As representatives for our entire team, we thank Jhaveri et al. (1) for their insightful comments on our recent study investigating the increased expression of programmed cell death protein-1 (PD1) in kidneys during aging and FSGS.(2) In our manuscript we showed that PD1 was predominantly increased in podocytes and kidney tubular epithelial cells in both mice and humans. Moreover, in humans, age-elevated glomerular PCDC1 (gene encoding human PD1) levels were associated with a lower eGFR, increased segmental glomerulosclerosis, and reduced arterial intima-to-lumen ratio. We also demonstrated a mechanistic link between increased PD1 levels in podocytes and their shortened lifespan. Finally, specifically antagonizing the PD1 pathway with a specific anti-PD1 antibody (similar to humanized Pembrolizumab or Nivolumab) in aged mice and mice with experimental FSGS had major benefits on kidney histology, podocyte life- and health-span, and tubular epithelial injury.(2) In their response, Jhaveri and colleagues, experts in onco-nephrology, eloquently discuss the clinical kidney-specific adverse events (AEs) when using immune checkpoint inhibitors (ICI) in cancer patients.(1) They provide important clinical insights and an up-to-date summary of the incidence and types of glomerular lesions, acute kidney injury and acute interstitial nephritis observed in patients receiving ICI for cancer treatment.(3,4) Importantly, complete or partial remission of kidney-specific AEs upon discontinuation of ICI treatment in a subset of patients suggests a causal link.(3, 4) We unreservedly agree with Jhaveri et al. that caution is warranted when using ICI clinically. In fact, we have not advocated the clinical use of anti-PD1 treatment to limit or reverse kidney aging, nor to be used as a therapy for FSGS. The clinical data highlighted by Jhaveri et al. underscore the importance of gaining a better understanding of the mechanism(s) underlying kidney complications in patients. While T cell activation, proliferation and subsequent kidney infiltration is the leading hypothesis,(3, 4) how this cumulates into kidney dysfunction is unknown. ICIs block the CTLA-4 and/or PD1 pathways. CTLA-4 acts early in tolerance induction, stopping potentially autoreactive T cells at the initial stage of naive T-cell activation, while PD1 acts late to maintain long-term tolerance, primarily in peripheral tissues.(5) Typically a lower incidence of AEs is associated with PD1 blockade compared with CTLA-4 blockade.(3, 4) Interestingly, in our study mice Ctla4 mRNA levels in contrast to PD1 were not elevated in podocytes with age. There are also several differences between humans and mice that may influence the response to anti-PD1 treatments. To reconcile these, one needs to experimentally align the animal studies with the therapeutic scenario in human cancer patients. Possible considerations include: (i) the duration of therapy - in our study mice received 8 weeks of treatment, while human patients typically receive a 13-week median drug exposure before glomerular disease is first detected; (ii) the presence of comorbid conditions is oftentimes present in humans (e.g., patients receiving additional medications or already exhibiting altered kidney function before receiving ICI agents), but was absent in our mice; (iii) sex and age – the median age of patients developing glomerular disease after ICI treatment is 63 years and 75% thereof are male,(4) while our mouse study was based on males only; (iv) drug dosing – the therapeutic doses of ICIs used in humans might be much higher than the doses of the mouse-specific anti-PD1 antibody yielding beneficial effects in mouse podocytes; (v) finally, genetic variation in humans may influence the response to anti-PD1 treatments, while mice strains are genetically very homogenous. We believe that our study has provided some exciting new considerations that has moved us ahead scientifically. First, the PD1 signaling is a new pathway contributing to the aging of podocytes and other kidney epithelial cells, as well as the response of podocytes in disease. Second, podocyte aging and diseased-induced podocyte injury share a new common pathway – PD1. This raises the possibility that PD1 signaling is one of the pathways responsible for the more severe kidney injury when FSGS is superimposed on an aged kidney. Third, the effects of the anti-PD1 antibody treatment are not restricted to the kidney, but also reduced some aspects of liver aging. This suggests that it might be a common aging pathway, that needs to be studied further. Fourth, the unexpected discovery of PD1 signaling in aging leads us to predict that there will be additional surprises in new pathways contributing to kidney aging and disease that will translate into new druggable targets.
Stuart J. Shankland, Jeffrey W. Pippin, Oliver Wessely
22q11.2 deletion syndrome (22q11.2DS) is the most common human chromosomal microdeletion, causing developmentally linked congenital malformations; thymus hypoplasia, hypoparathyroidism and/or cardiac defects. Thymus hypoplasia leads to T cell lymphopenia, which most often results in mild SCID. Despite decades of research, the molecular underpinnings leading to thymus hypoplasia in 22q11.2DS remain unknown. Comparing embryonic thymuses from mouse models of 22q11.2DS (Tbx1neo2/neo2) revealed similar proportions of mesenchymal-, epithelial- and hematopoietic- cell types as controls. Yet, the small thymuses were growth restricted in fetal organ cultures. Replacement of Tbx1neo2/neo2 thymus mesenchymal cells with normal ones restored tissue growth. Comparative single cell RNA sequencing of embryonic thymuses uncovered 17 distinct cell subsets, with transcriptome differences predominant in the 5 mesenchymal subsets from the Tbx1neo2/neo2 line. Transcripts impacted include extracellular matrix (ECM) proteins, consistent with increased collagen deposition seen in the small thymuses. Attenuating collagen cross-links with minoxidil restored thymus tissue expansion for hypoplastic lobes. In colony forming assays, the Tbx1neo2/neo2-derived mesenchymal cells had reduced expansion potential, contrasting the normal growth of thymus epithelial cells. These findings suggest that mesenchymal cells are causal to the small embryonic thymuses in 22q11.2DS mouse models, correctable by substituting with normal mesenchyme.
Pratibha Bhalla, Qiumei Du, Ashwani Kumar, Chao Xing, Angela Moses, Igor Dozmorov, Christian A. Wysocki, Ondine B. Cleaver, Timothy J. Pirolli, Mary Louise Markert, M. Teresa de la Morena, Antonio Baldini, Nicolai S.C. van Oers
BACKGROUND. Anti-neutrophil cytoplasmic autoantibody (ANCA)-associated vasculitidies (AAV) are life-threatening systemic autoimmune conditions. ANCA directed against proteinase 3 (PR3) or myeloperoxidase (MPO) bind their cell surface-presented antigen, activate neutrophils and cause vasculitis. An imbalance between PR3 and its major inhibitor α1-antitrypsin (AAT) was proposed to underlie PR3- but not MPO-AAV. We measured AAT and PR3 in healthies and AAV patients and studied protective AAT effects pertaining to PR3- and MPO-ANCA. METHODS. Plasma and blood neutrophils were assessed for PR3 and AAT. Wild-type, mutant, and oxidation-resistant AAT species were produced to characterize AAT-PR3 interactions by flow cytometry, immunoblotting, FRET assays, and surface plasmon resonance measurements. Neutrophil activation was measured using the ferricytochrome C assay and AAT methionine-oxidation by Parallel Reaction Monitoring. RESULTS. We found significantly increased PR3 and AAT pools in both PR3- and MPO-AAV patients, however, only in PR3-AAV did the PR3 pool correlate with ANCA titer, inflammatory response and disease severity. Mechanistically, AAT prevented PR3 from binding to CD177, thereby reducing neutrophil surface antigen for ligation by PR3-ANCA. Active PR3-AAV patients showed critical methionine-oxidation in plasma AAT that was recapitulated by ANCA-activated neutrophils. The protective PR3-related AAT effects were compromised by methionine-oxidation in the AAT reactive center loop but preserved when two critical methionines were substituted by valine and leucine. CONCLUSION. Pathogenic differences between PR3- and MPO-AAV are related to AAT regulation of membrane-PR3, attenuating neutrophil activation by PR3- rather than MPO-ANCA. Oxidation-resistant AAT could serve as adjunctive therapy in PR3-AAV.
Maximilian J.P. Ebert, Uwe Jerke, Claudia Eulenberg-Gustavus, Lovis Kling, Dieter E. Jenne, Marieluise Kirchner, Philipp Mertins, Markus Bieringer, Saban Elitok, Kai-Uwe Eckardt, Adrian Schreiber, Alan D. Salama, Ralph Kettritz
A fundamental issue in regenerative medicine is whether there exist endogenous regulatory mechanisms that limit the speed and efficiency of the repair process. We report the existence of a maturation checkpoint during muscle regeneration which pauses myofibers at a neonatal stage. This checkpoint is regulated by the mitochondrial protein mitofusin 2 (Mfn2), whose expression is activated in response to muscle injury. Mfn2 is required for growth and maturation of regenerating myofibers; in the absence of Mfn2, new myofibers arrested at a neonatal stage, characterized by centrally nucleated myofibers and loss of H3K27me3 repressive marks at the neonatal myosin heavy chain gene. A similar arrest at the neonatal stage was observed in infantile cases of human centronuclear myopathy. Mechanistically, Mfn2 upregulation suppressed expression of Hypoxia-induced Factor 1α (Hif1α), which is induced in the setting of muscle damage. Sustained Hif1α signaling blocked maturation of new myofibers at the neonatal-to-adult fate transition, revealing the existence of a checkpoint that delays muscle regeneration. Correspondingly, inhibition of Hif1α allowed myofibers to bypass the checkpoint, thereby accelerating the repair process. We conclude that skeletal muscle contains a regenerative checkpoint which regulates the speed of myofiber maturation in response to Mitofusin 2 and Hif1α activity.
Xun Wang, Yuemeng Jia, Jiawei Zhao, Nicholas P. Lesner, Cameron J. Menezes, Spencer D. Shelton, Siva Sai Krishna Venigalla, Jian Xu, Chunyu Cai, Prashant Mishra
Pediatric high-grade gliomas (pHGGs) are the leading cause of cancer-related deaths in children in the USA. Sixteen percent of hemispheric pediatric and young adult HGGs encode Gly34Arg/Val substitutions in the histone H3.3 (H3.3-G34R/V). The mechanisms by which H3.3-G34R/V drive malignancy and therapeutic resistance in pHGGs remain unknown. Using a syngeneic, genetically engineered mouse model (GEMM) and human pHGG cells encoding H3.3-G34R, we demonstrate that this mutation leads to downregulation of the DNA repair pathways. This leads to enhanced susceptibility to DNA damage and inhibition of the DNA damage response (DDR). We demonstrate that genetic instability resulting from improper DNA repair in G34R-mutant pHGG leads to accumulation of extrachromosomal DNA, which activates the cGAS-STING pathway, inducing the release of immune-stimulatory cytokines. We treated H3.3-G34R pHGG-bearing mice with a combination of radiotherapy (RT) and DNA damage response inhibitors (DDRi) (i.e., the blood-brain barrier permeable PARP inhibitor, pamiparib, and the cell cycle checkpoint CHK1/2 inhibitor, AZD7762), and these combinations resulted in approximately 50% long-term survivors. Moreover, the addition of a STING agonist (diABZl) enhanced the therapeutic efficacy of these treatments. Long-term survivors developed immunological memory, preventing pHGG growth upon rechallenge. These results demonstrate that DDRi and STING agonists in combination with RT induce immune-mediated therapeutic efficacy in G34-mutant pHGG.
Santiago Haase, Kaushik Banerjee, Anzar A. Mujeeb, Carson S. Hartlage, Fernando M. Nunez, Felipe J. Nuñez, Mahmoud S. Alghamri, Padma Kadiyala, Stephen Carney, Marcus Barissi, Ayman W. Taher, Emily K. Brumley, Sarah Thompson, Justin T. Dreyer, Caitlin T. Alindogan, Maria B. Garcia-Fabiani, Andrea Comba, Sriram Venneti, Visweswaran Ravikumar, Carl Koschmann, Angel M. Carcaboso, Maria Vinci, Arvind Rao, Jennifer S. Yu, Pedro R. Lowenstein, Maria G. Castro
BACKGROUND. Cytochrome P450 Family 8 Subfamily B Member 1 (CYP8B1) generates 12α-hydroxylated bile acids (BAs) which were associated with insulin resistance in humans. METHODS. To determine if reduced CYP8B1 activity improves insulin sensitivity, we sequenced CYP8B1 in individuals without diabetes and identified carriers of complete loss-of-function (CLOF) mutations utilizing functional assays. RESULTS. Mutation carriers had lower plasma 12α-hydroxylated:non-12α-hydroxylated BA and cholic acid (CA):chenodeoxycholic acid (CDCA) ratios compared to age-, gender- and BMI-matched controls. During insulin clamps, hepatic glucose production was suppressed to a similar magnitude by insulin, but glucose infusion rates to maintain euglycemia were higher in mutation carriers, indicating increased peripheral insulin sensitivity. Consistently, a polymorphic CLOF CYP8B1 mutation associated with lower fasting insulin in the AMP-T2D-GENES study. Exposure of primary human muscle cells to carrier CA:CDCA ratios demonstrated increased FOXO1 activity, and upregulation of both insulin signaling and glucose uptake, which were mediated by increased CDCA. Inhibition of FOXO1 attenuated the CDCA-mediated increase in muscle insulin signaling and glucose uptake. We find that reduced CYP8B1 activity associates with increased insulin sensitivity in humans. CONCLUSION. Our findings suggest that increased circulatory CDCA due to reduced CYP8B1 activity increases skeletal muscle insulin sensitivity, contributing to increased whole-body insulin sensitization. FUNDING. This study was funded by BMRC/NMRC Bench and Bedside grant (BnB13Dec011) to HCT and RRS.
Shiqi Zhong, Raphael Chevre, David Castaño Mayan, Maria Corlianò, Blake J. Cochran, Kai Ping Sem, Theo H. van Dijk, Jianhe Peng, Liang Juin Tan, Siddesh V. Hartimath, Boominathan Ramasamy, Peter Cheng, Albert K. Groen, Folkert Kuipers, Julian L. Goggi, Chester Drum, Rob M. van Dam, Ru-San Tan, Kerry-Anne Rye, Michael R. Hayden, Ching-Yu Cheng, Shaji Chacko, Jason Flannick, Xueling Sim, Hong Chang Tan, Roshni R. Singaraja
Cell surface receptors, ligands and adhesion molecules underlie development, circuit formation and synaptic function of the central nervous system and represent important therapeutic targets for many neuropathologies. The functional contributions of interactions between cell surface proteins of neurons and non-neuronal cells have not been fully addressed. Using an unbiased protein-protein interaction screen, we showed that the human immunomodulatory ligand, B7-1 (hB7-1), interacts with the p75 neurotrophin receptor (p75NTR), and that the B7-1:p75NTR interaction is a recent evolutionary adaptation present in humans and other primates, but absent in mice, rats, and other lower mammals. The surface of hB7-1 that engages p75NTR overlapped with the hB7-1 surface involved in CTLA-4/CD28 recognition, and these molecules directly competed for binding to p75NTR. Soluble or membrane bound hB7-1 altered dendritic morphology of cultured hippocampal neurons, with loss of the postsynaptic protein PSD95 in a p75NTR-dependent manner. Abatacept, an FDA-approved therapeutic (CTLA-4-hFc fusion) inhibited these processes. In vivo injection of hB7-1 into the murine subiculum, a hippocampal region affected in Alzheimer’s Disease, resulted in p75NTR-dependent pruning of dendritic spines. Collectively, we have reported the biochemical interaction between B7-1 and p75NTR, described biological effects on neuronal morphology, and identified a therapeutic opportunity for treatment of neuroinflammatory diseases.
Nicholas C. Morano, Roshelle S. Smith, Victor Danelon, Ryan Schreiner, Uttsav Patel, Natalia G. Herrera, Carla Smith, Steven M. Olson, Michelle K. Laerke, Alev Celikgil, Scott J. Garforth, Sarah C. Garrett-Thomson, Francis S. Lee, Barbara L. Hempstead, Steven C. Almo
A major complication of hemophilia A therapy is the development of alloantibodies (inhibitors) that neutralize intravenously administered coagulation factor VIII (FVIII). Immune tolerance induction therapy (ITI) by repetitive FVIII injection can eradicate inhibitors, and thereby reduce morbidity and treatment costs. However, ITI success is difficult to predict and the underlying immunological mechanisms are unknown. Here, we demonstrated that immune tolerance against FVIII under non-hemophilic conditions was maintained by programmed death (PD) ligand 1 (PD-L1)-expressing regulatory T cells (Treg) that ligated PD-1 on FVIII-specific B cells, causing them to undergo apoptosis. FVIII-deficient mice injected with FVIII lacked such Treg and developed inhibitors. Using an ITI mouse model, we found that repetitive FVIII injection induced FVIII-specific PD-L1+ Tregs and re-engaged removal of inhibitor-forming B cells. We demonstrated the existence of FVIII-specific Tregs also in humans and showed that such Tregs upregulated PD-L1 after successful ITI. Simultaneously, FVIII-specific B cells upregulated PD-1 and became killable by Tregs. In summary, we showed that PD-1-mediated B cell tolerance against FVIII operated in healthy individuals and in hemophilia A patients without inhibitors, and that ITI re-engaged this mechanism. These findings may impact monitoring of ITI success and treatment of hemophilia A patients.
Janine Becker-Gotot, Mirjam Meissner, Vadim Kotov, Blanca Jurado-Mestre, Andrea Maione, Andreas Pannek, Thilo Albert, Chrystel Flores, Frank A. Schildberg, Paul A. Gleeson, Birgit M. Reipert, Johannes Oldenburg, Christian Kurts
The stomach corpus epithelium is organized into anatomical units that consist of glands and pits and contain different specialized secretory cells. Acute and chronic injury of the corpus are associated with characteristic changes of cellular differentiation and proliferation. Processes that control cellular differentiation under homeostatic conditions and upon injury are not well understood. R-spondin 3 (Rspo3) is a Wnt signalling enhancer secreted by gastric stromal cells, which controls stem cell homeostasis in different organs. Here we investigated the function of Rspo3 in the corpus during homeostasis, acute injury, and H. pylori infection. Using organoid culture and conditional mouse models, we demonstrate that RSPO3 is a critical driver of secretory cell differentiation in the corpus gland towards parietal and chief cells, while its absence promoted pit cell differentiation. Acute loss of chief and parietal cells induced by high dose tamoxifen - or merely the depletion of LGR5+ chief cells – caused an upregulation of RSPO3 expression, which was required for the initiation of a coordinated regenerative response via the activation of yes-associated protein (YAP) signaling. This response enabled a rapid recovery of the injured secretory gland cells. However, in the context of chronic H. pylori infection, the R-spondin-driven regeneraton was maintained long-term, promoing severe glandular hyperproliferation and the development of premalignant metaplasia.
Anne-Sophie Fischer, Stefanie Müllerke, Alexander Arnold, Julian Heuberger, Hilmar Berger, Manqiang Lin, Hans-Joachim Mollenkopf, Jonas Wizenty, David Horst, Frank Tacke, Michael Sigal
Spinal muscular atrophy (SMA) is a neuromuscular disorder due to degeneration of spinal cord motor neurons caused by the deficiency of the ubiquitously expressed SMN protein. Here, we present a retinal vascular defect in patients, recapitulated in SMA transgenic mice, driven by failure of angiogenesis and maturation of blood vessels. Importantly, the retinal vascular phenotype was rescued by early, systemic SMN restoration therapy in SMA mice. We also demonstrate in patients an unfavourable imbalance between endothelial injury and repair, as indicated by increased circulating endothelial cell counts and decreased endothelial progenitor cell counts in blood circulation. The cellular markers of endothelial injury were associated with disease severity and improved following SMN restoration treatment in cultured endothelial cells from patients. Finally, we demonstrated autonomous defects in angiogenesis and blood vessel formation, secondary to SMN deficiency in cultured human and mouse endothelial cells, as the underlying cellular mechanism of microvascular pathology. Our cellular and vascular biomarkers findings indicate microvasculopathy as a fundamental feature of SMA. Our findings provide mechanistic insights into previously described SMA microvascular complications, and highlight the functional role of SMN in the periphery, including the vascular system, where deficiency of SMN can be addressed by systemic SMN-restoring treatment.
Haiyan Zhou, Ying Hong, Mariacristina Scoto, Alison Thomson, Emma Pead, Tom MacGillivray, Elena Hernandez-Gerez, Francesco Catapano, Jinhong Meng, Qiang Zhang, Gillian Hunter, Hannah K. Shorrock, Thomas K. Ng, Abedallah Hamida, Mathilde Sanson, Giovanni Baranello, Kevin Howell, Thomas H. Gillingwater, Paul Brogan, Dorothy A. Thompson, Simon H. Parson, Francesco Muntoni
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