Adoptive transfer of immune regulatory cells can prevent or ameliorate graft-versus-host disease (GVHD), which remains the main cause of non-relapse mortality after allogeneic hematopoietic stem-cell transplantation. Mucosal-associated invariant T cells (MAITs) were recently associated with tissue repair capacities and with lower rates of GVHD in humans. Here, we analyzed the immunosuppressive effect of MAITs in an in vitro model of alloreactivity and explored their adoptive transfer in a preclinical xenogeneic-GVHD model. We found that MAIT cells, whether freshly purified or shortly expanded, dose-dependently inhibited proliferation and activation of alloreactive T cells. In immunodeficient mice injected with human PBMCs, MAITs strongly delayed GVHD onset and severity when transferred early after PBMC injection, but could also control ongoing GVHD when transferred at delayed time points. This effect was associated with decreased proliferation and effector function of human T cells infiltrating tissues of diseased mice and was correlated with lower circulating IFN-γ and TNF-α levels, and increased IL-10 levels. MAITs acted partly in a contact-dependent manner, which likely required direct interaction of their TCR with MR1 induced on host-reactive T cells. These results support the setup of clinical trials using MAITs as universal therapeutic tools to control severe GVHD or mucosal inflammatory disorders.
Nana Talvard-Balland, Marion Lambert, Mathieu F. Chevalier, Norbert Minet, Marion Salou, Marie Tourret, Armelle Bohineust, Idan Milo, Véronique Parietti, Thomas Yvorra, Gérard Socié, Olivier Lantz, Sophie Caillat-Zucman
Rare diseases are underrepresented in biomedical research, leading to insufficient awareness. Zhu-Tokita-Takenouchi-Kim (ZTTK) syndrome is a rare disease caused by genetic alterations that result in heterozygous loss-of-function of SON. While ZTTK syndrome patients suffer from numerous symptoms, the lack of model organisms hampers our understanding of SON and this complex syndrome. Here, we developed Son haploinsufficiency (Son+/−) mice as a model of ZTTK syndrome and identified the indispensable roles of Son in organ development and hematopoiesis. Son+/− mice recapitulated clinical symptoms of ZTTK syndrome, including growth retardation, cognitive impairment, skeletal abnormalities, and kidney agenesis. Furthermore, we identified hematopoietic abnormalities in Son+/− mice, including leukopenia and immunoglobulin deficiency, similar to those observed in human patients. Surface marker analyses and single-cell transcriptome profiling of hematopoietic stem and progenitor cells revealed that Son haploinsufficiency shifts cell fate more toward the myeloid lineage but compromises lymphoid lineage development by reducing genes required for lymphoid and B-cell lineage specification. Additionally, Son haploinsufficiency causes inappropriate activation of erythroid genes and impaired erythropoiesis. These findings highlight the importance of the full gene expression of Son in multiple organs. Our model serves as an invaluable research tool for this rare disease and related disorders associated with SON dysfunction.
Lana Vukadin, Bohye Park, Mostafa Mohamed, Huashi Li, Amr Elkholy, Alex Torrelli-Diljohn, Jung-Hyun Kim, Kyuho Jeong, James M. Murphy, Caitlin A. Harvey, Sophia Dunlap, Leah Gehrs, Hanna Lee, Hyung-Gyoon Kim, Jay Prakash Sah, Seth N. Lee, Denise Stanford, Robert A. Barrington, Jeremy B. Foote, Anna G. Sorace, Robert S. Welner, Blake E. Hildreth III, Ssang-Taek Steve Lim, Eun-Young Erin Ahn
Pseudohypoparathyroidism type 1B (PHP1B) is caused by aberrant genomic imprinting at the GNAS gene. Defining the underlying genetic cause in new patients is challenging because various genetic alterations (e.g., deletions, insertions) within the GNAS genomic region, including the neighboring STX16 gene, can cause PHP1B, and the genotype-epigenotype correlation has not been clearly established. Here, by analyzing PHP1B patients with a wide variety of genotypes and epigenotypes, we identified a GNAS differentially methylated region (DMR) of distinct diagnostic value. This region, GNAS AS2, was hypomethylated in patients with genetic alterations located centromeric but not telomeric of this DMR. The AS2 methylation status was captured by a single probe of the methylation-sensitive multiplex ligation-dependent probe amplification (MS-MLPA) assay utilized to diagnose PHP1B. In human embryonic stem cells, where NESP55 transcription regulates GNAS methylation status on the maternal allele, AS2 methylation depended on two imprinting control regions (STX16-ICR and NESP-ICR) essential for NESP55 transcription. These results suggest that the AS2 methylation status in PHP1B patients reflects the position at which the genetic alteration affects NESP55 transcription during an early embryonic period. Therefore, AS2 methylation levels enable mechanistic PHP1B categorization based on genotype-epigenotype correlation and thus help identify the underlying molecular defect in patients.
Yorihiro Iwasaki, Monica Reyes, Harald Jüppner, Murat Bastepe
A robust sterile inflammation underlies myocardial ischemia and reperfusion injury (MIRI). Several subsets of B-cells possess the immune-regulatory capacity that limits tissue damage, yet the role of B-cells in MIRI remains elusive. Here, we sought to elucidate the contribution of B-cells to the MIRI by transient ligation of the left anterior descending in the B-cell depleted or deficient mice. Following ischemia and reperfusion (I/R), regulatory B-cells are rapidly recruited to the heart. B-cell-depleted or deficient mice exhibited exacerbated tissue damage, adverse cardiac remodeling, and an augmented inflammatory response after I/R. Rescue and chimeric experiments indicated that the cardioprotective effect of B-cells was not solely dependent on IL10. Coculture experiments demonstrated that B-cells induced neutrophil apoptosis through contact-dependent interaction, subsequently promoting reparative macrophage polarization by facilitating the phagocytosis of neutrophils by macrophages. The in-vivo cardioprotective effect of B-cells was absent in absence of neutrophils after I/R. Mechanistically, ligand-receptor imputation identified FCER2A as a potential mediator of interactions between B-cells and neutrophils. Blocking FCER2A on B-cells resulted in a reduction in the percentage of apoptotic neutrophils, contributing to the deterioration of cardiac remodeling. Our findings unveil a potential cardioprotective role of B-cells in myocardial I/R through mechanisms involving FCER2A, neutrophil, and macrophage.
Fangyang Huang, Jialiang Zhang, Hao Zhou, Tianyi Qu, Yan Wang, Kexin Jiang, Yutong Liu, Yuan Ning Xu, Mao Chen, Li Chen
Colon cancer affects people of all ages. However, its frequency, as well as the related morbidity and mortality, are high among older adults. The complex physiological changes in the aging gut substantially limit the development of cancer therapies. Here, we identify a unique intestinal microenvironment that is linked with an increased risk of colon cancer in older adults. Our findings show that aging markedly influences persistent fucosylation of the apical surfaces of intestinal epithelial cells, which results in a favorable environment for tumor growth. Furthermore, our findings shed light on the importance of the host-commensal interaction, which facilitates the dysregulation of fucosylation and promotes tumor growth as people get older. We analyzed colonic microbial populations at the species level to find changes associated with aging that could contribute to the development of colon cancer. Analysis of scRNAseq from previous publication datasets identifies distinct epithelial cell subtypes involved in dysregulated fucosylation in older adults. Overall, our study provides compelling evidence that excessive fucosylation is associated with the development of colon cancer, that age-related changes increase vulnerability to colon cancer, and that a dysbiosis in microbial diversity and metabolic changes in the homeostasis of older mice dysregulate fucosylation levels with age.
Zhihan Wang, Pan Gao, Kai Guo, Grace Schirrick, Jappreet Singh Gill, Jett Weis, Abby Lund Da Costa, Mansib Rahman, Het Mehta, Julia Fleecs, Shilpi Jain, Trishna Debnath, Junguk Hur, Nadeem Khan, Robert Sticca, Holly M. Brown-Borg, Donald A. Jurivich, Ramkumar Mathur
Regulatory T (Treg) cells can facilitate transplant tolerance and attenuate autoimmune- and inflammatory diseases. Therefore, it is clinically relevant to stimulate Treg cell expansion and function in vivo and to create therapeutic Treg cell products in vitro. We report that TNF receptor 2 (TNFR2) is a unique costimulus for naïve, thymus-derived (t)Treg cells from human blood that promotes their differentiation into non-lymphoid tissue (NLT)-resident effector Treg cells, without Th-like polarization. In contrast, CD28 costimulation maintains a lymphoid tissue (LT)-resident Treg cell phenotype. We base this conclusion on transcriptome and proteome analysis of TNFR2- and CD28-costimulated CD4+ tTreg cells and conventional T (Tconv) cells, followed by bioinformatic comparison with published transcriptomic Treg cell signatures from NLT and LT in health and disease, including autoimmunity and cancer. These analyses illuminated that TNFR2 costimulation promotes tTreg cell capacity for survival, migration, immunosuppression and tissue regeneration. Functional studies confirmed improved migratory ability of TNFR2-costimulated tTreg cells. Flow cytometry validated the presence of the TNFR2-driven tTreg cell signature in effector/memory Treg cells from the human placenta as opposed to blood. Thus, TNFR2 can be exploited as driver of NLT-resident tTreg cell differentiation for adoptive cell therapy or antibody-based immunomodulation in human disease.
Mark Mensink, Lotte J. Verleng, Ellen Schrama, George M.C. Janssen, Rayman T.N. Tjokrodirijo, Peter A. van Veelen, Qinyue Jiang, M. Fernanda Pascutti, Marie-Louise van der Hoorn, Michael Eikmans, Sander de Kivit, Jannie Borst
Hemorrhagic cystitis may be induced by infection, radiation therapy, medications, or may be idiopathic. Along with hemorrhagic features, symptoms include urinary urgency and frequency, dysuria (painful urination) and visceral pain. Cystitis-induced visceral pain is one of the most challenging types of pain to treat and an effective treatment would address a major unmet medical need. We assessed the efficacy of a purine nucleoside phosphorylase (PNPase) inhibitor, 8-aminoguanine (8-AG) for the treatment of hemorrhagic/ulcerative cystitis. Lower urinary tract (LUT) function and structure were assessed in adult Sprague Dawley rats, treated chronically with cyclophosphamide (CYP; sacrificed day 8) and randomized to daily oral treatment with 8-AG (begun 14 days prior to CYP induction) or its vehicle. CYP-treated rats exhibited multiple abnormalities including increased urinary frequency and neural mechanosensitivity, reduced bladder levels of inosine, urothelial inflammation/damage, and activation of spinal cord microglia, associated with pain hypersensitivity. 8-AG treatment of CYP-treated rats normalized all observed histological, structural, biochemical, and physiological abnormalities. In cystitis 8-AG improved function and reduced both pain and inflammation likely by increasing inosine, a tissue-protective purine metabolite. These findings demonstrate that 8-AG has translational potential for reducing pain and preventing bladder damage in cystitis-associated LUT dysfunctions.
Amanda Wolf-Johnston, Youko Ikeda, Irina V. Zabbarova, Anthony J. Kanai, Sheldon Bastacky, Robert Moldwin, Joel N.H. Stern, Edwin K. Jackson, Lori A. Birder
A distinct adipose tissue distribution pattern was observed in patients with methylmalonyl-CoA mutase deficiency, an inborn error of branched-chain amino acid (BCAA) metabolism, characterized by centripetal obesity with proximal upper and lower extremities fat deposition and paucity of visceral fat, that resembles familial multiple lipomatosis syndrome. To explore brown and white fat physiology in methylmalonic acidemia (MMA), body composition, adipokines and inflammatory markers were assessed in 46 MMA subjects and 99 matched controls. Fibroblast growth factor-21 (FGF21) levels were associated with acyl-coenzyme A accretion, aberrant methylmalonylation in adipose tissue, and an attenuated inflammatory cytokine profile. In parallel, brown and white fat were examined in a liver-specific transgenic MMA mouse model (Mmut-/-;TgINS-Alb-Mmut). The MMA mice exhibited abnormal non-shivering thermogenesis with whitened brown fat and had an ineffective transcriptional response to cold stress. Treatment of the MMA mice with bezafibrates led to clinical improvement with beiging of subcutaneous fat depots, which resembled the distribution seen in the patients. These studies defined what we believe to be a novel lipodystrophy phenotype in patients with defects in the terminal steps of BCAA oxidation and demonstrated that beiging of subcutaneous adipose tissue in MMA could readily be induced with small molecules.
Irini Manoli, Justin R. Sysol, PamelaSara E. Head, Madeline W. Epping, Oksana Gavrilova, Melissa K. Crocker, Jennifer L. Sloan, Stefanos A. Koutsoukos, Cindy X. Wang, Yiouli P. Ktena, Sophia Mendelson, Alexandra R. Pass, Patricia M. Zerfas, Victoria J. Hoffmann, Hilary J. Vernon, Laura A. Fletcher, James C. Reynolds, Maria G. Tsokos, Constantine A. Stratakis, Stephan D. Voss, Kong Y. Chen, Rebecca J. Brown, Ada Hamosh, Gerard T. Berry, Xiaoyuan Chen, Jack A. Yanovski, Charles P. Venditti
High grade serous carcinoma (HGSC) is the most lethal gynecological malignancy in the United States. Late diagnosis and the emergence of chemoresistance have prompted studies into how the tumor microenvironment, and more recently tumor innervation, may be leveraged for HGSC prevention and interception. In addition to biobehavioral sources, concentrations of the sympathetic neurotransmitter norepinephrine (NE) in the ovary increase during ovulation and after menopause. Importantly, NE exacerbates advanced HGSC progression. However, little is known about the role of NE in early disease pathogenesis. Here, we investigated the role of NE in instigating anchorage independence and micrometastasis of preneoplastic lesions from the fallopian tube epithelium (FTE) to the ovary, an essential step in HGSC onset. We found that in the presence of NE, FTE cell lines are able to survive in ultra-low attachment (ULA) culture in a beta-adrenergic receptor (β-AR)-dependent manner. Importantly, spheroid formation and cell viability conferred by treatment with physiological sources of NE can be abrogated using the beta-adrenergic receptor blocker propranolol. We have also identified that NE-mediated anoikis resistance may be attributable to downregulation of colony stimulating factor 2 (CSF2). These findings provide mechanistic insight and identify targets that may be regulated by ovarian-derived NE in early HGSC.
Hunter D. Reavis, Stefan M. Gysler, Grace B. McKenney, Matthew Knarr, Hannah J. Lusk, Priyanka Rawat, Hannah S. Rendulich, Marilyn A. Mitchell, Dara S. Berger, Jamie S. Moon, Suyeon Ryu, Monica Mainigi, Marcin P. Iwanicki, Dave S.B. Hoon, Laura M. Sanchez, Ronny Drapkin
Genetic modifications leading to pain insensitivity phenotypes are rare but can provide invaluable insights into the molecular biology of pain and reveal novel targets for analgesic drugs. Pain insensitivity typically results from Mendelian loss-of-function mutations in genes expressed in nociceptive (pain-sensing) dorsal root ganglion (DRG) neurons that connect the body to the spinal cord. We document a novel pain insensitivity mechanism arising from gene overexpression in individuals with the rare 7q11.23 duplication syndrome (Dup7), who have three copies of the approximately 1.5 megabase Williams syndrome (WS) critical region. Based on parental accounts and pain ratings, people with Dup7, mainly children in this study, are pain insensitive following serious injury to skin, bones, teeth, or viscera. In contrast, diploid siblings (two copies) and people with WS (one copy) show standard reactions to painful events. A converging series of human assessments and cross-species cell biological and transcriptomic studies identified one likely candidate in the WS critical region, STX1A, as underlying the pain insensitivity phenotype. STX1A codes for the synaptic vesicle fusion protein Syntaxin1A and neuropeptide release studies from nociceptive DRG neurons, show that excess syntaxin1A compromises exocytosis which when extrapolated to Dup7 individuals, produces a “genetic analgesia” and new potential routes to pain control.
Michael J. Iadarola, Matthew R. Sapio, Amelia J. Loydpierson, Carolyn B. Mervis, Jill C. Fehrenbacher, Michael R. Vasko, Dragan Maric, Daniel P. Eisenberg, Tiffany A. Nash, J. Shane Kippenhan, Madeline H. Garvey, Andrew J. Mannes, Michael D. Gregory, Karen F. Berman
Vascular calcification is a severe complication of cardiovascular diseases. Previous studies demonstrated that endothelial lineage cells transitioned into osteoblast-like cells and contributed to vascular calcification. Here, we found that inhibition of cyclin-dependent kinase (CDK) prevented endothelial lineage cells from transitioning to osteoblast-like cells and reduced vascular calcification. We identified a robust induction of CDK1 in endothelial cells (ECs) in calcified arteries and showed that endothelial-specific gene deletion of CDK1 decreased the calcification. We found that limiting CDK1 induced E-twenty-six specific sequence variant 2 (ETV2), which was responsible for blocking endothelial lineage cells from undergoing osteoblast differentiation. We also found that inhibition of CDK1 reduced vascular calcification in a diabetic mouse model. Together, the results highlight the importance of CDK1 suppression and suggest CDK1 inhibition as a potential option for treating vascular calcification.
Yan Zhao, Yang Yang, Xiuju Wu, Li Zhang, Xinjiang Cai, Jaden Ji, Sydney Chen, Abigail Vera, Kristina I. Boström, Yucheng Yao
Autosomal Dominant Polycystic Kidney Disease (ADPKD) is a monogenic disorder accounting for approximately 5% of patients with renal failure. Yet, therapeutics for the treatment of ADPKD remain limited. ADPKD tissues display abnormalities in the biogenesis of the centrosome, a defect that can cause genome instability, aberrant ciliary signaling, and secretion of pro-inflammatory factors. Cystic cells form excess centrosomes via a process termed centrosome amplification (CA), which causes abnormal multipolar spindle configurations, mitotic catastrophe, and reduced cell viability. However, cells with CA can suppress multipolarity via “centrosome clustering,” a key mechanism by which cells circumvent apoptosis. Here, we demonstrate that inhibiting centrosome clustering can counteract the proliferation of renal cystic cells with high incidences of CA. Using ADPKD human cells and mouse models, we show that preventing centrosome clustering with two inhibitors, CCB02 and PJ34, blocks cyst initiation and growth in vitro and in vivo. Inhibiting centrosome clustering activates a p53-mediated surveillance mechanism leading to apoptosis, reduced cyst expansion, interstitial fibrosis, and improved kidney function. Transcriptional analysis of kidneys from treated mice identified pro-inflammatory signaling pathways implicated in CA-mediated cystogenesis and fibrosis. Our results demonstrate that centrosome clustering is a cyst-selective target for the improvement of renal morphology and function in ADPKD.
Tao Cheng, Aruljothi Mariappan, Ewa Langner, Kyuhwan Shim, Jay Gopalakrishnan, Moe R. Mahjoub
SARS-CoV-2 spike-based vaccines are used to control the COVID-19 pandemic. However, emerging variants became resistant to antibody neutralization and further mutations may lead to full resistance. We tested whether T cells alone could provide protection without antibodies. We designed a T cell-based vaccine in which SARS-CoV-2 spike sequences were rearranged and attached to ubiquitin. Immunization of mice with the vaccine induced no specific antibodies but strong specific T cell responses. We challenged mice with SARS-CoV-2 wild-type strain or an Omicron variant after the immunization and monitored survival or viral titers in the lungs. The mice were significantly protected against death and weight loss caused by SARS-CoV-2 wild-type strain, and the viral titers in the lungs of mice challenged with SARS-CoV-2 wild-type or the Omicron variant were significantly reduced. Importantly, depletion of CD4+ or CD8+ T cells led to significant loss of the protection. Our analyses of spike protein sequences of the variants indicated that fewer than 1/3 presented by dominant HLA alleles were mutated and that most of the mutated epitopes were in subunit 1 region. As subunit 2 region is conservative, the vaccines targeting spike protein are expected to protect against future variants due to the T cell responses.
Juan Shi, Jian Zheng, Xiujuan Zhang, Wanbo Tai, Ryan Compas, Jack C. Deno, Natalie Jachym, Abhishek K. Verma, Gang Wang, Xiaoqing Guan, Abby E. Odle, Yushun Wan, Fang Li, Stanley Perlman, Liang Qiao, Lanying Du
Innate immune cells are important in the initiation and potentiation of alloimmunity in transplantation. Immediately upon organ anastomosis and reperfusion, recipient monocytes enter the graft from circulation and differentiate to inflammatory macrophages to promote allograft inflammation. However, factors that drive their differentiation to inflammatory macrophages are not understood. Here, we showed that the receptor tyrosine kinase AXL was a key driver of early intragraft differentiation of recipient infiltrating monocytes to inflammatory macrophages in the presence of allogeneic stimulation and cell-cell contact. In this context, the differentiated inflammatory macrophages were capable of efficient alloantigen presentation and allo-stimulation of T cells of the indirect pathway. Consequently, early and transient AXL inhibition with the pharmacological inhibitor bemcentinib resulted in a profound reduction of initial allograft inflammation and a significant prolongation of allograft survival in a murine heart transplant model. Our results support further investigation of AXL inhibition as part of an induction regimen for transplantation.
Collin Z. Jordan, Matthew Tunbridge, Irma Husain, Hiroki Kitai, Miriam E. Dilts, Olivia K. Fay, Koki Abe, Catherine Xiang, Jean Kwun, Tomokazu Souma, Edward B. Thorp, Xunrong Luo
BACKGROUND. T cell responses are impaired in Staphylococcus aureus-infected children, highlighting a potential mechanism of immune evasion. This study tested the hypotheses that toxin-specific antibodies protect immune cells from bacterial killing and are associated with improved T cell function following infection. METHODS.S. aureus-infected and healthy children (n = 33 each) were prospectively enrolled. During acute infection and convalescence, we quantified toxin-specific IgG levels by ELISA, antibody function using a cell-killing assay, and functional T cell responses by ELISpot. RESULTS. There were no differences in toxin-specific IgG levels or ability to neutralize toxin-mediated immune cell killing between healthy and acutely-infected children, but antibody levels and function increased following infection. Similarly, T cell function, which was impaired during acute infection, improved following infection. However, the response to infection was highly variable; up to half of children did not have improved antibody or T cell function. Serum from children with higher ɑ-hemolysin (Hla)-specific IgG levels more strongly protected immune cells against toxin-mediated killing. Importantly, children whose serum more strongly protected against toxin-mediated killing also had stronger immune responses to infection, characterized by more elicited antibody and greater improvement in T cell function following infection. CONCLUSIONS. This study demonstrates that, despite T cell impairment during acute infection, S. aureus elicits toxin-neutralizing antibodies. Individual antibody responses and T cell recovery are variable. These findings also suggest that toxin-neutralizing antibodies protect antigen-presenting cells and T cells, thereby promoting immune recovery. Finally, failure to elicit toxin-neutralizing antibodies may identify children at risk for prolonged T cell suppression. FUNDING. NIAID R01AI125489 and Nationwide Children’s Hospital.
Maureen Kleinhenz, Zhaotao Li, Usha V. Chidella, Walissa Picard, Amber Wolfe, Jill Popelka, Robin Alexander, Christopher P. Montgomery
Hypercapnia, elevation of the partial pressure of CO2 in blood and tissues, is a risk factor for mortality in patients with severe acute and chronic lung diseases. We previously showed that hypercapnia inhibits multiple macrophage and neutrophil antimicrobial functions, and that elevated CO2 increases the mortality of bacterial and viral pneumonia in mice. Here, we show that normoxic hypercapnia downregulates innate immune and antiviral gene programs in alveolar macrophages (AMØs). We also show that zinc finger homeobox 3 (Zfhx3), a mammalian ortholog of zfh2, which mediates hypercapnic immune suppression in Drosophila, is expressed in mouse and human macrophages. Deletion of Zfhx3 in the myeloid lineage blocked the suppressive effect of hypercapnia on immune gene expression in AMØs and decreased viral replication, inflammatory lung injury and mortality in hypercapnic mice infected with influenza A virus. Our results establish Zfhx3 as the first known mammalian mediator of CO2 effects on immune gene expression and lay the basis for future studies to identify therapeutic targets to interrupt hypercapnic immunosuppression in patients with advanced lung disease.
S. Marina Casalino-Matsuda, Fei Chen, Francisco J. Gonzalez-Gonzalez, Hiroaki Matsuda, Aisha Nair, Hiam Abdala-Valencia, G.R. Scott Budinger, Jin-Tang Dong, Greg J. Beitel, Peter H.S. Sporn
AMP-activated protein kinase (AMPK) plays a crucial role in maintaining ATP homeostasis in photoreceptor neurons. AMPK is a heterotrimeric protein consisting of alpha, beta, and gamma subunits. The independent functions of the two isoforms of the catalytic alpha subunit, PRKAA1 and PRKAA2, are uncharacterized in specialized neurons such as photoreceptors. Here we demonstrate in mice that rod photoreceptors lacking PRKAA2, but not PRKAA1, show altered levels of cGMP, GTP, and ATP, suggesting isoform-specific regulation of photoreceptor metabolism. Furthermore, PRKAA2 deficient mice display visual functional deficits on electroretinography and photoreceptor outer segment structural abnormalities on transmission electron microscopy consistent with neuronal dysfunction, but not neurodegeneration. Phosphoproteomics identified inosine monophosphate dehydrogenase (IMPDH) as a molecular driver of PRKAA2-specific photoreceptor dysfunction, and inhibition of IMPDH improved visual function in Prkaa2 rod photoreceptor knockout mice. These findings highlight a novel, therapeutically targetable PRKAA2 isoform-specific function of AMPK in regulating photoreceptor metabolism and function through a previously uncharacterized mechanism affecting IMPDH activity.
Tae Jun Lee, Yo Sasaki, Philip A. Ruzycki, Norimitsu Ban, Joseph B. Lin, Hung-Ting Wu, Andrea Santeford, Rajendra S. Apte
Circular RNAs (circRNAs) are highly expressed in the mammalian intestinal epithelium, but their functions remain largely unknown. Here we identified the circRNA Cdr1as as a repressor of intestinal epithelial regeneration and defense. Cdr1as levels increase in mouse intestinal mucosa after colitis and septic stress, as well as in human intestinal mucosa from patients with inflammatory bowel diseases and sepsis. Ablation of the Cdr1as locus from the mouse genome enhances renewal of the intestinal mucosa, promotes injury-induced epithelial regeneration, and protects the mucosa against colitis. We found approximately 40 microRNAs, including microRNA miR-195, differentially express between intestinal mucosa of Cdr1as knockout (–/–) versus littermate mice. Increasing the levels of Cdr1as inhibits intestinal epithelial repair after wounding in cultured cells and represses growth of intestinal organoids cultured ex vivo, but this inhibition is abolished by miR-195 silencing. The reduction in miR-195 levels in the Cdr1as–/– intestinal epithelium is the result of reduced stability and processing of the precursor miR-195. These findings indicate that Cdr1as reduces proliferation and repair of the intestinal epithelium at least in part via interaction with miR-195 and highlight a role for induced Cdr1as in the pathogenesis of unhealed wounds and disrupted renewal of the intestinal mucosa.
Hee Kyoung Chung, Lan Xiao, Naomi Han, Jason Chen, Vivian Yao, Cassandra M. Cairns, Benjamin Raufman, Jaladanki N. Rao, Douglas J. Turner, Rosemary Kozar, Myriam Gorospe, Jian-Ying Wang
The deposition of anti-podocyte auto-antibodies in the glomerular subepithelial space induces primary membranous nephropathy (MN), the leading cause of nephrotic syndrome worldwide. Taking advantage of the glomerulus-on-a-chip system, we modeled human primary MN induced by anti-PLA2R antibodies. Here we show that exposure of primary human podocytes expressing PLA2R to MN serum results in IgG deposition and complement activation on their surface, leading to loss of the chip permselectivity to albumin. C3a receptor (C3aR) antagonists as well as C3AR gene silencing in podocytes reduced oxidative stress induced by MN serum and prevented albumin leakage. In contrast, inhibition of the formation of the membrane-attack-complex (MAC), previously thought to play a major role in MN pathogenesis, did not affect permselectivity to albumin. In addition, treatment with a C3aR antagonist effectively prevented proteinuria in a mouse model of MN, substantiating the chip findings. In conclusion, using a combination of pathophysiologically relevant in vitro and in vivo models, we established that C3a/C3aR signaling plays a critical role in complement-mediated MN pathogenesis, indicating an alternative therapeutic target for MN.
Qi Zhang, Sofia Bin, Kelly L. Budge, Astgik Petrosyan, Valentina Villani, Paola Aguiari, Coralien H. Vink, Jack Wetzels, Hasmik Soloyan, Gaetano La Manna, Manuel Alfredo Podestà, Paolo Molinari, Sargis Sedrakyan, Kevin V. Lemley, Roger E. De Filippo, Laura Perin, Paolo Cravedi, Stefano Da Sacco
Functional avidity is supposed to critically shape the quality of immune responses, thereby impacting host protection against infectious agents including SARS-CoV2. Here we show that after human SARS-CoV2 vaccination, a large portion of high-avidity spike-specific CD4+ T cells lose CD3 expression after in vitro activation. The CD3- subset is enriched for cytokine positive cells, including elevated per-cell expression levels, and shows increased polyfunctionality. Assessment of key metabolic pathways by flow cytometry revealed that superior functionality is accompanied by a shift towards fatty acid-synthesis at the expense of their oxidation, whereas glucose transport and glycolysis were similarly regulated in SARS-CoV2-specific CD3- and CD3+ subsets. As opposed to their CD3+ counterparts, frequencies of vaccine-specific CD3- T cells positively correlate with both the size of the naïve CD4+ T cell pool and vaccine-specific IgG levels. Moreover, their frequencies negatively correlate with advancing age and are impaired in patients under immunosuppressive therapy. Typical recall-antigen-reactive T cells show a comparable segregation into functionally and metabolically distinct CD3+ and CD3- subsets, but are quantitatively maintained upon ageing, likely due to earlier recruitment in life. In summary, our data identify CD3- T helper cells as correlates of high quality immune responses that are impaired in at-risk populations.
Arne Sattler, Stefanie Gamradt, Vanessa Proß, Linda Marie Laura Thole, An He, Eva Vanessa Schrezenmeier, Katharina Jechow, Stefan M. Gold, Soeren Lukassen, Christian Conrad, Katja Kotsch