Sparsentan improves glomerular hemodynamics, cell functions, and tissue repair in a mouse model of FSGS
Gyarmati et al. report on mechanisms underlying the glomeruloprotective and tissue remodeling activity of sparsentan, a dual endothelin-1 and angiotensin II receptor antagonist. The cover shows an immunofluorescence image of p57+ podocytes (red) in glomeruli of a TRPC6-transgenic mouse kidney with focal segmental glomerulosclerosis, indicating the preservation of podocyte number by renoprotective therapies. Image credit: Georgina Gyarmati.
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Research Articles
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Abstract
Transmission of HIV-1 to newborns and infants remains high, with 130,000 new infections in 2022 in resource-limited settings. Half of HIV-infected newborns, if untreated, progress to disease and death within 2 years. While immunologic immaturity likely promotes pathogenesis and poor viral control, little is known about immune damage in newborns and infants. Here we examined pathologic, virologic, and immunologic outcomes in rhesus macaques exposed to pathogenic simian-human immunodeficiency virus (SHIV) at 1–2 weeks, defined as newborns, or at 4 months of age, considered infants. Kinetics of plasma viremia and lymph node seeding DNA were indistinguishable in newborns and infants, but levels of viral DNA in gut and lymphoid tissues 6–10 weeks after infection were significantly higher in newborns versus either infant or adult macaques. Two of 6 newborns with the highest viral seeding required euthanasia at 25 days. We observed age-dependent alterations in leukocyte subsets and gene expression. Compared with infants, newborns had stronger skewing of monocytes and CD8+ T cells toward differentiated subsets and little evidence of type I interferon responses by transcriptomic analyses. Thus, SHIV infection reveals distinct immunological alterations in newborn and infant macaques. These studies lay the groundwork for understanding how immune maturation affects pathogenesis in pediatric HIV-1 infection.
Authors
Mariya B. Shapiro, Tracy Ordonez, Shilpi Pandey, Eisa Mahyari, Kosiso Onwuzu, Jason Reed, Heather Sidener, Jeremy Smedley, Lois M. Colgin, Amanda Johnson, Anne D. Lewis, Benjamin Bimber, Jonah B. Sacha, Ann J. Hessell, Nancy L. Haigwood
×Abstract
The complexity of the mechanisms underlying metabolic dysfunction–associated steatotic liver disease (MASLD) progression remains a significant challenge for the development of effective therapeutics. miRNAs have shown great promise as regulators of biological processes and as therapeutic targets for complex diseases. Here, we study the role of hepatic miR-33, an important regulator of lipid metabolism, during the progression of MASLD and the development of hepatocellular carcinoma (HCC). We report that miR-33 was elevated in the livers of humans and mice with MASLD and that its deletion in hepatocytes (miR-33 HKO) improved multiple aspects of the disease, including steatosis and inflammation, limiting the progression to metabolic dysfunction–associated steatotic hepatitis (MASH), fibrosis, and HCC. Mechanistically, hepatic miR-33 deletion reduced lipid synthesis and promoted mitochondrial fatty acid oxidation, reducing lipid burden. Additionally, absence of miR-33 altered the expression of several known miR-33 target genes involved in metabolism and resulted in improved mitochondrial function and reduced oxidative stress. The reduction in lipid accumulation and liver injury resulted in decreased YAP/TAZ pathway activation, which may be involved in the reduced HCC progression in HKO livers. Together, these results suggest suppressing hepatic miR-33 may be an effective therapeutic approach to temper the development of MASLD, MASH, and HCC in obesity.
Authors
Pablo Fernández-Tussy, Magdalena P. Cardelo, Hanming Zhang, Jonathan Sun, Nathan L. Price, Nabil E. Boutagy, Leigh Goedeke, Martí Cadena-Sandoval, Chrysovalantou E. Xirouchaki, Wendy Brown, Xiaoyong Yang, Oscar Pastor-Rojo, Rebecca A. Haeusler, Anton M. Bennett, Tony Tiganis, Yajaira Suárez, Carlos Fernández-Hernando
×Abstract
T follicular regulatory (TFR) cells are found in the germinal center (GC) response and, along with T follicular helper (TFH) cells, help to control the development of high-affinity antibodies (Ab). Although TFR cells are generally thought to repress GC B cells and the Ab response, we have previously shown that in a mouse food allergy model, TFR cells produce IL-10 and play an essential helper role such that in the absence of TFR cells, IgE responses are diminished. Here we show that in this food allergy response, TFR cells produced IL-4 that promotes the generation of antigen-specific IgE. We show that food allergy–primed TFR cells specifically upregulate IL-4 gene transcription and produce functional IL-4 that promoted IgE responses both in vitro and in vivo. We determined that IgE responses are dependent on a high level of IL-4 produced by follicular T cells in the GC, explaining the need for IL-4 produced by TFR cells in the food allergy response. Overall, our findings have demonstrated that in food allergy, TFR cells can produce IL-4 and regulate IgE in a manner that augments the role of TFH cells in IgE responses.
Authors
Qiang Chen, Abdullahi M. Abdi, Wei Luo, Xue Yuan, Alexander L. Dent
×Abstract
Dual endothelin-1 (ET-1) and angiotensin II (AngII) receptor antagonism with sparsentan has strong antiproteinuric actions via multiple potential mechanisms that are more pronounced, or additive, compared with current standard of care using angiotensin receptor blockers (ARBs). Considering the many actions of ET-1 and AngII on multiple cell types, this study aimed to determine glomeruloprotective mechanisms of sparsentan compared to the ARB losartan by direct visualization of its effects in the intact kidney in focal segmental glomerulosclerosis (FSGS) using intravital multiphoton microscopy. In both healthy and FSGS models, sparsentan treatment increased afferent/efferent arteriole diameters; increased or preserved blood flow and single-nephron glomerular filtration rate; attenuated acute ET-1 and AngII–induced increases in podocyte calcium; reduced proteinuria; preserved podocyte number; increased both endothelial and renin lineage cells and clones in vasculature, glomeruli, and tubules; restored glomerular endothelial glycocalyx; and attenuated mitochondrial stress and immune cell homing. These effects were either not observed or of smaller magnitude with losartan. The pleiotropic nephroprotective effects of sparsentan included improved hemodynamics, podocyte and endothelial cell functions, and tissue repair. Compared with losartan, sparsentan was more effective in the sustained preservation of kidney structure and function, which underscores the importance of the ET-1 component in FSGS pathogenesis and therapy.
Authors
Georgina Gyarmati, Urvi Nikhil Shroff, Audrey Izuhara, Sachin Deepak, Radko Komers, Patricia W. Bedard, Janos Peti-Peterdi
×Abstract
Tumor-educated platelets (TEPs) are a potential method of liquid biopsy for the diagnosis and monitoring of cancer. However, the mechanism underlying tumor education of platelets is not known, and transcripts associated with TEPs are often not tumor-associated transcripts. We demonstrated that direct tumor transfer of transcripts to circulating platelets is an unlikely source of the TEP signal. We used CDSeq, a latent Dirichlet allocation algorithm, to deconvolute the TEP signal in blood samples from patients with glioblastoma. We demonstrated that a substantial proportion of transcripts in the platelet transcriptome are derived from nonplatelet cells, and the use of this algorithm allows the removal of contaminant transcripts. Furthermore, we used the results of this algorithm to demonstrate that TEPs represent a subset of more activated platelets, which also contain transcripts normally associated with nonplatelet inflammatory cells, suggesting that these inflammatory cells, possibly in the tumor microenvironment, transfer transcripts to platelets that are then found in circulation. Our analysis suggests a useful and efficient method of processing TEP transcriptomic data to enable the isolation of a unique TEP signal associated with specific tumors.
Authors
Jerome M. Karp, Aram S. Modrek, Ravesanker Ezhilarasan, Ze-Yan Zhang, Yingwen Ding, Melanie Graciani, Ali Sahimi, Michele Silvestro, Ting Chen, Shuai Li, Kwok-Kin Wong, Bhama Ramkhelawon, Krishna P.L. Bhat, Erik P. Sulman
×Abstract
BACKGROUND The mechanism(s) responsible for the efficacy of WHO-recommended malaria vaccine RTS,S/AS01 are not completely understood. We previously identified RTS,S vaccine–induced Plasmodium falciparum circumsporozoite protein–specific (PfCSP-specific) antibody measures associated with protection from controlled human malaria infection (CHMI). Here, we tested the protection-predicting capability of these measures in independent CHMI studies.METHODS Vaccine-induced total serum antibody (immunoglobulins, Igs) and subclass antibody (IgG1 and IgG3) responses were measured by biolayer interferometry and the binding antibody multiplex assay, respectively. Immune responses were compared between protected and nonprotected vaccinees using univariate and multivariate logistic regression.RESULTS Blinded prediction analysis showed that 5 antibody binding measures, including magnitude-avidity composite of serum Ig specific for PfCSP, major NANP repeats and N-terminal junction, and PfCSP- and NANP-specific IgG1 subclass magnitude, had good prediction accuracy (area under the receiver operating characteristic curves [ROC AUC] > 0.7) in at least 1 trial. Furthermore, univariate analysis showed a significant association between these antibody measures and protection (odds ratios 2.6–3.1). Multivariate modeling of combined data from 3 RTS,S CHMI trials identified the combination of IgG1 NANP binding magnitude plus serum NANP and N-junction Ig binding magnitude-avidity composite as the best predictor of protection (95% confidence interval for ROC AUC 0.693–0.834).CONCLUSION These results reinforce our previous findings and provide a tool for predicting protection in future trials.TRIAL REGISTRATION ClinicalTrials.gov NCT03162614, NCT03824236, NCT01366534, and NCT01857869.FUNDING This study was supported by Bill & Melinda Gates Foundation’s Global Health-Discovery Collaboratory grants (INV-008612 and INV-043419) to GDT.
Authors
Rachel L. Spreng, Kelly E. Seaton, Lin Lin, Sir’Tauria Hilliard, Gillian Q. Horn, Milite Abraha, Aaron W. Deal, Kan Li, Alexander J. Carnacchi, Elizabeth Feeney, Siam Shabbir, Lu Zhang, Valerie Bekker, Sarah V. Mudrak, Sheetij Dutta, Laina D. Mercer, Scott Gregory, C. Richter King, Ulrike Wille-Reece, Erik Jongert, Neville K. Kisalu, Georgia D. Tomaras, S. Moses Dennison
×Abstract
Immune cell–mediated inflammation is important in normal tissue regeneration but can be pathologic in diabetic wounds. Limited literature exists on the role of CD4+ T cells in normal or diabetic wound repair; however, the imbalance of CD4+ Th17/Tregs has been found to promote inflammation in other diabetic tissues. Here, using human tissue and murine transgenic models, we identified that the histone methyltransferase Mixed-lineage-leukemia-1 (MLL1) directly regulates the Th17 transcription factor RORγ via an H3K4me3 mechanism and increases expression of Notch receptors and downstream Notch signaling. Furthermore, we found that Notch receptor signaling regulates CD4+ Th cell differentiation and is critical for normal wound repair, and loss of upstream Notch pathway mediators or receptors in CD4+ T cells resulted in the loss of CD4+ Th cell differentiation in wounds. In diabetes, MLL1 and Notch-receptor signaling was upregulated in wound CD4+ Th cells, driving CD4+ T cells toward the Th17 cell phenotype. Treatment of diabetic wound CD4+ T cells with a small molecule inhibitor of MLL1 (MI-2) yielded a significant reduction in CD4+ Th17 cells and IL-17A. This is the first study to our knowledge to identify the MLL1-mediated mechanisms responsible for regulating the Th17/Treg balance in normal and diabetic wounds and to define the complex role of Notch signaling in CD4+ T cells in wounds, where increased or decreased Notch signaling both result in pathologic wound repair. Therapeutic targeting of MLL1 in diabetic CD4+ Th cells may decrease pathologic inflammation through regulation of CD4+ T cell differentiation.
Authors
William J. Melvin, Tyler M. Bauer, Kevin D. Mangum, Christopher O. Audu, James Shadiow, Emily C. Barrett, Amrita D. Joshi, Jadie Y. Moon, Rachael Bogle, Purba Mazumder, Sonya J. Wolf, Steven L. Kunke, Johann E. Gudjonsson, Frank M. Davis, Katherine A. Gallagher
×Abstract
This study aimed at defining the role of the B cell adaptor protein BANK1 in the appearance of age-associated B cells (ABCs) in 2 SLE mouse models (TLR7.tg6 and imiquimod-induced mice), crossed with Bank1–/– mice. The absence of Bank1 led to a significant reduction in ABC levels, also affecting other B cell populations. To gain deeper insights into their differentiation pathway and the effect of Bank1 on B cell populations, a single-cell transcriptome assay was performed. In the TLR7.tg6 model, we identified 10 clusters within B cells, including an ABC-specific cluster that was decreased in Bank1-deficient mice. In its absence, ABCs exhibited an antiinflammatory gene expression profile, while being proinflammatory in Bank1-sufficient lupus-prone mice. Trajectory analyses revealed that ABCs originated from marginal zone and memory-like B cells, ultimately acquiring transcriptional characteristics associated with atypical memory cells and long-lived plasma cells. Also, Bank1 deficiency normalized the presence of naive B cells, which were nearly absent in lupus-prone mice. Interestingly, Bank1 deficiency significantly reduced a distinct cluster containing IFN-responsive genes. These findings underscore the critical role of Bank1 in ABC development, affecting early B cell stages toward ABC differentiation, and the presence of IFN-stimulated gene–containing B cells, both populations determinant for autoimmunity.
Authors
Gonzalo Gómez Hernández, Toro Domínguez, Georgina Galicia, María Morell, Marta E. Alarcón-Riquelme
×Abstract
Ataxia telangiectasia and Rad3-related protein (ATR) is a key DNA damage response protein that facilitates DNA damage repair and regulates cell cycle progression. As such, ATR is an important component of the cellular response to radiation, particularly in cancer cells, which show altered DNA damage response and aberrant cell cycle checkpoints. Therefore, ATR’s pharmacological inhibition could be an effective radiosensitization strategy to improve radiotherapy. We assessed the ability of an ATR inhibitor, AZD6738, to sensitize cancer cell lines of various histologic types to photon and proton radiotherapy. We found that radiosensitization took place through persistent DNA damage and abrogated G2 cell cycle arrest. We also found that AZD6738 increased the number of micronuclei after exposure to radiotherapy. We found that combining radiation with AZD6738 led to tumor growth delay and prolonged survival relative to radiation alone in a breast cancer model. Combining AZD6738 with photons or protons also led to increased macrophage infiltration at the tumor microenvironment. These results provide a rationale for further investigation of ATR inhibition in combination with radiotherapy and with other agents such as immune checkpoint blockade.
Authors
Scott J. Bright, Mandira Manandhar, David B. Flint, Rishab Kolachina, Mariam Ben Kacem, David K.J. Martinus, Broderick X. Turner, Ilsa Qureshi, Conor H. McFadden, Poliana C. Marinello, Simona F. Shaitelman, Gabriel O. Sawakuchi
×Abstract
Glioblastoma (GBM) is the most aggressive form of glioma with a high rate of relapse despite intensive treatment. Tumor recurrence is tightly linked to radio-resistance, which in turn is associated with hypoxia. Here, we discovered a strong link between hypoxia and local complement signaling using publicly available bulk, single-cell, and spatially resolved transcriptomic data from patients with GBM. Complement component 3 (C3) and the receptor C3AR1 were both associated with aggressive disease and shorter survival in human glioma. In a genetically engineered mouse model of GBM, we found C3 specifically in hypoxic tumor areas. In vitro, we found an oxygen level–dependent increase in C3 and C3AR1 expression in response to hypoxia in several GBM and stromal cell types. C3a induced M2 polarization of cultured microglia and macrophages in a C3aR-dependent fashion. Targeting C3aR using the antagonist SB290157 prolonged survival of glioma-bearing mice both alone and in combination with radiotherapy while reducing the number of M2-polarized macrophages. Our findings establish a strong link between hypoxia and complement pathways in GBM and support a role of hypoxia-induced C3a/C3aR signaling as a contributor to glioma aggressiveness by regulating macrophage polarization.
Authors
Rebecca Rosberg, Karolina I. Smolag, Jonas Sjölund, Elinn Johansson, Christina Bergelin, Julia Wahldén, Vasiliki Pantazopoulou, Crister Ceberg, Kristian Pietras, Anna M. Blom, Alexander Pietras
×Abstract
The prevalence of chronic kidney disease (CKD) varies by race because of genetic and environmental factors. The Glu504Lys polymorphism in aldehyde dehydrogenase 2 (ALDH2), commonly observed among East Asian people, alters the enzyme’s function in detoxifying alcohol-derived aldehydes, affecting kidney function. This study investigated the association between variations in ALDH2 levels within the kidney and the progression of kidney fibrosis. Our clinical data indicate that diminished ALDH2 levels are linked to worse CKD outcomes, with correlations between ALDH2 expression, estimated glomerular filtration rate, urinary levels of acrolein — an aldehyde metabolized by ALDH2 — and fibrosis severity. In mouse models of unilateral ureteral obstruction and folic acid nephropathy, reduced ALDH2 levels and elevated acrolein were observed in kidneys, especially in ALDH2 Glu504Lys–knockin mice. Mechanistically, acrolein modifies pyruvate kinase M2, leading to its nuclear translocation and coactivation of HIF-1α, shifting cellular metabolism to glycolysis, disrupting mitochondrial function, and contributing to tubular damage and the progression of kidney fibrosis. Enhancing ALDH2 expression through adeno-associated virus vectors reduced acrolein and mitigated fibrosis in both WT and Glu504Lys-knockin mice. These findings underscore the potential therapeutic significance of targeting the dynamic interaction between ALDH2 and acrolein in CKD.
Authors
Szu-Yuan Li, Ming-Tsun Tsai, Yu-Ming Kuo, Hui-Min Yang, Zhen-Jie Tong, Hsiao-Wei Cheng, Chih-Ching Lin, Hsiang-Tsui Wang
×Abstract
Lung transplantation (LTx) outcomes are impeded by ischemia/reperfusion injury (IRI) and subsequent chronic lung allograft dysfunction (CLAD). We examined the undefined role of receptor Mer tyrosine kinase (MerTK) on monocytic myeloid-derived suppressor cells (M-MDSCs) in efferocytosis to facilitate resolution of lung IRI. Single-cell RNA sequencing of lung tissue and bronchoalveolar lavage (BAL) from patients after LTx were analyzed. Murine lung hilar ligation and allogeneic orthotopic LTx models of IRI were used with BALB/c (WT), Cebpb–/– (MDSC-deficient), Mertk–/–, or MerTK–cleavage-resistant mice. A significant downregulation in MerTK-related efferocytosis genes in M-MDSC populations of patients with CLAD was observed compared with healthy individuals. In the murine IRI model, a significant increase in M-MDSCs, MerTK expression, and efferocytosis and attenuation of lung dysfunction was observed in WT mice during injury resolution that was absent in Cebpb–/– and Mertk–/– mice. Adoptive transfer of M-MDSCs in Cebpb–/– mice significantly attenuated lung dysfunction and inflammation. Additionally, in a murine orthotopic LTx model, increases in M-MDSCs were associated with resolution of lung IRI in the transplant recipients. In vitro studies demonstrated the ability of M-MDSCs to efferocytose apoptotic neutrophils in a MerTK-dependent manner. Our results suggest that MerTK-dependent efferocytosis by M-MDSCs can substantially contribute to the resolution of post-LTx IRI.
Authors
Victoria Leroy, Denny J. Manual Kollareth, Zhenxiao Tu, Jeff Arni C. Valisno, Makena Woolet-Stockton, Biplab Saha, Amir M. Emtiazjoo, Mindaugas Rackauskas, Lyle L. Moldawer, Philip A. Efron, Guoshuai Cai, Carl Atkinson, Gilbert R. Upchurch Jr., Ashish K. Sharma
×Abstract
Our objective was to interrogate mesenchymal stem cell (MSC) lipid metabolism and gestational exposures beyond maternal body mass that may contribute to child obesity risk. MSCs were cultured from term infants of mothers with obesity (n = 16) or normal weight (n = 15). In MSCs undergoing myogenesis in vitro, we used lipidomics to distinguish phenotypes by unbiased cluster analysis and lipid challenge (24-hour excess fatty acid [24hFA]). We measured MSC AMP-activated protein kinase (AMPK) activity and fatty acid oxidation (FAO), and a composite index of maternal glucose, insulin, triglycerides, free fatty acids, TNF-α, and high-density lipoprotein and total cholesterol in fasting blood from mid and late gestation (~17 and ~27 weeks, respectively). We measured child adiposity at birth (n = 29), 4–6 months (n = 29), and 4–6 years (n = 13). Three MSC clusters were distinguished by triacylglycerol (TAG) stores, with greatest TAGs in Cluster 2. All clusters increased acylcarnitines and TAGs with 24hFA, although Cluster 2 was more pronounced and corresponded to AMPK activation and FAO. Maternal metabolic markers predicted MSC clusters and child adiposity at 4–6 years (both highest in Cluster 3). Our data support the notion that MSC phenotypes are predicted by comprehensive maternal metabolic milieu exposures, independent of maternal BMI, and suggest utility as an at-birth predictor for child adiposity, although validation with larger longitudinal samples is warranted.
Authors
Lauren E. Gyllenhammer, Vincent Zaegel, Allison M. Duensing, Manoel E. Lixandrao, Dana Dabelea, Bryan C. Bergman, Kristen E. Boyle
×Abstract
Hidden hearing loss (HHL), a recently described auditory neuropathy characterized by normal audiometric thresholds but reduced sound-evoked cochlear compound action potentials, has been proposed to contribute to hearing difficulty in noisy environments in people with normal hearing thresholds and has become a widespread complaint. While most studies on HHL pathogenesis have focused on inner hair cell (IHC) synaptopathy, we recently showed that transient auditory nerve (AN) demyelination also causes HHL in mice. To test the effect of myelinopathy on hearing in a clinically relevant model, we studied a mouse model of Charcot-Marie-Tooth type 1A (CMT1A), the most prevalent hereditary peripheral neuropathy in humans. CMT1A mice exhibited the functional hallmarks of HHL together with disorganization of AN heminodes near the IHCs with minor loss of AN fibers. These results support the hypothesis that mild disruptions of AN myelination can cause HHL and that heminodal defects contribute to the alterations in the sound-evoked cochlear compound action potentials seen in this mouse model. Furthermore, these findings suggest that patients with CMT1A or other mild peripheral neuropathies are likely to suffer from HHL. Furthermore, these results suggest that studies of hearing in patients with CMT1A might help develop robust clinical tests for HHL, which are currently lacking.
Authors
Luis R. Cassinotti, Lingchao Ji, M. Caroline Yuk, Aditi S. Desai, Nathan D. Cass, Zahara A. Amir, Gabriel Corfas
×Abstract
BACKGROUND Two coding alleles within the APOL1 gene, G1 and G2, found almost exclusively in individuals genetically similar to West African populations, contribute substantially to the pathogenesis of chronic kidney disease (CKD). The APOL gene cluster on chromosome 22 contains a total of 6 APOL genes that have arisen as a result of gene duplication.METHODS Using a genome-first approach in the Penn Medicine BioBank, we identified 62 protein-altering variants in the 6 APOL genes with a minor allele frequency of >0.1% in a population of participants genetically similar to African reference populations and performed population-specific phenome-wide association studies.RESULTS We identified rs1108978, a stop-gain variant in APOL3 (p.Q58*), to be significantly associated with increased CKD risk, even after conditioning on APOL1 G1/G2 carrier status. These findings were replicated in the Veterans Affairs Million Veteran Program and the All of Us Research Program. APOL3 p.Q58* was also significantly associated with a number of quantitative traits linked to CKD, including decreased kidney volume. This truncating variant contributed the most risk for CKD in patients monoallelic for APOL1 G1/G2, suggesting an epistatic interaction and a potential protective effect of wild-type APOL3 against APOL1-induced kidney disease.CONCLUSION This study demonstrates the utility of targeting population-specific variants in a genome-first approach, even in the context of well-studied gene-disease relationships.FUNDING National Heart, Lung, and Blood Institute (F30HL172382, R01HL169378, R01HL169458), Doris Duke Foundation (grant 2023-2024), National Institute of Biomedical Imaging and Bioengineering (P41EB029460), and National Center for Advancing Translational Sciences (UL1-TR-001878).
Authors
David Y. Zhang, Michael G. Levin, Jeffrey T. Duda, Latrice G. Landry, Walter R. Witschey, Scott M. Damrauer, Marylyn D. Ritchie, Daniel J. Rader
×Abstract
Embryo implantation is crucial for ensuring a successful pregnancy outcome and subsequent child health. The intrauterine environment during the peri-implantation period shows drastic changes in gene expression and cellular metabolism in response to hormonal stimuli and reciprocal communication with embryos. Here, we performed spatial transcriptomic analysis to elucidate the mechanisms underlying embryo implantation. Transcriptome data revealed that lipid metabolism pathways, especially arachidonic acid–related (AA-related) ones, were enriched in the embryo-receptive luminal epithelia. Cyclooxygenases (COXs), rate-limiting enzymes involved in prostaglandin production by AA, were spatiotemporally regulated in the vicinity of embryos during implantation, but the role of each COX isozyme in the uterus for successful pregnancy was unclear. We established uterine-specific COX2-knockout (uKO) and COX1/uterine COX2-double-KO (COX1/COX2-DKO) mice. COX2 uKO caused deferred implantation with failed trophoblast invasion, resulting in subfertility with reduced pregnancy rates and litter sizes. COX1/COX2 DKO induced complete infertility, owing to abrogated embryo attachment. These results demonstrate that both isozymes have distinct roles during embryo implantation. Spatial transcriptome and lipidome analyses revealed unique profiles of prostaglandin synthesis by each COX isozyme and spatiotemporal expression patterns of downstream receptors throughout the endometrium. Our findings reveal previously unappreciated roles of COXs at the fetomaternal interface to establish early pregnancy.
Authors
Shizu Aikawa, Mitsunori Matsuo, Shun Akaeda, Yukihiko Sugimoto, Makoto Arita, Yosuke Isobe, Yuki Sugiura, Shu Taira, Rae Maeda, Ryoko Shimizu-Hirota, Norihiko Takeda, Daiki Hiratsuka, Xueting He, Chihiro Ishizawa, Rei Iida, Yamato Fukui, Takehiro Hiraoka, Miyuki Harada, Osamu Wada-Hiraike, Yutaka Osuga, Yasushi Hirota
×Abstract
Pulmonary veno-occlusive disease (PVOD) is a rare but severe form of pulmonary hypertension characterized by the obstruction of pulmonary arteries and veins, causing increased pulmonary artery pressure and leading to right ventricular (RV) heart failure. PVOD is often resistant to conventional pulmonary arterial hypertension (PAH) treatments and has a poor prognosis, with a median survival time of 2–3 years after diagnosis. We previously showed that the administration of a chemotherapy agent mitomycin C (MMC) in rats mediates PVOD through the activation of the eukaryotic initiation factor 2 (eIF2) kinase protein kinase R (PKR) and the integrated stress response (ISR), resulting in the impairment of vascular endothelial junctional structure and barrier function. Here, we demonstrate that aged rats over 1 year exhibit more severe vascular remodeling and RV hypertrophy than young adult rats following MMC treatment. This is attributed to an age-associated elevation of basal ISR activity and depletion of protein phosphatase 1, leading to prolonged eIF2 phosphorylation and sustained ISR activation. Pharmacological blockade of PKR or ISR mitigates PVOD phenotypes in both age groups, suggesting that targeting the PKR/ISR axis could be a potential therapeutic strategy for PVOD.
Authors
Amit Prabhakar, Meetu Wadhwa, Rahul Kumar, Prajakta Ghatpande, Aneta Gandjeva, Rubin M. Tuder, Brian B. Graham, Giorgio Lagna, Akiko Hata
×Abstract
Bone contains multiple pools of skeletal stem/progenitor cells (SSPCs), and SSPCs in periosteal compartments are known to exhibit higher regenerative potential than those in BM and endosteal compartments. However, the in vivo identity and hierarchical relationships of periosteal SSPCs (P-SSPCs) remain unclear due to a lack of reliable markers to distinguish BM SSPCs and P-SSPCs. Here, we found that periosteal mesenchymal progenitor cells (P-MPs) in periosteum can be identified based on Postn-CreERT2 expression. Postn-expressing periosteal subpopulation produces osteolineage descendants that fuel bones to maintain homeostasis and support regeneration. Notably, Postn+ P-MPs are likely derived from Gli1+ skeletal stem cells (SSCs). Ablation of Postn+ cells results in impairments in homeostatic cortical bone architecture and defects in fracture repair. Genetic deletion of Igf1r in Postn+ cells dampens bone fracture healing. In summary, our study provides a mechanistic understanding of bone regeneration through the regulation of region-specific Postn+ P-MPs.
Authors
Bei Yin, Fangyuan Shen, Qingge Ma, Yongcheng Liu, Xianglong Han, Xuyu Cai, Yu Shi, Ling Ye
×Abstract
Drugs that inhibit HIV transcription and/or reactivation of latent HIV have been proposed as a strategy to reduce HIV-associated immune activation or to achieve a functional cure, yet comparative studies are lacking. We evaluated 26 drugs, including drugs previously reported to inhibit HIV transcription (inhibitors of Tat-dependent HIV transcription, Rev, HSF-1/PTEF-b, HSP90, Jak/Stat, or SIRT1/Tat deacetylation) and other agents that were not tested before (inhibitors of PKC, NF-κB, SP-1, or histone acetyltransferase; NR2F1 agonists), elongation (inhibitors of CDK9/ PTEF-b), completion (inhibitors of PolyA-polymerase), or splicing (inhibitors of human splice factors). To investigate if those drugs would vary in their ability to affect different blocks to HIV transcription, we measured levels of initiated, elongated, midtranscribed, completed, and multiply spliced HIV RNA in PBMCs from antiretroviral therapy–suppressed individuals following ex vivo treatment with each drug and subsequent T cell activation. We identified new drugs that prevent HIV reactivation, including CDK and splicing inhibitors. While some drugs inhibited 1 or 2 steps, other drugs (CDK inhibitors, splicing inhibitors, tanespimycin, and triptolide) inhibited multiple stages of HIV transcription and blocked the production of supernatant viral RNA. These drugs and targets deserve further study in strategies aimed at reducing HIV-associated immune activation or achieving a functional cure.
Authors
Julie Janssens, Peggy Kim, Sun Jin Kim, Adam Wedrychowski, Gayatri N. Kadiyala, Peter W. Hunt, Steven G. Deeks, Joseph K. Wong, Steven A. Yukl
×Abstract
The cytoplasmic peptide:N-glycanase (NGLY1) is ubiquitously expressed and functions as a de–N-glycosylating enzyme that degrades misfolded N-glycosylated proteins. NGLY1 deficiency due to biallelic loss-of-function NGLY1 variants is an ultrarare autosomal recessive deglycosylation disorder with multisystemic involvement; the neurological manifestations represent the main disease burden. Currently, there is no treatment for this disease. To develop a gene therapy, we first characterized a tamoxifen-inducible Ngly1-knockout (iNgly1) C57BL/6J mouse model, which exhibited symptoms recapitulating human disease, including elevation of the biomarker GlcNAc-Asn, motor deficits, kyphosis, Purkinje cell loss, and gait abnormalities. We packaged a codon-optimized human NGLY1 transgene cassette into 2 adeno-associated virus (AAV) capsids, AAV9 and AAV.PHPeB. Systemic administration of the AAV.PHPeB vector to symptomatic iNgly1 mice corrected multiple disease features at 8 weeks after treatment. Furthermore, another cohort of AAV.PHPeB-treated iNgly1 mice were monitored over a year and showed near-complete normalization of the neurological aspects of the disease phenotype, demonstrating the durability of gene therapy. Our data suggested that brain-directed NGLY1 gene replacement via systemic delivery is a promising therapeutic strategy for NGLY1 deficiency. Although the superior CNS tropism of AAV.PHPeB vector does not translate to primates, emerging AAV capsids with enhanced primate CNS tropism will enable future translational studies.
Authors
Ailing Du, Kun Yang, Xuntao Zhou, Lingzhi Ren, Nan Liu, Chen Zhou, Jialing Liang, Nan Yan, Guangping Gao, Dan Wang
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Abstract
Excessive fructose intake is a risk factor for the development of obesity and its complications. Targeting ketohexokinase (KHK), the first enzyme of fructose metabolism, has been investigated for the management of MASLD. We compared the effects of systemic, small molecule inhibitor of KHK enzymatic activity to hepatocyte-specific, GalNAc-siRNA mediated knockdown of KHK in mice on a HFD. We measured KHK enzymatic activity, extensively quantified glycogen accumulation, performed RNAseq analysis, and enumerated hepatic metabolites using mass spectrometry. Both KHK siRNA and KHK inhibitor led to an improvement in liver steatosis, however, via substantially different mechanisms. KHK knockdown decreased the de novo lipogenesis pathway, whereas the inhibitor increased the fatty acid oxidation pathway. Moreover, KHK knockdown completely prevented hepatic fructolysis and improved glucose tolerance. Conversely, the KHK inhibitor only partially reduced fructolysis, but it also targeted triokinase, mediating the third step of fructolysis. This leads to the accumulation of fructose-1 phosphate, resulting in glycogen accumulation, hepatomegaly, and impaired glucose tolerance. Overexpression of wild-type, but not kinase-dead KHK in cultured hepatocytes increased hepatocyte injury and glycogen accumulation when treated with fructose. The differences between KHK inhibition and knockdown are, in part, explained by the kinase-dependent and independent effects of KHK on hepatic metabolism.
Authors
Se-Hyung Park, Taghreed Fadhul, Lindsey R. Conroy, Harrison A. Clarke, Ramon C. Sun, Kristina Wallenius, Jeremie Boucher, Gavin O'Mahony, Alessandro Boianelli, Marie Persson, Sunhee Jung, Cholsoon Jang, Analia S. Loria, Genesee J. Martinez, Zachary A. Kipp, Evelyn A. Bates, Terry D. Hinds, Jr., Senad Divanovic, Samir Softic
Abstract
Systemic sclerosis (SSc) is characterized by immune system failure, vascular insult, autoimmunity, and tissue fibrosis. Transforming growth factor-beta (TGF-β) is a crucial mediator of persistent myofibroblast activation and aberrant extracellular matrix production in SSc. The factors responsible for this are unknown. By amplifying pattern recognition receptor signaling, Triggering Receptor Expressed on Myeloid Cells 1 (TREM-1) is implicated in multiple inflammatory conditions. In this study, we used novel ligand-independent TREM-1 inhibitors in order to investigate the pathogenic role of TREM-1 in SSc, using preclinical models of fibrosis, and explanted SSc skin fibroblasts. Selective pharmacological TREM-1 blockade prevented and reversed skin fibrosis induced by bleomycin in mice and mitigated constitutive collagen synthesis and myofibroblast features in SSc fibroblasts in vitro. Our results implicate aberrantly activated TREM-1 signaling in SSc pathogenesis, identify a unique approach to TREM-1 blockade, and suggest a potential therapeutic benefit for TREM-1 inhibition.
Authors
Swarna Bale, Priyanka Verma, Bharath Yalavarthi, Matija Bajželj, Syed A.M. Hasan, Jenna N. Silverman, Katherine Broderick, Kris A. Shah, Timothy Hamill, Dinesh Khanna, Alexander B. Sigalov, Swati Bhattacharyya, John Varga
Abstract
Despite proven therapy options for estrogen receptor (ER)-positive breast tumors, a substantial number of ER+ cancer patients exhibit relapse with associated metastasis. Loss of expression of RasGAPs leads to poor outcomes in several cancers, including breast cancer. Mining the TCGA breast cancer RNA-sequencing dataset revealed that low expression of the RasGAP DAB2IP was associated with a significant decrease in relapse-free survival in Luminal A breast cancer patients. Immunostaining demonstrated that DAB2IP loss occurred in grade 2 tumors and higher. Consistent with this, genes upregulated in DAB2IP-low Luminal A tumors were shared with more aggressive tumor subtypes and were associated with proliferation, metastasis, and altered ER signaling. Low DAB2IP expression in ER+ breast cancer cells was associated with increased proliferation, enhanced stemness phenotypes, and activation of IKK, the upstream regulator of the transcription factor NF-kB. Integrating cell-based ChIP-sequencing with motif analysis and TCGA RNA-seq data, we identified a set of candidate NF-kB target genes upregulated with loss of DAB2IP linked with several oncogenic phenotypes, including altered RNA processing. This study provides insight into mechanisms associated with aggressiveness and recurrence within a subset of the typically less aggressive Luminal A breast cancer intrinsic subtype.
Authors
Angana Mukherjee, Rasha T. Kakati, Sarah C. Van Alsten, Tyler Laws, Aaron L. Ebbs, Daniel P. Hollern, Philip M. Spanheimer, Katherine A. Hoadley, Melissa A. Troester, Jeremy M. Simon, Albert S. Baldwin
Abstract
Rheumatoid Arthritis (RA) is an immune-mediated, chronic inflammatory condition. With modern therapeutics and evidence-based management strategies, achieving sustained remission is increasingly common. To prevent complications associated with prolonged use of immunosuppressants, drug tapering or withdrawal is recommended. However, due to the lack of tools that define immunological remission, disease flares are frequent, highlighting the need for a more precision medicine-based approach. Utilising high dimensional phenotyping platforms, we set out to define peripheral blood immunological signatures of sustained remission in RA. We identified that CD8+CD57+KIR2DL1+ NK cells are associated with sustained remission. Functional studies uncovered an NK cell subset characterized by normal degranulation responses and reduced pro-inflammatory cytokine expression, which was elevated in sustained remission. Furthermore, flow cytometric analysis of NK cells from synovial fluid combined with interrogation of a publicly available single cell RNA-seq dataset of synovial tissue from active RA identified a deficiency of the phenotypic characteristics associated with this NK cell remission signature. In summary, we have uncovered a novel RA remission signature associated with compositional changes in NK cell phenotype and function that has implications for understanding the impact of sustained remission on host immunity and distinct features which may define operational tolerance in RA.
Authors
Carl Coyle, Margaret Ma, Yann Abraham, Christopher B. Mahony, Kathryn Steel, Catherine Simpson, Nadia Guerra, Adam P. Croft, Stephen Rapecki, Andrew Cope, Rowann Bowcutt, Esperanza Perucha
Abstract
Human periosteal skeletal stem cells (P-SSCs) are critical for cortical bone maintenance and repair. However, their in vivo identity, molecular characteristics, and specific markers remain unknown. Here, single-cell sequencing revealed human periosteum contains SSC clusters expressing known SSC markers, PDPN and PDGFRA. Notably, human P-SSCs, but not bone marrow SSCs (BM-SSCs), selectively expressed newly identified markers, LRP1 and CD13. These LRP1+CD13+ human P-SSCs were perivascular cells with high osteochondrogenic but minimal adipogenic potential. Upon transplantation into bone injuries in mice, they preserved self-renewal capability in vivo. Single-cell analysis of mouse periosteum further supported the preferential expression of LRP1 and CD13 in Prx1+ P-SSCs. When Lrp1 was conditionally deleted in Prx1-lineage cells, it led to severe bone deformity, short statue, and periosteal defects. By contrast, local treatment with a LRP1 agonist at the injury sites induced early P-SSC proliferation and bone healing. Thus, human and mouse periosteum contains unique osteochondrogenic stem cell subsets, and these P-SSCs express specific markers, LRP1 and CD13, with regulatory mechanism through LRP1 that enhances P-SSC function and bone repair.
Authors
Youngjae Jeong, Lorenzo R. Deveza, Laura Ortinau, Kevin Lei, John R. Dawson, Dongsu Park
