue metabolites in diffuse glioma and their modulations by IDH1 mutation, histology, and tumor treatments in 101 tissue samples from 73 diffuse glioma patients (24 astrocytoma, 17 oligodendroglioma, 32 glioblastoma), investigated by NMR-based metabolomics and supported by RNA-Seq. We discovered comparison-specific metabolites and pathways modulated by IDH1 (IDH1 mutation status cohort) and tumor entity. The Longitudinal investigation cohort provides metabolic profiles of untreated and corresponding treated glioma samples at first progression. Most interestingly, univariate and multivariate cox regressions and Kaplan-Meier analyses revealed that tissue metabolites correlate with progression-free and overall survival. Thus, this study introduces potentially novel candidate prognostic and surrogate metabolite biomarkers for future prospective clinical studies, aiming at further refining patient stratification in diffuse glioma. Furthermore, our data will facilitate the generation of so-far–unanticipated hypotheses for experimental studies to advance our molecular understanding of glioma biology.
Christoph Trautwein, Laimdota Zizmare, Irina Mäurer, Benjamin Bender, Björn Bayer, Ulrike Ernemann, Marcos Tatagiba, Stefan J. Grau, Bernd J. Pichler, Marco Skardelly, Ghazaleh Tabatabai
Mosaic loss of chromosome Y (mLOY) in blood cells is one of the most frequent chromosome alterations in adult males. It is strongly associated with clonal hematopoiesis, hematopoietic malignancies, and other hematopoietic and nonhematopoietic diseases. However, whether there is a causal relationship between mLOY and human diseases is unknown. Here, we generated mLOY in murine hematopoietic stem and progenitor cells (HSPCs) with CRISPR/Cas9 genome editing. We found that mLOY led to dramatically increased DNA damage in HSPCs. Interestingly, HSPCs with mLOY displayed significantly enhanced reconstitution capacity and gave rise to clonal hematopoiesis in vivo. mLOY, which is associated with AML1-ETO translocation and p53 defects in patients with acute myeloid leukemia (AML), promoted AML in mice. Mechanistically, loss of KDM5D, a chromosome Y–specific histone 3 lysine 4 demethylase in both humans and mice, partially recapitulated mLOY in DNA damage and leukemogenesis. Thus, our study validates mLOY as a functional driver for clonal hematopoiesis and leukemogenesis.
Qi Zhang, Lei Zhao, Yi Yang, Shujun Li, Yu Liu, Chong Chen
Remodeling of injured sympathetic nerves on the heart after myocardial infarction (MI) contributes to adverse outcomes such as sudden arrhythmic death, yet the underlying structural mechanisms are poorly understood. We sought to examine microstructural changes on the heart after MI and to directly link these changes with electrical dysfunction. We developed a high-resolution pipeline for anatomically precise alignment of electrical maps with structural myofiber and nerve-fiber maps created by customized computer vision algorithms. Using this integrative approach in a mouse model, we identified distinct structure-function correlates to objectively delineate the infarct border zone, a known source of arrhythmias after MI. During tyramine-induced sympathetic nerve activation, we demonstrated regional patterns of altered electrical conduction aligned directly with altered neuroeffector junction distribution, pointing to potential neural substrates for cardiac arrhythmia. This study establishes a synergistic framework for examining structure-function relationships after MI with microscopic precision that has potential to advance understanding of arrhythmogenic mechanisms.
Ching Zhu, Pradeep S. Rajendran, Peter Hanna, Igor R. Efimov, Guy Salama, Charless C. Fowlkes, Kalyanam Shivkumar
CD8+ tumor-infiltrating lymphocytes (TILs) are associated with improved survival in triple-negative breast cancer (TNBC) yet have no association with survival in estrogen receptor–positive (ER+) BC. The basis for these contrasting findings remains elusive. We identified subsets of BC tumors infiltrated by CD8+ T cells with characteristic features of exhausted T cells (TEX). Tumors with abundant CD8+ TEX exhibited a distinct tumor microenvironment marked by amplified interferon-γ signaling–related pathways and higher programmed death ligand 1 expression. Paradoxically, higher levels of tumor-infiltrating CD8+ TEX associated with decreased overall survival of patients with ER+ BC but not patients with TNBC. Moreover, high tumor expression of a CD8+ TEX signature identified dramatically reduced survival in premenopausal, but not postmenopausal, patients with ER+ BC. Finally, we demonstrated the value of a tumor TEX signature score in identifying high-risk premenopausal ER+ BC patients among those with intermediate Oncotype DX Breast Recurrence Scores. Our data highlight the complex relationship between CD8+ TILs, interferon-γ signaling, and ER status in BC patient survival. This work identifies tumor-infiltrating CD8+ TEX as a key feature of reduced survival outcomes in premenopausal patients with early-stage ER+ BC.
Colt A. Egelston, Weihua Guo, Jiayi Tan, Christian Avalos, Diana L. Simons, Min Hui Lim, Yinghui J. Huang, Michael S. Nelson, Arnab Chowdhury, Daniel B. Schmolze, John H. Yim, Laura Kruper, Laleh Melstrom, Kim Margolin, Joanne E. Mortimer, Yuan Yuan, James R. Waisman, Peter P. Lee
The duodenum is a major site of HIV persistence during suppressive antiretroviral therapy despite harboring abundant tissue-resident memory (Trm) CD8+ T cells. The role of duodenal Trm CD8+ T cells in viral control is still not well defined. We examined the spatial localization, phenotype, and function of CD8+ T cells in the human duodenal tissue from people living with HIV (PLHIV) and healthy controls. We found that Trm (CD69+CD103hi) cells were the predominant CD8+ T cell population in the duodenum. Immunofluorescence imaging of the duodenal tissue revealed that CD103+CD8+ T cells were localized in the intraepithelial region, while CD103–CD8+ T cells and CD4+ T cells were mostly localized in the lamina propria (LP). Furthermore, HIV-specific CD8+ T cells were enriched in the CD69+CD103–/lo population. However, the duodenal HIV-specific CD8+ Trm cells rarely expressed canonical molecules for potent cytolytic function (perforin and granzyme B) but were more polyfunctional than those from peripheral blood. Taken together, our results show that duodenal CD8+ Trm cells possess limited perforin-mediated cytolytic potential and are spatially separated from HIV-susceptible LP CD4+ T cells. This could contribute to HIV persistence in the duodenum and provides critical information for the design of cure therapies.
Leonard Mvaya, Trevor Khaba, Agness E. Lakudzala, Thandeka Nkosi, Ndaru Jambo, Innocent Kadwala, Anstead Kankwatira, Priyanka D. Patel, Melita A. Gordon, Tonney S. Nyirenda, Kondwani C. Jambo, Zaza M. Ndhlovu
Standard radiation therapy (RT) does not reliably provide locoregional control for women with multinode-positive breast cancer and triple-negative breast cancer (TNBC). We hypothesized that CDK4/6 inhibition (CDK4/6i) would increase the radiosensitivity not only of estrogen receptor–positive (ER+) cells, but also of TNBC that expresses retinoblastoma (RB) protein. We found that CDK4/6i radiosensitized RB WT TNBC (n = 4, radiation enhancement ratio [rER]: 1.49–2.22) but failed to radiosensitize RB-null TNBC (n = 3, rER: 0.84–1.00). RB expression predicted response to CDK4/6i + RT (R2 = 0.84), and radiosensitization was lost in ER+/TNBC cells (rER: 0.88–1.13) after RB1 knockdown in isogenic and nonisogenic models. CDK4/6i suppressed homologous recombination (HR) in RB WT cells but not in RB-null cells or isogenic models of RB1 loss; HR competency was rescued with RB reexpression. Radiosensitization was independent of nonhomologous end joining and the known effects of CDK4/6i on cell cycle arrest. Mechanistically, RB and RAD51 interact in vitro to promote HR repair. CDK4/6i produced RB-dependent radiosensitization in TNBC xenografts but not in isogenic RB1-null xenografts. Our data provide the preclinical rationale for a clinical trial expanding the use of CDK4/6i + RT to difficult-to-control RB-intact breast cancers (including TNBC) and nominate RB status as a predictive biomarker of therapeutic efficacy.
Andrea M. Pesch, Nicole H. Hirsh, Anna R. Michmerhuizen, Kassidy M. Jungles, Kari Wilder-Romans, Benjamin C. Chandler, Meilan Liu, Lynn M. Lerner, Charles A. Nino, Connor Ward, Erin F. Cobain, Theodore S. Lawrence, Lori J. Pierce, James M. Rae, Corey W. Speers
Systemic hypoxia is characterized by peripheral vasodilation and pulmonary vasoconstriction. However, the system-wide mechanism for signaling hypoxia remains unknown. Accumulating evidence suggests that hemoglobin (Hb) in RBCs may serve as an O2 sensor and O2-responsive NO signal transducer to regulate systemic and pulmonary vascular tone, but this remains unexamined at the integrated system level. One residue invariant in mammalian Hbs, β-globin cysteine93 (βCys93), carries NO as vasorelaxant S-nitrosothiol (SNO) to autoregulate blood flow during O2 delivery. βCys93Ala mutant mice thus exhibit systemic hypoxia despite transporting O2 normally. Here, we show that βCys93Ala mutant mice had reduced S-nitrosohemoglobin (SNO-Hb) at baseline and upon targeted SNO repletion and that hypoxic vasodilation by RBCs was impaired in vitro and in vivo, recapitulating hypoxic pathophysiology. Notably, βCys93Ala mutant mice showed marked impairment of hypoxic peripheral vasodilation and developed signs of pulmonary hypertension with age. Mutant mice also died prematurely with cor pulmonale (pulmonary hypertension with right ventricular dysfunction) when living under low O2. Altogether, we identify a major role for RBC SNO in clinically relevant vasodilatory responses attributed previously to endothelial NO. We conclude that SNO-Hb transduces the integrated, system-wide response to hypoxia in the mammalian respiratory cycle, expanding a core physiological principle.
Rongli Zhang, Alfred Hausladen, Zhaoxia Qian, Xudong Liao, Richard T. Premont, Jonathan S. Stamler
Background Adenovirus-vectored (Ad-vectored) vaccines are typically administered via i.m. injection to humans and are incapable of inducing respiratory mucosal immunity. However, aerosol delivery of Ad-vectored vaccines remains poorly characterized, and its ability to induce mucosal immunity in humans is unknown. This phase Ib trial evaluated the safety and immunogenicity of human serotype-5 Ad-vectored tuberculosis (TB) vaccine (AdHu5Ag85A) delivered to humans via inhaled aerosol or i.m. injection.Methods Thirty-one healthy, previously BCG-vaccinated adults were enrolled. AdHu5Ag85A was administered by single-dose aerosol using Aeroneb Solo Nebulizer or by i.m. injection. The study consisted of the low-dose (LD) aerosol, high-dose (HD) aerosol, and i.m. groups. The adverse events were assessed at various times after vaccination. Immunogenicity data were collected from the peripheral blood and bronchoalveolar lavage samples at baseline, as well as at select time points after vaccination.Results The nebulized aerosol droplets were < 5.39 μm in size. Both LD and HD of AdHu5Ag85A administered by aerosol inhalation and i.m. injection were safe and well tolerated. Both aerosol doses, particularly LD, but not i.m., vaccination markedly induced airway tissue–resident memory CD4+ and CD8+ T cells of polyfunctionality. While as expected, i.m. vaccination induced Ag85A-specific T cell responses in the blood, the LD aerosol vaccination also elicited such T cells in the blood. Furthermore, the LD aerosol vaccination induced persisting transcriptional changes in alveolar macrophages.Conclusion Inhaled aerosol delivery of Ad-vectored vaccine is a safe and superior way to elicit respiratory mucosal immunity. This study warrants further development of aerosol vaccine strategies against respiratory pathogens, including TB and COVID-19.Trial registration ClinicalTrial.gov, NCT02337270.Funding The Canadian Institutes for Health Research (CIHR) and the Natural Sciences and Engineering Research Council of Canada funded this work.
Mangalakumari Jeyanathan, Dominik K. Fritz, Sam Afkhami, Emilio Aguirre, Karen J. Howie, Anna Zganiacz, Anna Dvorkin-Gheva, Michael R. Thompson, Richard F. Silver, Ruth P. Cusack, Brian D. Lichty, Paul M. O’Byrne, Martin Kolb, Maria Fe C. Medina, Myrna B. Dolovich, Imran Satia, Gail M. Gauvreau, Zhou Xing, Fiona Smaill
Aortic dissection and rupture are triggered by decreased vascular wall strength and/or increased mechanical loads. We investigated the role of mTOR signaling in aortopathy using a well-described model of angiotensin II–induced dissection, aneurysm, or rupture of the suprarenal abdominal aorta in Apoe-deficient mice. Although not widely appreciated, nonlethal hemorrhagic lesions present as pseudoaneurysms without significant dissection in this model. Angiotensin II–induced aortic tears result in free rupture, contained rupture with subadventitial hematoma (forming pseudoaneurysms), dilatation, or healing, while the media invariably thickens regardless of mural tears. Medial thickening results from smooth muscle cell hypertrophy and extracellular matrix accumulation, including matricellular proteins. Angiotensin II activates mTOR signaling in vascular wall cells, and inhibition of mTOR signaling by rapamycin prevents aortic rupture but promotes dissection. Decreased aortic rupture correlates with decreased inflammation and metalloproteinase expression, whereas extensive dissection correlates with induction of matricellular proteins that modulate adhesion of vascular cells. Thus, mTOR activation in vascular wall cells determines whether aortic tears progress to dissection or rupture. Previous mechanistic studies of aortic aneurysm and dissection by angiotensin II in Apoe-deficient mice should be reinterpreted as clinically relevant to pseudoaneurysms, and mTOR inhibition for aortic disease should be explored with caution.
Changshun He, Bo Jiang, Mo Wang, Pengwei Ren, Sae-Il Murtada, Alexander W. Caulk, Guangxin Li, Lingfeng Qin, Roland Assi, Constantinos J. Lovoulos, Martin A. Schwartz, Jay D. Humphrey, George Tellides
Takotsubo syndrome (TTS) is an acute, stress-induced cardiomyopathy that occurs predominantly in women after extreme physical and/or emotional stress. To date, our understanding of the molecular basis for TTS remains unknown and, consequently, specific therapies are lacking. Myocardial infiltration of monocytes and macrophages in TTS has been documented in clinical studies. However, the functional importance of these findings remains poorly understood. Here, we show that a single high dose of isoproterenol (ISO) in mice induced a TTS-like cardiomyopathy phenotype characterized by female predominance, severe cardiac dysfunction, and robust myocardial infiltration of macrophages. Single-cell RNA-Seq studies of myocardial immune cells revealed that TTS-like cardiomyopathy is associated with complex activation of innate and adaptive immune cells in the heart, and macrophages were identified as the dominant immune cells. Global macrophage depletion (via clodronate liposome administration) or blockade of macrophage infiltration (via a CCR2 antagonist or in CCR2-KO mice) resulted in recovery of cardiac dysfunction in ISO-challenged mice. In addition, damping myeloid cell activation by HIF1α deficiency or exposure to the immunomodulatory agent bortezomib ameliorated ISO-induced cardiac dysfunction. Collectively, our findings identify macrophages as a critical regulator of TTS pathogenesis that can be targeted for therapeutic gain.
Xudong Liao, Eugene Chang, Xinmiao Tang, Ippei Watanabe, Rongli Zhang, Hyun-Woo Jeong, Ralf H. Adams, Mukesh K. Jain