Issue published November 8, 2023
- Volume 8, Issue 21
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Bocquet et al. utilize human genetics, Xenopus loss-of-function studies, and patient iPSC-derived retinal models to demonstrate that TBC1D32 is a causative gene for retinitis pigmentosa (RP). The cover image shows a 3D reconstruction of an iPSC-derived retinal pigment epithelium monolayer (white), with elongated primary cilia (in color) of a patient with RP carrying TBC1D32 variants. Image credit: Hassan Boukhaddaoui and Nejla Erkilic.
On the cover: TBC1D32 variants disrupt retinal ciliogenesis and cause retinitis pigmentosa
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Research Articles
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Abstract
Chronic lung allograft dysfunction (CLAD) is a major complication after lung transplantation that results from a complex interplay of innate inflammatory and alloimmune factors, culminating in parenchymal and/or obliterative airway fibrosis. Excessive IL-17A signaling and chronic inflammation have been recognized as key factors in these pathological processes. Herein, we developed a model of repeated airway inflammation in mouse minor alloantigen-mismatched single-lung transplantation. Repeated intratracheal LPS instillations augmented pulmonary IL-17A expression. LPS also increased acute rejection, airway epithelial damage, and obliterative airway fibrosis, similar to human explanted lung allografts with antecedent episodes of airway infection. We then investigated the role of donor and recipient IL-17 receptor A (IL-17RA) in this context. Donor IL-17RA deficiency significantly attenuated acute rejection and CLAD features, whereas recipient IL-17RA deficiency only slightly reduced airway obliteration in LPS allografts. IL-17RA immunofluorescence positive staining was greater in human CLAD lungs compared with control human lung specimens, with localization to fibroblasts and myofibroblasts, which was also seen in mouse LPS allografts. Taken together, repeated airway inflammation after lung transplantation caused local airway epithelial damage, with persistent elevation of IL-17A and IL-17RA expression and particular involvement of IL-17RA on donor structural cells in development of fibrosis.
Authors
Tatsuaki Watanabe, Stephen C. Juvet, Gregory Berra, Jan Havlin, Wenshan Zhong, Kristen Boonstra, Tina Daigneault, Miho Horie, Chihiro Konoeda, Grace Teskey, Zehong Guan, David M. Hwang, Mingyao Liu, Shaf Keshavjee, Tereza Martinu
×Abstract
Nonalcoholic steatohepatitis (NASH) is a leading cause for chronic liver diseases. Current therapeutic options are limited due to an incomplete mechanistic understanding of how steatosis transitions to NASH. Here we show that the TRIM21 E3 ubiquitin ligase is induced by the synergistic actions of proinflammatory TNF-α and fatty acids in livers of humans and mice with NASH. TRIM21 ubiquitinates and degrades ChREBP, SREBP1, ACC1, and FASN, key regulators of de novo lipogenesis, and A1CF, an alternative splicing regulator of the high-activity ketohexokinase-C (KHK-C) isoform and rate-limiting enzyme of fructose metabolism. TRIM21-mediated degradation of these lipogenic activators improved steatosis and hyperglycemia as well as fructose and glucose tolerance. Our study identifies TRIM21 as a negative regulator of liver steatosis in NASH and provides mechanistic insights into an immunometabolic crosstalk that limits fatty acid synthesis and fructose metabolism during metabolic stress. Thus, enhancing this natural counteracting force of steatosis through inhibition of key lipogenic activators via TRIM21-mediated ubiquitination may provide a therapeutic opportunity to treat NASH.
Authors
Kostas C. Nikolaou, Svenja Godbersen, Muthiah Manoharan, Stefan Wieland, Markus H. Heim, Markus Stoffel
×Abstract
Poly (ADP-ribose) polymerase inhibitors (PARPis) are approved for cancer therapy according to their synthetic lethal interactions, and clinical trials have been applied in non–small cell lung cancer. However, the therapeutic efficacy of PARPis in lung adenocarcinoma (LUAD) is still unknown. We explored the effect of a mutated retinoblastoma gene (RB1) on PARPi sensitivity in LUAD. Bioinformatic screening was performed to identify PARPi-sensitive biomarkers. Here, we showed that viability of LUAD cell lines with mutated RB1 was significantly decreased by PARPis (niraparib, rucaparib, and olaparib). RB1 deficiency induced genomic instability, prompted cytosolic double-stranded DNA (dsDNA) formation, activated the cGAS/STING pathway, and upregulated downstream chemokines CCL5 and CXCL10, triggering immune cell infiltration. Xenograft experiments indicated that PARPi treatment reduced tumorigenesis in RB1-KO mice. Additionally, single-cell RNA sequencing analysis showed that malignant cells with downregulated expression of RB1 had more communications with other cell types, exhibiting activation of specific signaling such as GAS, IFN response, and antigen-presenting and cytokine activities. Our findings suggest that RB1 mutation mediates the sensitivity to PARPis through a synthetic lethal effect by triggering the cGAS/STING pathway and upregulation of immune infiltration in LUAD, which may be a potential therapeutic strategy.
Authors
Qi Dong, Tong Yu, Bo Chen, Mingyue Liu, Xiang Sun, Huiying Cao, Kaidong Liu, Huanhuan Xu, Yuquan Wang, Shuping Zhuang, Zixin Jin, Haihai Liang, Yang Hui, Yunyan Gu
×Cholestanol accelerates α-synuclein aggregation and spreading by activating asparagine endopeptidase
Abstract
Cerebrotendinous xanthomatosis (CTX), an autosomal recessive disorder characterized by high levels of cholestanol in the blood and accumulation of cholestanol in multiple tissues, especially the brain, often presents in parkinsonism. However, it remains unknown whether cholestanol plays a role in the pathogenesis of sporadic Parkinson’s disease (PD). Here, we show that the levels of serum cholestanol in patients with sporadic PD are higher than those in control participants. Cholestanol activates the protease asparagine endopeptidase (AEP) and induces the fragmentation of α-synuclein (α-syn) and facilitates its aggregation. Furthermore, cholestanol promotes the spreading of α-syn pathology in a mouse model induced by intrastriatal injection of α-syn fibrils. KO of AEP or administration of an AEP inhibitor ameliorates α-syn pathology, degeneration of the nigrostriatal dopaminergic pathway, and PD-like motor symptoms. These results not only indicate that cholestanol contributes to the aggregation and spreading of α-syn by activating AEP but also reveal an opportunity for treating PD with AEP inhibitors.
Authors
Ting Yu, Shuke Nie, Lihong Bu, Miao Liu, Juanfeng He, Xuan Niu, Hongyan Feng, Jifeng Guo, Beisha Tang, Zhaohui Zhang, Keqiang Ye, Haiqiang Jiang, Liam Chen, Zhentao Zhang
×Abstract
New medicines are urgently required to treat the fatal neuromuscular disease Duchenne muscular dystrophy (DMD). Dimethyl fumarate (DMF) is a potent immunomodulatory small molecule nuclear erythroid 2-related factor 2 activator with current clinical utility in the treatment of multiple sclerosis and psoriasis that could be effective for DMD and rapidly translatable. Here, we tested 2 weeks of daily 100 mg/kg DMF versus 5 mg/kg standard-care prednisone (PRED) treatment in juvenile mdx mice with early symptomatic DMD. Both drugs modulated seed genes driving the DMD disease program and improved force production in fast-twitch muscle. However, only DMF showed pro-mitochondrial effects, protected contracting muscles from fatigue, improved histopathology, and augmented clinically compatible muscle function tests. DMF may be a more selective modulator of the DMD disease program than PRED, warranting follow-up longitudinal studies to evaluate disease-modifying impact.
Authors
Cara A. Timpani, Stephanie Kourakis, Danielle A. Debruin, Dean G. Campelj, Nancy Pompeani, Narges Dargahi, Angelo P. Bautista, Ryan M. Bagaric, Elya J. Ritenis, Lauren Sahakian, Didier Debrincat, Nicole Stupka, Patricia Hafner, Peter G. Arthur, Jessica R. Terrill, Vasso Apostolopoulos, Judy B. de Haan, Nuri Guven, Dirk Fischer, Emma Rybalka
×Abstract
Glioblastoma (GBM) is the most lethal brain cancer with a dismal prognosis. Stem-like GBM cells (GSCs) are a major driver of GBM propagation and recurrence; thus, understanding the molecular mechanisms that promote GSCs may lead to effective therapeutic approaches. Through in vitro clonogenic growth-based assays, we determined mitogenic activities of the ligand molecules that are implicated in neural development. We have identified that semaphorin 3A (Sema3A), originally known as an axon guidance molecule in the CNS, promotes clonogenic growth of GBM cells but not normal neural progenitor cells (NPCs). Mechanistically, Sema3A binds to its receptor neuropilin-1 (NRP1) and facilitates an interaction between NRP1 and TGF-β receptor 1 (TGF-βR1), which in turn leads to activation of canonical TGF-β signaling in both GSCs and NPCs. TGF-β signaling enhances self-renewal and survival of GBM tumors through induction of key stem cell factors, but it evokes cytostatic responses in NPCs. Blockage of the Sema3A/NRP1 axis via shRNA-mediated knockdown of Sema3A or NRP1 impeded clonogenic growth and TGF-β pathway activity in GSCs and inhibited tumor growth in vivo. Taken together, these findings suggest that the Sema3A/NRP1/TGF-βR1 signaling axis is a critical regulator of GSC propagation and a potential therapeutic target for GBM.
Authors
Hye-Min Jeon, Yong Jae Shin, Jaehyun Lee, Nakho Chang, Dong-Hun Woo, Won Jun Lee, Dayna Nguyen, Wonyoung Kang, Hee Jin Cho, Heekyoung Yang, Jin-Ku Lee, Jason K. Sa, Yeri Lee, Dong Geon Kim, Benjamin W. Purow, Yeup Yoon, Do-Hyun Nam, Jeongwu Lee
×Abstract
MTORC1 integrates signaling from the immune microenvironment to regulate T cell activation, differentiation, and function. TSC2 in the tuberous sclerosis complex tightly regulates mTORC1 activation. CD8+ T cells lacking TSC2 have constitutively enhanced mTORC1 activity and generate robust effector T cells; however, sustained mTORC1 activation prevents generation of long-lived memory CD8+ T cells. Here we show that manipulating TSC2 at Ser1365 potently regulated activated but not basal mTORC1 signaling in CD8+ T cells. Unlike nonstimulated TSC2-KO cells, CD8+ T cells expressing a phosphosilencing mutant TSC2-S1365A (TSC2-SA) retained normal basal mTORC1 activity. PKC and T cell receptor (TCR) stimulation induced TSC2 S1365 phosphorylation, and preventing this with the SA mutation markedly increased mTORC1 activation and T cell effector function. Consequently, SA CD8+ T cells displayed greater effector responses while retaining their capacity to become long-lived memory T cells. SA CD8+ T cells also displayed enhanced effector function under hypoxic and acidic conditions. In murine and human solid-tumor models, SA CD8+ T cells used as adoptive cell therapy displayed greater antitumor immunity than WT CD8+ T cells. These findings reveal an upstream mechanism to regulate mTORC1 activity in T cells. The TSC2-SA mutation enhanced both T cell effector function and long-term persistence/memory formation, supporting an approach to engineer better CAR-T cells for treating cancer.
Authors
Chirag H. Patel, Yi Dong, Navid Koleini, Xiaoxu Wang, Brittany L. Dunkerly-Eyring, Jiayu Wen, Mark J. Ranek, Laura M. Bartle, Daniel B. Henderson, Jason Sagert, David A. Kass, Jonathan D. Powell
×Abstract
Emerging evidence shows that KRAS-mutant colorectal cancer (CRC) depends on glutamine (Gln) for survival and progression, indicating that targeting Gln metabolism may be a promising therapeutic strategy for KRAS-mutant CRC. However, the precise mechanism by which Gln metabolism reprogramming promotes and coordinates KRAS-mutant CRC progression remains to be fully investigated. Here, we discovered that solute carrier 25 member 21 (SLC25A21) expression was downregulated in KRAS-mutant CRC, and that SLC25A21 downregulation was correlated with poor survival of KRAS-mutant CRC patients. SLC25A21 depletion selectively accelerated the growth, invasion, migration, and metastasis of KRAS-mutant CRC cells in vitro and in vivo, and inhibited Gln-derived α-ketoglutarate (α-KG) efflux from mitochondria, thereby potentiating Gln replenishment, accompanied by increased GTP availability for persistent KRAS activation in KRAS-mutant CRC. The restoration of SLC25A21 expression impaired the KRAS-mutation-mediated resistance to cetuximab in KRAS-mutant CRC. Moreover, the arrested α-KG efflux that occurred in response to SLC25A21 depletion inhibited the activity of α-KG–dependent DNA demethylases, resulting in a further decrease in SLC25A21 expression. Our studies demonstrate that SLC25A21 plays a significant role as a tumor suppressor in KRAS-mutant CRC by antagonizing Gln-dependent anaplerosis to limit GTP availability for KRAS activation, which suggests potential alternative therapeutic strategies for KRAS-mutant CRC.
Authors
Sha-Sha Hu, Yue Han, Tian-Yuan Tan, Hui Chen, Jia-Wen Gao, Lan Wang, Min-Hui Yang, Li Zhao, Yi-Qing Wang, Yan-Qing Ding, Shuang Wang
×Abstract
Variation in the preservation of β cell function in clinical trials in type 1 diabetes (T1D) has emphasized the need to define biomarkers to predict treatment response. The T1DAL trial targeted T cells with alefacept (LFA-3–Ig) and demonstrated C-peptide preservation in approximately 30% of new-onset T1D individuals. We analyzed islet antigen–reactive (IAR) CD4+ T cells in PBMC samples collected prior to treatment from alefacept- and placebo-treated individuals using flow cytometry and single-cell RNA sequencing. IAR CD4+ T cells at baseline had heterogeneous phenotypes. Transcript profiles formed phenotypic clusters of cells along a trajectory based on increasing maturation and activation, and T cell receptor (TCR) chains showed clonal expansion. Notably, the frequency of IAR CD4+ T cells with a memory phenotype and a unique transcript profile (cluster 3) were inversely correlated with C-peptide preservation in alefacept-treated, but not placebo-treated, individuals. Cluster 3 cells had a proinflammatory phenotype characterized by expression of the transcription factor BHLHE40 and the cytokines GM-CSF and TNF-α, and shared TCR chains with effector memory–like clusters. Our results suggest IAR CD4+ T cells as a potential baseline biomarker of response to therapies targeting the CD2 pathway and warrant investigation for other T cell–related therapies.
Authors
Elisa Balmas, Janice Chen, Alex K. Hu, Hannah A. DeBerg, Mario G. Rosasco, Vivian H. Gersuk, Elisavet Serti, Cate Speake, Carla J. Greenbaum, Gerald T. Nepom, Peter S. Linsley, Karen Cerosaletti
×Abstract
The penetration of allergens through the epithelial layer is the initial step in the development of allergic conjunctivitis. Although pollinosis patients manifest symptoms within minutes after pollen exposure, the mechanisms of the rapid transport of the allergens remain unclear. In the present study, we found that the instillation of pollen shells rapidly induces a large number of goblet cell–associated antigen passages (GAPs) in the conjunctiva. Antigen acquisition by stromal cells, including macrophages and CD11b+ dendritic cells, correlated with surface GAP formation. Furthermore, a substantial amount of antigen was transported to the stroma during the first 10 minutes of pollen exposure, which was sufficient for the full induction of an allergic conjunctivitis mouse model. This inducible, rapid GAP formation and antigen acquisition were suppressed by topical lidocaine or trigeminal nerve ablation, indicating that the sensory nervous system plays an essential role. Interestingly, pollen shell–stimulated GAP formation was not suppressed by topical atropine, suggesting that the conjunctival GAPs and intestinal GAPs are differentially regulated. These results identify pollen shell–induced GAP as a therapeutic target for allergic conjunctivitis.
Authors
Meiko Kimura, Tomoaki Ando, Yasuharu Kume, Saaya Fukase, Moe Matsuzawa, Kosuke Kashiwagi, Kumi Izawa, Ayako Kaitani, Nobuhiro Nakano, Keiko Maeda, Hideoki Ogawa, Ko Okumura, Shintaro Nakao, Akira Murakami, Nobuyuki Ebihara, Jiro Kitaura
×Abstract
Retinitis pigmentosa (RP) is the most common inherited retinal disease (IRD) and is characterized by photoreceptor degeneration and progressive vision loss. We report 4 patients presenting with RP from 3 unrelated families with variants in TBC1D32, which to date has never been associated with an IRD. To validate TBC1D32 as a putative RP causative gene, we combined Xenopus in vivo approaches and human induced pluripotent stem cell–derived (iPSC-derived) retinal models. Our data showed that TBC1D32 was expressed during retinal development and that it played an important role in retinal pigment epithelium (RPE) differentiation. Furthermore, we identified a role for TBC1D32 in ciliogenesis of the RPE. We demonstrated elongated ciliary defects that resulted in disrupted apical tight junctions, loss of functionality (delayed retinoid cycling and altered secretion balance), and the onset of an epithelial-mesenchymal transition–like phenotype. Last, our results suggested photoreceptor differentiation defects, including connecting cilium anomalies, that resulted in impaired trafficking to the outer segment in cones and rods in TBC1D32 iPSC-derived retinal organoids. Overall, our data highlight a critical role for TBC1D32 in the retina and demonstrate that TBC1D32 mutations lead to RP. We thus identify TBC1D32 as an IRD-causative gene.
Authors
Béatrice Bocquet, Caroline Borday, Nejla Erkilic, Daria Mamaeva, Alicia Donval, Christel Masson, Karine Parain, Karolina Kaminska, Mathieu Quinodoz, Irene Perea-Romero, Gema Garcia-Garcia, Carla Jimenez-Medina, Hassan Boukhaddaoui, Arthur Coget, Nicolas Leboucq, Giacomo Calzetti, Stefano Gandolfi, Antonio Percesepe, Valeria Barili, Vera Uliana, Marco Delsante, Francesca Bozzetti, Hendrik P.N. Scholl, Marta Corton, Carmen Ayuso, Jose M. Millan, Carlo Rivolta, Isabelle Meunier, Muriel Perron, Vasiliki Kalatzis
×Abstract
Calponin 2 (CNN2) is a prominent actin stabilizer. It regulates fatty acid oxidation (FAO) by interacting with estrogen receptor 2 (ESR2) to determine kidney fibrosis. However, whether CNN2 is actively involved in acute kidney injury (AKI) remains unclear. Here, we report that CNN2 was induced in human and animal kidneys after AKI. Knockdown of CNN2 preserved kidney function, mitigated tubular cell death and inflammation, and promoted cell proliferation. Distinct from kidney fibrosis, proteomics showed that the key elements in the FAO pathway had few changes during AKI, but we identified that 3-hydroxymethylglutaryl-CoA synthase 2 (Hmgcs2), a rate-limiting enzyme of endogenous ketogenesis that promotes cell self-renewal, was markedly increased in CNN2-knockdown kidneys. The production of ketone body β-hydroxybutyrate and ATP was increased in CNN2-knockdown mice. Mechanistically, CNN2 interacted with ESR2 to negatively regulate the activities of mitochondrial sirtuin 5. Activated sirtuin 5 subsequently desuccinylated Hmgcs2 to produce energy for mitigating AKI. Understanding CNN2-mediated discrete fine-tuning of protein posttranslational modification is critical to optimize organ performance after AKI.
Authors
Yuan Gui, Zachary Palanza, Priya Gupta, Hanwen Li, Yuchen Pan, Yuanyuan Wang, Geneva Hargis, Donald L. Kreutzer, Yanlin Wang, Sheldon I. Bastacky, Yansheng Liu, Silvia Liu, Dong Zhou
×Abstract
Application of classic liver-directed gene replacement strategies is limited in genetic diseases characterized by liver injury due to hepatocyte proliferation, resulting in decline of therapeutic transgene expression and potential genotoxic risk. Wilson disease (WD) is a life-threatening autosomal disorder of copper homeostasis caused by pathogenic variants in copper transporter ATP7B and characterized by toxic copper accumulation, resulting in severe liver and brain diseases. Genome editing holds promise for the treatment of WD; nevertheless, to rescue copper homeostasis, ATP7B function must be restored in at least 25% of the hepatocytes, which surpasses by far genome-editing correction rates. We applied a liver-directed, nuclease-free genome editing approach, based on adeno-associated viral vector–mediated (AAV-mediated) targeted integration of a promoterless mini-ATP7B cDNA into the albumin (Alb) locus. Administration of AAV-Alb–mini-ATP7B in 2 WD mouse models resulted in extensive liver repopulation by genome-edited hepatocytes holding a proliferative advantage over nonedited ones, and ameliorated liver injury and copper metabolism. Furthermore, combination of genome editing with a copper chelator, currently used for WD treatment, achieved greater disease improvement compared with chelation therapy alone. Nuclease-free genome editing provided therapeutic efficacy and may represent a safer and longer-lasting alternative to classic gene replacement strategies for WD.
Authors
Agnese Padula, Michele Spinelli, Edoardo Nusco, Xabier Bujanda Cundin, Filomena Capolongo, Severo Campione, Claudia Perna, Amy Bastille, Megan Ericson, Chih-Chieh Wang, Shengwen Zhang, Angela Amoresano, Mariana Nacht, Pasquale Piccolo
×Abstract
Mucopolysaccharidosis VI (MPS VI) is a rare lysosomal disease arising from impaired function of the enzyme arylsulfatase B (ARSB). This impairment causes aberrant accumulation of dermatan sulfate, a glycosaminoglycan (GAG) abundant in cartilage. While clinical severity varies along with age at first symptom manifestation, MPS VI usually presents early and strongly affects the skeleton. Current enzyme replacement therapy (ERT) does not provide effective treatment for the skeletal manifestations of MPS VI. This lack of efficacy may be due to an inability of ERT to reach affected cells or to the irreversibility of the disease. To address the question of reversibility of skeletal phenotypes, we generated a conditional by inversion (COIN) mouse model of MPS VI, ArsbCOIN/COIN, wherein Arsb is initially null and can be restored to WT using Cre. We restored Arsb at different times during postnatal development, using a tamoxifen-dependent global Cre driver. By restoring Arsb at P7, P21, and P56–P70, we determined that skeletal phenotypes can be fully rescued if Arsb restoration occurs at P7, while only achieving partial rescue at P21 and no significant rescue at P56–P70. This work has highlighted the importance of early intervention in patients with MPS VI to maximize therapeutic impact.
Authors
Elizabeth Hwang-Wong, Gabrielle Amar, Nanditha Das, Xiaoli Zhang, Nina Aaron, Kirsten Gale, Nyanza Rothman, Massimo Fante, Andrew Baik, Ajay Bhargava, Arun Fricker, Michelle McAlister, Jeremy Rabinowitz, John Lees-Shepard, Kalyan Nannuru, Aris N. Economides, Katherine D. Cygnar
×Abstract
Osteogenesis imperfecta (OI), or brittle bone disease, is a disorder characterized by bone fragility and increased fracture incidence. All forms of OI also feature short stature, implying an effect on endochondral ossification. Using the Aga2+/– mouse, which has a mutation in type I collagen, we show an affected growth plate primarily due to a shortened proliferative zone. We used single-cell RNA-Seq analysis of tibial and femoral growth plate tissues to understand transcriptional consequences on growth plate cell types. We show that perichondrial cells, which express abundant type I procollagen, and growth plate chondrocytes, which were found to express low amounts of type I procollagen, had ER stress and dysregulation of the same unfolded protein response pathway as previously demonstrated in osteoblasts. Aga2+/– proliferating chondrocytes showed increased FGF and MAPK signaling, findings consistent with accelerated differentiation. There was also increased Sox9 expression throughout the growth plate, which is expected to accelerate early chondrocyte differentiation but reduce late hypertrophic differentiation. These data reveal that mutant type I collagen expression in OI has an impact on the cartilage growth plate. These effects on endochondral ossification indicate that OI is a biologically complex phenotype going beyond its known impacts on bone to negatively affect linear growth.
Authors
Jennifer Zieba, Lisette Nevarez, Davis Wachtell, Jorge H. Martin, Alexander Kot, Sereen Wong, Daniel H. Cohn, Deborah Krakow
×Abstract
Epithelial Na+ channels (ENaCs) control extracellular fluid volume by facilitating Na+ absorption across transporting epithelia. In vitro studies showed that Cys-palmitoylation of the γENaC subunit is a major regulator of channel activity. We tested whether γ subunit palmitoylation sites are necessary for channel function in vivo by generating mice lacking the palmitoylated cysteines (γC33A,C41A) using CRISPR/Cas9 technology. ENaCs in dissected kidney tubules from γC33A,C41A mice had reduced open probability compared with wild-type (WT) littermates maintained on either standard or Na+-deficient diets. Male mutant mice also had higher aldosterone levels than WT littermates following Na+ restriction. However, γC33A,C41A mice did not have reduced amiloride-sensitive Na+ currents in the distal colon or benzamil-induced natriuresis compared to WT mice. We identified a second, larger conductance cation channel in the distal nephron with biophysical properties distinct from ENaC. The activity of this channel was higher in Na+-restricted γC33A,C41A versus WT mice and was blocked by benzamil, providing a possible compensatory mechanism for reduced prototypic ENaC function. We conclude that γ subunit palmitoylation sites are required for prototypic ENaC activity in vivo but are not necessary for amiloride/benzamil-sensitive Na+ transport in the distal nephron or colon.
Authors
Andrew J. Nickerson, Stephanie M. Mutchler, Shaohu Sheng, Natalie A. Cox, Evan C. Ray, Ossama B. Kashlan, Marcelo D. Carattino, Allison L. Marciszyn, Aaliyah Winfrey, Sebastien Gingras, Annet Kirabo, Rebecca P. Hughey, Thomas R. Kleyman
×Abstract
CXCR4 is a key regulator of the development of NK cells and DCs, both of which play an important role in early placental development and immune tolerance at the maternal-fetal interface. However, the role of CXCR4 in pregnancy is not well understood. Our study demonstrates that adult-induced global genetic CXCR4 deletion, but not uterine-specific CXCR4 deletion, was associated with increased pregnancy resorptions and decreased litter size. CXCR4-deficient mice had decreased NK cells and increased granulocytes in the decidua, along with increased leukocyte numbers in peripheral blood. We found that CXCR4-deficient mice had abnormal decidual NK cell aggregates and NK cell infiltration into trophoblast areas beyond the giant cell layer. This was associated with low NK cell expression of granzyme B, a NK cell granule effector, indicative of NK cell dysfunction. Pregnancy failure in these mice was associated with abnormalities in placental vascular development and increased placental expression of inflammatory genes. Importantly, adoptive BM transfer of WT CXCR4+ BM cells into CXCR4-deficient mice rescued the reproductive deficits by normalizing NK cell function and mediating normal placental vascular development. Collectively, our study found an important role for maternal CXCR4 expression in immune cell function, placental development, and pregnancy maintenance.
Authors
Fang Lyu, Chase Burzynski, Yuan yuan Fang, Aya Tal, Alice Y. Chen, Jacqueline Kisa, Kriti Agrawal, Yuval Kluger, Hugh S. Taylor, Reshef Tal
×Abstract
Myosin heavy chains encoded by MYH7 and MYH2 are abundant in human skeletal muscle and important for muscle contraction. However, it is unclear how mutations in these genes disrupt myosin structure and function leading to skeletal muscle myopathies termed myosinopathies. Here, we used multiple approaches to analyze the effects of common MYH7 and MYH2 mutations in the light meromyosin (LMM) region of myosin. Analyses of expressed and purified MYH7 and MYH2 LMM mutant proteins combined with in silico modeling showed that myosin coiled coil structure and packing of filaments in vitro are commonly disrupted. Using muscle biopsies from patients and fluorescent ATP analog chase protocols to estimate the proportion of myosin heads that were super-relaxed, together with x-ray diffraction measurements to estimate myosin head order, we found that basal myosin ATP consumption was increased and the myosin super-relaxed state was decreased in vivo. In addition, myofiber mechanics experiments to investigate contractile function showed that myofiber contractility was not affected. These findings indicate that the structural remodeling associated with LMM mutations induces a pathogenic state in which formation of shutdown heads is impaired, thus increasing myosin head ATP demand in the filaments, rather than affecting contractility. These key findings will help design future therapies for myosinopathies.
Authors
Glenn Carrington, Abbi Hau, Sarah Kosta, Hannah F. Dugdale, Francesco Muntoni, Adele D’Amico, Peter Van den Bergh, Norma B. Romero, Edoardo Malfatti, Juan Jesus Vilchez, Anders Oldfors, Sander Pajusalu, Katrin Õunap, Marta Giralt-Pujol, Edmar Zanoteli, Kenneth S. Campbell, Hiroyuki Iwamoto, Michelle Peckham, Julien Ochala
×Abstract
The organic anion transporter OAT1 (SLC22A6, originally identified as NKT) is a multispecific transporter responsible for the elimination by the kidney of small organic anions that derive from the gut microbiome. Many are uremic toxins associated with chronic kidney disease (CKD). OAT1 is among a group of “drug” transporters that act as hubs in a large homeostatic network regulating interorgan and interorganismal communication via small molecules. The Remote Sensing and Signaling Theory predicts that genetic deletion of such a key hub in the network results in compensatory interorganismal communication (e.g., host-gut microbe dynamics). Recent metabolomics data from Oat1-KO mice indicate that some of the most highly affected metabolites derive from bacterial tyrosine, tryptophan, purine, and fatty acid metabolism. Functional metagenomic analysis of fecal 16S amplicon and whole-genome sequencing revealed that loss of OAT1 was impressively associated with microbial pathways regulating production of urate, gut-derived p-cresol, tryptophan derivatives, and fatty acids. Certain changes, such as alterations in gut microbiome urate metabolism, appear compensatory. Thus, Oat1 in the kidney appears to mediate remote interorganismal communication by regulating the gut microbiome composition and metabolic capability. Since OAT1 function in the proximal tubule is substantially affected in CKD, our results may shed light on the associated alterations in gut-microbiome dynamics.
Authors
Vladimir S. Ermakov, Jeffry C. Granados, Sanjay K. Nigam
×Abstract
Intratumoral B cell responses are associated with more favorable clinical outcomes in human pancreatic ductal adenocarcinoma (PDAC). However, the antigens driving these B cell responses are largely unknown. We sought to discover these antigens by using single-cell RNA sequencing (scRNA-Seq) and immunoglobulin (Ig) sequencing of tumor-infiltrating immune cells from 7 primary PDAC samples. We identified activated T and B cell responses and evidence of germinal center reactions. Ig sequencing identified plasma cell (PC) clones expressing isotype-switched and hypermutated Igs, suggesting the occurrence of T cell–dependent B cell responses. We assessed the reactivity of 41 recombinant antibodies that represented the products of 235 PCs and 12 B cells toward multiple cell lines and PDAC tissues and observed frequent staining of intracellular self-antigens. Three of these antigens were identified: the filamentous actin (F-actin), the nucleic protein RuvB like AAA ATPase 2 (RUVBL2), and the mitochondrial protein heat shock protein family D (Hsp60) member 1 (HSPD1). Antibody titers against F-actin and HSPD1 were substantially elevated in the plasma of patients with PDAC compared with healthy donors. Thus, PCs in PDAC produce autoantibodies reacting with intracellular self-antigens, which may result from promotion of preexisting, autoreactive B cell responses. These observations indicate the chronic inflammatory microenvironment of PDAC can support the adaptive immune response.
Authors
Min Yao, Jonathan Preall, Johannes T.-H. Yeh, Darryl Pappin, Paolo Cifani, Yixin Zhao, Sophia Shen, Philip Moresco, Brian He, Hardik Patel, Amber N. Habowski, Daniel A. King, Kara Raphael, Arvind Rishi, Divyesh Sejpal, Matthew J. Weiss, David Tuveson, Douglas T. Fearon
×Abstract
SARS-CoV-2 is the third zoonotic coronavirus to cause a major outbreak in humans in recent years, and many more SARS-like coronaviruses with pandemic potential are circulating in several animal species. Vaccines inducing T cell immunity against broadly conserved viral antigens may protect against hospitalization and death caused by outbreaks of such viruses. We report the design and preclinical testing of 2 T cell–based pan-sarbecovirus vaccines, based on conserved regions within viral proteins of sarbecovirus isolates of human and other carrier animals, like bats and pangolins. One vaccine (CoVAX_ORF1ab) encoded antigens derived from nonstructural proteins, and the other (CoVAX_MNS) encoded antigens from structural proteins. Both multiantigen DNA vaccines contained a large set of antigens shared across sarbecoviruses and were rich in predicted and experimentally validated human T cell epitopes. In mice, the multiantigen vaccines generated both CD8+ and CD4+ T cell responses to shared epitopes. Upon encounter of full-length spike antigen, CoVAX_MNS-induced CD4+ T cells were responsible for accelerated CD8+ T cell and IgG Ab responses specific to the incoming spike, irrespective of its sarbecovirus origin. Finally, both vaccines elicited partial protection against a lethal SARS-CoV-2 challenge in human angiotensin-converting enzyme 2–transgenic mice. These results support clinical testing of these universal sarbecovirus vaccines for pandemic preparedness.
Authors
Jeroen van Bergen, Marcel G.M. Camps, Iris N. Pardieck, Dominique Veerkamp, Wing Yan Leung, Anouk A. Leijs, Sebenzile K. Myeni, Marjolein Kikkert, Ramon Arens, Gerben C. Zondag, Ferry Ossendorp
×Abstract
Tissue-resident lymphocytes (TRLs) are critical for local protection against viral pathogens in peripheral tissue. However, it is unclear if TRLs perform a similar role in transplanted organs under chronic immunosuppressed conditions. In this study, we aimed to characterize the TRL compartment in human kidney transplant nephrectomies and examine its potential role in antiviral immunity. The TRL compartment of kidney transplants contained diverse innate, innate-like, and adaptive TRL populations expressing the canonical residency markers CD69, CD103, and CD49a. Chimerism of donor and recipient cells was present in 43% of kidney transplants and occurred in all TRL subpopulations. Paired single-cell transcriptome and T cell receptor (TCR) sequencing showed that donor and recipient tissue–resident memory T (TRM) cells exhibit striking similarities in their transcriptomic profiles and share numerous TCR clonotypes predicted to target viral pathogens. Virus dextramer staining further confirmed that CD8 TRM cells of both donor and recipient origin express TCRs with specificities against common viruses, including CMV, EBV, BK polyomavirus, and influenza A. Overall, the study results demonstrate that a diverse population of TRLs resides in kidney transplants and offer compelling evidence that TRM cells of both donor and recipient origin reside within this TRL population and may contribute to local protection against viral pathogens.
Authors
Daphne M. Hullegie-Peelen, Hector Tejeda Mora, Dennis A. Hesselink, Eric M.J. Bindels, Thierry P.P. van den Bosch, Marian C. Clahsen-van Groningen, Marjolein Dieterich, Sebastiaan Heidt, Robert C. Minnee, Georges M.G.M. Verjans, Martin J. Hoogduijn, Carla C. Baan
×Abstract
The mechanisms underlying susceptibility to recurrent herpes simplex virus type 2 (HSV-2) meningitis remain incompletely understood. In a patient experiencing multiple episodes of HSV-2 meningitis, we identified a monoallelic variant in the IKBKE gene, which encodes the IKKε kinase involved in induction of antiviral IFN genes. Patient cells displayed impaired induction of IFN-β1 (IFNB1) expression upon infection with HSV-2 or stimulation with double-stranded DNA (dsDNA) and failed to induce phosphorylation of STING, an activation marker of the DNA-sensing cyclic GMP-AMP synthase/stimulator of IFN genes (cGAS/STING) pathway. The patient allele encoded a truncated IKKε protein with loss of kinase activity and also capable of exerting dominant-negative activity. In stem cell–derived microglia, HSV-2–induced expression of IFNB1 was dependent on cGAS, TANK binding kinase 1 (TBK1), and IKBKE, but not TLR3, and supernatants from HSV-2–treated microglia exerted IKBKE-dependent type I IFN–mediated antiviral activity upon neurons. Reintroducing wild-type IKBKE into patient cells rescued IFNB1 induction following treatment with HSV-2 or dsDNA and restored antiviral activity. Collectively, we identify IKKε to be important for protection against HSV-2 meningitis and suggest a nonredundant role for the cGAS/STING pathway in human antiviral immunity.
Authors
Azadeh Reyahi, Marie Studahl, Morten K. Skouboe, Stefanie Fruhwürth, Ryo Narita, Fanghui Ren, Moa Bjerhem Viklund, Marie B. Iversen, Mette Christiansen, Alexandra Svensson, Trine H. Mogensen, Kristina Eriksson, Søren R. Paludan
×Abstract
BACKGROUND Slow-flow vascular malformations frequently harbor activating mutations in the PI3K/AKT/mTOR cascade. Phase II trials pinpointed sirolimus effectiveness as a drug therapy. Efficacy and safety of sirolimus thus need to be evaluated in large prospective phase III trials.METHODS The Vascular Anomaly-Sirolimus-Europe (VASE) trial, initiated in 2016, is a large multicentric prospective phase III trial (EudraCT 2015-001703-32), which evaluates efficacy and safety of sirolimus for 2 years in pediatric and adult patients with symptomatic slow-flow vascular malformations. In this interim analysis, we studied all patients enrolled up to October 2021 who received sirolimus for 12 or more months or who prematurely stopped the treatment.RESULTS Thirty-one pediatric and 101 adult patients were included in this analysis; 107 completed 12 or more months of sirolimus, including 61 who were treated for the whole 2-year period. Sirolimus resulted in a clinical improvement in 85% of patients. The efficacy appeared within the first month for the majority of them. Grade 3–4 adverse events were observed in 24 (18%) patients; all resolved after treatment interruption/arrest. Sirolimus increased feasibility of surgery or sclerotherapy in 20 (15%) patients initially deemed unsuitable for intervention. Among the 61 patients who completed the 2-year treatment, 33 (54%) reported a recurrence of symptoms after a median follow-up of 13 months after sirolimus arrest. While there was no difference in efficacy, clinical improvement was faster but subsided more rapidly in PIK3CA-mutated (n = 24) compared with TIE2-mutated (n = 19) patients.CONCLUSION Sirolimus has a high efficacy and good tolerance in treatment of slow-flow vascular malformations in children and adults.TRIAL REGISTRATION ClinicalTrials.gov NCT02638389 and EudraCT 2015-001703-32.FUNDING The Fonds de la Recherche Scientifique (FNRS grants T.0247.19, P.C005.22, T.0146.16, and P.C013.20), the Fund Generet managed by the King Baudouin Foundation (grant 2018-J1810250-211305), the Walloon Region through the FRFS-WELBIO strategic research programme (WELBIO-CR-2019C-06), the MSCA-ITN network V.A. Cure no. 814316, the Leducq Foundation Networks of Excellence Program grant “ReVAMP” (LFCR grant 21CVD03), the European Union’s Horizon 2020 research and innovation programme under grant agreement no. 874708 (Theralymph), the Swiss National Science Foundation under the Sinergia project no. CRSII5_193694, and a Pierre M. fellowship.
Authors
Emmanuel Seront, An Van Damme, Catherine Legrand, Annouk Bisdorff-Bresson, Philippe Orcel, Thomas Funck-Brentano, Marie-Antoinette Sevestre, Anne Dompmartin, Isabelle Quere, Pascal Brouillard, Nicole Revencu, Martina De Bortoli, Frank Hammer, Philippe Clapuyt, Dana Dumitriu, Miikka Vikkula, Laurence M. Boon
×Abstract
Microcephalic osteodysplastic primordial dwarfism type II (MOPDII) is caused by biallelic loss-of-function variants in pericentrin (PCNT), and premature coronary artery disease (CAD) is a complication of the syndrome. Histopathology of coronary arteries from patients with MOPDII who died of CAD in their 20s showed extensive atherosclerosis. Hyperlipidemic mice with smooth muscle cell–specific (SMC-specific) Pcnt deficiency (PcntSMC–/–) exhibited significantly greater atherosclerotic plaque burden compared with similarly treated littermate controls despite similar serum lipid levels. Loss of PCNT in SMCs induced activation of heat shock factor 1 (HSF1) and consequently upregulated the expression and activity of HMG-CoA reductase (HMGCR), the rate-limiting enzyme in cholesterol biosynthesis. The increased cholesterol biosynthesis in PcntSMC–/– SMCs augmented PERK signaling and phenotypic modulation compared with control SMCs. Treatment with the HMGCR inhibitor, pravastatin, blocked the augmented SMC modulation and reduced plaque burden in hyperlipidemic PcntSMC–/– mice to that of control mice. These data support the notion that Pcnt deficiency activates cellular stress to increase SMC modulation and plaque burden, and targeting this pathway with statins in patients with MOPDII has the potential to reduce CAD in these individuals. The molecular mechanism uncovered further emphasizes SMC cytosolic stress and HSF1 activation as a pathway driving atherosclerotic plaque formation independently of cholesterol levels.
Authors
Suravi Majumder, Abhijnan Chattopadhyay, Jamie M. Wright, Pujun Guan, L. Maximilian Buja, Callie S. Kwartler, Dianna M. Milewicz
×Abstract
Primary graft dysfunction (PGD) limits clinical benefit after lung transplantation, a life-prolonging therapy for patients with end-stage disease. PGD is the clinical syndrome resulting from pulmonary ischemia-reperfusion injury (IRI), driven by innate immune inflammation. We recently demonstrated a key role for NK cells in the airways of mouse models and human tissue samples of IRI. Here, we used 2 mouse models paired with human lung transplant samples to investigate the mechanisms whereby NK cells migrate to the airways to mediate lung injury. We demonstrate that chemokine receptor ligand transcripts and proteins are increased in mouse and human disease. CCR5 ligand transcripts were correlated with NK cell gene signatures independently of NK cell CCR5 ligand secretion. NK cells expressing CCR5 were increased in the lung and airways during IRI and had increased markers of tissue residency and maturation. Allosteric CCR5 drug blockade reduced the migration of NK cells to the site of injury. CCR5 blockade also blunted quantitative measures of experimental IRI. Additionally, in human lung transplant bronchoalveolar lavage samples, we found that CCR5 ligand was associated with increased patient morbidity and that the CCR5 receptor was increased in expression on human NK cells following PGD. These data support a potential mechanism for NK cell migration during lung injury and identify a plausible preventative treatment for PGD.
Authors
Jesse Santos, Ping Wang, Avishai Shemesh, Fengchun Liu, Tasha Tsao, Oscar A. Aguilar, Simon J. Cleary, Jonathan P. Singer, Ying Gao, Steven R. Hays, Jeffrey A. Golden, Lorriana Leard, Mary Ellen Kleinhenz, Nicholas A. Kolaitis, Rupal Shah, Aida Venado, Jasleen Kukreja, S. Sam Weigt, John A. Belperio, Lewis L. Lanier, Mark R. Looney, John R. Greenland, Daniel R. Calabrese
